responses of the halophyte atriplex nummularia to non ... · seasonally but the salinity of the...

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Responses of the halophyte Atriplex nummularia to non-uniform salinities in the root-zone

Nadia Bazihizina

This thesis is presented for the degree of Doctor of Philosophy

School of Plant Biology

Faculty of Natural and Agricultural Sciences

The University of Western Australia

July 2010

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Abstract

Salinity of the soil solution in soils growing halophytes can reach extreme values, but

salinity is often not uniform across sites. There is a surprising deficiency in knowledge

of halophyte physiology under non-uniform salinity; almost all experiments have

applied uniform root-zone treatments. Atriplex nummularia was used in this thesis as a

model halophytic plant to elucidate responses to non-uniform salinity in the root-zone.

A split-root system with two pots was used to expose roots of plants for 21 days to

either uniform or two different levels of salinity. The aims were to: (a) determine how

growth, water and ion relations are affected in plants subject to laterally non-uniform

moderate to extreme (up to 1500 mM NaCl) salinity; (b) understand water uptake

patterns, and in particular determine whether water uptake from the high-salt side is

maintained; (c) evaluate under non-uniform salinities whether various physiological

parameters (shoot and root growth, stomatal conductance, water relations, and Na+, K+

and Cl- concentrations in leaves) are determined mostly by the low- or high-salt sides,

or an average of the two salt concentrations in the root-zone.

Overall, the research shows that A. nummularia was able to grow with up to 1500 mM

NaCl in one root half, a level that when uniform in the root-zone completely inhibited

shoot and root growth. Growth (shoot elongation and ethanol-insoluble dry mass), leaf

gas exchange and leaf Na+ and Cl- concentrations responded to the ‘root-weighted

average’ salinity of the root-zone (i.e. mean NaCl concentration ‘root-weighted’ for root

ethanol-insoluble dry mass in the low and high-salt sides). As a consequence of A.

nummularia having optimal growth in the 10–400 mM NaCl range, when the ‘root-

weighted average’ salinity in the root-zone was in the 120 to 340 mM NaCl range, shoot

and root growth were both similar to that of control plants with 10 mM NaCl in both

root halves. Interestingly, as compensatory root growth in the 10 mM NaCl side (40%

increase in ethanol-insoluble dry mass) was observed when the high-salt side contained

1500 mM NaCl, the root-weighted average salinity (316 mM) in the root-zone was still

within the optimal zone, and therefore growth was similar to that of control plants

(uniform 10 mM NaCl). This split-root treatment contrasted markedly to plants with

uniform 1500 mM NaCl in the root-zone, for which there was no ethanol-insoluble dry

mass increase and most leaves showed chlorosis by 21 days.

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One parameter that did not respond to the root-weighted average salinity of the root-

zone was shoot water potential. Under non-uniform salinity plants took up water mostly

from the low-salt side and, consequently, midday shoot water potentials under non-

uniform conditions resembled those of plants grown under uniform conditions at the

lower salinity level. Despite taking up most water from the low-salt side, there was a

small amount of water uptake from high-salt side (% total water uptake: 10-15% with

10–120 mM in the low-salt side; 22-23% with 230–450 mM NaCl in the low-salt side).

This water uptake from the high-salt side occurred despite shoot water potential being

up to 1 MPa higher (i.e. less negative) than the osmotic potential of the external solution

on the high-salt side. With a simple water uptake model, it was hypothesized that this

water uptake could be related to a decline in xylem osmotic potentials (due to solute

accumulation) and/or decreases in root reflection coefficients.

Overall, A. nummularia was able to maintain growth with one root half exposed to low

salinity, even when the other half was exposed to 1500 mM NaCl, a level that prevents

growth and damages tissues when at uniform concentrations in the root-zone. As A.

nummularia, like most dicotyledonous halophytes, showed optimal growth in the 10-

400 mM NaCl range, if the root-weighted average salinities in the root-zone are within

that range, plants can express optimal growth even with severe salinities in one root

half. Given the intrinsic heterogeneity of saline landscapes, preferential root growth and

water uptake in less saline areas could potentially explain the presence of halophytic

vegetation on sites even with high to extreme areas of soil salinity. This study adds to

the knowledge of halophyte physiology, which has previously been studied over a range

of uniform salinities in the root-zone.

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Acknowledgments

Firstly, I would like to thank my supervisors Tim Colmer and Ed Barrett-Lennard for

their excellent guidance during my PhD. Thank you for encouraging me and providing

critical advice on my experiments during these last years and carefully editing this

thesis. All your help and enthusiasm made possible for me to initiate the exploitation of

the “gold mine” of non-uniform salinity. Thank you also for all the assistance with the

administrative requirements, especially to obtain my SIRF scholarship, and throughout

my PhD. Thank you Ed also for your help in the use the ‘explosive’ pressure bomb.

I am grateful to the Endeavour Europe Award, and the SIRF scholarship funded by the

Faculty of Natural and Agricultural Sciences, the School of Plant Biology and the

ARWA Center for Ecohydrology. I thank, for the provision of operating funds for my

research, the ARWA Center for Ecohydrology and School of Plant Biology. Thanks

also to the Future Farm Industries CRC for professional development and financial

support for my overseas conference travel. Conference travel funds have also been

provided by the Australian Society of Plant Scientists travel award, School of Plant

Biology travel award and the Graduate Research School travel award.

Special thanks to Em. Prof. Hank Greenway for all his advice and long discussions on

water uptake and hydraulic redistribution. Thanks to Prof. Erik Veneklaas for the

technical advice given and discussions when drafting the protocol for the experiment on

the hydraulic redistribution. Thanks to Dr. Simone Godoi for her assistance with the

hydraulic redistribution experiment, and mostly for surviving with me those predawn

water potential measurements. Thanks also to Dr. Grzegorz Skrzypek for providing the

methods and equations to calculate stable isotope enrichment, and for the stable isotope

deuterium measurements in the UWA Stable Isotope Center. Thanks to Christiane

Ludwig at CSIRO for assisting me with the use of the dew-point osmometer.

Special thanks also to Eli Bradbury, Lalith Suriyagoda, and Srinivan Samineni, for their

help during my various plant harvests. Thanks to Meir Altman for teaching me how to

prepare and grow saltbush cuttings. Thanks to Sarah Rich for taking care of my plants

during my holidays. Special thanks also to Prof. Hans Lambers, the Plant Biology office

staff, and the Graduate Research School staff, for assisting me with all the

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administrative requirements of my PhD. Thanks to Gary, Elizabeth, Hai, Perry and all

the glasshouse staff for their assistance for my experimental work.

Thank you all the past (Imran, Ghazi, Kirsten) and present members of Tim Colmer’s

Laboratory (too many to name) for assisting me to learn the new equipment and

techniques, and for many fun discussions. I feel very privileged to have worked in such

an enthusiastic group of people. Special thanks to past (Raphael, Eli, Sanjhuta) and

present students (Brian, Fazilah, Srinivasan) with whom I shared the office and pleasant

hours in the weekends. Thanks also to Alea, Elefteria, Leida, Kongit, Sara, Raphael and

Marie for all the hours spent together drinking coffees and sharing with me all the high

and lows of these years.

Thanks to my Mum and Dad, my “European donors”, for supporting me in many

countless ways and being there when I most needed them. Finally, but by no means the

least, thanks to David for putting up with me in these years, the numerous trips to and

from the university and for all the time spent helping me during this period. Without his

support, patience and encouragement, it would not have been possible for me to finish

my PhD.

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Table of contents

Content ........................................................................................................ Start page #

Abstract ......................................................................................................................... iii

Acknowledgments .......................................................................................................... v

Table of contents .......................................................................................................... vii

List of Figures................................................................................................................. x

List of Tables............................................................................................................... xiii

Thesis Declaration .........................................................................................................xv Chapter 1: General Introduction ................................................................................. 1

1.1 Salinity in the field .......................................................................................... 2

1.2 Thesis outline and aims ................................................................................... 3

Chapter 2: Literature Review ...................................................................................... 5

2.1 Introduction..................................................................................................... 6

2.2 Heterogeneity in saline soils ............................................................................ 6

2.3 Plant responses to non-uniform salinities....................................................... 11

2.3.1. Root growth............................................................................................... 11

2.3.2. Water uptake ............................................................................................. 14

2.3.3. Shoot growth, water and ion relations under non-uniform salinity.............. 17

2.3.3.1. Shoot growth ......................................................................................... 17

2.3.3.2. Water and ion relations .......................................................................... 18

2.3.3.3. Stomatal conductance and long distance signalling ................................ 21

2.4 Salinity tolerance of Atriplex nummularia..................................................... 24

2.4.1. Growth under uniform salinities ................................................................24

2.4.2. Ion relations and osmotic adjustment with uniform salinities ..................... 26

2.4.3. Leaf gas exchange ..................................................................................... 28

2.5 Summary....................................................................................................... 30

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Chapter 3: Responses to Moderate to Severe Non-uniform Salinity: Growth,

Stomatal Conductance, Water and Ion Relations...................................................... 33

3.1 Abstract ......................................................................................................... 34

3.2 Introduction................................................................................................... 34

3.3 Materials and Methods................................................................................... 36

3.4 Results........................................................................................................... 42

3.5 Discussion ..................................................................................................... 50

Chapter 4: Responses to Extreme Non-Uniform Salinity: Compensatory Root

Growth in, and Preferential Water Uptake from, the Least Saline Side .................. 57

4.1 Abstract ......................................................................................................... 58

4.2 Introduction................................................................................................... 58


4.4 Results........................................................................................................... 66

4.5 Discussion ..................................................................................................... 77

Chapter 5: Effects of Increasing the Salinity on the Low-Salt Side: Most Plant

Physiological Parameters Respond to the Mean Salinity of the Root-Zone.............. 83

5.1 Abstract ......................................................................................................... 84

5.2 Introduction................................................................................................... 85


5.4 Results........................................................................................................... 93

5.5 Discussion ................................................................................................... 108

Chapter 6: Concluding Discussion ........................................................................... 117

6.1 Summary of key findings............................................................................. 118

6.1.1. Plant responses to non-uniform salinity in the root-zone .......................... 118

6.1.2. Ion relations under non-uniform salinities ................................................ 119

6.1.3. Water uptake and potential gradients under non-uniform salinity ............. 122

6.1.4. Growth under uniform salinities............................................................... 125

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6.2 Implications of the thesis for saltland capability assessment .........................128

6.3 Limitations and future studies.......................................................................129

6.4 Conclusion ...................................................................................................132

Literature Cited .........................................................................................................133

Appendix: Hydraulic redistribution in Atriplex nummularia under non-uniform

salinities......................................................................................................................155

A.1 Introduction..................................................................................................156

A.2 Materials and Methods .................................................................................157

A.3 Results and Discussion ................................................................................161

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List of Figures Figure 2.1. Soil water content (% dry mass) and Cl- (% dry mass) changes with seasons (summer vs. winter) and depth in the soil profile……………………………………... 10 Figure 2.2. Water sources of Rhizophora mangle, Sesuvium portulacastrum and Cladium jamaicense in an ecotone where the salinity of the shallow water varies seasonally but the salinity of the water deeper in the soil profile remains more constant between seasons………………………………………………………………………...20 Figure 2.3. Growth response of Atriplex nummularia to increasing uniform NaCl in the root-zone (% dry mass compared to plants growing in 0 mM NaCl) ………………….29 Figure 3.1. Schematic diagram of the split-root system used in this study …………....41 Figure 3.2. Responses of ethanol-insoluble dry mass of shoots and roots of Atriplex nummularia with uniform or non-uniform NaCl in the root-zone…………………..…45 Figure 3.3. Responses of net photosynthetic rate and stomatal conductance of the young fully expanded leaves of Atriplex nummularia with uniform or non-uniform NaCl in the root-zone…………………………………………………………………………...…...46 Figure 3.4. Responses of shoot predawn water potential, osmotic potential of expressed sap of expanding, and expanded, leaves of Atriplex nummularia with uniform or non-uniform NaCl in the root-zone …………………………………………………………48 Figure 3.5. Concentrations on a tissue water basis (mM) of Na+, K+ and Cl- of different plant parts of Atriplex nummularia grown with uniform or non-uniform salinity in the root-zone……………………...………………………………………………………...54 Figure 4.1. Responses to uniform and non-uniform NaCl treatments in the root-zone of shoot extension, leaf area, and the ethanol-insoluble dry mass of the shoot, entire root system and roots in each side of the split-root system…………………………..……..69 Figure 4.2. Responses to uniform and non-uniform NaCl treatments in the root-zone of net photosynthetic rate, stomatal conductance, intercellular CO2 concentration and maximum quantum efficiency of the PSII of young fully expanded leaves of Atriplex nummularia……………………………………………………………………………..70 Figure 4.3. Concentration of total soluble sugars (hexose equivalents) on a tissue water basis in the young fully expanded leaves of Atriplex nummularia exposed for 0 and 21 days to uniform and non-uniform NaCl treatments in the root-zone………………..…71 Figure 4.4. Responses to uniform and non-uniform NaCl treatments in the root-zone of midday shoot water potential, osmotic potential of expressed sap and leaf water content of young fully expanded leaves of Atriplex nummularia……………………..………..74 Figure 4.5. Responses to uniform and non-uniform NaCl treatments in the root-zone of concentrations on a tissue water basis (mM) of Na+, K+, and Cl- of the young fully expanded leaves of Atriplex nummularia.…………………………………..………….75

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Figure 5.1. Responses of shoot growth parameters of Atriplex nummularia to uniform or non-uniform NaCl treatments: shoot extension, leaf area, and shoot ethanol-insoluble dry mass………………………………………………………………………………...97 Figure 5.2. Responses of root ethanol-insoluble dry mass of Atriplex nummularia to uniform or non-uniform NaCl treatments: entire root system, and roots in each side of the non-uniform treatments…………………………………………………………….98 Figure 5.3. Responses of leaf gas exchange parameters of Atriplex nummularia to uniform or non-uniform NaCl treatments: net photosynthetic rate, and stomatal conductance, both for young fully expanded leaves…………………………………..101 Figure 5.4. Concentration of total soluble sugars (tissue water basis) in young fully expanded leaves of Atriplex nummularia exposed to uniform or non-uniform NaCl treatments…………………………………..………………………………………….102 Figure 5.5. Responses of shoot water relations parameters in Atriplex nummularia to uniform or non-uniform NaCl treatments: midday shoot water potential, osmotic potential of expressed leaf sap including salt bladders, and leaf water content…...….103 Figure 5.6. Ion concentrations (tissue water basis) in Atriplex nummularia exposed to uniform or non-uniform NaCl treatments: Na+, K+, and Cl-, all in young fully expanded leaves………………………………..………………………………………………...105 Figure 5.7. Responses of plant water uptake by Atriplex nummularia to uniform 670 mM NaCl or non-uniform NaCl treatments in the root-zone: whole-plant water uptake, and water uptake rate on a root surface area basis from the low and high-salt sides…………………………………………………………………………………...106 Figure 5.8. Schematic diagram of the pathway of water flow from the external medium to the xylem across a plant root………………………………………………..……...115 Figure 5.9. Relationship between Jvtotal/Lp, root reflection coefficients and xylem osmotic potential……………………………………………………………………....116 Figure 6.1. Responses to different methods of expressing salinity in the root-zone of: whole plant ethanol-insoluble dry mass and stomatal conductance expressed as % of the uniform 10 mM NaCl treatment in Atriplex nummularia exposed to uniform or non-uniform NaCl treatments………………………………………...……………………121 Figure 6.2. Growth response curve (whole plant ethanol-insoluble expressed as % of the uniform 10 mM NaCl) of Atriplex nummularia exposed to uniform NaCl treatments……………………………………………………………………………...127 Figure A1. Calculated water movement from the side where roots were exposed to deuterium enriched solution to the other sides where roots where exposed to non-enriched solution in Atriplex nummularia grown for 21 days in a split-root system…164 Figure A2. Changes in soil water potential in the first 7–13 cm of the soil columns with Atriplex nummularia over 5 days of treatments………………………………..……..167

xii

Figure A3. Soil water content at different depths 5 days after imposing the treatments in Atriplex nummularia……………………………………………..……………………168 Figure A4. Changes in deuterium concentration (atom%) in roots of Atriplex nummularia and the closely adhering soil in the first 15 cm of the soil column…..….169

xiii

List of Tables Table 2.1. Variation for a range of saline environments in: the electrical conductivity of the saturation extract or dissolved salt in the soil solution, the depth to the watertable, and the electrical conductivity of the groundwater……………………………………...9 Table 2.2. Root dry mass allocation in the salt-free compartment for non-halophytes exposed to non-uniform salinity (vertical or lateral) in the root-zone……...…………..15 Table 3.1. Treatments imposed on Atriplex nummularia for 21 days………,,………...40 Table 3.2. Response of whole plant water use and water uptake rates expressed on root surface area basis of Atriplex nummularia grown under uniform and non-uniform NaCl concentrations in the root-zone………………………………………………...……….55 Table 4.1. Estimated rate of delivery of Na+, K+ and Cl- to the shoot in Atriplex nummularia exposed to uniform and non-uniform salinities in the root-zone………....76 Table 4.2. Responses to uniform and non-uniform NaCl concentrations in the root-zone of whole-plant water use and water uptake expressed on a root surface area basis………………………………………………………………………………...…..82 Table 5.1. R2 and P values of regression lines and curves fitted to the entire uniform and non-uniform data set, with non-uniform data either plotted against the lowest salinity in the root-zone or against the mean salinity in the root-zone…………………….………99 Table 5.2. Depletion rates of K+ in the treatment solutions for the period 8 to 15 days in Atriplex nummularia grown under uniform or non-uniform salinities…………...…...107 Table A1. Shoot dry mass, leaf area and root dry mass in each side of the split-root pots of Atriplex nummularia exposed for 21 days to uniform and non-uniform salinities in the root-zone………………………………………………………….…………….....163 Table A2. Predawn and midday water potentials of Atriplex nummularia after 0 and 5 days of treatments……………………………………..………………………………166

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Thesis Declaration

The work carried out in this thesis is entirely mine with one exception: deuterium concentrations in soil and nutrient solution samples (Appendix) were determined at the UWA Stable Isotopes Center. To the best of my knowledge all the sources have been acknowledged. The thesis was completed during the course of my enrolment in a PhD degree at UWA and has not been previously accepted for a degree at this or any other institution.

Sections of this thesis (Chapters 3 and 4 – bibliographical details listed below) have been published elsewhere. All experimental work and writing was conducted by N. Bazihizina under the supervision of TD Colmer and EG Barrett-Lennard.

Journal Publication:

Bazihizina N, Colmer TD, Barrett-Lennard EG. 2009. Response to non-uniform salinity in the root zone of the halophyte Atriplex nummularia: growth, photosynthesis, water relations and tissue ion concentrations. Annals of Botany 104: 737-745.

Conference abstracts:

Bazihizina N, Colmer TD, Barrett-Lennard EG. Water relations and growth of Atriplex nummularia in a split-root system with unequal salt concentrations. In: Proceedings of the Combio 2008 conference, Canberra, Australia, 21st-25th September 2008.

Bazihizina N, Colmer TD, Barrett-Lennard EG. Response of the halophyte Atriplex nummularia to non-uniform root zone salinity. In Proceedings of the SEB Annual Main Meeting 2010, Prague, Czech Republic, 30th June - 3rd July 2010.

Chapter 1 General Introduction

Chapter 1

General Introduction

Chapter 1: General Introduction


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1.1 Salinity in the field

Among the many factors restricting the productivity of agricultural systems, salinity has

been identified as one of the major threats because of its degrading effects on

landscapes (Ghassemi et al., 1995). Moreover, as the world population is rising, the

need to increase food production has pushed agriculture into marginal, salt-affected

lands (Läuchli and Grattan, 2007). In Australia alone, around 2.5 Mha are subject to

secondary, or human induced, salinity, caused by the clearing of land for agriculture that

has lead to the rise of watertables and mobilization of salt stored in the soil profile

(Barrett-Lennard et al., 2003; Pannell and Ewing, 2006), and by 2050 the area affected

by secondary salinity is predicted to rise to 17 Mha (NLWRA, 2001).

One solution that has received increasing attention in past decades for the productive

use of salt-affected land is revegetation with perennial plants (Bennett et al., 2009;

Smith and Malcolm, 1959). Nevertheless, despite the early optimism regarding the use

of perennial species to revegetate saline lands, doubts on the sustainability of this

solution have emerged (Thorburn, 1996). There is evidence of an increasing build up of

salts in the plant root-zone due to the absorption of saline groundwater and exclusion of

most salts at the root surface (Archibald et al., 2006; Barrett-Lennard, 2002; Barrett-

Lennard and Malcolm, 1999; Thorburn, 1999). This salt accumulation reduces the soil

water potential and therefore the ability of the plants to absorb water and, in the long-

term, could cause plant death. On the other hand, the presence of established stands of

halophytic shrubs for at least 50 years in saline landscapes suggests that long-term

persistence can be achieved in at least some situations (Malcolm, 2000).

Salt concentrations in the soil solution up to several times higher than in seawater can

be found in some saline landscapes (Bleby et al., 1997; Slavich et al., 1999; Mensforth

& Walker, 1996; see Table 2.1), so the question arises as to how vegetation copes in

these extreme conditions. However, salinity in the field is rarely uniform (see Table

2.1), and the magnitude of the spatial heterogeneity in salt concentrations likely to be

experienced by individual plants may differ widely. In past studies on plant responses to

salinity, little attention has been given to the fact that salts are not likely to be uniform

in the root-zone. There is very limited knowledge on the response of plants to non-

uniform salinity. This lack of information on the physiological responses of the plants to


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non-uniform salinities is even more surprising for halophytes, species that have evolved

in saline environments.

1.2 Thesis outline and aims

Atriplex nummularia has been used in this thesis as a model halophytic plant to

elucidate responses to non-uniform salinities in the root-zone. A. nummularia was

chosen as: (i) it tolerates prolonged periods with high root-zone salinity (Ashby and

Beadle, 1957), (ii) it has an extensive and deep root system (Jones and Hodgkinson,

1969) and so is likely to encounter different salinities in different soil regions, and (iii)

Atriplex (saltbush) species have been used for decades (Smith and Malcolm, 1959) to

revegetate saline land for livestock forage (Atiq-ur-Rehman et al., 1999; Barrett-

Lennard et al., 2003; Lefroy, 2002; Ostyina et al., 1983). In the thesis I used the

commercial clone of Atriplex nummularia, “Eyres Green” (Tamlin’s Nursery, South

Australia) for the following reasons: it provided consistent material for the series of

experiments conducted over 3 years; it is a readily available clone that would be

accessible to other researchers; some prior knowledge existed on this clone; and it is a

priority plant for the agricultural industry in Western Australia.

The overall aim of this thesis was to understand how the halophyte Atriplex nummularia

responds physiologically to spatially non-uniform salinities in the root-zone and how

this is likely to affect productivity (e.g. shoot growth). As halophytes are able to tolerate

high salinities in their root-zone (Flowers and Colmer, 2008), ranges of salinities used

in this thesis will be defined as: low salinity (10–125 mM NaCl); moderate salinity

(125–250 mM NaCl); high salinity (250–500 mM NaCl); severe salinity (500–1000 mM

NaCl); and extreme salinity (more than 1000 mM NaCl). This thesis had initially 2

parallel streams of investigation; however, as results from work on hydraulic

redistribution were not promising for developing this topic further (see Appendix), only

results from the second stream of investigation are reported in the thesis. More

specifically, the thesis aims were to:

1. Determine how growth, water and ion relations are affected when A. nummularia is

subject to laterally non-uniform moderate to severe salinities (Chapter 3).


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2. Determine how extreme non-uniform salinity, sufficient to completely inhibit

growth when present uniformly, affects the physiology and growth of A. nummularia

(Chapter 4).

3. Determine how plant growth and physiology (water and ion relations) under non-

uniform salinities with increasing (low to moderate) salinities in the low-salt side and

severe salinity in the high-salt side, affect plants compared to those in uniform salinities

(Chapter 5).

4. Understand water uptake patterns when A. nummularia is subject to laterally non-

uniform salinities, varying from low to extreme salinities (Chapters 3, 4 and 5); in

particular determine whether water uptake from the high-salt side is maintained, and if

not, whether there is compensatory water uptake from the low-salt side.

5. Based on the acquired information on A. nummularia responses (Chapters 3, 4 and

5) determine, under non-uniform salinities, which side most affects: (i) shoot and root

growth; (ii) stomatal conductance; (iii) water relations, and (iv) leaf Na+, K+ and Cl-

concentrations (General Discussion, Chapter 6).

More specific hypotheses for the various experiments conducted are given in the

relevant experimental chapters (Chapters 3, 4 and 5).

Chapter 2 Literature Review

Chapter 2

Literature Review

2. Literature Review

Chapter 2 Literature review

6

2.1 Introduction

The habitat for plants in saline environments is rarely uniform, and although there is

now a good understanding of plant physiological responses to uniform salinities (e.g.

Flowers and Colmer, 2008; Munns and Tester, 2008), information on how plants

respond to temporal and spatial heterogeneity in salinity is largely lacking (Homaee and

Schmidhalter, 2008). This deficiency in knowledge is particularly striking given the

extent of the variation of salinity in the field (described below). This review will focus

on: (i) the patterns of root growth in media and soils with non-uniform salinities; (ii) the

preferential water uptake from soil regions with the most favourable (i.e. least negative)

water potentials, and finally (iii) the current understanding of plant physiological

responses to non-uniform salinities based mainly on the few previous studies conducted

on non-halophytes and halophytes. As Atriplex nummularia has been used in this thesis

as a model halophytic plant to elucidate responses to non-uniform salinities in the root-

zone, this review also summarises knowledge on salt tolerance in Atriplex species.

2.2 Heterogeneity in saline soils

Temporal and spatial variations in soil salinity are present in most landscapes affected

by salinity (Mass, 1993), and from agricultural soils to salt marshes the variation in the

salinity of the soil solution can be as large as four times the salinity of seawater1 (Table

2.1). In a dryland saline environment with duplex soil, soil salinity2 (ECe) in the upper

10 cm of the profile varied in space from 3 to 65 dS m-1 over a distance of 10 m

(Davidson et al., 1996). Similarly on a semi-arid floodplain in South Australia, large

variations in salinity in the first 1.2 m of the soil profile, were found in space (2–54 dS

m-1) and time (3–43 dS m-1) (estimated from total soil Cl- concentrations and converted

to the concentration in the soil solution using the gravimetric water content and

1 Seawater has approximately 500 mM NaCl and an electrical conductivity of 55 dS m-1 (Wyn Jones and Gorham, 2002). 2 The three main methods of measuring soil salinity cited in this thesis are: (a) measuring the electrical conductivity (EC) of the water separated from a saturated soil paste extract, created by adding water to dry soil (ECe, expressed in dS m-1); (b) measuring the electrical conductivity of a 1:5 dry soil : water mixture (EC1:5, dS m-1); (c) direct estimation of the concentration or osmotic potential of dissolved salts in the soil water (Barrett-Lennard et al., 2003). The electrical conductivity of the soil solution can be expected to be ~2 times the ECe for soils at field capacity, and ~4 times the ECe for soils at wilting point (Richards, 1954).


7

assuming all salts were NaCl; Akeyord et al., 1998). In other environments, such as salt

marshes, these salinity variations can be even more extreme (Álvarez-Rogel et al.,

2001; Silvestri et al., 2005; Table 2.1). An example of extreme temporal variation is

from a saline swamp where the salinity of the soil solution varied between 36 dS m-1 in

winter and 215 dS m-1 in summer (estimated from soil water content and Cl-

concentrations, Mensforth and Walker, 1996).

Seasonal and spatial changes in soil salinity result from complex interactions between

topography, leaching, soil properties (e.g., texture, surface mulches), plant water use,

evaporation and the presence of fluctuating saline watertables. The salinity experienced

by roots (i.e. the salinity of the soil solution) will depend on the concentrations of ions

and water in the soil (Bennett et al., 2009). Fluctuations of saline watertables are a key

factor affecting seasonal and spatial dynamics in soil salinity (Jackson et al., 1956;

Silvestri et al., 2005). When watertables are shallow there is an upward movement of

water and ions through the soil profile by capillarity. The capillary movement of ions

occurs mostly during dry seasons when there is high evaporative demand (spring and

summer in Mediterranean environments); this leads to the accumulation of salts in the

upper layers of the soil profile (Jackson et al., 1956; Northey et al., 2006). In a non-

irrigated soil in south-eastern Turkey characterized by a high content of clay, the

increase in soil salinity (ECe) in the upper 20 cm of the profile from February (11 dS m-

1) to November (28 dS m-1) was a result of the high evaporative impact on the capillary

movement of ions from the shallow groundwater (Çullu et al., 2009). The critical

watertable depth that results in salt accumulation in the upper regions of a soil profile

will vary according to the soil texture, with values that range about from about 180 cm

in clays to about 240 cm in medium textured soils (Gutteridge, Haskins and Davey Pty

Ltd, 1970).

Soil moisture, mostly in the superficial soil layers, has strong seasonal dynamics (Fig.

2.1), with soils in Mediterranean environments becoming drier in summer. Plant

available water will also be influenced by soil proprieties (Alessio et al., 2004), such as

the low water holding capacity of sandy soils or the high water binding capability of

clay soils. Therefore, the combination of rainfall, which leaches salts out of the

superficial layers, and the alternation of wet and dry periods, exacerbates the temporal

and spatial fluctuations in salinity of the soil solution (Álvarez-Rogel et al., 1997, 2001;

Bleby et al., 1997; Bennett et al., 2009; Mensforth and Walker, 1996; Carter, 2004).


8

Temporal and spatial changes in soil salinity can also be driven by plant transpirational

demands. The accumulation or depletion of a given ion at the root surface will depend

upon the rate of ion transport from the bulk soil to the root surface (mass flow and

diffusion) and on the net uptake rate of that ion by the roots (Sinha and Singh, 1974). In

saline environments, with Na+ and Cl- being high, plants effectively partition water from

the salt solution and exclude most (generally > 90%) of the Na+ and Cl- at the root

surface (Munns et al., 1983). Therefore mass flow associated with high transpiration

rates can cause a large flux of Na+ and Cl- towards roots and a rapid accumulation of

these ions at the soil-root interface (Hamza and Aylmore, 1992; Sinha and Singh, 1974,

1976). Increases in Na+ and Cl- concentrations were found in soil closely adhering to

roots in Triticum aestivum and Zea mays, and ion accumulation near roots was found to

increase with time and at higher transpirational demands (Sinha and Singh, 1974, 1976).

For example, for Zea mays at low transpirational demand, there was no difference in the

Cl- concentration at the root surface between 7 and 14 days, but when plants were

subject to high transpirational demand, the concentration of Cl- at the root surface

doubled after 7 days and tripled after 14 days (Sinha and Singh, 1974). Salt

accumulation in the root-zone was also found in field plots of Atriplex species. The

accumulation was proportional to the leaf density (ratio leaf weight to soil surface area);

with a leaf density of 0.139 kg m-2 soil Cl- concentrations at depths of 0.4–0.8 m were

twice those of plots with a leaf density of 0.072 kg m-2 (Barrett-Lennard and Malcolm,

1999).


9

Table 2.1. Variation for a range of saline environments in: the electrical conductivity of the saturation extract (ECe; dS m-1) or dissolved salt in the soil solution (estimated from the water EC, osmotic potentials and soil Cl- concentration), the depth to the watertable (m), and the electrical conductivity

Environment Method of measurements

Maximal groundwater fluctuations

(m)

Maximal variation in groundwater salinity

(dS m-1) ReferenceSpatial Temporal

Agricultural soil - not irrigated 9 - 21 n.d. ECe 0.9 - 1.8 8 - 30 Çullu et al ., 2009

Agricultural soil - irrigated 2 - 10 n.d. ECe 1.5 - 1.9 7 - 7.5 Çullu et al ., 2009

Dryland - halophytic shrub plantation 3 - 65 n.d. ECe 0.2 - 1.4 n.d. Davidson et al. , 1996

Dryland - halophytic shrub plantation 46 - > 150 49 - > 150 Soil Cl- 0 - 1 n.d. Slavich et al ., 1999Dryland - grassland 15 - 129 0 - 129 Estimated OP from soil Cl- 0 - 1.6 n.d. Bleby et al. , 1997Semi-arid floodplain 4 - 54 4 - 43 Soil Cl- 1.7 - 3.5 n.d. Akeyord et al. , 1998Semi-arid floodplain 26 - 97 n.d. Soil Cl- n.d. n.d. Jolly et al ., 1993Floodplain 23 - 106 n.d. Estimated OP from soil Cl- n.d. n.d. Holland et al ., 2006Floodplain <1 - 64 n.d. Soil Cl- n.d. n.d. Thorburn et al ., 1993aMangrove forest 5 - 35 24 - 35 OP soil water n.d. n.d. Hao et al ., 2009Mangrove forest 0 - 105 15 – 105 Dissolved salt in water n.d. n.d. Lin and Sternberg, 1994 Mangrove forest 62 - 186 39 - 186 Dissolved salt in water n.d. n.d. Lambs et al ., 2008Salt marsh 56 - 107 35 - 107 ECe 0.5 - 0.9 n.d. Bornman et al ., 2002Salt marsh 12 - 280 n.d. Dissolved salt in water n.d. n.d. Silvestri et al ., 2004Salt marsh 30 - 110 n.d. ECe n.d. n.d. Alvarez Rogel et al ., 2001Swamp forest n.d. 36 - 215 Soil Cl- 0.3 - 1.3 50 - 73 Mensforth and Walker, 1996Wetland 36 - 67 0 - 67 Dissolved salt in water n.d. 25 - 36 Ewe et al. , 2007

Maximal changes in salinity observed in field studies

(dS m-1)

Soil salinity (ECe) is expressed in units of dS m-1, and, where appropriate, conversions have been made from ppm, ppt or PSU assuming all salts were NaCl, assuming that 1 ppt = 1.551724138 dS m-1 (www.iep.ca.gov/suisun/facts/salin/salinityConversion.jpg). The electrical conductivity of the soil solution could be calculated from the ECe, as the electrical conductivity of the soil solution is expected to be ~2 times the ECe for soils at field capacity, and ~4 times the ECe for soils at wilting point (Richards, 1954). n.d. = not determined.


10

Moisture content (%)

0 5 10 15 20 25

Dep

th (

cm)

0-2.5

2.5-15

30-35

45-50

50-55

Chloride (% )0 1 2 3 4

Water - winterWater - summer

Chloride - winterChloride - summer

Figure 2.1. Soil water content (% dry mass) and Cl- (% dry mass) changes with seasons (summer vs. winter) and depth in the soil profile (from Jackson et al., 1956).


11

2.3 Plant responses to non-uniform salinities

2.3.1. Root growth

Plant root systems are highly plastic and can vary their development in response to

many environmental cues (Malamy, 2005; Sun et al., 2008). Generally under non-

uniform conditions in the root-zone, proliferation in favourable patches is an obvious

adaptive response, with the preferential deployment of roots in favourable patches and

reduced growth of roots in the less favourable areas (Drew, 1975). As root development

and architecture depend upon both genetic and environmental conditions, the ability to

proliferate roots in favourable patches, also called root foraging, will vary between

species and the environments in which they have evolved (Malamy, 2005; Bauerle et al.,

2008). Although root proliferation in favourable patches can confer advantages to plants

in non-uniform environments, there are costs involved in the formation of new roots

(Fitter, 1994). For example if the species environment is characterized by infrequent

patches with high soil moisture content in the upper soil profile, the investment of new

roots in those patches might not be repaid, and under these conditions, it can better for

plants to adopt a different genotypically fixed response, such as the formation of roots

deeper in the soil profile where there is a more stable source of water (Dawson and

Ehleringer, 1991; Fitter, 1994; Bauerle et al., 2008; see example below on water uptake

patterns in Eucalyptus spp. in a saline landscape, Thorburn et al., 1993a). For example,

when two genetically identical shoots of Vitis vinifera were grafted onto two genetically

different root systems, one associated with high shoot vigour (HSV) and the other with

low shoot vigour (LSV), it was found that growth predisposition influenced root growth

strategies (Bauerle et al., 2008). With vertical moisture heterogeneity, i.e. unirrigated

soil plots where the top soil was allowed to dry, both HSV and LSV plants produced a

similar proportion of roots, 25–30% of the total root produced, at soil depths > 60 cm.

However, HSV and LSV showed different root growth in response to seasonal patterns

of water availability. Compared to root production in June and July (early summer),

HSV plants doubled the production of new fine roots deeper in the soil profile in August

when surface soil layers dried out. On the other hand, LSV plants, independently of the

moisture in the upper surface, grew 87% of its new roots earlier in the season (June–

July).


12

Similarly, in saline soils root growth can be expected to vary in response to

heterogeneous soil conditions. An example that would support such a view is given in a

study where the dynamics of root growth in the upper 50 cm of the soil profile were

investigated in Melaleuca halmatorum over 21 months in a saline swamp (Mensforth

and Walker, 1996). At the end of the winter when groundwater was 0.3 m from the soil

surface and the soil water potential was -2 MPa, most root growth occurred in the upper

10 cm of the soil surface. As the soil profile dried out in spring and summer, and the

water potential in the top 10 cm of the soil profile declined to values as low as -10.9

MPa, most of the root growth occurred below 30 cm where the soil water potential was

between -2.8 and -3.8 MPa.

Under non-uniform saline conditions, there is a wide range of responses amongst non-

halophyte species in root dry mass allocation between saline and non-saline areas (Table

2.2). The impact of spatially heterogeneous salinity has been studied in vertical and

lateral split-root systems, where roots are divided either vertically or horizontally into

two or more portions. In Zea mays, root systems were divided into 3 vertical

compartments of 24 cm depth, each separated with wax layers; these different

compartments could then be irrigated with fresh or saline (12 dS m-1) water. Four weeks

after imposing vertically non-uniform salinities there was no increased root growth in

the non-saline compartments. On the contrary, salinisation of the lower 1/3 or 2/3 of root

systems caused an 8–31% decline in root growth in the non-salinized upper root

compartment (Bingham and Garber, 1970). As shoot growth was maintained or even

increased (up to 20% increase when 1/3 of the root system was salinized) this decline in

root mass in the non-salinized layers could possibly reflect a different biomass

partitioning, as root/shoot ratio declined under non-uniform conditions. By contrast, in

Lycopersicon esculentum grown for 8 days in a lateral split-root system with one root

half exposed to a saline medium (total of 75 mM Cl-) and the other half to a NaCl-free

medium, root growth in the NaCl-free side increased by 60% compared to that in plants

growing with both root halves in the salt-free medium (Flores et al., 2002). In Citrus

aurantium there was only a 5% compensatory root growth 4 months after imposing

laterally non-uniform salinity (8.8 dS m-1) (Zekri and Parsons, 1990). These

contradictory results raise the question whether under non-uniform salinity

compensatory root growth is exclusively related to species specific phenotypic

plasticity, or is perhaps also related to the salinities in each root portion and how these

locally affect root growth (i.e. levels of salt tolerance).


13

A single root system should be viewed as an array of different roots, such as tap root vs.

laterals or seminal vs. nodal roots, all with varying morphological and physiological

characteristics (Doussan et al, 2009) and the degree of growth reduction can be expected

to be affected by root type. For example, with the non-halophyte Opuntia ficus-indica,

exposure to 100 mM NaCl had differing effects on the growth of the main root axis and

primary lateral roots (Gersani et al., 1993). After 28 days, the dry mass of the main root

axis was reduced by 54% but the dry mass of the primary lateral roots was reduced by

95%, compared with root growth in a NaCl-free medium.

Root distributions in soil profiles with non-uniform salinity will be affected by the fact

that generally roots of non-halophytes do not penetrate readily into more saline layers

(Homaee and Schmidhalter, 2008). In general, the degree of root penetration can be

expected to decrease with increased salinity, but this effect will be influenced by the

degree of salinity tolerance of the species (Wadleigh et al., 1947). As an example, the

rooting depth of the non-halophytes Phaseolus vulgaris, Medicago sativa, Zea mays and

Gossypium hirsutum were all decreased when the layers to be penetrated were saline

(Wadleigh et al., 1947). When growing in non-saline soil columns, both P. vulgaris and

G. hirsutum had roots that penetrated to the base of 0.9 m deep soil columns. However,

when the soil columns were packed with 5 soil layers of increasing salinity with depth

(from 0 to 0.25% dry mass NaCl), only a few roots of P. vulgaris penetrated the layer

containing 0.1% NaCl and no roots occurred in the layers with NaCl concentrations

higher than 0.15% NaCl. By contrast, the roots of G. hirsutum, a more salt tolerant non-

halophyte, penetrated all saline soil layers in the columns.

The reduced penetration of roots into saline layers has been found to be caused not only

by a decline in root elongation rate but also by changes in root curvature, indicating that

the gravitropic response of the stressed plant roots is reduced under salinity (Sun et al.,

2008). In order to assess how salinity affected the gravitropic responses of primary roots

in the non-halophyte Arabidopsis thaliana, seedling were initially placed between pairs

of agar layers orientated vertically; in one treatment, both layers were free of NaCl and

in the other the two layers contained different NaCl concentrations (one withouth NaCl

and the other with 25 to 150 mM NaCl). After the plants were established, the agar

layers were rotated by 90° so that the non-saline agar layer was above the layer of

varying salinity. When plants were grown with both layers free of NaCl or with the

lower layer containing 25 mM NaCl, roots grew downward into the lower layer,


14

showing a normal gravitropic response. However, if the lower layer contained 50 mM

NaCl there was a decline in the number of roots growing downward, and at 150 mM

NaCl more than 85% of the seedlings showed a negative gravitropic response (growing

at an angle > 45° from the direction of the gravity).

In conclusion, understanding root systems is a fundamental part of the evaluation of a

species’ ability to perform in saline lands (Hoffmann et al., 2003). The survival of a

perennial species in a saline environment will depend not only on that plant’s tolerance

to water stress and/or high soil salinity, but also on its ability to preferentially grow into

and exploit those regions of the soil with the most available water (i.e. least negative

water potential).

2.3.2. Water uptake

Water uptake rates are proportional to the water potential gradient across shoot, roots

and soil, and are inversely proportional to the resistances to water flow in each

component of the soil-plant-atmosphere continuum where water moves as a liquid

(Nobel, 1991). Conceptually this means that plants will tend to use the most accessible

water sources (i.e. least negative water potential) in contact with roots, according to the

soil water potential. As early as 1911, it was suggested that the presence of fresh-water

species in saline marshes could be explained by the fact that, while surface soils were

strongly saline, these plants had roots in the subsoil with access to non-saline water

(Harshberger, 1911). Several species, from non-halophytic Eucalyptus spp. to

halophytes such as Rhizophora spp., have the ability to change their major sources of

water in the soil profile from saline groundwater to rain-derived non-saline water,

between dry and wet seasons (e.g. Bleby et al., 1997; Ewe et al., 2007; Lin and

Sternberg, 1992; Mensforth and Walker, 1996; Slavich et al., 1999).


15

Table 2.2. Root dry mass allocation in the salt-free compartment for non-halophytes exposed to non-uniform salinity (vertical or lateral) in the root-zone.

SpeciesVertical or lateral non-uniform salinity

Root biomass in non-saline side

(% compared to control)Days of

treatment Reference

Zea mays A Vertical -13% to -23% 4 weeks Bingham & Garber, 1970Zea mays B Vertical - 23% to -28% 4 weeks Bingham & Garber, 1970

Phaseolus vulgaris Lateral +345%* 16 days Kirkham et al ., 1969Hordeum vulgare Lateral +5%* 16 days Kirkham et al ., 1969Citrus aurantium Lateral +5% 4 months Zekri & Parsons, 1990Lycopersicon esculentum Lateral +56% 8 days Flores et al ., 2002

Differences between root biomass are expressed as the percentage difference in dry mass between roots uniformly at low salinity and the same portion of the roots in the low salinity compartment under vertical or lateral non-uniform salinity. A= one third of the root system salinized, B= two thirds of the root system salinized, * refers to one replicate only as dry mass was not measured in the other replications.


16

In some environments the natural concentrations of the heavy stable isotope 2H and/or

that of 18O varies in the water of different soil layers, so it has therefore been possible to

obtain information on which soil layers water transpired has been sourced (Lambers et

al., 2008). In the coastal Everglades of Southern Florida, seasonal water use patterns of

the dominant macrophytes were investigated by comparing the stable isotope signatures

(2H and 18O) of the plant xylem water with those of the possible water sources (Ewe et

al., 2007). The alternative water sources were shallow soil water and groundwater; the

shallow soil water had an ECw (dissolved salt in the soil water) that varied between 0 dS

m-1 and 67 dS m-1 while the groundwater had a more stable ECw that varied between 25

and 36 dS m-1. In winter all plants mainly used the non-saline shallow water, but in

summer, when there was an inversion of the salinity between the shallow water and

groundwater, different water uptake patterns occurred amongst the dominant

macrophytes in the ecotone (Fig. 2.2). Rhizophora mangle, which had roots occurring

throughout the soil profile, switched to a soil-groundwater mix (∼55% groundwater),

whereas Sesuvium portulacastrum and Cladium jamaicense, that had shallower root

systems, only used shallow water, independently of the soil solution salinity (Fig. 2.2).

Similar general effects were also reported in a coastal marsh in Florida where different

water uptake patterns were observed between R. mangle growing on marsh fringes

compared with those at more elevated areas (Lin and Sternberg, 1992). R. mangle that

occurred in areas of higher elevation were able to use rainfall derived non-saline water

during the wet season whereas in the dry summer, when precipitation decreased, plants

in most cases only had access to saline tidal waters. By contrast, fringe R. mangle, which

had most of their roots continuously inundated with tidal water, were mainly limited to

the use of saline tidal waters throughout the year.

Interestingly in a study conducted at four sites in a floodplain forest, for two of the

studied sites, Eucalyptus largiflorens did not respond to the rainfall derived non-saline

water available in the upper soil profile and, despite the salinity of the groundwater

being elevated (11–25 dS m-1), saline groundwater remained the only water source. This

lack of response to the availability of non-saline water was hypothesized to be related to

differences in the ability of this species to reactivate dormant surface roots (see above;

Mensforth and Walker, 1996; Thorburn et al., 1993a). It was hypothesized that if these

plants had adapted to environments where there is a constant source of water (e.g.

groundwater) at a certain depth, these plants would preferentially grow more roots

deeper in the soil, regardless of soil moisture conditions at the surface (Dawson and


17

Ehleringer, 1991; Bauerle et al., 2008). By extension of this principle, the contrasting

behaviours observed between plants in response to temporal changes in soil salinity are

also likely to be related to the ability of plants to maintain or shed roots when some soil

portions become too saline and quickly form new roots when conditions are more

favourable (Mensforth and Walker, 1996; Thorburn et al., 1993a).

2.3.3. Shoot growth, water and ion relations under non-uniform salinity

2.3.3.1.Shoot growth

Plants are generally able to withstand and grow with salinities in one root half that

would have strongly affected growth when applied to the entire root system (Bingham

and Graber, 1970; Zekri and Parsons, 1990). In non-halophytes, under non-uniform

salinity, there were variations in the growth responses between species (e.g. Flores et al.,

2002; Lycoskoufis et al., 2005; Zekri and Parsons, 1990). For Lycopersicon esculentum

growing for 8 days in a lateral split-root system with one root half exposed to a saline

medium (total of 75 mM Cl-) and the other half to a NaCl-free medium, there were no

effects of the non-uniform salinities on shoot dry mass compared to plants with both

root halves in the NaCl-free medium. On the other hand, in Citrus aurantium exposed to

laterally non-uniform salinity, with one root half exposed to 8.8 dS m-1, shoot growth

was only reduced by 21% compared with control plants in a non-saline medium (Zekri

and Parsons, 1990). When both root halves were exposed to salinity, shoot growth was

reduced by 81%. In contrast to these results, in Capsicum annuum grown with a laterally

split-root system, despite having one root half exposed to non-saline water, non-uniform

salinity (conductance saline solution – 8 dS m-1) severely inhibited shoot growth, with a

decline similar to that observed when the entire root system was salinized (Lycoskoufis

et al., 2005). It is however important to stress that in this study, the severe decrease in

growth in C. annuum with non-uniform salinities may have an explanation unrelated to

the uptake of Na+ and/or Cl-. In this experiment, the roots on the NaCl-free side were

grown in tap water instead of a standard nutrient solution (as for the other treatments).

Ordinarily, plants growing with high external Na+ have inhibited K+ uptake (Hajji et al.,

2001). Therefore the plants exposed to non-uniform salinities could well have suffered


18

from K+ deficiency, being unable to take up K+ from the high-salt side (because of Na+

competition) and also unable to take up K+ from the low-salt side (as it was not present).

In general it appears that growth responses of halophytic plants to non-uniform salinity

are more similar across species, although only 3 halophytes have been examined to date.

Under non-uniform salinity shoot growth was maintained or increased compared with

uniform salinities. For Crithmum maritimum, having one root half exposed to 300 mM

NaCl did not affect shoot dry mass compared to the dry mass of plants with uniform 0

mM NaCl, whereas when plants were grown with both root halves at 300 mM NaCl

shoot dry mass declined by more than 50% (Hamed et al., 2008). In Sesuvium

portulacastrum and Batis maritima, when both species had one root half exposed to 800

mM NaCl and the other to 0 mM NaCl, shoot dry mass increased to 1.2 and 2 times the

shoot dry mass when both root halves were at 0 mM NaCl, respectively (Hamed et al.,

2008; Messedi et al., 2004). However, for these halophytes, the observed growth

enhancements were likely due to the mass of ions accumulated in the shoots (in

dicotyledonous halophytes ions may contribute up to 30–50% of the dry mass; Flowers

et al., 1986) and the fact that these dicotyledonous halophytes need ion uptake for

maximal growth on an organic weight basis (Yeo and Flowers, 1980; Flowers and

Colmer, 2008).

2.3.3.2. Water and ion relations

Under non-uniform salinity, midday water potentials indicate that plants mostly take up

the most accessible water in terms of water potential. In Citrus aurantium, shoot midday

water potential with one root half exposed to a saline solution (8.8 dS m-1; -0.35 MPa)

only decreased by 0.1 MPa compared with plants with both root halves in non-saline

solution (Zekri and Parsons, 1990). On the other hand, when saline solution was applied

to both root halves, the water potential of the shoots declined by 0.4 MPa. Similar trends

were found for Phaseolus vulgaris and Hordeum vulgare growing under non-uniform

salinities (Kirkham et al., 1969). In P. vulgaris the water potential of shoots exposed for

16 days to non-uniform salinity (0 and -0.4 MPa; 0 and 80 mM NaCl) declined by 0.1

MPa compared to the values in plants in NaCl-free solutions; by contrast water

potentials declined by -0.7 MPa when both root halves were exposed to salinity (the

time of measurement was not indicated in this study but it seems likely that they were

taken at midday). Although no studies have examined predawn water potentials under


19

non-uniform salinities, it is reasonable to assume that under such conditions plants

would have similar responses to those observed in plants exposed to non-uniform soil

moisture in the root-zone. Under non-uniform moisture, plants equilibrated with the root

portion exposed to the least negative water potential (e.g., Bouteloua gracilis, Sala et al.,

1981; Quercus spp., Bréda et al., 1995; Betula pendula, Fort et al., 1998; Castanea

sativa, Maurel et al., 2004), provided that there were enough roots in the side with the

least negative water potential to enable the equilibration to occur overnight (Améglio et

al., 1999).

To this date no information is available on shoot water relations (shoot/leaf water

potentials and leaf osmotic potentials) for halophytes under non-uniform salinity.

However, by extension of the general principle that plants equilibrate with the root

portion exposed to the least negative water potential (e.g. Sala et al., 1981; Bréda et al.,

1995; Fort et al., 1998; Maurel et al., 2004), it is expected that the shoot water potentials

of halophytes will also be influenced mainly by the least negative water potential of the

root-zone.

Under non-uniform conditions there is an increase in shoot ion concentrations, possibly

associated with ion uptake from the high-salt side (non-halophyte: Hajji et al., 2001;

Lycoskoufis et al., 2005; halophytes: Messedi et al., 2004; Hamed et al., 2008). Despite

most of the water coming from the low salt side, some water uptake still occurs from the

high salt side and this flow of water through the xylem from the high-salt side may be

partly responsible for the observed increases in shoot ion concentrations (Kirkham et al.,

1969, 1972). Despite the fact that more than 90% of Na+ and Cl- are usually excluded at

the root surface (Yeo, 2007; Munns et al., 1983), there is clearly some uptake of these

ions during water uptake from the high-salt side, and these then move via the xylem

vessels to the shoot (c.f. Flowers and Yeo, 2007). In the non-halophytes Hordeum

vulgare, Phaseolus vulgaris and Nerium oleander non-uniform salinities (0/100 and

0/200 mM NaCl) were associated, respectively, with a leaf Na+ concentrations 58, 34

and 57 times that of plants with both root halves in the non-saline solution (Hajji et al.,

2001).


20

-1 0 1 2 3 4 5 6

WinterSummer

δδδδ18181818ΟΟΟΟ (‰)

Sesuviumportulacastrum

Cladium jamaicense

Rhizophora mangle

Spe

cies

B C ADA

Figure 2.2. Water sources of Rhizophora mangle, Sesuvium portulacastrum and Cladium jamaicense in an ecotone where the salinity of the shallow water varies seasonally but the salinity of the groundwater remains more constant between seasons. The sources of plant water were assessed by comparing the isotopic composition of the non-photosynthetic tissues (filled and empty bars) with the differences in the isotopic composition of the two water sources in each season. Line A is the δ18O of the shallow water in winter; line B is the δ18O of the groundwater in the soil profile in winter; line C is the δ18O of the shallow water in summer; line D is the δ18O of the groundwater in the soil profile in winter. Data are from Ewe et al. (2007).


21

In halophytes also, increases in leaf Na+ and Cl- can be associated with a non-uniform

distribution of salts in the root-zone (Hamed et al., 2008; Messedi et al., 2004). In

Sesuvium portulacastrum and Batis maritima growing with 0 mM NaCl in one root half

and 800 mM NaCl in the other half, the shoot Na+ concentration was in-between

concentrations in the plants in which both root halves were in the non-saline (0 mM

NaCl) medium or in the saline (800 mM NaCl) medium (Hamed et al., 2008; Messedi et

al., 2004). Under non-uniform salinity shoot Na+ concentrations in S. portulacastrum

and B. maritima were 20 and 13 times those of control plants, respectively (Messedi et

al., 2004; Hamed et al., 2008). The above increases in shoot Na+ are large for halophytes

and are likely to have been caused by the use of a NaCl-free solution in the controls.

Completely Na+-free solutions can result in ion deficiency in dicotyledonous halophytes

(Flowers and Colmer, 2008).

2.3.3.3.Stomatal conductance and long distance signalling

Under non-uniform salinity there is always a certain degree of reduction in stomatal

conductance. For example, stomatal conductance decreased by 19% with Citrus

aurantium exposed for 4 months to a non-uniform salinity of 0/8.8 dS m-1 and by 90%

with Phaseolus vulgaris exposed to a non-uniform salinity of 0/~80 mM NaCl (Zekri

and Parsons, 1990; Kirkham et al., 1972). It would appear from the existing studies that

the reductions in stomatal conductance do occur without changes in leaf water status,

thus implying that chemical or electrical signals from the root in the salinized portion

are involved in stomatal regulation. In theory, chemical signals could be transported

from the roots in the saline side to the shoot as some water uptake is maintained from

the high-salt side under spatially non-uniform salinities (Bingham and Graber, 1970;

Zekri and Parsons, 1990). This might then explain the reduced stomatal conductance

without changes in leaf water potentials.

Stomatal regulation under drought conditions is affected by both hydraulic and non-

hydraulic (e.g. chemical) signals (reviewed by Comstock, 2002). The relative

importance of these two types of signal on stomatal regulation is controversial, with one

type of signal appearing to dominate over the other depending on the species and

experimental design (Liu et al., 2003; Rodrigues et al., 2008; Comstock, 2002; Fuchs

and Livingston, 1996; Ren et al., 2007). However under non-uniform soil moisture

deficit there is a large body of evidence that suggests that stomatal conductance is


22

mainly controlled by non-hydraulic signals, as stomatal closure can be observed

independent of changes in leaf water status (Lovisolo et al., 2002; Sobeih et al., 2004;

and reviews by Davies and Zhang, 1991; Dodd, 2005). In a split-root study conducted

with Lycopersicon esculentum it was found that both stomatal conductance and leaf

elongation rates declined when water was withheld from one root half (a treatment also

called partial root drying - PRD), compared to well watered plants (Sobeih et al., 2004).

In this study, the leaf water potential in PRD plants remained equal to well watered

plants throughout the entire experiment, but a 34% reduction in stomatal conductance

was seen on the third day after imposing the treatments, and a 43% reduction in leaf

elongation rate was seen on the fifth day. Further evidence that there are signals from

the root in the dry side that trigger stomatal closure and/or reduction in leaf elongation

rates in PRD comes from experiments where the roots in the dry soil were excised (Saab

and Sharp, 1989; Gowing et al., 1990). In Malus domestica, when water was withheld

from one root half, there was almost a 40% decline in leaf area over 24 days compared

to well watered plants, despite leaf water potentials remaining unaffected. However

shoot growth recovered when the root half growing in the drying soil was excised.

Similar results were found in Zea mays where excising one root half with adequate

water had no effect on leaf elongation whereas exposing one root half to drying soil

resulted in a 25% inhibition of the leaf elongation rates compared to well-watered plants

(Saab and Sharp, 1989).

The nature of the chemical signal responsible for stomatal closure under PRD is still

controversial. Many reports indicate that abscisic acid (ABA) is the predominant

hormone involved in the chemical regulation under PRD (Khalil and Grace, 1993;

Jokhan et al., 1996; Dodd et al., 2006; Dood, 2007; reviewed by Jiang and Hartung,

2008; Schatman and Goodger, 2008). For example, in Acer pseudoplatanus subject to

PRD, a decline in stomatal conductance coincided with an increase in ABA xylem sap

concentrations, which was 2 times higher after 6 days of treatment than initially (Khalil

and Grace, 1993). Moreover, there was a partial recovery of the stomatal conductance

on the 7th day of treatment, when a marked reduction in ABA was observed in the xylem

sap, possibly due to a decline in water supply from the dry side.

On the other hand, there are also studies where the stomatal closure observed under PRD

did not require root sourced ABA and PRD did not cause an increase in xylem ABA

(e.g. Zea mays, Blackman and Davies, 1985; Lycopersicon esculentum, Holbrook et al.,


23

2002 and Sobeih et al., 2004). When ABA deficient (flacca) and wild type (sitiens)

Lycopersicon esculentum roots were grafted to a common shoot and subject to PRD, a

reduction in stomatal conductance was observed in response to withholding the water

from one root half, independently of the root genotype that was allowed to dry, i.e.

regardless of the ability of the roots to produce ABA (Holbrook et al., 2002). Hence

other type of signals, such as the alkalinisation of xylem pH (Sobeih et al., 2004), have

been related to stomatal closure under partial root drying.

Increases in xylem sap pH (in non stressed plants xylem pH is generally around 5–6.5,

Wilkinson, 1999) have been found to occur in plants subject to drought or PRD and

these changes were generally detected with no changes in leaf or shoot water potentials

(Wilkinson et al., 1998; Sobeih et al., 2004). Stomatal regulation by xylem pH appears

to be mediated through the accumulation of the ABA, already present in the leaves, in

the apoplast of the guard cells, thus promoting stomatal closure (Wilkinson and Davies,

1997; Jia and Davies, 2007; Ren et al., 2007). Artificial xylem sap buffered at pH 7 fed

to detached leaves of Commelina communis caused a ∼50% decrease in transpiration

rates compared to the transpiration rate in leaves fed with artificial sap buffered at pH 6

(Wilkinson and Davies, 1997). However as mentioned above, stomatal regulation

through pH requires the presence of ABA (Wilkinson et al., 1998). When artificial sap

buffered to pH 7.75 was fed to a wild type or ABA deficient Lycopersicon esculentum, it

was found that only the wild type had reduced transpiration rates and it was only when

exogenous ABA (0.03 mM – the concentration found in well-watered plants) was added

to the sap that a reduction in transpiration was observed in ABA deficient L.

esculentum leaves (Wilkinson et al., 1998).

Electrical signals have also been found to be involved in stomatal regulation and might

therefore be involved in stomatal regulation under non-uniform conditions. In a study

conducted under uniform conditions in the root-zone, a decline in CO2 uptake rate and

transpiration rate in Zea mays was detected 6 minutes after applying PEG to the root

system, and after 12 minutes the rates of CO2 uptake and transpiration had declined by

ca. 20% and 35%, respectively (Fromm and Fei, 1998). This reduction in stomatal

conductance corresponded with propagating signals that began in the roots and moved

up the phloem by a rapid depolarization of the membrane potential of the phloem. In the

same study, a similar response was observed by applying NaCl to the roots. Another

study showed that both electrical and hydraulic signals are involved in the rapid opening


24

of the stomata after the re-watering of droughted Zea mays (Grams et al., 2007).

Electrical signals, propagated independently of the hydraulic signals, were detected in

the leaves within less than 40 s after re-watering and the initiation of the stomatal

opening was observed ~60 s after re-watering. As salinity has been found to cause rapid

membrane depolarization in roots (Chen et al., 2005; Cakirlar and Bowling, 1981;

Shabala et al., 2003), it is plausible that electrical signals might be involved in stomatal

regulation under non-uniform conditions. Clearly, more detailed time-course studies of

the changes in stomatal conductance and leaf water potentials are required under non-

uniform salinity to understand whether hydraulic or non-hydraulic signals, or

combinations of these, are involved in stomatal regulation and ultimately in plant water

use.

Given the temporal and spatial salinity variations in most field situations, the plant’s

ability to take up the most available water within its root-zone will undoubtedly

positively affect plant productivity under field conditions in saline environments. The

fact that plants will access the most available water within the active root system also

implies that the plant’s ability to perform in the field will not necessarily be affected by

the salinity in the upper soil profile, and measuring salinity at shallow depths and at one

time may give a quite misleading impression of the suitability of a site for a given

species (Bennett et al., 2009).

2.4 Salinity tolerance of Atriplex nummularia

2.4.1. Growth under uniform salinities

Atriplex species are able to tolerate high NaCl concentrations in the root-zone and some

species have been reported to grow at concentrations higher than 500 mM NaCl (e.g. A.

inflata and A. nummularia at 600 mM NaCl, Ashby and Beadle, 1957; A. canescens at

900 mM NaCl, Glenn et al., 1996; A. halimus at 800 mM NaCl, Boughalleb et al.,

2009). In general for Atriplex spp. there is a growth enhancement in the 25–200 mM

NaCl range (Fig. 2.3; e.g. +30% dry mass in A. nummularia at 100 mM NaCl compared

to dry mass at 1 mM NaCl, Greenway, 1968; +248% dry mass in A. inflata at 50 and

200 mM compared to dry mass at 0 mM, Ashby and Beadle, 1957; +50% relative


25

growth rate in A. portulacoides at 200 mM compared to relative growth rate at 0 mM

NaCl, Redondo-Gómez et al., 2007); this is consistent with the fact that dicotyledonous

halophytes require ions to osmotically adjust and grow optimally (Yeo and Flowers,

1980; Flowers and Colmer, 2008). Indeed, the sub-optimal growth of dicotyledonous

halophytes with the absence of salt has caused some researchers to criticize the use of

salt-free solutions as controls (Yeo and Flowers, 1980; Flowers and Colmer, 2008).

The enhancement in plant dry mass at higher salinity is due to three factors: (i) changes

in the amount of water per unit dry mass, that is increased succulence at 200–300 mM

NaCl (e.g. A. amnicola, Aslam et al., 1986; A. nummularia, Silveira et al., 2009; A.

patula and A. lentiformis, Glenn and O’Leary, 1984); (ii) substantial ion accumulation

that can represent almost half of the shoot dry mass in some situations (Flowers and

Colmer, 2008); and (iii) an increase in production of macromolecules (i.e. structural

biomass, albeit also including any starch if present) that is considered to be genuine

“growth”.

With increasing salinities, there is an increase in the mineral (ash) fraction, mainly in the

leaves (Ashby and Beadle, 1957; Redondo-Gómez et al., 2007; Wang et al., 1997; Khan

et al., 2000). In A. portulacoides, the leaf ash concentration increased linearly with

increasing salinity, reaching approximately 40% of leaf dry mass at 700 mM NaCl

(Redondo-Gómez et al., 2007). In A. nummularia grown at 600 mM NaCl the leaf ash

fraction was almost 50% of leaf dry mass and was 2 times higher than the ash fraction of

leaves grown with no salt in the root medium (Ashby and Beadle, 1957). Nevertheless

increases in structural biomass can be seen when growth is expressed as ethanol-

insoluble dry mass. In A. griffithii there was a 30% increase in whole plant ethanol

insoluble dry mass when plants were grown at 90 mM NaCl compared to 0 mM NaCl

(Khan et al., 2000). Growth enhancement in Atriplex spp. at low to moderate salinities

could be caused by the following factors:

(i) With no Na+ in the substrate, the plants might compensate for the Na+ deficiency

by accumulating K+ for osmoregulation. In the halophyte Salicornia bigelovii, there was

a reduced growth at 5 mM NaCl compared to 200 mM NaCl, which may have been

associated with increased concentrations of K+, Ca2+ and Mg2+ in shoot tissues; these

ions were about 4 times the levels adequate for shoots (Ayala and O’Leary, 1995).

Similar results were found in Atriplex amnicola grown in nutrient solutions with a


26

mixture of KCl and NaCl with a final concentration of 400 mM (K+ + Na+) Cl- and

different K+/Na+ ratios; an increase in the K+/Na+ in the external solution from 0.025 to

1.0 decreased relative growth rate by 38% (Aslam et al., 1988). This decline in plant

growth with a K+/Na+ of 1.0 was associated with increased K+ concentrations in the

expanded leaves, 2.1–12.5 times those in plants growing with lower K+/Na+ in the

external solution. These observations lead the authors to hypothesize that high K+ in the

leaves could lead to toxic cytoplasmic concentrations of K+. This hypothesis has

credence as it had been previously found that the halophyte Suaeda maritima has a poor

ability to retain K+ in the vacuoles compared to Na+ (Yeo, 1981).

(ii) When no NaCl is present externally, halophytes lack the Na+ and Cl- necessary

for osmotic adjustment and the generation of turgor pressure in elongating cells (Yeo

and Flowers, 1980). An example that would support such a view is given in a study

where the effects of a high (69%) or low (27%) relative humidity where determined for

Atriplex halimus grown with different NaCl concentrations in the root-zone (Gale et al.,

1970). With plants exposed to high (69%) relative humidity, highest growth occurred

with the NaCl-free medium, and whole-plant dry mass declined by 30% when the NaCl

concentration in the root-zone was increased up to 480 mM. On the other hand, when

relative humidity was lower (27%), plants had the highest dry mass at 120 mM NaCl,

and with the NaCl-free solution in the root-zone whole-plant dry mass was only about

8% of that with 120 mM NaCl. The interpretation of these results is that under low

humidity, where plants had higher transpirational demands, the plants had a high

requirement for Na+ and Cl- for osmotic adjustment and the generation of turgor, but

these ions were simply not available with plants grown with 0 mM NaCl; this loss of

turgor then resulted in a growth depression. By contrast, with high relative humidity,

transpirational demands were low, shoot water relations were less affected, and Na+ and

Cl- were therefore not required for osmotic adjustment.

2.4.2. Ion relations and osmotic adjustment with uniform salinities

To overcome the low osmotic potentials in saline soils, Atriplex spp. tend to accumulate

a large concentration of inorganic ions, mainly Na+ and Cl- (Albert and Popp, 1977;

Bennert and Schmidt, 1984). In general leaf Na+ and Cl- concentrations, expressed on a


27

leaf dry mass or leaf water basis, increase in an asymptotic trend with increasing

salinities in the root media, with the steepest increase over the range from 0 to 200-400

mM, while at higher salinities, values tend to remain more or less stable (Greenway,

1968; Ashby and Beadle, 1957; Redondo-Gómez et al., 2007; Zhu and Meinzer, 1999).

In A. nummularia exposed to 150–600 mM NaCl, Na+ and Cl- contributed to 60% of the

leaf osmolality (Silveira et al., 2009).

As enzymes in halophytes have the same sensitivity to NaCl as the corresponding

enzymes in non-halophytes (Flowers et al., 1977), it is assumed that part of Na+ and Cl-

ions are compartmentalized mainly in vacuoles to maintain low concentrations in the

cytoplasm (Storey et al., 1983; Wyn Jones and Gorham, 2002; Flowers and Colmer,

2008). To counterbalance the osmotic potential of the vacuoles, halophytes synthesize

metabolically compatible solutes in the cytoplasm, and for Atriplex spp. the main

compatible solute is glycinebetaine (GB) (e.g. A. spongiosa, Storey and Wyn Jones,

1978; A. prostrata, Egan et al., 2001). In the leaves of A. nummularia exposed to 0–600

mM NaCl, GB was the only organic solute that showed a salt-induced accumulation and

had the highest concentrations compared to all other studied organic fractions (Silviera

et al., 2009). In this study, GB was twice the concentration of total soluble sugars and

more than 200 times larger than the concentration of proline.

Although Na+ and Cl- are required for osmotic adjustment, the amount of these ions

delivered to the leaves via transpiration must exceed demand, as leaves of Atriplex spp.

possess salt bladders that remove ions from the leaves (Mozafar and Goodin, 1970;

Karimi and Ungar, 1986). In A. amnicola the salt bladders were of greatest importance

in the young leaves and, at 200 and 400 mM NaCl, bladders in young leaves accounted

for 81–86% of the leaf Na+ content whereas in older leaves bladders only accounted for

≤ 10% of the leaf Na+ content (Aslam et al., 1986).

In Atriplex spp. there are usually substantial decreases in shoot K+ concentrations as Na+

concentrations increase in the root medium. For example in A. griffithii, K+

concentrations in the leaves decreased from 311 µmol g-1 dry mass with 0 mM NaCl to

144 µmol g-1 dry mass with 90 mM NaCl in the external medium. By contrast, there was

no decline in K+ concentrations in roots with the same treatments (Khan et al., 2000).

Similar results were found for A. portulacoides (Redondo-Gómez et al., 2007) and A.


28

canescens (Glenn et al., 1996). The declines in K+ uptake, as Na+ concentrations

increase, result in sharp declines in the ratio of K+:Na+ in shoot tissues. For example in

the young leaves of A. amnicola, K+:Na+ ratio was 1.98:1 at 25 mM NaCl and this

decreased to 0.48:1 at 400 mM NaCl. Similar low ratios of K+:Na+ have been reported

for other halophytes, (e.g. Sarcocornia, Naidoo and Rughunanan, 1990; Halosarcia

pergranulata subsp. pergranulata, Short and Colmer, 1999); nevertheless the

concentration of K+ in the cytoplasm is likely to be adequate, as halophytes

compartmentalize the bulk of the Na+ in the vacuoles and the K+:Na+ ratio in the

cytoplasm of the cells is undoubtedly higher than in the tissues as a whole (Short and

Colmer, 1999).

2.4.3. Leaf gas exchange

In Atriplex spp., photosynthesis and stomatal conductance tend to decline with

increasing salinities in the root-zone (e.g. A. centralasiatica, Qiu et al., 2003; A.

lentiformis, Zhu and Menzeir, 1999). In A. centralasiatica at 400 mM NaCl, CO2

assimilation rates were half those measured with no NaCl in the root medium (Qiu et al.,

2003). The observed reductions in photosynthesis are in part due to stomatal limitations.

In halophytes exposed to salinity, stomatal conductance on a leaf area basis can be

related to a change in leaf thickness and to changes in stomatal frequency caused by

succulence (Flowers, 1985). For example, in Suaeda maritima grown either with no salt

or with 340 mM NaCl, there was a 35% decrease in transpiration caused by NaCl

(Hajibagheri et al., 1983). This decreased transpiration was associated with a 60% and a

90% increase in the thickness of the cuticle and the epidermal cell wall respectively.

Moreover, there is evidence that suggests that salinity does not damage the

photosynthetic apparatus in halophytic Atriplex spp. (Qiu et al., 2003; Boughalleb et al.,

2009). In A. centralasiatica the salinity of the root-zone (0–400 mM NaCl) had no effect

on the maximal efficiency of PSII photochemistry (Qiu et al., 2003). Similar results

were found for A. halimus exposed to 0–800 mM NaCl (Boughalleb et al., 2009).

However salt stress has been found to decrease net photosynthetic performance of the C4

species A. lentiformis by increasing the leakiness of the bundle sheath, thereby reducing

the inherent efficiency of the C4 CO2 concentrating system (Zhu and Menzeir, 1999). In

addition, in the same study increasing leaf Na+ concentrations associated with increased

salinity also caused a decline in Rubisco activity.


29

NaCl(mM)

0 100 200 300 400 500 600

% d

ry m

ass

0

50

100

150

200Sub-optimal

Optimal Supra-optimal

Figure 2.3. Growth response of Atriplex nummularia to increasing uniform NaCl in the root-zone (% dry mass compared to plants growing in 0 mM NaCl). Whole plant dry mass data were taken from: • Araújo et al. (2006) and ♦Dunn and Neales (1993).

W

hole

pla

nt d

ry m

ass

(%

zer

o sa

linity

)


30

2.5 Summary

The salinity of soil growing halophytic vegetation can reach extreme values, as high as

2–4 times that of seawater (Lambs et al., 2008; Silvestri et al., 2005; Slavich et al.,

1999). These extreme salinities can be above the levels endured by most halophytes. The

presence of halophytic vegetation in these hostile environments therefore raises the

question of how plants can survive such salinities. The habitat for plants in saline

environments is rarely uniform but there is a surprising deficiency in the knowledge of

halophyte physiology under non-uniform conditions.

Plant tolerance to salinity has been mostly investigated with uniform salt treatments.

However, heterogeneity in field soils implies that plant root systems will be exposed to

different salinities in time and space, and this is likely to affect plant performance in the

field. The fact that plants can have access to combinations of water of low and high

salinity in time and space within their rooting zones opens up the following questions:

(i) How is root growth affected by spatially non-uniform salinity?

(ii) Does the root presence, per se, in various soil layers give accurate information on

plant water and nutrient uptake?

(iii) Will the least saline region of the soil profile accessed by the root system ultimately

be the factor that influences total plant water use and plant productivity (e.g. shoot

growth)?

(iv) Does the root portion in the high salt zone affect plant physiology, e.g. stomatal

conductance?

(v) Despite preferential water uptake from the least saline zone, will the high salt portion

cause an increase in shoot ions? If there is an increase in ion accumulation in the

shoots what are the implications in terms of plant productivity in species with high

Na+ tissue tolerance, such as halophytes?


31

It is important to understand whether there are physiological mechanisms triggered by a

non-uniform distribution of salts in root systems, such as compensatory root growth in

the least saline portion, that are likely to mitigate the negative effects of high, even

extreme, salinities in one root portion. Understanding these mechanisms in conjunction

with knowledge on halophytic species’ salinity tolerances, will improve inferences about

species’ ability to perform in saline fields.


32

Chapter 3 Responses to moderate and severe non-uniform salinities

33

Chapter 3

Responses to Moderate to Severe Non-Uniform Salinity: Growth,

Stomatal Conductance, Water and Ion Relations

This chapter has been published:

Bazihizina N, Barrett-Lennard ED, Colmer TD (2009). Response to non-uniform

salinity in the root-zone of the halophyte Atriplex nummularia Lindl.: growth,

photosynthesis, water relations and tissue ion concentrations. Annals of Botany 104:

737-745.

3. Chapter 3: Growth, Stomatal Conductance, Water and Ion Relations in Atriplex nummularia Exposed to Moderate to Severe Salinities in One Root Half and Low Salinity in the Other One


34

3.1 Abstract

Soil salinity is often heterogeneous, yet the physiology of halophytes has typically been

studied with uniform salinity treatments. Growth, net photosynthesis, water use, water

relations and tissue ions in the halophytic shrub Atriplex nummularia Lindl. were

evaluated in response to non-uniform root-zone salinity. A. nummularia was grown in a

split-root system for 21 days, with either the same or two different NaCl concentrations

in the root-zone (ranging from 10 to 670 mM), in aerated nutrient solution bathing each

root half. Non-uniform salinity, with high salinity in one root half (up to 670 mM) and

10 mM in the other half, had no effect on shoot ethanol-insoluble dry mass, net

photosynthesis nor shoot predawn water potential. By contrast, a modest effect occurred

for leaf osmotic potential (up to 30% more solutes compared with uniform 10 mM NaCl

treatment). With non-uniform salinity (10/670 mM), 90% of water was absorbed from

the low salt side, and the reduction in water use from the high salt side caused whole

plant water use to decrease by about 30%; there was no compensatory water uptake

from the low salt side. In the uniform 670 mM treatment Na+ and Cl- concentrations in

expanding and expanded leaves were 1.9 to 2.3 times those of the non-uniform 10/670

mM treatment. On the other hand, in the 10/670 mM NaCl treatment, K+ concentrations

in expanding and expanded leaves were 1.2 to 2.0 times those of the uniform 670 mM

NaCl treatment. A. nummularia maintained net photosynthesis, shoot growth and shoot

water potential with non-uniform salinity in the root-zone, even when one root half was

exposed to 670 mM NaCl, a concentration that inhibits growth by 65% when uniform in

the root-zone.

3.2 Introduction

Saline environments can be highly variable in soil salinity in both space and time (e.g.,

Davidson et al., 1996; Mensforth and Walker, 1996; Bleby et al., 1997; Barrett-Lennard

and Malcolm, 1999; Slavich et al., 1999). The factors contributing to this variability are

the complex interactions of climate, topography, soil properties (e.g., texture, surface

mulches) and the presence of fluctuating groundwater. Halophytes in field situations

with distinct sources of water differing in salinity are able to take up water mainly from


35

the least saline source (e.g., Suaeda fruticosa, Anabasis articulata, Atriplex halimus,

Tamarix nilotica – Yakir and Yechieli, 1995).

Split-root experiments, where a root system is divided into two or more portions that are

exposed to different conditions, have been useful for studies of how plants respond to

heterogeneous soil conditions; e.g., partial root drying (Lawlor, 1973; Sobeih et al.,

2004) and heterogeneous nutrient distribution (Arredondo and Johnson, 1999; Drew and

Saker, 1978; Paterson et al., 2006). A split-root approach has also been used to study

physiological responses (i.e. growth, water relations, water use) of some non halophytes

to non-uniform root-zone salinity (e.g., Jeschke and Wolf, 1988; Kirkham et al., 1969;

Lycoskoufis et al., 2005; Shani et al., 1993; Zekri and Parsons, 1990). For halophytes,

there have only been two papers that have used split-root systems with non-uniform

salinity, and the purpose of these was to study nutrient uptake (Messedi et al., 2004;

Hamed et al., 2008). However, as these studies used a salt-free solution as the uniform

low salt ‘control’, growth enhancement in non-uniform salinity (zero on one side and

300–800 mM NaCl on the other side) was likely due to the ion requirements in these

dicotyledonous halophytes for maximal growth (Yeo and Flowers, 1980). For

halophytes, the absence of salt results in sub-optimal growth owing to ion deficiency,

and so use of NaCl-free solutions as controls has been criticized (Yeo and Flowers,

1980; Flowers and Colmer, 2008).

To study the physiological responses of a halophytic species when exposed to non-

uniform salinity, experiments were conducted on Atriplex nummularia Lindl. (old man

saltbush). This is a deep-rooted perennial C4 shrub that occurs naturally on saline lands

in the semi-arid zone of Australia, and has been established on salt-affected agricultural

lands for grazing livestock (Barrett-Lennard et al., 2003). Atriplex spp. occur in habitats

that are often characterized by seasonally- and spatially-variable soil salinities (Sharma

and Tongway, 1973; Barrett-Lennard and Malcolm, 1999; Slavich et al., 1999) and

water sources can change within the soil profile depending on seasonal changes in soil

water availability (Slavich et al., 1999). In the present experiments, A. nummularia had

its root system simultaneously exposed to a range of uniform salinities (10–670 mM

NaCl) or to non-uniform salinities, with one root half exposed to 10 mM NaCl and the

other root half to higher salinities (230–670 mM). Control plants were grown in 10 mM

NaCl, so as to avoid ion deficiencies (cf. Yeo and Flowers, 1980).


36

Experiments were conducted with A. nummularia in a split-root system to test four

hypotheses, that with high salinity in one root half: (i) there will be little or no adverse

effect on shoot ethanol-insoluble dry mass accumulation or net photosynthetic rate, (ii)

most of the water will be drawn from the low-salt side, (iii) plants with high salinity

only in one root half will have similar shoot water potential, leaf osmotic potential and

stomatal conductance as plants exposed to uniform low salinity, and (iv) plants will have

ion (Na+, K+, Cl-) concentrations in shoots intermediate to those in the low salinity

controls and plants at uniform high salinity.

3.3 Materials and Methods

A commercial clone of Atriplex nummularia, “Eyres Green” (Tamlin’s Nursery, South

Australia), was used. Stem cuttings (10 cm long) with about 5 nodes and leaves on the

upper two nodes were taken from a mother plant. Cuttings were propagated in a

glasshouse with day/night temperatures of 25/15 °C. The stem cuttings were moistened

at the base, dipped in a hormonal rooting powder (‘Richgro Root Strike’, containing 3 g

kg-1 indole-butyric acid) and planted into drained containers filled with washed white

sand. The containers were flushed daily with water until small roots were visible at the

shoot base, and then were irrigated with 0.1-strength nutrient solution for 4 days,

followed by 0.5-strength for 7 days, and thereafter full-strength solution was used. The

full-strength nutrient solution consisted of (mM): 4.7 K2SO4, 9.3 CaCl2, 5.0 Na2SO4, 1.0

MgSO4, 0.7 Ca(NO3)2, 0.3 K2HPO4, 0.2 NH4H2PO4; and (µM): 80 Fe EDDHA

(‘Sequestrene 138’), 23 H3BO3, 2 MnSO4, 2 ZnSO4, 0.5 CuSO4, 0.5 Na2MoO4. The

nutrient solution was adjusted to pH 6, using KOH.

After 4-6 weeks, when roots were about 2 cm long, established cuttings were transferred

to a naturally-sunlit phytotron with day/night temperatures of 20/15 °C. Cuttings were

washed free of sand and transferred to 4.5 L plastic pots containing aerated full-strength

nutrient solution. This solution contained the same nutrient concentrations reported

above, except that 0.1 mM Na2O3Si and 1.0 mM MES were added; pH was again

adjusted to 6, using KOH. There were 4 plants per 4.5 L pot and nutrient solutions were

topped up with deionized water as required and renewed weekly.

In experiment 1, two weeks after transferring the cuttings to nutrient solution culture,

plants were selected for shoot and root uniformity, and transferred into split-root pots


37

(one plant per pot, with 1.2 L per side; the split-root pots are described below). After a

further 4 days, NaCl was increased in both sides of all the split-pots in increments of 55

mM every 12 hours, until NaCl concentrations reached 670 mM. Three days after

reaching this concentration, all treatments were imposed with a single step down from

670 mM NaCl to the required level in each side. Plants were all exposed to 670 mM

NaCl before applying treatments in order to mimic seasonal dynamics in soil salinity in

the field, where there is salt accumulation after periods of high evapotranspiration

demands (summer) and, in autumn and winter, rainfall can then leach salts out of the

upper soil (Mensforth and Walker, 1996). In experiment 2, plants were grown the same

way, except that they were provided with full strength nutrient solution for 2 weeks

longer before being transferred into the split-root pots. This was done in order to have

larger plants to enhance measurements of water uptake over 12 hour periods.

Split-root system and water use measurement. Roots were divided into two

approximately equal halves, with each positioned in a split-root pot, so that the two root

halves could be exposed, at the same time, to different NaCl concentrations (Fig. 3.1).

To prevent mixing of the solutions between each root side, roots were laid in a

lengthwise-cut plastic T-piece (length: 6 cm; height: 6 cm; diameter: 3 cm). Inverted T-

pieces were sealed and then placed over two cylindrical plastic containers with a notch

into the top, cut to fit the T-piece, so that each root half was in a cylindrical container.

Each container was filled with 1.2 L of nutrient solution. A similar split-root system was

also designed to measure water use in each container of the split-root pot without

removing the roots from the pots. For water use measurements, each pot of the split-root

system had two electric wires glued on the inside that allowed re-filling of the pots to a

precise and constant level, with a precision of 10 µL, indicated by the presence or

absence of electrical conductivity between the wires.

Experiment 1

The experiment was conducted to assess growth, ion concentrations and water relations

of Atriplex nummularia when exposed to uniform or non-uniform salt concentrations, at

a range of NaCl concentrations in a split-root system. The experiment was conducted in

a naturally-lit phytotron (20/15°C day/night with an average PAR at midday during the

experimental period of 870 µmol m-2 s-1). The experiment tested 7 treatments (Table 3.1)

with 5 replicates in a completely randomized block design. In four treatments, the two


38

halves of the root system were both exposed to the same NaCl concentrations (mM): 10,

230, 450 or 670. The remaining three treatments had the two halves of the root system

exposed to two different NaCl concentrations, in each case with one side at 10 mM

NaCl, and the other at 230, 450 or 670 mM NaCl. Osmotic potentials of the solutions

were determined with a freezing point osmometer (Fiske Associates, Needham Heights,

Massachusetts, USA).

Leaf gas exchange. Leaf gas exchange measurements were taken on day 19 of

treatments on three randomly chosen plants in each treatment between 1100 and 1300

hours. Leaves on each side of the shoot, directly above each root side in treatments

10/230, 10/450 and 10/670, were measured separately. Measurements of net

photosynthetic rate and stomatal conductance were determined on young fully expanded

leaves using a LI-COR 6400 Photosynthesis System (LI-COR, Inc., Lincoln, NE, USA)

at ambient relative humidity (50-60%), a reference CO2 of 380 µmol mol-1, flow rate of

500 µmol s-1 and PAR of 1500 µmol m-2 s-1.

Harvests. Plants were harvested on days 0 and 21 after the commencement of

treatments. Leaf area, shoot and root fresh mass were determined. Leaf area was

measured with a portable leaf area meter (Li-Cor LI-3100, Lincoln, NE, USA). In order

to assess any differences between sides, the two halves of the root system and each side

of the shoot above each of the root halves were harvested separately (Fig. 1). The

portion of the stem that was central was also sampled separately. Each shoot side

directly above each root half was then subdivided into expanding leaves, expanded

leaves and side branches with leaves removed. Roots were washed for 2 min in 3

changes of iso-osmotic mannitol solution, also containing 9 mM CaSO4, and blotted dry.

Fresh mass was recorded. All samples were snap-frozen in liquid N2, stored at -80°C,

and then freeze-dried.

Measurement of predawn water potential and osmotic potential of expressed leaf sap.

Predawn shoot water potential was measured on excised shoot segments using a

Scholander pressure chamber between 400 and 500 hours. Leaf tissue osmotic potential

was measured on expanding leaves (leaves at the 3rd or 4th node in which leaf area varied

from 20 to 40% of the size of fully expanded leaves) and on fully expanded leaves.

Predawn shoot water potential, and the osmotic potential of expanding and fully

expanded leaves were measured on three randomly chosen plants from each treatment


39

on day 21 after commencement of treatments. Shoot segments and leaves from both

sides of the shoot, directly above each root half, were measured. As no differences

between predawn shoot water potential, osmotic potential of expanding and fully

expanded leaves in the two shoot sides directly above each root half were found, data

presented are the average values for the data pooled from the two shoot sides.

Ethanol-insoluble dry mass. To determine ethanol-insoluble dry mass, ground plant

tissues were extracted twice with 80% ethanol, refluxed twice for 20 minutes,

centrifuged for 10 minutes at 9,335 g (IEC micromax ventilated microcentrifuge

OM3590, Needham Heights, MA, USA), and the insoluble-fraction was dried at 70°C

for 24 hours and weighed.

Measurement of ion concentrations. Ground tissue samples were extracted with 0.5 M

HNO3 by shaking in vials for 48 hours. Diluted extracts were analyzed for Na+, K+

(Jenway PFP7 Flame Photometer, Essex, UK) and Cl- (Buchler-Cotlove Chloridometer

662201, Fort-Lee, New Jersey, USA). The reliability of the methods was confirmed by

analyses of a reference tissue (broccoli, ASPAC Plant number 85) taken through the

same procedures.

Experiment 2

The experiment was conducted to assess water use patterns of Atriplex nummularia

when exposed to non-uniform salinity in the root-zone. The plants were raised in a

naturally-lit phytotron (20/15°C day/night with an average PAR at midday during the

experimental period of 790 µmol m-2 s-1). The experiment consisted of 3 treatments with

4 replicates, and two times of measurement, in a randomized block design. The

treatments were (mM NaCl): 10/10; 10/670; 670/670. The first measurement of water

use was performed 7 days after treatments commenced, as root halves were expected to

be approximately equal in surface area early after the commencement of treatments. The

morning after the water use measurements, plants were harvested (H1). Water use was

also measured for a second complete set of replicate plants 21 days after imposing the

treatments. Plants were again harvested the morning after the water use measurements.

Midday shoot water potentials were measured for the 10/10 and 10/670 treatments as

described for experiment 1.


40

Table 3.1. Treatments imposed on Atriplex nummularia for 21 days (experiment 1). Cuttings were raised, stepped up in salinity and grown for 3 days at the highest salinity (670 mM) in both sides of the split-root system, and then treatments were imposed. Osmotic potentials of the applied solutions were measured with a freezing point osmometer.

Treatments (mM NaCl)

Osmotic potential of applied solution

(MPa)

10*/10* -0.15/-0.15 230/230 -1.15/-1.15 450/450 -2.16/-2.16 670/670 -3.18/-3.18 10*/230 -0.15/-1.15 10*/450 -0.15/-2.16 10*/670 -0.15/-3.18

*Values from full strength nutrient solution; the concentration of Na+ (from Na2SO4 and Na2O3Si) was 10.2 mM, and of Cl- was 18.6 mM (from CaCl2).


41

Figure 3.1. Schematic diagram of the split-root system used in this study. The root system of Atriplex nummularia raised from stem cuttings was exposed either to uniform salinity (equal salinity in sides A and B) or to non-uniform salinity (different salinities in sides A and B) for 21 days. In experiment 1, in the uniform salinity treatments, both root halves were exposed to 10, 230, 450 or 670 mM NaCl and, in non-uniform treatments, one root half was exposed to 10 mM NaCl and the other half to 230, 450 or 670 mM NaCl. In experiment 2, there were only 3 treatments: uniform salinity with 10 or 670 mM NaCl and non-uniform salinity where one root half was exposed to 10 mM NaCl and the other to 670 mM NaCl.

Expanding leaves

Roots

Side branch Central stem

Expanded leaves


42

Water use measurements. 48 hours prior to water use measurements, plants were

transferred to a split-root system designed for water use measurements (described

above) in a controlled-environment room (20/15°C day/night, 12 hours day/night,

average relative humidity 70%, with an average PAR at shoot height of 310 µmol m-2 s-

1). All containers were bubbled with pre-humidified air and three blank pots (i.e. without

plants) were used to determine any background evaporative losses. To measure water

use, each pot containing nutrient solution was topped up with deionized water at 600

hours and then again at 1800 hours (commencement and end of the 12 hours light

period) to the point where both electrical wires (described above) were in contact with

the nutrient solution, and the volumes added were recorded.

Harvests. Root and shoot fresh mass and leaf areas were measured as described in

experiment 1. The two halves of the root system were harvested separately as in

experiment 1, but no separations were made between shoot sides. Total leaf area was

measured with a portable leaf area meter (Li-Cor LI-3100, Lincoln, NE, USA) and

stems and leaves were oven-dried at 60°C to determine dry masses. Roots were blotted

to remove excess surface moisture, sealed in plastic bags and stored at 4°C for 12 hours.

Root systems were scanned for surface area using a WinRhizo root scanner (Regent

Instruments Inc., Quebec). Roots were oven-dried at 60°C to determine dry mass.

Statistical analyses. Statistical analyses were conducted using Genstat for Windows 10th

Edition (Genstat software, VSN International, Hemel Hempsted, UK). ANOVA was

used to identify overall significant differences between treatments and between sides

within treatments, depending on the data set. When significant differences were found,

mean-separations were calculated using Duncan’s multiple range test. Unless otherwise

stated, the significance level was P ≤ 0.05.

3.4 Results

Shoot and root growth (experiment 1)

Growth was measured as ethanol-insoluble dry mass, as in dicotyledonous halophytes

ions may contribute up to 30 to 50% of the dry mass (Flowers et al., 1986). In addition,


43

as in in many stress situations sugars accumulate as a mere consequence of the inhibited

growth (i.e. Kameli and Lösel, 1996; Barrett-Lennard et al., 1988), ethanol-insoluble dry

mass (i.e. structural biomass with soluble sugars removed, including any starch if

present) rather than total dry mass has been used to assess growth (Barrett-Lennard et

al., 1988; Colmer and Greenway, 2010). The ratio of ethanol-insoluble dry mass to dry

mass ranged from 0.92 (central stem at 230 mM NaCl) to 0.55 (young leaves at 670 mM

NaCl) (data not shown).

Under uniform conditions, no differences in shoot ethanol-insoluble dry mass were

found at 10 and 230 mM NaCl, but at 450 and 670 mM NaCl shoot dry mass declined to

be 58% and 35% respectively of that with uniform 10 mM NaCl (Fig. 3.2A). When only

one root half was exposed to these NaCl concentrations, shoot growth was equal to the

growth observed in plants with treatment 10/10 mM NaCl. The difference between

uniform and non-uniform salt treatments was most apparent at the highest NaCl

concentration, where the shoot ethanol-insoluble dry mass of plants at 10/670 mM NaCl

was 2.3 times that of plants grown in uniform 670 mM NaCl.

By contrast with shoot ethanol-insoluble dry matter accumulation, uniform and non-

uniform treatments had no significant effects on total root ethanol-insoluble dry mass

(Fig 3.2B). There were no significant differences in root ethanol-insoluble dry mass

between the high and low-salt sides in any of the non-uniform treatments.

Leaf gas exchange parameters (experiment 1)

All gas exchange parameters in non-uniform treatments were measured for both shoot

sides directly above each shoot half. A significant difference (P < 0.05) was found in the

treatment 10/450 mM NaCl, where all gas exchange parameters in the shoot side above

450 mM were 74–80% of those measured in the shoot side above the 10 mM NaCl (data

not shown). However, as no differences between shoot sides were observed in the other

treatments, the data presented are the average values of the measurements from the two

shoot sides.

In uniform treatments, net photosynthetic rate (Fig. 3.3A) and stomatal conductance

(Fig. 3.3B) declined as salinity in the medium increased, and at 670 mM NaCl net


44

photosynthetic rate and stomatal conductance were 28% and 23% respectively of those

in uniform 10 mM NaCl. In plants subject to non-uniform salinities, net photosynthetic

rate was similar to that of plants uniformly subject to 10 mM NaCl. However, applying

450 and 670 mM NaCl to one root half caused a 21–24 % decline of stomatal

conductance compared with the uniform 10 mM treatment (Fig. 3.3B). Uniform high

salinity was, however, more inhibitory compared with non-uniform salinity, and the

difference between uniform and non-uniform salt treatments was most apparent at the

highest NaCl treatment, where net photosynthetic rate and stomatal conductance in

plants growing with only one root half at 670 mM NaCl were increased to 3.2 and 3.5

times respectively of the values for plants with both root halves at 670 mM NaCl (Fig.

3.3).

Shoot water potential and leaf osmotic potential (experiment 1)

Independently of the NaCl concentration in the high-salt sides, the predawn shoot water

potential of plants exposed to non-uniform salinities in the root-zone were equal to those

of plants exposed uniformly to 10 mM NaCl (Fig. 3.4A). In uniform treatments, the

predawn water potential decreased as NaCl concentrations in the medium increased,

with the value at 450 mM NaCl almost 2 times lower (i.e. more negative) than that for

plants at 10 mM. Values for plants growing at 670 mM NaCl were not obtained as

shoots were succulent, balancing pressures applied were particularly high and plants

burst out of the Scholander chamber gasket at around -2.7 MPa.

In uniform treatments, the osmotic potential of expanding and fully expanded leaves

declined significantly as NaCl concentrations in the root medium increased (Fig. 3.4B

and 3.4C). At 670 mM NaCl, the osmotic potential of expanding leaves was 2 times

lower (i.e. more negative) than the osmotic potential of expanding leaves of plants

exposed to uniform 10 mM NaCl. Similar results were found for the osmotic potential of

fully expanded leaves of plants growing in uniform 670 mM NaCl. Non-uniform salinity

had small effects on leaf osmotic potentials, and, compared to leaf osmotic potentials in

the 10/10 treatment, the osmotic potential of expanding leaves was 1.4 and 1.2 times

lower (i.e. more negative) in treatments 10/450 and 10/670, respectively, and the

osmotic potential of expanded leaves was 1.15 times lower in treatment 10/450 (Fig.

3.4B and 3.4C).


45

A

0

1

2

3

4

B

NaCl (mM)

0 100 200 300 400 500 600 7000

1

2

******

Sho

ot e

than

ol-is

nolu

ble

dry

mas

s(g

)

Roo

t eth

anol

-inso

lubl

e dr

y m

ass

(g)

Figure 3.2. Responses of ethanol-insoluble dry mass of (A) shoots and (B) roots of Atriplex nummularia with uniform ( ) or non-uniform ( ) NaCl in the root-zone (experiment 1). A. nummularia cuttings were grown for 21 days in a split-root system with uniform and non-uniform salinities. In non-uniform treatments one root half was exposed to 10 mM NaCl and the other half to 230, 450 or 670 mM NaCl. Initial values of shoot and root ethanol-insoluble dry mass (g) were: 0.74 ± 0.08 and 0.18 ± 0.02. Values are means (n = 5) ± S.E. Asterisks above symbols indicate where means of non-uniform treatments are significantly different from the means of the corresponding uniform treatments: *** (P≤ 0.001). Note different scale in (A) and (B).


46

B

NaCl(mM)

0 100 200 300 400 500 600 700

(mm

ol m

-2 s

-1)

0

50

100

150

200

250

300

A

( m

ol m

-2 s

-1)

0

5

10

15

20

25

Sto

mat

al c

ondu

ctan

ceN

et p

hoto

synt

hetic

rat

e

*

***

***

*

µµ µµ

Figure 3.3. Responses of (A) net photosynthetic rate and (B) stomatal conductance of the young fully expanded leaves of Atriplex nummularia with uniform ( ) or non-uniform ( ) NaCl in the root-zone (experiment 1). In non-uniform treatments one root half was exposed to 10 mM NaCl and the other half to 230, 450 or 670 mM NaCl. Measurements were taken 19 days after imposing treatments with PAR of 1500 µmol m-2 s-1, ambient relative humidity of 50-60%, reference CO2 of 380 µmol mol-1 and flow rate of 500 µmol s-1. Compared to uniform low salt control (10/10 mM NaCl), declines in stomatal conductance by 21–24% in the non-uniform 10/450 and 10/670 mM treatments were significant (P≤ 0.05). Values are means (n = 3) ± S.E. Asterisks above symbols indicate where means of non-uniform treatments are significantly different from the means of the corresponding uniform treatments: * (P≤ 0.05); *** (P≤ 0.001).


47

The contributions of Na+, K+ and Cl− to the sap osmotic potential of the leaves were

calculated from the tissue ion data (discussed below); these ions were estimated to

contribute 73–89% of the sap osmotic potential in leaves of plants grown under uniform

and non-uniform salinities. Estimates of "bulk turgor" in the leaves could have been

calculated from osmotic and water potential data; however, this approach does not take

into account the partitioning of water and solutes between the apoplastic and symplastic

compartments (Wardlaw, 2005). This indirect calculation of turgor may therefore lead to

erroneous estimates, and would be further compromised in the present study by ions in

salt bladders contributing to leaf sap osmotic potential.

Tissue ion concentrations (experiment 1)

Ion concentrations were measured in roots, expanding and expanded leaves, and are

expressed on a tissue water basis (Fig. 3.5). In plants growing with non-uniform

salinities, differences in ion concentrations could be found between the two root halves

and thus data for each side are presented. Differences in K+ concentrations were also

found in leaves above each root half in non-uniform treatments; K+ concentrations in the

side above the 10 mM half were 23–52 % higher compared to those in leaves above the

high salt side. As Na+ and Cl- concentrations in non-uniform treatments were not

significantly different between shoot sides, the data presented are the average values for

the data pooled from the two shoot sides.

Roots. In uniform treatments, Na+ and Cl- concentrations in root tissues increased with

increasing NaCl concentrations in the medium, with Na+ and Cl- concentrations at 670

mM being 5.1 and 5.8 times respectively of those in plants grown at 10 mM (Fig. 3.5A

and 3.5G). In non-uniform treatments, Na+ and Cl- concentrations in roots in each side

were similar to the values measured in roots in uniform treatments at the same NaCl

concentrations (Fig. 3.5A and 3.5G). There were little or no differences in K+

concentrations on an ethanol-insoluble dry mass basis in roots (data not shown), but as

tissue water content declined at higher NaCl concentrations in the root-zone (data not

shown), K+ concentrations on a tissue water basis increased at 450 and 670 mM (Fig.

3.5D).


48

C

NaCl (mM)

0 100 200 300 400 500 600 700

-5

-4

-3

-2

-1

0

B

Leaf

sap

os

mot

ic p

oten

tial

expa

ndin

g le

aves

(MP

a)

-5

-4

-3

-2

-1

0

A

-5

-4

-3

-2

-1

0S

hoot

pre

daw

nw

ater

pot

entia

l(M

Pa)

**

** **

***

*

*

**

*

Leaf

sap

os

mot

ic p

oten

tial

expa

nded

leav

es(M

Pa)

Figure 3.4. Responses of (A) shoot predawn water potential, (B) osmotic potential of expressed sap of expanding, and (C) expanded, leaves of Atriplex nummularia with uniform ( ) or non-uniform ( ) NaCl in the root-zone (experiment 1). In non-uniform treatments one root half was exposed to 10 mM NaCl and the other half to 230, 450 or 670 mM NaCl. Predawn water potentials and osmotic potentials were determined 21 days after imposing the treatments. Expanding leaves were 20–40% the size of the expanded leaves, and in both cases included salt bladders. No differences were found between the measurements taken on each shoot side directly above each root half, therefore data from the two shoot sides were averaged. Water potential data are not available for uniform 670 mM treatment as succulent shoot tissues burst out of the Scholander chamber at pressures > 2.7 MPa. Values are means (n = 3) ± S.E. Asterisks above symbols indicate where means of non-uniform treatments are significantly different from the means of the corresponding uniform treatments: ** ( P≤ 0.01); *** (P≤ 0.001).


49

Shoots. In uniform treatments the concentration of Na+ in both expanding (Fig. 3.5B)

and expanded leaves (Fig. 3.5C) increased with the external NaCl concentration. In

plants at uniform 670 mM NaCl, Na+ concentrations in expanding and expanded leaves

increased to 3.3 times and 2.8 times respectively of the concentrations with uniform 10

mM NaCl. However with non-uniform salinity treatments, concentrations of Na+ in

leaves were relatively constant across non-uniform salt treatments and increased to only

1.30–1.75 times the concentration in the uniform 10 mM control. For leaf Cl- the general

trends were similar to those observed for Na+ (Fig. 3.5H and 3.5I). Potassium

concentrations in leaves were affected by salinity, uniform/non-uniform salt treatment

and in the non-uniform treatments by the differences in salinity of each root half. In

leaves of plants exposed to uniform salinities, K+ concentrations were highest at 10 mM

NaCl and declined by 55–60% at 230 mM and then remained relatively constant at NaCl

concentrations up to 670 mM (Fig. 3.5E and 3.5F). In all non-uniform treatments, leaf

K+ concentrations were generally higher than in uniform treatments with the same high

salt concentration.

Ion concentrations in lateral branches and central stems followed similar trends as

expanding and expanded leaves (data not shown).

Water use, water use efficiency and midday shoot water potential (experiment 2)

The patterns of water use across treatments were similar after 7 and 21 days of

treatment; the data presented here are for day 21 (Table 3.2). In all treatments, whole

plant water use after 21 days was about 1.5 times higher than that measured after 7 days

of treatment. Whole plant water use in uniform 670 mM NaCl and in non-uniform

10/670 was 23% and 69% of that in uniform 10 mM NaCl, respectively. Under non-

uniform conditions, at both times, most water uptake was from the side with 10 mM

NaCl but there was still some water uptake (7–13% of total water use) from the side at

670 mM. There was no compensatory increase in water uptake from the low-salt side.

The differences in water uptake between treatments were not mediated by changes in

root or shoot growth; similar patterns in water use between treatments also occurred

when the data were expressed on root surface area (Table 3.2) or leaf area (data not

shown) bases.


50

With whole plant water use over 12 h data and accumulation of ethanol-insoluble dry

mass between day 7 and 21 of treatments, water use efficiency (ethanol-insoluble dry

mass accumulated over 14 days / whole plant water use over 12 h) was estimated.

Although there was an increase in water use efficiency with 10/670 and uniform 670

mM NaCl compared with uniform 10 mM NaCl, differences between all treatments

were not significant (average values: 10/10: 0.12 g ml-1; 10/670: 0.24 g ml-1; 670/670:

0.21 g ml-1). It is important to note that water use measurements were only limited to a

12 hour period at the end of the experiment and thus did not represent the whole plant

water use over the experimental period.

Midday shoot water potential was also measured in experiment 2, and for treatments

10/10 and 10/670, average values were: -1.43 ± 0.7 MPa and -1.67 ± 0.3 MPa,

respectively.

3.5 Discussion

The results of the present chapter show that A. nummularia with a non-uniform salt

distribution in the root-zone (i.e. 10 mM NaCl in one half, up to 670 mM in the other

half) maintained shoot growth and photosynthesis relative to the uniform low salt

control (Fig. 3.2A and 3.3A). This occurred even with NaCl in one half of the root

system of 670 mM, a concentration sufficient to decrease shoot ethanol-insoluble dry

mass by 65% in a uniform treatment. The present results contrast with previous studies

where halophytes exposed to non-uniform salinity had 4 to 104% increases in shoot dry

mass compared with control plants growing in NaCl-free conditions (Messedi et al.,

2004; Hamed et al., 2008). The present methodology used in this thesis for A.

nummularia would seem more appropriate for understanding halophyte physiology, as

unlike the two previous split-root experiments with halophytes, the use of the NaCl-free

solutions that can cause ion deficiency in dicotyledonous halophytes was avoided (cf.

Yeo and Flowers, 1980).

In accord with our second hypothesis, under non-uniform salinity most of the water

(90%) came from the low-salt side (Table 3.2). However, compared with the low salt

control, there was no increase in water uptake from the low-salt side to compensate for


51

the decrease in water uptake from the high-salt side, as it had been previously reported

for some non halophytes exposed to non-uniform salinities in the root-zone (West, 1978;

Zekri and Parsons, 1990). Non-uniform salinity had no effect on the partitioning of root

growth between the high and low-salt sides during the 21 days of treatment. Previous

experiments with Atriplex spp. have reported no effect of NaCl at concentrations up to

360 mM applied for 1 month on root dry mass, whereas shoot growth declined (e.g., A.

nummularia, Greenway, 1968; A. griffithii var. stocksii, Khan et al., 2000). Thus, the

effects on water use are not caused by changes in the partitioning of root growth

between the two sides.

The 30% reduction in water use by plants exposed to non-uniform 10/670 mM NaCl

(calculated from Table 3.2, experiment 2) was generally consistent with the declines of

21–24% in stomatal conductance in leaves of plants exposed to 10/450 and 10/670 mM

non-uniform NaCl, as compared to the uniform low salt controls (Fig. 3.3B, experiment

1). For some halophytes at uniformly high salinity (700 mM), root signals have been

suggested as the cause for stomatal closure (e.g., Atriplex portulacoides and Sarcocornia

fructicosa; Redondo-Gómez et al., 2006, 2007). The slight decrease in stomatal

conductance between plants exposed to non-uniform salinities (10/450, 10/670 mM) and

uniform low salt controls referred to above in A. nummularia provides some evidence

for root-to-shoot signalling from the root portion at high salinity. Such a signal could

have conceivably been transported out of the high salinity root half via the xylem as

some water uptake occurred from this side.

Irrespective of the salinity on the high-salt side, all plants with non-uniform salinities

had predawn shoot water potentials similar to those of the uniform low-salt control,

suggesting that, under non-uniform salinities, predawn water potentials were influenced

mainly by the water potential of the low-salt side. It is generally expected that predawn

water potential will be in equilibrium with the less negative soil water potentials around

active roots (i.e. as shown for a range of non halophytic species with non-uniform soil

moisture e.g., Bouteloua gracilis, Sala et al., 1981; Quercus spp., Bréda et al., 1995;

Betula pendula, Fort et al., 1998). This would occur provided that there are enough roots

in the least negative water potential side to enable the equilibration to occur (Améglio et

al., 1999). It is, however, important to note that with uniform 10 mM NaCl, and also

with non-uniform salinities, there was a discrepancy between the predawn shoot water

potential and the water potential of the medium in the least saline portion of the root-


52

zone. This difference, also called predawn disequilibrium (Donovan et al., 2001), has

been previously reported for several non-halophytes and halophytes, and has been

related to: poor hydraulic soil-root contact; the build-up of solutes at the root surface

and/or apoplast; low plant hydraulic conductance; growth; and hydraulic redistribution

(Donovan et al., 1999; Donovan et al., 2001; James et al., 2006; Stirzaker and Passioura,

1996). In the present study, for uniform 10 mM NaCl and all non-uniform treatments,

the predawn disequilibrium was ∼ 1 MPa and then declined at higher salinities, as

previously noted for the halophytic shrub Sarcobatus vermiculatus (James et al., 2006).

As plant were grown in an aerated nutrient solution, poor soil-medium contact,

accumulation of solutes at the root surface and hydraulic redistribution (at least under

uniform 10 mM NaCl) can be excluded; therefore this observed predawn disequilibrium

observed could have been caused by: low plant hydraulic conductivity; nighttime

transpiration (as shoots were not bagged to prevent this); growth; and also a possible

accumulation of solutes in the root apoplast (Donovan et al., 1999; Donovan et al.,

2001; James et al., 2006; Stirzaker and Passioura, 1996).

Plants subject to non-uniform salinity (10/670 mM NaCl) had also similar midday water

potentials to those of plants exposed to uniform 10 mM NaCl. Despite the limitation that

stomatal conductances and midday water potentials were not measured on the same

treatments at the same time, it is highly probable that the decline in stomatal

conductance observed in plants subject to non-uniform salinities compared to the

uniform low controls (experiment 1) was sufficient to maintain similar midday water

potentials in plants subject to non-uniform salinity in experiment 2.

Interestingly, leaf osmotic potentials under non-uniform salinities were not substantially

affected by the level of salinity on the high-salt side; even with 670 mM NaCl in one

root side, the leaf osmotic potential was only 20% more negative than that of the

uniform low-salt control, and was approximately 20% less negative than the osmotic

potential of the nutrient solution on the high-salt side (670 mM NaCl = -3.07 MPa).

Given these data on osmotic potential, and that midday shoot water potential was also

less negative than that of the 670 mM NaCl treatment solution, it is unclear from this

experiment how these plants maintained water uptake from the high-salt side (up to 13%

of the total whole plant water use)3. The similarity in leaf osmotic and water potential of

3 With the availability of further data in Chapters 4 and 5, this issue has been re-considered in the Concluding Discussion (Chapter6).


53

plants exposed to non-uniform salinity and zero-salt controls has also been reported for

some non halophytes, but in these cases leaf osmotic and water potentials were always

more negative than those of the saline solutions (Kirkham et al., 1969; Zekri and

Parsons, 1990).

Under non-uniform conditions, the root half exposed to high salinity had high

concentrations of Na+ and Cl-, but there were not large increases in these ions in the

shoot tissues, even when one root half was exposed to 670 mM NaCl (Fig. 3.5). In non-

uniform salinity treatments, leaf Na+ and Cl- concentrations remained substantially

below those in uniform treatments at 450 and 670 mM. Across all non-uniform

treatments, plants maintained relatively constant Na+ and Cl- concentrations in leaves,

which indicate that A. nummularia can well-regulate its internal ion concentrations

under non-uniform conditions. Regulation of leaf ions is essential for salt tolerance, as

adverse effects can result if concentrations become too high, even in halophytes

(Flowers and Colmer, 2008).

In conclusion, the present Chapter shows that non-uniform salinity is not damaging to

the halophytic shrub A. nummularia, even when the NaCl concentration in the high-salt

side does impede growth when applied to the whole root system. Although the results

documented here are only applicable to the commercial clone of A. nummularia, “Eyres

Green”, and therefore these results should be verified on other ecotypes of A.

nummularia and other halophytes, the results of this Chapter undoubtedly add to

knowledge on halophyte physiology (reviewed by Flowers and Colmer, 2008), which

previously has generally only been studied with uniform salinities in the root-zone.

Given the temporal and spatial salinity variations in most field situations (see

Introduction), the responses described here to non-uniform salinity would undoubtedly

contribute positively to the growth and physiology of halophytes in saline environments.


54

NaCl treatment(mM)

0 200 400 600

C

Ion co

ncen

tratio

ns on a

tissu

e water

bas

is

(mM

)

K+

0 200 400 600

B

F E

I H

Cl-

***

***

***

***

*** *** ***

******

* ** * *** **

**

0

100

200

300

400

500

0 200 400 6000

200

400

600

800

1000

A

Na+

0

200

400

600

800

1000

Roots Expanding leaves Expanded leaves

D

G

High salinity side

Low salinity side

High salinity side

Low salinity side

High salinity side

Low salinity side

*

Figure 3.5. Concentrations on a tissue water basis (mM) of (A, B, C) Na+, (D, E, F) K+ and (G, H, I) Cl- of different plant parts of Atriplex nummularia grown with uniform ( ) or non-uniform ( ) salinity in the root-zone (experiment 1). Uniform salinity treatments had in both root halves 10, 230, 450 and 670 mM NaCl. In non-uniform treatments one root half was exposed to 10 mM NaCl and the other half to 230, 450 or 670 mM NaCl. In non-uniform treatments, the two root halves showed different ion concentrations, and thus means for each side (labelled with arrows) are presented. With the exception of K+, ion concentrations did not differ in leaves from above each root half in non-uniform treatments, hence data presented are the averages for values pooled for the two sides of each replicate plant. K+ concentrations (D, E, F) are also averages for the two shoot sides, although values for leaves directly above the low salinity side were 23–52 % higher than those above the high salinity side. Values are the mean (n = 5) ± S.E. Asterisks above symbols indicate where means of non-uniform treatments are significantly different from the means of the corresponding uniform treatments: * (P≤ 0.05); ** (P≤ 0.01); *** (P≤ 0.001). Note different scales for (D), (E) and (F).


55

Table 3.2. Response of whole plant water use and water uptake rates expressed on root surface area basis of Atriplex nummularia grown under uniform and non-uniform NaCl concentrations in the root-zone (experiment 2).

Low salinity side High salinity side Low salinity side High salinity side

10/1010/670 16.90 ± 1.38a 1.94 ± 0.49b 0.19 ± 0.03a 0.03 ± 0.01b

670/670

13.68 ± 0.63a* 0.23 ± 0.04a

3.21 ± 0.36b* 0.08 ± 0.01b*

NaCl (mM)

Whole-plant water useWater uptake on root surface area

basis(mL) (mmol m-2 s-1)

Plants were exposed to the NaCl treatments for 21 d. Water uptake was measured over a 12 hours light period (600 hours – 1800 hours) on the final day of treatments. Values for each root half are shown separately for the non-uniform treatment. Water uptake rates on a leaf area basis (mmol m-2 s-1) were: 0.85 ± 0.05 in treatment 10/10, 0.53 ± 0.06 in treatment 10/670, and 0.31 ± 0.04 in treatment 670/670. Values are means (n = 4) ± S.E. and different lower indicate significant differences (P≤ 0.05). * as under uniform 10 and 670 mM NaCl no differences were found between means in each side, data for the two sides were averaged.

Chapter 4 Responses to extreme non-uniform salinity

56


57

Chapter 4

Responses to Extreme Non-Uniform Salinity: Compensatory Root

Growth in, and Preferential Water Uptake from, the Least Saline Side

4. Chapter 4: Responses to Extreme Non-Uniform Salinity: Compensatory Root Growth in, and Preferential Water Uptake from, the Least Saline Side


58

4.1 Abstract

Salinity in soils is often heterogeneous, yet the physiology of halophytes has typically

been studied in media with uniform salinity. The growth and physiology of the

halophyte Atriplex nummularia was assessed when exposed to severe and extreme non-

uniform salinities in the root-zone. Six-week-old cuttings were exposed either to

uniform (10, 500 or 1500 mM NaCl) or non-uniform salinities (one half in 10 and the

other in 500 or 1500 mM NaCl). Shoot growth was severely inhibited by 1500 mM

NaCl when applied to both root halves. By contrast, in both non-uniform treatments,

shoot dry mass was similar to that of control plants (uniform 10 mM NaCl). In the non-

uniform treatment 10/500, root dry mass was no different from that of uniform

treatments at 10 and 500 mM NaCl. However, with 10/1500 there was an increased

allocation (40% higher than in uniform 10 mM NaCl) of root dry mass to the low-salt

side. Despite most water being taken up from the low-salt side, in both non-uniform

treatments, a reduction in stomatal conductance was observed, and this reduction was

more pronounced at 10/1500. Midday water potentials in non-uniform treatments were

similar to those of control plants, but leaf osmotic potentials became more negative due

to solute accumulation. With non-uniform salinities, shoot Na+ and Cl- concentrations

more than doubled compared with uniform 10 mM NaCl, with part of these ions being

most likely used for osmotic adjustment. On the other hand, the water necessary for

growth was taken up from the low-salt side. In conclusion compensatory root growth in

the low-salt side and water uptake from the low-salt side were likely to be the main

factors that enabled the observed shoot growth under extreme non-uniform salinity.

4.2 Introduction

Temporal and spatial variations in soil salinity occur in most landscapes affected by

salinity (Mass, 1993). Examples from agricultural soils to salt marshes show that the

salinities in the soil solution over distances accessible by the roots of single plants can

vary from one-tenth to four times seawater1 (Akeyord et al., 1998; Archibald et al.,

2006; Bleby et al., 1997; Çullu et al., 2009; Ewe et al., 2007; Hao et al., 2009; Lin and

Sternberg, 1994). One example of this comes from a dryland saline environment 1 Seawater has approximately 500 mM NaCl and an electrical conductivity of 55 dS m-1 (Wyn Jones and Gorham, 2002)


59

growing the halophyte Atriplex amnicola; at this location, the ECe of the soil2 in the

upper 10 cm of the soil profile varied from 3 to 65 dS m-1 over a distance of 10 m

(Davidson et al., 1996). Salinity can also reach extreme levels; in a salt marsh growing

halophytic vegetation (e.g. Suaeda maritima and Sarcocornia fruticosa), the salinity in

the upper 20 cm of the soil profile varied from 70 ppt (approximately 108 dS m-1) to 180

ppt (approximately 280 dS m-1) over a distance of less than 15 m (Silvestri et al., 2005).

The presence of halophytic vegetation in these environments with extreme salinities

warrants studies of responses to extreme non-uniform salinities in the root-zone.

I studied the growth of halophytes – plants that are able to tolerate far higher salinities in

their root-zone than non-halophytes (Flowers and Colmer, 2008). In the previous

chapter, when the halophyte Atriplex nummularia had one root half exposed to 670 mM

and the other half exposed to 10 mM NaCl, shoot growth was maintained, although

when 670 mM NaCl was uniformly applied to the entire root system there was a 65%

decline in shoot ethanol-insoluble dry mass (Chapter 3). On the other hand, root growth

was not affected by salinity, in both uniform and non-uniform treatments. Previous work

has also shown that growing halophytes (Sesuvium portulacastrum, Batis maritima and

Crithmum maritimum) with non-uniform salinities in the root-zone and with up to 800

mM NaCl in one root half does not affect growth (Messedi et al., 2004; Hamed et al.,

2008; Chapter 3). However, although shoot growth was not affected, non-uniform

salinities lead to higher concentrations of Na+ and Cl- and lower concentrations of K+ in

leaves compared to plants in uniform low salinity or NaCl-free media (Messedi et al.,

2004; Hamed et al., 2008; Chapter 3). In A. nummularia, there was a 30–75% increase

in leaf Na+ and Cl- concentrations whereas leaf K+ concentrations were ~25% lower

compared to plants in uniform low salinity. The fact that the growth of plants under non-

uniform salinities were not affected by these increases in Na+ and Cl- concentrations is

consistent the fact that part of these ions are sequestred in the bladders (Mozafar and

Goodin, 1970; Karimi and Ungar, 1986; Aslam et al., 1986) and with

compartmentalization of Na+ and Cl- mainly in vacuoles, to maintain low concentrations

in the cytoplasm and contribute to the osmotic adjustment of the plants (Storey et al.,

1983; Wyn Jones and Gorham, 2002; Flowers and Colmer, 2008)

2 As ECe are measured on a saturated soil paste, if soil is at field capacity,salinity of the soil solution could be estimated to be double that measured with the ECe (Richards, 1954)


60

Under non-uniform conditions in the root-zone, the preferential deployment of roots in

favourable patches and the reduced growth of roots in less favourable patches appears to

be an adaptive response (Drew, 1975). Root growth dynamics in the upper 50 cm of the

soil profile of Melaleuca halmatorum in a saline field with fluctuating saline

groundwater do indeed support the view that new roots are preferentially formed in the

soil regions with the least negative water potentials (Mensforth and Walker, 1996). As

the soil profile dried in spring and summer, and the surface soil water potential declined

to values as low as -10.9 MPa, most of the root growth occurred below 30 cm, whereas

in the winter prior to this most root growth occurred in the upper 10 cm of the soil. In

view of this result, it is surprising that in Chapter 3 there was no compensatory root

growth on the low-salt side when A. nummularia was exposed to non-uniform moderate

(230, 450 mM NaCl) or severe (670 mM NaCl) salinities (Chapter 3). On the other

hand, compensatory root growth has been observed in the few other studies of non-

halophytes subject to non-uniform lateral salinities (Zekri and Parsons, 1990; Flores et

al., 2002). In Citrus aurantium subject to non-uniform salinity, with a solution with an

osmotic potential of -0.35 MPa (ca. 80 mM NaCl) in one root half and a non-saline

solution in the other half, there was a 5% increase in root growth in the non-saline side

compared to root growth in plants with both root halves in the NaCl-free medium (Zekri

and Parsons, 1990). By contrast, there was more than a 50% increase in root growth in

Lycopersicon esculentum exposed to non-uniform 75 mM Cl- (Flores et al., 2002). It is

not clear whether compensatory root growth is related to the salinities used in each study

or more generally related to species-specific abilities to proliferate roots in favourable

patches.

Under non-uniform salinity there is always a certain degree of reduction in stomatal

conductance, but in general the stomata do not close to the same extent as in plants

under uniform salinity (non-halophytes: Zekri and Parsons, 1990; Kirkham et al., 1972;

halophyte: Chapter 3). In the non-halophytes Citrus aurantium and Phaseolus vulgaris,

the stomatal conductance decreased by 19% and 90%, respectively, when one root half

was exposed to salinity compared to plants growing in NaCl-free media (Zekri and

Parsons, 1990; Kirkham et al., 1972). In C. aurantium, the reductions in stomatal

conductance were more pronounced when both root halves were exposed to NaCl (50%

compared to uniform non-saline), whereas in P. vulgaris the reduction in non-uniform

plants was similar to when both root halves were in the saline solution. The question

remains for extreme non-uniform salinity, will stomatal conductance be affected even if


61

there is compensatory root growth in the low-salt side? Hypothetically, compensatory

root growth in the low-salt side should cause compensatory water uptake from the low-

salt side and this should sustain the plant’s transpirational demands; under these

conditions it might be expected that there would be no need for stomatal conductance to

decline. However, if it is the high-salt side that controls stomatal conductance (as

suggested for several species under partial root drying – Lovisolo et al., 2002; Sobeih et

al., 2004; and reviewed in Davies and Zhang, 1991; Dodd, 2005), the above hypothesis

would not hold, and stomatal conductance should be reduced under non-uniform

extreme salinity, independent of any compensatory root growth.

Previous studies on the growth of halophytes (Sesuvium portulacastrum, Batis maritima

and Crithmum maritimum) under non-uniform salinities have only focussed on the range

of salinities that could be considered as being within the NaCl range that still allowed

growth (up to 800 mM NaCl) (Messedi et al., 2004; Hamed et al., 2008; Chapter 3). It is

unknown how halophytes would acclimate if the salinity in one root half was sufficient

to completely inhibit plant growth if applied uniformly to the root system. In such

extreme conditions, would the plant’s growth be substantially affected by the salinity on

the high-salt side, leading to toxic Na+ and Cl- concentrations, and/or K+ deficiency in

the shoot tissues? Alternatively, would the growth of the plants with non-uniform

salinity be driven by the least salinized root portion, independent of the salinity of the

other root half?

To test these questions, A. nummularia was grown with uniform and non-uniform severe

and extreme salinities in the root-zone. Previous work with another Atriplex spp., A.

canescens, did show that 16 accessions had a positive relative growth rate at salinities up

to 900 mM NaCl, but only half of these accessions had a positive or nil relative growth

rates at 1500 mM NaCl (Glenn et al., 1996). 1500 mM NaCl was therefore selected as a

salinity at which A. nummularia would probably not grow. Using a split-root system as

in Chapter 3 with a high and a low (10 mM NaCl) salt side, the following five

hypotheses were tested; that with non-uniform salinities up to 1500 mM NaCl in the

high-salt side: (i) plants will still be able to maintain shoot growth, whereas plants in

uniformly extreme salinity will likely not grow; (ii) root growth in the high-salt side will

be inhibited, but there will be compensatory root growth in the low-salt side; (iii)

stomatal conductance will be reduced, (iv) despite compensatory root growth, there will

be no compensatory water uptake from the low-salt side; and (v) tissue Na+ and Cl- and


62

K+ concentrations under non-uniform conditions will be intermediate to those in plants

in uniform low and uniform extreme salinities.


As previously described (Chapter 3): (a) rooted plants were established from cuttings of

a commercial clone of Atriplex nummularia (“Eyres Green”, Tamlin’s Nursery, South

Australia), (b) these cuttings were raised in pots of washed white sand irrigated with a

gradually increasing concentration of nutrient solution, and (c) after 6 weeks, these

cuttings were transferred to aerated nutrient solution cultures.

Six weeks after transferring the cuttings to the nutrient solutions, plants were selected

for shoot and root uniformity, and transferred into split-root pots (one plant per pot, with

0.6 L per side; the split-root pots are described below) and transferred to a controlled-

environment room (20/15°C day/night, 12 hours day/night, average RH 56%, with an

average PAR at shoot height of 460 µmol m-2 s-1). After a further 7 days, plants for each

treatment were selected. The experiment tested 5 treatments with 4 replicates in a

completely randomized block design. In three treatments, the two halves of the root

systems were both exposed to the same NaCl concentrations (mM): 10, 500 or 1500.

The remaining two treatments had the two halves of the root system exposed to two

different NaCl concentrations, with one side at 10 mM NaCl and the other at 500 or

1500 mM NaCl. NaCl was increased only for plants in uniform treatments with 500 and

1500 mM NaCl and in the high-salt side of both non-uniform treatments. NaCl was

increased in solutions in increments of 50 mM every 12 hours until the designated

concentrations were reached. Twelve hours after reaching the highest salinity (1500 mM

NaCl) was considered as day 0 of the treatments, and an initial harvest was taken

(described below).

Split-root systems. Roots were divided into two approximately equal halves, with each

positioned in a split-root pot, so that the two root halves could be exposed, at the same

time, to different NaCl concentrations (see Fig. 3.1 in Chapter 3). To prevent mixing of

solution, roots were laid in a lengthwise-cut plastic T-piece (length: 6 cm; height: 6 cm;

diameter: 3 cm). Inverted T-pieces were joined and then placed over two cylindrical

plastic containers with a notch into the top, cut to fit the T-piece, so that each root half

was in a cylindrical container. Each container was filled with 0.6 L of aerated nutrient


63

solution. A similar split-root system was also designed to measure water use in each

container of the split-root pot without removing the roots from the pots. For water use

measurements, each pot of the split-root system had two wires glued on the inside that

allowed re-filling of the pots to a precise and constant level, with a precision of 10 µL,

indicated by presence of electrical conductivity when the solution was re-filled to the

original height. Conductivity was measured only when it was time to replace the water

used during the water use measurements (see below) by connecting a voltage meter to

the wires and current only flowed when both wires touched the solution.

Water use measurements. On the 19th day of treatments, 48 hours prior to the harvest,

water use was measured in three treatments (uniform 10/10 and both non-uniform

treatments) on three randomly chosen plants from each treatment (there was no capacity

to measure more plants). Plants were transferred to the split-root system designed for

water use measurements (described above) and all containers were bubbled with pre-

humidified air. Three blank pots (i.e. without plants) were used to determine any

background evaporative losses. To measure water use, each pot containing nutrient

solution was topped up with deionized water at 600 hours and then again at 1800 hours

(commencement and end of the 12 hours light period) to the point where both wires

were in contact with the nutrient solution, and the volumes added were recorded.

Leaf gas exchange and chlorophyll fluorescence. Leaf gas exchange measurements were

taken on day 20 of treatments on three randomly chosen plants in each treatment. Under

non-uniform treatments, leaves on each side of the shoot, directly above each root side,

were measured separately, but as there were no differences between sides, data

presented are the average values for the data pooled from the two shoot sides.

Measurements of net photosynthetic rate and stomatal conductance were determined on

young fully expanded leaves using a LI-COR 6400 Photosynthesis System (LI-COR,

Inc., Lincoln, NE, USA) at ambient RH (50-60%), a reference CO2 of 380 µmol mol-1,

flow rate of 400 µmol s-1 and PAR of 1500 µmol m-2 s-1. The maximum quantum

efficiency of the PSII (Fv/Fm) was measured using a pulse amplitude modulation

fluorometer (PAM-2000, Heinz Walz GmbH, Effeltrich, Germany) equipped with the

leaf clip holder (model 2030-B, Heinz Walz GmbH, Effeltrich, Germany) on the same

leaves used for gas exchange measurement. Fluorescence measurements were taken at

the end of the night period (the whole plants were in dark for at least 11 h), before lights

were switched on in the controlled environment room. Maximal photochemical


64

efficiency of photosystem II was estimated by the fluorescence ratio Fv/Fm of dark-

adapted leaves, calculated from F0 (basal fluorescence) and Fm (maximal fluorescence),

with Fv being Fm – Fo. Leaves were initially exposed to a low modulated light (0.1 µmol

quanta m-2 s-1), which was sufficiently low not to induce any significant variable

fluorescence, to measure basal fluorescence. Successively the maximal fluorescence

level was measured by exposing leaves to a 0.8-s saturating pulse with a photon flux

density of about 15000 µmol quanta m-2 s-1.

Harvests. Plants were harvested on days 0 and 21 after the commencement of

treatments. For both harvests shoot and root fresh masses were determined, whereas

midday water potential and leaf area were only determined on day 21. In both harvests,

plants were processed between 1100 and 1400 hours. Shoot lengths were measured on

day 0 after reaching the highest salinity (1500 mM NaCl) and again on day 21, thus

enabling extension during the treatment period to be calculated. Leaf area was measured

with a portable leaf area meter (Li-Cor LI-3100, Lincoln, NE, USA), with values

adjusted for the weights of leaves already sampled as described above. Shoots and roots

were separated and, in order to assess any differences in root dry mass between sides,

the two halves of each root system were harvested separately. For the shoot, two

(uniform treatments) or four (non-uniform treatments) young fully expanded leaves

were selected at 1100 hours. One (uniform treatments) or two (non-uniform treatments)

leaf samples were used for the determination of leaf sap osmotic potential. The other one

(uniform treatments) or two (non-uniform treatments) leaf samples (the same leaves as

the ones used for leaf gas exchange measurements two days earlier in three replicates)

were selected for subsequent ion and total soluble sugars determinations. These leaf

tissues were snap-frozen in liquid N2, stored at -80°C, freeze-dried, and then stored at -

20°C. All remaining shoots were oven dried at 60°C to determine dry mass. For root

tissues, a subsample of the roots was excised for ethanol-insoluble dry mass

determination and snap-frozen in liquid N2, stored at -80°C, freeze-dried and then stored

at -20°C. In plants used for water use measurements, the remaining root system was

used to determine root surface area per unit root mass, so that total root surface area

could be estimated from total root mass. Roots were blotted to remove excess surface

moisture, sealed in plastic bags and stored at 4°C for 12 hours. Root systems were

scanned for surface area using a WinRhizo root scanner (Regent Instruments Inc.,

Quebec). These roots, and all remaining roots in plants that were not used for water use

measurements, were oven dried at 60°C to determine dry mass.


65

Measurement of midday water potential and osmotic potential of expressed leaf sap.

Midday shoot water potential was measured on excised shoots using a Scholander

pressure chamber between 1130 and 1230 hours, 21 days after reaching the highest

salinity (1500 mM NaCl). Midday water potentials for plants growing in uniform 1500

mM were not determined as potentials were expected to be low and in Chapter 3 shoots

burst out of the Scholander chamber gasket when high balancing pressures were applied.

The osmotic potential of sap pressed out of frozen/thawed young fully expanded leaves

collected at 1100 hours was measured using an osmometer (Fiske Micro-Osmometer

210, Fiske Associates, Massachusetts, USA). In non-uniform treatments, shoot segments

and leaves from both sides of the shoot, directly above the low- and the high-salt sides,

were measured. As no differences between midday water potential and leaf sap osmotic

potential in the two shoot sides directly above each root half were found, data presented

are the average values for the data pooled from the two shoot sides.

Ethanol-insoluble dry mass and measurements of total soluble sugars. To determine

ethanol-insoluble dry mass, ground plant tissues were extracted twice with boiling 80%

ethanol, refluxed for 20 minutes, centrifuged for 10 minutes at 9,335 g (IEC micromax

ventilated microcentrifuge OM3590, Needham Heights, MA, USA), and the insoluble-

fraction was dried at 60°C for 24 hours and weighed. For young fully expanded leaves,

supernatant was used to measure total sugars using anthrone (Yemm and Willis 1954).

Total sugar content (as hexose equivalents) was determined by measuring the

absorbance of the samples at 620 nm in an UV-visible spectrophotometer (UV-1601, uv-

visible spectrophotometer, Shimadzu, Kyoto, Japan), and by relating these values to a

standard curve for glucose. The reliability of this method was verified by determining

the recovery of known amounts of glucose added to additional tissue samples

immediately prior to extraction and to ethanol only. The recovery of glucose from these

samples was 108% (data presented here not adjusted). In non-uniform treatments, sugars

in young fully expanded leaves from both sides of the shoot, directly above the low- and

the high-salt sides, were measured. As no differences were found between the two shoot

sides, values for the two shoot sides were averaged for each replicate.

Measurement of ion concentrations. Ground tissue samples were extracted with 0.5 M

HNO3 by shaking in vials for 48 hours. Diluted extracts were analyzed for Na+, K+

(Flame Photometer 410, Sherwood, Cambridge, UK) and Cl- (Chloridometer 50CL,

SLAMED ING GmbH, Frankfurt, Germany). The reliability of the methods was


66

confirmed by analyses of a reference tissue (broccoli, ASPAC Plant number 85) taken

through the same procedures. The recovery from the reference tissue was: Na+, 98%; K+,

103% and Cl-, 102%. In non-uniform treatments, ions were measured in young fully

expanded leaves from both sides of the shoot, directly above the low and high-salt sides.

As no differences were found between the two shoot sides, data presented are the

average values for the data pooled from the two shoot sides. The estimated rate of

delivery to the shoot of Na+, K+ and Cl- were calculated using Equation 1 (Williams

1948, in this study only shoot ion contents, including dead leaves):

(1)

where shoot = shoot dry mass including dead leaves (g); U = shoot ion content including

dead leaves (µmol shoot-1); t = time (d); ln = natural logarithm; and the indices 1 and 2

refer to harvests 1 and 2, respectively.



used to identify overall significant differences between treatments and between sides

within treatments, depending on the data set. When significant differences were found,

mean-separations were calculated using Duncan’s multiple range test. Unless otherwise

stated, the significance level was P ≤ 0.05.

4.4 Results

Shoot and root growth

Under uniform conditions, there was a substantial decline in shoot extension rate and

leaf area with extreme (1500 mM NaCl) salinity in the root-zone (Fig 4.1A and 4.1B).

On day 21 after reaching 1500 mM NaCl, shoot extension was completely inhibited and

leaf area was 28% of that in the uniform 10 mM treatment. Shoot ethanol-insoluble dry

mass (Fig 4.1C) declined as salinity in the root-zone increased and, at 500 mM NaCl,

shoot dry mass was 70% of that with uniform 10 mM NaCl. For the uniform treatment

with 1500 mM NaCl shoot growth was completely suppressed; shoot ethanol-insoluble

dry masses 0 and 21 days after reaching the highest salinity were (g) 2.33 ± 0.27 and

2.27 ± 0.18, respectively. In the final measure of shoot ethanol-insoluble dry mass, dead

= ln (shoot2)-ln(shoot1)

t2 – t1

U2-U1

shoot2 - shoot1

Ion shoot delivery rate


67

leaves were not included. In uniform extreme salinity dead leaves represented 15% of

the shoot green dry mass while in all other treatments the dry mass of dead leaves was <

3% of shoot dry mass. Under non-uniform salinities, independently of the salinity in the

high-salt side, there were no significant differences in shoot extension rate (Fig 4.1A),

leaf area (Fig 4.1B) or shoot ethanol-insoluble dry mass (Fig 4.1C) after 21 days of

treatment compared to uniform 10 mM NaCl.

Under uniform salinities, with low (10 mM NaCl) and moderate (500 mM NaCl) salinity

there were no significant differences between total root ethanol-insoluble dry mass 21

days after reaching the highest salinity (Fig 4.1D), and over the 21 days of treatment

root ethanol-insoluble dry mass increased 2.7 and 2.0 times, respectively. However, with

the uniform 1500 mM NaCl treatment there was no increase in total root ethanol-

insoluble dry mass between days 0 and 21, and 21 days after reaching the highest

salinity total root ethanol-insoluble dry mass was 41% of the root ethanol-insoluble dry

mass in the uniform 10 mM treatment. Interestingly under both non-uniform treatments,

there was no significant reduction in total root ethanol-insoluble dry mass compared to

uniform 10 mM NaCl. With non-uniform 500 mM NaCl this was due to the fact that

there was no reduction in the dry mass of the roots on either side (Fig 4.1E). On the

other hand, with 1500 mM NaCl in one root half, most of the root ethanol-insoluble dry

mass (79%) was on the side with 10 mM NaCl. In non-uniform 10/1500, between days 0

and 21 after reaching the highest salinity, there was no increase in root ethanol-insoluble

dry mass on the side with 1500 mM NaCl, but there was compensatory root growth on

the side with 10 mM NaCl. In the low-salt side of non-uniform 10/1500, root ethanol-

insoluble dry mass was 3.3 times the dry mass at day 0 after reaching 1500 mM NaCl,

and that resulted in a 40% larger root ethanol-insoluble dry mass on the 10 mM side

compared with the average root ethanol-insoluble dry mass on each side with uniform

10 mM NaCl (Fig 4.1E).

One extra set of plants exposed to the non-uniform treatment 10/1500 (grown at the

same times and following the same procedure as the main experiment) was used to

conduct a short a recovery test (done by stepping down the salinity with a 250 mM NaCl

step-down every 12 h after 0, 5, 10 or 20 days of exposure to 1500 mM NaCl, data not

shown). This was done to qualitatively assess whether roots in the high salt-side, after 0,

5, 10 or 20 days of exposure to 1500 mM NaCl were still alive. New root formation was

observed 24 to 48 h after reaching 10 mM NaCl for all treatments.


68

Leaf gas exchange parameters, Fv/Fm and total soluble sugars

In the uniform treatments, 7 and 20 days after reaching the highest salinity, net

photosynthetic rate (Fig. 4.2A, E) and stomatal conductance (Fig. 4.2B, F) declined as

salinity in the medium increased. With uniform 1500 mM NaCl, net photosynthesis was

almost completely inhibited (net photosynthetic rate <1% of plants with uniform 10 mM

NaCl on day 20) and stomata were almost closed (stomatal conductance ~10% of plants

with uniform 10 mM NaCl on day 20). Associated with this reduced photosynthesis and

stomatal conductance, there was a substantial increase (1.8 times that of plants in

uniform 10 mM NaCl) in the intercellular CO2 concentration (Fig 4.2C, G) compared

with plants grown with uniform 10 mM NaCl. This indicates that with this uniform

extreme salinity there was a non-stomatal limitation to photosynthesis. This is consistent

with the sharp decline in maximum quantum efficiency of the PSII (Fv/Fm) under

uniform 1500 mM, already present 7 days after reaching the highest salinity (Fig 4.2D,

H). Declines in Fv/Fm under uniform extreme salinity were first seen before plants even

reached the 1500 mM NaCl treatment (60 hours prior for 2 replicates, 12 hours prior for

the third – data not shown). Under non-uniform salinities there was also a decline in net

photosynthetic rate and stomatal conductance compared with those at uniform 10 mM

NaCl. For example, 7 and 20 days after reaching the highest salinity, stomatal

conductance at 10/500 was 79% and 73% of that of plants in uniform 10 mM NaCl,

respectively. Furthermore, stomatal conductance was always significantly lower with

non-uniform 10/1500 than with non-uniform 10/500. After 20 days, stomatal

conductance in non-uniform 10/1500 plants was 61% of that in plants grown with

uniform 10 mM NaCl and 84% of that in plants grown with non-uniform 10/500. No

differences were found between intercellular CO2 concentrations within uniform 10 and

500 mM NaCl and non-uniform treatments (183.6 ± 5.9).

Twelve hours after reaching the highest salinity (1500 mM NaCl) the concentration of

total soluble sugars in young fully expanded leaves under uniform extreme salinity

increased to almost 3 times the concentration of that in plants in the uniform 10 mM

NaCl treatment (Fig. 4.3A). By contrast, there were no significant differences in total

soluble sugars of the young fully expanded leaves in any other treatment. After 21 days

no differences were found between any treatments (Fig. 4.3B).


69

NaCl(mM)

0 400 800 1200 1600

Roo

tset

hano

l-ins

olub

le

dry

mas

s(g

)

0

1

2

3

4

5

D

0

1

2

3

4

5

C

Sho

ot e

than

ol- in

solu

ble

dry

mas

s(g

)

0

2

4

6

8

B

Leaf

are

a

(cm

2 )

0

100

200

300

400

500

A

Sho

ot e

xten

sion

(mm

)

0

50

100

150

***

**

***

** ***

E

***

***

High salinity side

Low salinity side

Roo

ts in

eac

h si

deet

hano

l-ins

olub

ledr

y m

ass

(g)

Figure 4.1. Responses to uniform ( ) and non-uniform ( ) NaCl treatments in the root-zone of: (A) shoot extension, (B) leaf area, and the ethanol-insoluble dry mass of (C) the shoot, (D) the entire root system and (E) roots in each side of the split-root system. Atriplex nummularia cuttings were grown for 21 days in a split-root system after reaching the highest salinity (1500 mM NaCl). In uniform treatments, plants were exposed to 10, 500 or 1500 mM NaCl in both root halves. In non-uniform treatments one root half was exposed to 500 or 1500 mM NaCl (indicated on x-axis) and the other root half was exposed to 10 mM NaCl. Dead leaves were not included for total shoot ethanol-insoluble dry mass. Dead leaves at day 21 were < 3% of the total shoot green dry mass in all treatments expect for uniform 1500 mM NaCl, where dead leaves were 15% of the total shoot green dry mass. Initial (day 0) shoot ethanol-insoluble dry masses (g) were: 3.81 ± 0.35 (10/10); 3.09 ± 0.22 (500/500); 2.33 ± 0.27 (1500/1500); 3.50 ± 0.16 (10/500); 3.46 ± 0.25 (10/1500). Initial entire root ethanol-insoluble dry masses (g) were: 1.66 ± 0.24 (10/10); 1.85 ± 0.18 (500/500); 1.22 ± 0.23 (1500/1500); 2.11 ± 0.21 (10/500); 1.55 ± 0.25 (10/1500). Values are means (n = 4) ± S.E. Asterisks above symbols indicate where means of non-uniform treatments are significantly different from the means of the corresponding uniform treatments. ** (P ≤ 0.01). *** ( P ≤ 0.001).


70

D

NaCl(mM)

0 400 800 1200 1600

Fv/

Fm

afte

r ni

ght

0.0

0.2

0.4

0.6

0.8

C

Inte

rcel

lula

r C

O2

conc

entr

atio

n

( m

ol m

ol-1

)

0150

200

250

300

350

B

Sto

mat

al

cond

ucta

nce

(mm

ol m

-2 s

-1)

0

50

100

150

200

250

Day 7µµ µµ

A

Net

pho

tosy

nthe

tic

rate

( m

ol m

-2 s-1

)

0

5

10

15

20

H

NaCl(mM)

0 400 800 1200 1600

G

E

Day 20

F

µµ µµ

***

***

**

***

**

******

*** ***

******

Figure 4.2. Responses to uniform ( ) and non-uniform ( ) NaCl treatments in the root-zone of: (A, E) net photosynthetic rate, (B, F) stomatal conductance, (C, G) intercellular CO2 concentration, and (D, H) maximum quantum efficiency of the PSII of young fully expanded leaves of Atriplex nummularia. In uniform treatments plants were exposed to 10, 500 or 1500 mM NaCl in both root halves. In non-uniform treatments one root half was exposed to 500 or 1500 mM NaCl (indicated on x-axis) and the other root half was exposed to 10 mM NaCl. Measurements were taken 7 and 20 days after reaching the highest salinity (1500 mM NaCl) between 1100 and 1300 hours, with PAR of 1500 µmol m-2 s-1, ambient relative humidity of 50–60% and reference CO2 of 380 µmol mol-1. Values are means (n = 3) ± SE, with each replicate under non-uniform treatments being the mean of two measurements per plant from opposing leaves (i.e. one leaf above each split-root side). Asterisks above symbols indicate where means of non-uniform treatments are significantly different from the means of the corresponding uniform treatments: ** (P ≤ 0.01); *** (P ≤ 0.001). Fv/Fm = maximum quantum efficiency of the PSII.

en

d of

nig

ht


71

Day 0

NaCl(mM)

0 400 800 1200 1600

Hex

ose

equi

vale

nt(m

M)

0

20

40

60

80

100

120

Day 21

0 400 800 1200 1600

A B

***

Figure 4.3. Concentration of total soluble sugars (hexose equivalents) on a tissue water basis in the young fully expanded leaves of Atriplex nummularia exposed for 0 and 21 days to uniform ( ) and non-uniform ( ) NaCl treatments in the root-zone. Atriplex nummularia cuttings were grown for 21 days in a split-root system after reaching the highest salinity (1500 mM NaCl). In uniform treatments, plants were exposed to 10, 500 or 1500 mM NaCl in both root halves. In non-uniform treatments one root half was exposed to 500 or 1500 mM NaCl (indicated on x-axis) and the other root half was exposed to 10 mM NaCl. Leaf tissues were sampled at midday. Values are means (n = 4) ± SE, with each replicate under non-uniform treatments being the mean of two measurements per plant from opposing leaves (i.e. one leaf above each split-root side). Asterisks above symbols indicate where means of non-uniform treatments are significantly different from the means of the corresponding uniform treatments: *** (P ≤ 0.001).

H

exos

e eq

uiva

lent

s


72

Shoot water potential and leaf sap osmotic potential

In uniform treatments, shoot midday water potential decreased as salinity in the root-

zone increased. The midday water potential with uniform 500 mM NaCl was 1.6 times

lower than with uniform 10 mM NaCl (Fig. 4.4A). The midday water potentials of

plants exposed to the non-uniform salinities (10/500 and 10/1500), independently of the

NaCl concentration in the high-salt side, were not different to that of plants exposed

uniformly to 10 mM NaCl.

In uniform treatments the leaf sap osmotic potential declined as NaCl concentrations in

the root-zone increased, and with 500 and 1500 mM NaCl, leaf sap osmotic potential

was 1.6 and 2.9 times lower than that of plants exposed uniformly to 10 mM NaCl,

respectively (Fig. 4.4B). Also with 10/500 and 10/1500, leaf sap osmotic potential was

1.2 and 1.4 times lower than in uniform 10 mM, respectively. These declines in leaf sap

osmotic potential in non-uniform treatments were not associated with tissue dehydration

(Fig. 4.4C) but were caused by an accumulation of solutes in the tissue (discussed

below).

Tissue ions and uptake rates

Ion concentrations were calculated on a tissue water basis, which provides a more

physiologically relevant interpretation of the regulation of ion concentrations in a

succulent halophyte than expression on a dry mass basis (Short and Colmer, 1999). In

uniform treatments, the concentration of Na+ in young fully expanded leaves (Fig 4.5A)

increased linearly with the NaCl concentration in the root-zone. With uniform 500 and

1500 mM NaCl, Na+ concentrations increased to 1.9 and 4.1 times respectively of the

concentrations in plants grown with uniform 10 mM NaCl. However in non-uniform

treatments, concentrations of Na+ in leaves remained relatively constant between the

10/500 and the 10/1500 treatments, being only 1.7 to 1.9 times the concentrations in

plants grown with uniform 10 mM NaCl. Concentrations of Cl- in the young fully

expanded leaves followed a similar trend to Na+ for all treatments (Fig 4.5C). K+

concentrations in leaves were also affected by salinity (Fig 4.5B). K+ concentrations in

both non-uniform treatments were 53–60% of that with uniform 10 mM NaCl, whereas


73

in uniform 500 mM NaCl values were only 34 % of that with uniform 10 mM NaCl. As

a result of the NaCl-induced decreases in tissue K+ and increases in tissue Na+, the

K+:Na+ ratio in shoot tissue decreased from 1.2:1 with uniform 10 mM NaCl (K+:Na+

ratio in external solution = 1:1) to 0.4:1 – 0.5:1 in the non-uniform treatments. The

K+:Na+ ratio was 0.3:1 for both uniform 500 and 1500 mM.

Ion delivery rates to the shoot were calculated from the change in shoot ion contents and

shoot dry mass, including both live and dead leaves, 0 and 21 days after reaching 1500

mM NaCl (Table 4.1). With uniform 500 and 1500 mM NaCl, Na+ delivery rates to the

shoot were 3.3 and 2.1 times respectively of the rates in plants grown with uniform 10

mM NaCl. Rates of Na+ delivery to the shoot were similar between non-uniform

treatments, and increased to 2.3–2.4 times of the rates calculated with the uniform 10

mM NaCl treatment. Rates of delivery of Cl- to the shoot followed similar trends to

those observed for Na+. On the other hand, shoot K+ delivery rates decreased with

increasing salinity, and under uniform 1500 mM NaCl there was a net loss of K+. With

non-uniform 10/500 and 10/1500, shoot K+ delivery was 51% and 22% of that with the

uniform 10 mM NaCl treatment.

Water use and water use efficiency

With the non-uniform 10/500 and 10/1500 mM NaCl treatments, whole plant water use

decreased and was 73% and 61% of that for plants exposed to uniform 10 mM NaCl,

respectively (Table 4.2). In both non-uniform treatments, most (79–81%) water was

taken up from the 10 mM NaCl side, but despite the compensatory root growth in the

low-salt side with the 10/1500 treatment (Fig. 4.1E), there was no compensatory

increase in water uptake from the low-salt side. Given the background evaporative

losses and the errors associated with the determination of the water uptake (precision of

the system was 10 µL), it was not clear whether water had been taken up from the high-

salt side of the non-uniform 10/1500 treatment: there appeared to be a small amount of

net water uptake from 1 but not the other 2 replicates.


74

C

NaCl(mM)

0 400 800 1200 1600

Leaf

wat

er c

onte

nt(m

L g

-1 d

ry m

ass)

0

2

4

6

B

Leaf

sap

os

mot

ic p

oten

tial

(MP

a)

-10

-8

-6

-4

-2

0

AS

hoot

mid

day

wat

er p

oten

tial

(MP

a)

-10

-8

-6

-4

-2

0

n.d.

***

***

******

Figure 4.4. Responses to uniform ( ) and non-uniform ( ) NaCl treatments in the root-zone of: (A) midday shoot water potential, (B) osmotic potential of expressed sap, and (C) leaf water content of young fully expanded leaves of Atriplex nummularia. In uniform treatments, plants were exposed to 10, 500 or 1500 mM NaCl in both root halves. In non-uniform treatments one root half was exposed to 500 or 1500 mM NaCl (indicated on x-axis) and the other root half was exposed to 10 mM NaCl. Midday water potential and osmotic potentials were determined between 1200 and 1400 hours, 21 days after reaching the highest salinity concentration (1500 mM NaCl). Values are means (n = 4) ± SE, with each replicate under non-uniform treatments being the mean of two measurements per plant from opposing leaves (i.e. one leaf above each split-root side). Asterisks above symbols indicate where means of non-uniform treatments are significantly different from the means of the corresponding uniform treatments: * (P ≤ 0.05). n.d. = water potential data for uniform 1500 mM NaCl treatment not determined.


75

C

NaCl(mM)

0 400 800 1200 1600

Leaf

Cl-

(m

M)

0

500

1000

1500

2000

2500

B

Leaf

K+

(mM

)

0

200

400

600

A

Leaf

Na

+ (m

M)

0

500

1000

1500

2000

2500

***

***

**

**

***

***

Figure 4.5. Responses to uniform ( ) and non-uniform ( ) NaCl treatments in the root-zone of ion concentrations on a tissue water basis (mM) of the young fully expanded leaves of Atriplex nummularia: (A) Na+, (B) K+, and (C) Cl-. In uniform treatments, plants were exposed to 10, 500 or 1500 mM NaCl in both root halves. In non-uniform treatments one root half was exposed to 500 or 1500 mM NaCl (indicated on x-axis) and the other root half was exposed to 10 mM NaCl. Values are means (n= 4) ± SE, with each replicate under non-uniform treatments being the mean of two measurements per plant from opposing leaves (i.e. one leaf above each split-root side). Asterisks above symbols indicate where means of non-uniform treatments are significantly different from the means of the corresponding uniform treatments. ** (P ≤ 0.01); *** ( P ≤ 0.001). Note different scale for (B).


76

Table 4.1. Estimated rate of delivery of Na+, K+ and Cl- to the shoot in Atriplex nummularia exposed to uniform and non-uniform salinities in the root-zone.

Na+ K+ Cl-

10/10 25.2 ± 1.1a 31.2 ± 4.0a 19.2 ± 1.4a

500/500 84.3 ± 6.8b 11.1 ± 7.1b,c 73.8 ± 6.8b

1500/1500 53.9 ± 12.8c -0.2 ± 5.7c 56.3 ± 11.3c

10/500 59.6 ± 7.7c 15.9 ± 1.9b 50.6 ± 4.9c

10/1500 57.1 ± 5.2c 6.7 ± 2.4b,c 48.6 ± 5.1c

NaCl (mM)

Shoot delivery rate

(µmol g-1 shoot dry mass d-1)

Estimated shoot delivery of Na+, K+ and Cl- were calculated using Equation 1, as described in Materials and Methods. Values are means (n = 4) ± S.E. Different lower case letters within each column indicate significant differences (P ≤ 0.05).


77

With whole plant water use over 12 hours data and the accumulated ethanol-insoluble

dry mass over 21 days, water use efficiency (accumulated ethanol-insoluble dry mass

over 21 days/ whole plant water use over 12 h) was estimated. Water use efficiency

significantly increased under extreme non-uniform salinity, from 0.16 g mL-1 with

uniform 10 mM NaCl to 0.26 g mL-1 with non-uniform 10/1500. Differences between

non-uniform treatments (10/500 and 10/1500) and between uniform 10/10 and non-

uniform 10/500 (0.17 g mL-1) were not significant (P > 0.05). It is however important to

note that water use measurements were only limited to a 12 hour period at the end of the

experiment, and did not represent the whole plant water use over the experimental

period.

4.5 Discussion

Shoot growth of Atriplex nummularia was not inhibited under non-uniform salinities,

even with one root half exposed to the extreme salinity of 1500 mM NaCl. This

contrasts with uniform 1500 mM NaCl, where there was no biomass increase over 21

days and most leaves showed chlorosis. The severe inhibition of growth under uniform

1500 mM NaCl was also associated with an impaired leaf photochemistry that would

also explain the high intercellular CO2 value in these plants. Interestingly under extreme

non-uniform salinity, root growth on the high-salt side was completely inhibited but

there was compensatory root growth on the low-salt side. It is probably this root

acclimation that enabled the observed shoot growth under extreme non-uniform salinity.

The independence of midday shoot water potentials in plants subject to non-uniform

salinities to the salinity of the high-salt side was in accord with the fact that most of the

water came from the root half on the low-salt side. Finally, A. nummularia, under non-

uniform salinities, was able to take most of the water necessary for growth from the low-

salt side.

Under extreme non-uniform salinity, the restriction in root growth on the high-salt side

enhanced the growth of the roots on the low-salt side. As hypothesized, compensatory

root growth on the low-salt side (+40% ethanol-insoluble dry mass compared to plants

with uniform 10 mM NaCl) was only observed when one root half was exposed to an

extreme salinity (1500 mM NaCl) that completely inhibited root growth. In contrast,


78

there was no preferential root growth on the low-salt side when 500 mM NaCl was

applied to the high-salt side. Clearly for A. nummularia, preferential root growth on the

low-salt side is not only dependent on the salinity of the immediate surroundings of

those roots, but also depends on the salinities around other root portions and how these

salinities affect root growth. This interpretation is consistent with the results of Chapter

3 where A. nummularia was exposed to low and moderate (10 to 670 mM NaCl)

uniform and non-uniform salinities and no differences in root ethanol-insoluble dry

masses were found between treatments and sides. Various degrees of compensatory root

growth have been found in non-halophytic species exposed to non-uniform salinities (+

5% in Citrus aurantium, Zekri and Parsons, 1990; more than 50% increase in

Lycopersicon esculentum, Flores et al., 2002), or in plants where normal root

development was disturbed by removing part of the root system or by applying different

stresses to one root portion (e.g. temperature or partial root desiccation, Crossett et al.,

1975).

In support of the hypothesis that water uptake would likely be reduced under non-

uniform salinities, whole plant water uptake was reduced (Table 4.2). This occurred

even in plants subject to extreme non-uniform salinity where compensatory root growth

occurred in the low-salt side (Fig. 4.1.E). Declines in whole plant water uptake under

non-uniform salinities were associated with reduced stomatal conductance (as also seen

under partial root drying, i.e. Liu et al., 2006); it is not clear, however, whether reduced

stomatal conductances under non-uniform salinities were the cause, or a consequence, of

the reduced water uptake. Nevertheless, these declines in stomatal conductance could

account for the finding that water status was maintained in plants exposed to non-

uniform salinities; that is, why midday water potentials in plants subject to non-uniform

salinities were similar to that of plants exposed uniformly to 10 mM NaCl (Fig. 4.4.A).

Under uniform extreme salinity, as leaf photochemistry was already severely impaired

12 hours prior to reaching 1500 mM NaCl, the decline in photosynthetic rate was

probably due to both stomatal and non-stomatal limitations. Damage to the

photosynthetic apparatus was estimated by the Fv/Fm ratio; declines in this ratio can be

attributed either to photodamage to the reaction centres of PSII and/or the development

of slowly relaxing fluorescence quenching processes, both of which lead to long-term

disturbances in plant physiology (Hsu, 2007). On the other hand, the high sugar

concentrations, initially found under extreme salinity 12 hours after reaching 1500 mM


79

NaCl, could also have lead to a reduction in CO2 fixation and thus stomatal closure

through negative feedback (Munns, 1993). In non-uniform treatments, however, reduced

stomatal conductance was not associated with decreased growth or increased total

soluble sugars in the leaf tissues, and these factors can therefore be ruled out as sources

of negative feedback. Hence under non-uniform salinities there is a possibility that the

reduced stomatal conductances could have been caused by non-hydraulic signals (e.g.

ABA or changes in xylem pH) from the high-salt side (Dodd, 2005; Khalil and Grace,

1993; Gowing et al., 1990; Lovisolo et al., 2002; Saab and Sharp, 1989; Sobeih et al.,

2004). In addition, as electrical signals have also been found to be involved in stomatal

regulation under drought (Fromm and Fei, 1998; Grams et al., 2007) and salinity causes

a rapid root membrane depolarization (Chen et al., 2005; Cakirlar and Bowling, 1981;

Shabala et al., 2003), it is also possible that electrical signals might be involved in

stomatal regulation under non-uniform conditions.

Under high and extreme non-uniform salinities shoot Na+ and Cl- accumulation rates

more than doubled compared with uniform 10 mM NaCl (Table 4.1), with these ions

most likely being in part being sequestred in the bladders (Aslam et al., 1986) and in

part used for osmotic adjustment. These large ion accumulations in shoot tissues under

non-uniform conditions were associated with a 20–40% decline in leaf sap osmotic

potential. Most halophytes tend to accumulate constitutively high concentrations of ions

in the tissues, even at low NaCl concentrations (Flowers et al., 1977), and are able to use

the accumulation and sequestration of inorganic ions to adjust their osmotic potential

(Flowers and Yeo, 1986; Flowers and Colmer, 2008). Dicotyledonous halophytes, such

as A. nummularia, can be expected to mainly use Na+ and Cl- to lower their water

potential (c.f. the case for Atriplex hymenelytra, Bennert and Schmidt, 1984; reviewed

extensively for other halophytes in Flowers and Colmer, 2008). As 10 mM NaCl

solution was used in the low-salt side, in the present study it is not clear whether these

increases in shoot Na+ and Cl- accumulation rates under non-uniform conditions reflect

ion uptake from the roots exposed to the high salitinites, as a greater Na+ and Cl- uptake

could have occurred from the low-salt side compared to ion uptake in control plants

exposed uniformly to 10 mM NaCl. However increased shoot Na+ and Cl- under non-

uniform salinities have been previously observed in non-halophytes (Hajji et al., 2001)

and halophytes (Messedi et al., 2004; Hamed et al., 2008) exposed to non-uniform

salinities with NaCl-free solutions in the low-salt side, thus suggesting that the bulk of

net Na+ and Cl- uptake occurred from the high-salt side.


80

In both non-uniform treatments there was a 73–91% decline in the shoot K+:Na+ ratio

(from 1.12:1 with 10/10 to 0.3:1 with 10/500 and 0.1:1 in 10/1500) compared to that

with uniform 10 mM NaCl. These K+:Na+ ratios under non-uniform salinities are within

the range previously found in leaves of Atriplex amnicola (Aslam et al., 1986), although

lower K+:Na+ ratios have been reported for other halophytes (e.g. in shoots of

Sarcocornia natalensis at 500 mM NaCl it was 0.04:1 – Naidoo and Rughunanan, 1990;

and in succulent shoot tissues of Halosarcia pergranulata subsp. pergranulata at 800

mM NaCl it was 0.02:1 – Short and Colmer, 1999). These levels are likely to be

adequate as plant requirements for a high K+:Na+ ratio to sustain metabolic processes

would be met because of the substantial compartmentation of Na+ in the vacuoles (Short

and Colmer, 1999). Moreover, despite being essential for several metabolic processes in

the cytoplasm, K+ is in most plants under non-saline conditions largely localized in the

vacuole where it is used as an osmoticum to maintain turgor (Subbarao et al., 1999,

2003; Maathuis and Amtmann, 1999). In halophytes this function of K+ that can be

fulfilled with other ions (e.g. Na+), and the K+ localized in the vacuole could therefore

be replaced by Na+, and the spare K+ released to the cytoplasm for metabolic functions

(Flowers and Läuchli, 1983; Wyn Jones and Gorham, 2002; Subbaroa et al., 2003;

Karrenberg et al., 2006).

In this Chapter, roots in the high salt-side of the extreme non-uniform treatments

remained alive and (1, 2)The ability to maintain functional roots in the high-salt side

under extreme non-uniform salinities requires an adequate rate supply of K+ to the root

tips, as high cytoplasmic K+ is required in meristematic root tips (Jeschke and Wolf,

1988; Wyn Jones and Gorham, 2002). Therefore, as the capacity of the roots to take up

K+ from the high salt-side was likely to be adversely affected by the extreme NaCl

concentrations in the external medium (c.f. Maathuis and Amtmann, 1999), it is likely

that root tips were maintained functional thanks to recycling of K+ from the shoot to the

roots in the 1500 mM NaCl side, through the import of K+ together with assimilates and

amino via the phloem. It has been previously shown that external K+ supply is not

required for root growth under saline conditions provided that there is an available K+

source in another root portion (Jeschke and Wolfe, 1988). In that study, Ricinus

communis was grown in a split-root system, with one root half exposed to a solution

with K+ but not nitrate, and the other half exposed to a solution with nitrate but not K+.

The solutions in both root halves were either saline or non-saline. Despite the absence of

added external K+ and an external Na:K ratio of 10000:1, the roots in the K-free saline


81

solution were able to grow and maintain a cytoplasmic K+ (130 mM) similar to that of

roots exposed to non-saline or saline complete solutions (135 mM K+ in non-saline

complete solution and 143 mM K+ in the complete saline solution). Since external K+

was missing from the external solution, these results can only be attributed to a recycling

of K+ from the shoots, via phloem, to the roots. The recycling of K+ from the shoots

could be of great advantage under non-uniform salinities, as high salinity can be

associated with local depletion of K+, but with available K+ in non-saline areas of the

root-zone and the recycling of K+, plants would be able to maintain functional

meristematic root tips.

Salinity in the field is temporally and spatially highly heterogeneous and can reach

extreme values, especially in the upper soil layers (see Introduction), that are higher than

the levels normally tolerated by most halophytes. In this Chapter A. nummularia was

able to maintain optimal growth with one root half exposed to 1500 mM NaCl, a salinity

that when applied to the entire root system was toxic for this species, provided that the

other root half was exposed to low salinity. It is likely that compensatory root growth in

the low-salt side and water from the low-salt side were the main factors that enabled the

observed shoot growth under extreme non-uniform salinity.


82

Table 4.2. Responses to uniform and non-uniform NaCl concentrations in the root-zone on whole-plant water use and water uptake, measured over 12 h, expressed on a root surface area basis

Low salinity side High salinity side

10/1010/500 0.17 ± 0.03a 0.04 ± 0.01b

10/1500 0.12 ± 0.01a 0.03 ± 0.04b

(mmol m-2 s-1)

0.15 ± 0.01a*

NaCl (mM)

19.17 ± 1.31b

Water uptake on root surface area basis

Whole-plant water use

(mL)

31.25 ± 0.57a

22.81 ± 2.17b

Values are means (n = 3) ± S.E. Different lower case letters within each column indicate significant differences (P ≤ 0.05). * With uniform 10 mM NaCl no differences were found between the means on each side, so data for the two sides were averaged.

Chapter 5 Increasing salinity in high-salt side

83

Chapter 5

Effects of Increasing the Salinity on the Low-Salt Side: Most Plant

Physiological Parameters Respond to the Mean Salinity of the Root-Zone

5. Chapter 5: Effects of Increasing the Salinity on the Low-Salt Side: Most Plant Physiological Parameters Respond to the Mean Salinity of the Root-Zone


84

5.1 Abstract

There are two main hypotheses on how plants respond to non-uniform salinity:

hypothesis 1 (H1), that plant growth responds to the mean salinity of the root-zone; and

hypothesis 2 (H2) that plant growth responds to the lowest salinity in the root-zone. To

test whether various physiological parameters in A. nummularia under non-uniform

salinities respond to the mean salinity in the root-zone (H1) or to the lowest salinity

(H2), plants were grown in a split-root system. 6-week-old cuttings were exposed either

to uniform (10, 120, 230, 450 or 670 mM NaCl) or non-uniform salinities (one root half

at 670 and the other root half at 10, 120, 230 or 450 mM NaCl). Shoot growth

parameters (shoot ethanol-insoluble dry mass, shoot extension and leaf area), leaf gas

exchange, leaf osmotic potential and leaf ion concentrations (Na+, K+ and Cl-) responded

more closely to the mean salinity rather than to the lowest salinity of the root-zone. On

the other hand, the midday shoot water potential and the leaf water content under non-

uniform salinities were similar to those of plants exposed to the uniform low salinities,

indicating that these two parameters were mainly influenced by the water potential in the

root-zone from where most of the water was taken up. Under non-uniform salinities

most of the water was taken up from the low-salt side (% total water uptake: 85–88%

with 10–120 mM in the low-salt side; 77–78% with 230–450 mM NaCl in the low-salt

side). Water uptake occurred from the high-salt side despite the fact that midday shoot

water potentials under non-uniform salinities reflected the water potential of the low-salt

side. This water uptake was explained with a simple water uptake model; a flow of

water from the external medium on the high-salt side to the root would have been

possible with a decreased (i.e. more negative) xylem osmotic potential, presumably

through the uptake of ions. Given the heterogeneity in salinity in most field situations,

dicotyledonous halophytes, such as A. nummularia that have optimal growth up to 400

mM NaCl, have the potential to withstand severe salinity in part of the root system as

long as there are other root portions at lower salinities.


85

5.2 Introduction

Salinity in the field is rarely uniform, and the magnitude of the spatial heterogeneity in

salt concentrations likely to be experienced by individual plants may differ widely (see

Table 2.1, Chapter 2). On a 40 m transect of an area growing Atriplex amnicola, the soil

salinity (ECe) of the upper 10 cm of the soil profile had large changes (65 to 3 dS m-1)

over 10 m in part of the transect but only moderate differences (50 to 65 dS m-1) over 10

m distance in another part of the transect (Davidson et al., 1996). Changes in soil

salinity on this scale raise the question how individual plants integrate the variation in

salinity within their root-zones. Under non-uniform salinities, plants are generally able

to withstand and grow with a salinity in one root half that would severely reduce growth

if applied to the entire root system (non-halophytes – Bingham and Graber, 1970; Zekri

and Parsons, 1990; halophytes – Hamed et al., 2008; Messedi et al., 2004; Chapter 4).

All experiments with non-uniform salinity that have been conducted so far have focused

on the effects of no or low salinity on half of the plant’s roots, and moderate to extreme

salinity on the other half of the roots. However, there are a several important questions

about plant responses to non-uniform salinity that require a complementary approach.

This chapter examines the effects of severe (670 mM NaCl) salinity on half of the

plant’s roots, and low to high salinity on the other half of the roots with the halophyte

Atriplex nummularia.

Two main hypotheses on how plants respond to non-uniform salinity have been put

forward: hypothesis 1 (H1), that plant growth responds to the mean salinity in the root-

zone (non-halophyte – Shani et al., 1993; Kirkham et al., 1969); and hypothesis 2 (H2)

that plant growth responds to the lowest salinity in the root-zone (non-halophyte –

Bingham and Garber, 1970; Flores et al., 2002; Zekri and Parsons, 1990; halophytes –

Chapter 3; Hamed et al., 2008; Messedi et al., 2004). Few studies have been conducted

on the responses of halophytes to non-uniform salinities, although the three existing

studies present growth data consistent with H2 (Batis maritima, Hamed et al., 2008;

Sesuvium portulacastrum, Messedi et al., 2004; Atriplex nummularia, Chapter 3). In

Chapter 3 with A. nummularia with one root half exposed to 10 mM NaCl (low salinity)

and the other half exposed to 230–670 mM NaCl (high/severe salinity), shoot growth

was similar to that observed in plants grown with uniform 10 mM NaCl; this clearly

supports H2.


86

The apparent discrepancies between the growth responses of halophytes and non-

halophytes might be related to fact that many halophytes have a growth optimum

around 10–300 mM NaCl (Flowers and Colmer, 2008). For example, A. nummularia

has optimal growth at 10–300 mM NaCl (Araújo et al., 2006; Dunn and Neales, 1993;

Greenway, 1968). Hence, when A. nummularia was exposed to non-uniform salinities

(with 10 mM NaCl in one root half and up to 670 mM NaCl in the other half), the mean

salinities in the root-zone (120–340 mM NaCl) in these treatments would still have been

within the “optimal” range. The question of whether A. nummularia under non-uniform

salinity responds to the mean salinity or to the low salinity in the root-zone is therefore

still open. On the other hand, if the salinity of the low-salt side were to be increased,

raising the mean salinity of the root-zone to values greater than the optimum, then it

would be possible to test more conclusively whether A. nummularia under non-uniform

salinity does indeed respond to the mean salinity or to the lowest salinity of the root-

zone.

In general under non-uniform salinity there is an increase in shoot ion concentrations

(non-halophyte – Capsicum annum Lycoskoufis et al., 2005; halophytes – A.

nummularia, Chapter 3; B. maritima, Hamed et al., 2008; S. portulacastrum, Messedi et

al., 2004). In S. portulacastrum growing with 0 mM NaCl in one root half and 800 mM

NaCl in the other half, the shoot Na+ concentration was 21 times that of the shoot

tissues of plants with both root halves in the “NaCl-free” medium, increasing from 0.2

to 4.2 mmol g-1 dry mass (Messedi et al., 2004). Smaller relative increases in shoot Na+

and Cl- were found in A. nummularia exposed to non-uniform salinities compared to

plants exposed to uniform 10 mM NaCl (Chapter 3). When A. nummularia was exposed

simultaneously to 10 and 670 mM NaCl, the Na+ concentration in fully expanded leaves

was 40% higher than that in plants with both root halves in 10 mM NaCl (Chapter 3).

This would suggest that leaf Na+ and Cl- concentrations and leaf osmotic potentials

(which derive from the ionic concentrations) should be more consistent with H1, viz.

this parameter responds to the mean salinity of the root-zone. In regard to ion relations,

another question that arises from Chapters 3 and 4 is whether the low-salt side provides

most of the K+ essential for growth under non-uniform salinities. Previous work on non

halophytes and halophytes (Hajji et al., 2001; Hamed et al., 2008; Messedi et al., 2004)

has suggested that the low-salt side might be important in providing most of the K+

required by shoot tissues under non-uniform salinities. This hypothesis is mainly based

on the fact that under saline conditions K+ uptake is usually limited as K+ and Na+


87

compete for the same entry pathways (Maathuis and Amtmann, 1999); therefore it is

likely that most of the K+ under non-uniform salinities comes from the low-salt side.

The picture for shoot water potentials appears to be different; in A. nummularia with

one root half at 670 mM and the other root half at 10 mM NaCl, the shoot water

potential at midday was similar to that of plants with both root halves at 10 mM NaCl.

Similarly in the non-halophyte Citrus aurantium, the midday water potentials of plants

with one root half exposed to a saline solution (8.8 dS m-1; -0.35 MPa) and the other

root half in non-saline solution only decreased by 0.1 MPa compared with plants with

both root halves in the non-saline solution (Zekri and Parsons, 1990). By contrast, when

saline solution was applied to both root halves, water potentials declined by 0.4 MPa.

Similar responses for shoot water potential were found for the non-halophytes

Phaseolus vulgaris and Hordeum vulgare growing under non-uniform salinities

(Kirkham et al., 1969). Thus, it appears that for the shoot water relations of plants

growing under non-uniform salinities, the responses appear to be more consistent with

H2, i.e. responding to the lowest salinity in the root-zone independently of the salinities

in the other root half.

Another question that arises from data on water potentials in Chapter 3 concerns the

mechanism of water uptake from the high-salt side under non-uniform salinities. With

A. nummularia growing under non-uniform salinities (10 and 670 mM NaCl in the root-

zone), the midday shoot water potential was 1.4 MPa higher than the osmotic potential

of the external solution on the high-salt side; despite this the plants took up ca. 10% of

their water from this side. This chapter also considers this issue using a simple water

uptake model.

In summary, the first two experimental chapters of this thesis have focused on the

effects of non-uniform salinity with low concentrations on one side (10 mM NaCl) and

moderate to extreme concentrations on the other side (230–1500 mM NaCl). However,

a more complete understanding of the effects of non-uniform salinity in the root-zone

also requires tests of the effects of severe (i.e. 670 mM NaCl) salinity on one side and

low to high salinity on the other side. This chapter tests whether the various

physiological parameters in A. nummularia growing under non-uniform salinities

respond to the mean salinity (H1) or to the lowest salinity (H2) of the root-zone. These

questions were considered by exposing one root half to 670 mM NaCl and the other root


88

half to a range of salinities between 10 and 450 mM NaCl. Assessments were made of

the impacts of non-uniform and uniform salinities on shoot and root dry mass, leaf gas

exchange parameters, leaf Na+ and Cl- concentrations, leaf osmotic potential and shoot

water potential. The hypothesis that under non-uniform salinity K+ will be mostly taken

up from the low-salt side was also tested in a parallel experiment.


Rooted cuttings of Atriplex nummularia, “Eyres Green” were established and raised in

nutrient solution culture in a phytotron with day/night temperatures of 20/15°C as

previously described (Chapter 3).

Experiment 1

The first experiment was conducted to assess the impacts of increasing salinities on the

low-salt side under non-uniform and uniform salinity on shoot and plant dry mass, leaf

gas exchange parameters, leaf Na+ and Cl- concentrations, leaf osmotic potential and

shoot water potential.

About six weeks after transferring the cuttings to the nutrient solution, the plants were

assessed for shoot and root uniformity, stratified by size and assigned to treatments. The

plants were transferred into split-root pots (one plant per pair of compartments, with 0.6

L of nutrient solution per side). Plants were then moved to a controlled-environment

room (20/15°C day/night, 12/12 hours day/night, average RH 55 %, and an average

PAR at shoot height of 460 µmol m-2 s-1). This experiment tested 9 treatments with 5

replicates, in a completely randomized block design. In five treatments the two halves of

the root systems were both exposed to the same NaCl concentrations (mM): 10, 120,

230, 450 or 670. The remaining four treatments had the two halves of the root system

exposed to two different NaCl concentrations, with one side exposed to 670 mM NaCl

and the other to 10, 120, 230 or 450 mM NaCl. On the 4th day after transferring the

plants to the split-root pots, NaCl was increased in both sides of all split-pots in

increments of 55 mM every 12 hours, until NaCl concentrations reached 670 mM in

both sides of all pots. Three days after reaching this concentration, all treatments were

imposed with a single step down from 670 mM NaCl to the required level in each side.

This slow step-up, but rapid step-down procedure is ecologically sound as salinity in the


89

field generally tends to gradually increase during periods of high evaporative demand in

summer, but then rapidly falls again in autumn and winter through the leaching effects

of rainfall (Mensforth and Walker, 1996).

Split-root systems. Intact roots were divided into two approximately equal halves, with

each positioned in a split-root compartment, so that the two root halves could be

exposed, at the same time, to different NaCl concentrations (see Fig. 3.1, Chapter 3). To

prevent the mixing of solution from one side to another, the upper few centimetres of the

roots were laid in a lengthwise-cut plastic T-piece (length: 6 cm; height: 6 cm; diameter:

3 cm). The sides of these T-pieces were joined with tape, inverted so that the roots were

downwards and the shoot upwards, and then placed over two cylindrical plastic

containers, each filled with 0.6 L of nutrient solution, so that each root half was in a

separate solution. A similar split-root system was also used to measure plant water use.

For water use measurements, each side of the split-root system had two electrical wires

glued to the inside wall that allowed re-filling of the compartment to a precise and

constant level, with an accuracy of 10 µL, indicated by the presence of a closed

electrical circuit when the solution was re-filled to the original height. Conductivity was

measured by connecting a voltage meter to the electric wires at the time of re-filling

each pot and current only flowed when both electrodes in the pot touched the solution.

Leaf gas exchange and chlorophyll fluorescence. Leaf gas exchange measurements were

taken on day 19 of treatment on four randomly chosen plants per treatment. Under non-

uniform treatments, separate gas exchange measurements were made in shoots above

each root half to check whether leaves above the low or high-salt sides had different

values. However, as no significant differences between sides were found, under non-

uniform treatments each replicate is the mean of the two measurements per plant from

the opposing sides. Measurements of net photosynthetic rate and stomatal conductance

were determined on young fully expanded leaves using a LI-COR 6400 Photosynthesis

System (LI-COR, Inc., Lincoln, NE, USA) at ambient relative humidity (50–60%),

reference CO2 of 380 µmol mol-1, flow rate of 400 µmol s-1 and PAR of 1500 µmol m-2

s-1. The maximum quantum efficiency of the PSII (Fv/Fm) was measured using a PAM-

2000 (PAM-2000 Chlorophyll Fluorometer, Heinz Walz GmbH, Effeltrich, Germany)

on the same leaves used for gas exchange measurements. PAM measurements were

taken at the end of the night period (the plants had been in the dark for at least 11 h) just

before the lights were switched on in the controlled environment room.


90

Water use measurements. Water use was only measured for non-uniform treatments and

plants grown with uniform 10 mM NaCl on four randomly chosen plants within each

treatment; it was not possible to manage more treatments. Plants were transferred to the

split-root system designed for water use measurements (described above) 12 hours prior

to the harvest (day 20), and all containers were bubbled with pre-humidified air. Three

blank pots (i.e. without plants) were used to determine any background evaporative

losses. To measure water use, each pot containing nutrient solution was topped up with

deionized water at 0600 and then again at 1800 hours (i.e. at the start and end of the 12

hours light period) to the point where both electrodes were just in contact with the

nutrient solution, and the volumes added at 1800 hours were recorded.

Harvests. Shoot lengths were measured on days 0 and 21 after imposing treatments, thus

enabling extension during the treatment period to be calculated. Plants were harvested

between 1100 and 1400 hours on the day treatments were imposed (day 0, i.e. when the

step-downs from 670 mM NaCl occurred) and after 21 days of treatment. At each

harvest, plants were separated into shoots and roots on each side.

For the shoot, two (uniform treatments) or four (non-uniform treatments) young fully

expanded leaves were selected at 1100 hours. One (uniform treatments) or two (non-

uniform treatments) leaves were used for the determination of leaf sap osmotic potential

and the other one (uniform treatments) or two (non-uniform treatments) leaves were

snap-frozen in liquid N2, stored at -80°C, freeze-dried, and then stored at -20°C for

subsequent determination of ions and total soluble sugars. For the harvest of day 21, the

leaf area of all remaining shoots was measured with a portable leaf area meter (Li-Cor

LI-3100, Lincoln, NE, USA), with values adjusted for the weights of leaves already

sampled as described above. Tissues were oven dried at 60°C to determine dry mass.

Root tissues were washed for 2 min in 3 changes of iso-osmotic mannitol solution, also

containing 9 mM CaSO4, and a subsample of the roots was excised for ethanol-insoluble

dry mass determination; this was snap-frozen in liquid N2, stored at -80°C, freeze-dried

and then stored at -20°C. In plants used for water use measurements, the remaining root

system was used to determine root surface area per unit root mass, so that total root

surface area could be estimated from total root mass. Roots were blotted to remove

excess surface moisture, sealed in plastic bags and stored at 4°C for 12 hours. Root

systems were scanned for surface area using a WinRhizo root scanner (Regent


91

Instruments Inc., Quebec). These roots and all remaining roots from plants that were not

used for water use measurements were oven dried at 60°C to determine dry mass.

Measurement of midday water potential, xylem osmotic potential and osmotic potential

of expressed leaf sap. Midday shoot water potential was measured using a Scholander

pressure chamber between 1130 and 1230 hours on day 21 after imposing treatments.

Water potentials were measured on stems excised close to the base of the shoot, to

provide a thick, woody stem to insert into the pressure chamber, thus avoiding the loss

of shoot tissues previously observed with high balancing pressure in Chapter 3. Sap was

pressed out of frozen/thawed young fully expanded leaves and the osmotic potential of

this was measured using an osmometer (Fiske Micro-Osmometer 210, Fiske Associates,

Massachusetts, USA). In non-uniform treatments, midday water potential and the

osmotic potential of extracted leaf sap were measured in shoot segments and leaves from

both sides of the shoot, directly above the low- and high-salt sides. As there were no

differences between the midday water potential and leaf extracted sap osmotic potential

between the two shoot sides, the data presented are the average values. Xylem osmotic

potential was estimated during pressure bomb measurements by collecting the sap on

filter paper discs. To avoid contamination from the damaged cells of the cut end, the

first sap samples collected were discarded. The filter paper weights before and after

collecting the xylem sap were taken and after adding a known amount of MilliQ water,

the osmotic potential of the mixture of MilliQ water and xylem sap was measured with

an osmometer (Fiske Micro-Osmometer 210, Fiske Associates, Massachusetts, USA).

The osmotic potential of the collected sap was then calculated taking into account the

amount of MilliQ water added. Blank filter papers were used to determine the osmotic

potentials associated with the filter papers alone.

Ethanol-insoluble dry mass and measurements of total soluble sugars. To determine

ethanol-insoluble dry mass, ground plant tissues were extracted twice with boiling 80%

ethanol, refluxed for 20 minutes, centrifuged for 10 minutes at 9,335 g (IEC micromax

ventilated microcentrifuge OM3590, Needham Heights, MA, USA), and the insoluble-

fraction was dried at 60°C for 24 hours and weighed. For the young fully expanded

leaves, the supernatant was used to measure total sugars using the anthrone method

(Yemm and Willis 1954). Total sugar content (as hexose equivalents) was determined

by measuring the absorbance of the samples at 620 nm in an UV-visible

spectrophotometer (UV-1601, uv-visible spectrophotometer, Shimadzu, Kyoto, Japan),


92

and by relating these values to a standard curve for glucose. The reliability of this

method was verified by determining the recovery of known amounts of glucose added to

additional tissue samples immediately prior to extraction and to ethanol only. The

recovery of glucose from these samples was 108%, so the data presented here have not

been adjusted. In non-uniform treatments, sugars were measured in young fully

expanded leaves from the sides of the shoot directly above the low and high-salt sides.

As there were no differences between these measurements, the values were averaged for

each replicate.

Measurement of ion concentrations. Ground tissue samples (25 to 100 mg dry mass)

were extracted with 5 to 10 mL 0.5 M HNO3 by shaking in vials for 48 hours. Diluted

extracts were analyzed for Na+, K+ (Flame Photometer 410, Sherwood, Cambridge, UK)

and Cl- (Chloridometer 50CL, SLAMED ING GmbH, Frankfurt, Germany). The

reliability of the methods was confirmed by the analysis of a reference tissue (broccoli,

ASPAC Plant number 85) taken through the same procedures; recoveries were: Na+

98%, K+ 103% and Cl- 102%. In non-uniform treatments, ions were measured in young

fully expanded leaves from both sides of the shoot, directly above the low- and high-salt

sides. As there were no differences between these measurements, the values for each ion

were averaged for each replicate.

Experiment 2

The second experiment was conducted to assess whether, under non-uniform salinities,

most of the K+ was taken up from the low-salt side. Plants were grown as in experiment

1 except that this experiment tested only 3 treatments and plants were grown for 15

days. In two treatments the two halves of the root systems were both exposed to the

same NaCl concentrations (mM): 10 or 450. The remaining treatment had the two

halves of the root system exposed to two different NaCl concentrations, with one side

exposed to 10 mM NaCl and the other to 450 mM NaCl. 8 days after imposing the

treatments, solutions were changed in all the split-root pots and, successively, samples

of solutions from each side were collected 7 days later to assess K+ depletion rates from

the low-salt side solution. Before these samples were collected, each pot was topped up

with deionized water to the original level. K+ concentrations were measured using a

flame photometer (Flame Photometer 410, Sherwood, Cambridge, UK). Plants were

harvested on days 0 and 15 after commencement of treatments, and shoot and root fresh


93

mass were determined. Knowing root fresh mass after 0 and 15 days of treatments,

relative growth rates were estimated using equation 1 (Hunt, 1982) and root fresh mass

after 8 days was estimated from the calculated relative growth rate. Estimated K+ uptake

rates were calculated using equation 2 (Williams, 1948).

where root = root fresh mass (g); U = ion content in the solution (µmol); t = time (d);

and the subscript indices 1 and 2 refer to times 1 and 2, respectively.



used to identify significant differences between treatments and between sides within

treatments, depending on the data set. When significant differences were found, mean-

separations were calculated using Duncan’s multiple range test. Unless otherwise stated,

the significance level was P ≤ 0.05. Linear or polynomial (quadratic or cubic) regression

analysis was performed as described with SigmaPlot 11.0 (Systat Software Inc.; Version

11.0, Chicago, IL, USA).

5.4 Results

Shoot and root growth

With uniform salinities in the root-zone there were no decreases in any of the shoot

growth parameters (shoot extension, Fig. 5.1A, leaf area, Fig. 5.1B, shoot ethanol-

insoluble dry mass, Fig. 5.1C) at concentrations up to 450 mM NaCl compared with

control plants at 10 mM NaCl. However with uniform 670 mM NaCl, shoot extension,

leaf area and shoot ethanol-insoluble dry mass were 58%, 73% and 71% respectively of

that in plants with uniform 10 mM NaCl.

= ln (root2) – ln(root1)

t2 – t1RGR

= ln (root2) – ln(root1)

t2 – t1

U2 – U1

root2 – root1

Ion uptake rate

(1)

(2)


94

With non-uniform salinities, there were no significant differences in shoot ethanol-

insoluble dry mass compared with the corresponding uniform salinity treatments

(10/670 vs. 10/10, 120/670 vs. 120/120; 230/670 vs. 230/230 or 450/670 vs. 450/450).

On the other hand, shoot extension and leaf area were generally reduced compared with

the corresponding uniform treatments. As examples, with 10/670, 120/670 and 230/670

shoot extension was 86%, 66% and 72% of the values with uniform 10, 120 and 230

mM NaCl, respectively. No difference (P > 0.05) was found for 450/670 compared with

uniform 450 mM.

To determine whether the three shoot parameters responded more closely to the mean

salinity (H1) or the lowest salinity (H2) of the root-zone, a regression analysis was

performed and regression curves (cubic relationships) were fitted to the combined

uniform and non-uniform data sets (shoot extension, Fig. 5.1D, leaf area, Fig. 5.1E,

shoot ethanol-insoluble dry mass, Fig. 5.1F). The regression curves for these three

parameters had best fit when the non-uniform data were plotted against the mean salinity

rather than the lowest salinity of the root-zone (see Table 5.1 for R2 and P-values of the

regression analyses).

Under uniform salinity, there was no difference in root ethanol-insoluble dry mass

between the 10 and 670 mM NaCl treatments (Fig 5.2A), but the mass tended to

increase in the range 120–450 mM NaCl (although not significantly, P > 0.05, compared

to uniform 10 mM NaCl). Root ethanol-insoluble dry mass at uniform 670 mM NaCl

was 49–53% of the dry mass at 120–450 mM NaCl.

Under non-uniform treatments, with NaCl concentrations on the low-salt side ≥ 120

mM, there were declines in root ethanol-insoluble dry mass compared to the

corresponding uniform treatments (Fig. 5.2A). The total root ethanol-insoluble dry mass

of the non-uniform 120/670, 230/670 and 450/670 treatments was 66%, 54% and 60%

of the respective values for uniform 120, 230 and 450 mM NaCl (Fig. 5.2A). In all non-

uniform treatments, the total root ethanol-insoluble dry mass was not different from the

ethanol-insoluble dry mass of roots grown at uniform 670 mM NaCl. The decreases in

total root ethanol-insoluble dry mass in non-uniform treatments compared with the

corresponding uniform treatments were caused by decreases in ethanol-insoluble dry

mass in the low-salt side (Fig 5.2B), compared with the ethanol-insoluble dry mass


95

averaged across both sides of the uniform treatments. For example at 230/670 and

450/670, the root ethanol-insoluble dry mass on the low-salt side was 59–66% of the

ethanol-insoluble dry mass averaged across both sides of the uniform 230 and 450 mM

NaCl treatments respectively. These reductions in root ethanol-insoluble dry mass in

non-uniform treatments with NaCl concentrations on the low-salt side ≥ 120 mM caused

a decrease in the root/shoot ratio in non-uniform treatments compared with the

corresponding uniform treatments; with 120/670, 230/670 and 450/670 the ratio

root/shoot was 74%, 60% and 65% respectively of the ratio with uniform 120, 230 and

450 mM NaCl. Furthermore, the ratios of root/shoot for these non-uniform treatments

were not different at that of the uniform 670 mM NaCl treatment.

Leaf gas exchange parameters

In uniform treatments, the net photosynthetic rate decreased as salinity in the medium

increased, with a sharp decline at 670 mM NaCl (Fig. 5.3A). In plants grown with

uniform 670 mM NaCl, the net photosynthetic rate was 49% of that of plants grown

with uniform 10 mM NaCl. There were also effects of increasing uniform salinity on

stomatal conductance. Stomatal conductance was not affected by salinity up to 230 mM

NaCl, but was decreased at 450 and 670 mM NaCl to 71% and 37% respectively of the

value of the uniform 10 mM NaCl treatment (Fig. 5.3B). Salinity (concentration and

distribution in the root-zone) had no effect on the maximum quantum efficiency of the

PSII (estimated Fv/Fm values, data not shown). The average Fv/Fm of A. nummularia

leaves from plants grown with uniform 10 mM and 670 mM NaCl was 0.74 ± 0.001

(S.E.) and 0.74 ± 0.004 (S.E.) respectively.

In non-uniform treatments, having one root half exposed to 670 mM NaCl, did not affect

net photosynthetic rate compared to the corresponding uniform treatments, except for

treatment 10/670 were values where 80% of those with the uniform 10 mM NaCl

treatment (Fig. 5.3A). On the other hand, stomatal conductances were decreased by non-

uniform treatments. For plants exposed to 120/670 and 230/670 stomatal conductances

were both 70% of those of plants grown with uniform 120 and 230 mM NaCl. However,

for all non-uniform treatments net photosynthetic rate and stomatal conductance were

1.6 to 2.0 times the values of plants grown with uniform 670 mM NaCl. Differences in

stomatal conductance between uniform and non-uniform treatments were not caused by


96

increases in total soluble sugars (Fig. 5.4) ruling out any possible negative feedback due

to reduced growth (cf. Munns, 1993).

Regression analyses were performed to determine whether these leaf gas exchange

parameters responded more closely to the salinity of the low-side or the mean salinity of

the root-zone, and the regressions (quadratic relationships) were of best fit for both net

photosynthetic rate (Fig. 5.3C) and stomatal conductance (Fig. 5.3D) when plotted

against the mean salinity of the root-zone (see Table 5.1 for R2 and P values for these

analyses).

Shoot water potential and leaf osmotic potential

In uniform treatments, shoot midday water potentials were relatively constant with

increasing salinity up to 230 mM NaCl, and then decreased with further increases above

that threshold. With 10–230 mM NaCl, the midday water potential was ~ -2.0 MPa, but

this decreased to -2.6 MPa at 450 mM and to -3.7 MPa at 670 mM NaCl (Fig. 5.5A).

The osmotic potential of the expressed sap of the young fully expanded leaves collected

at midday followed a similar trend to shoot water potential (Fig. 5.5B). With 10–230

mM NaCl, the leaf osmotic potential was -3.8 MPa, but this decreased to -4.2 MPa at

450 mM and to -5.6 MPa at 670 mM NaCl.

Under non-uniform treatments, midday shoot water potentials were all similar (within

0.1 MPa) to their corresponding uniform treatments (Fig. 5.5A). However, there were

some differences to this general picture with leaf osmotic potentials. With non-uniform

salinities between 10/670 and 230/670 mM NaCl, leaf osmotic potentials were not

different from those of the corresponding uniform treatments. However, at 450/670 mM

the leaf osmotic potential was 0.8 MPa lower than the uniform 450 mM treatment.

Compared with the uniform 670 mM treatment, leaf osmotic potentials were 1.2–1.5

MPa higher (i.e. less negative) with the 10/670 and 230/670 mM, and 0.5 MPa higher

with the 450/670 mM NaCl treatment (Fig. 5.5B). Under non-uniform salinity leaf water

contents mirrored those of the corresponding uniform treatments (Fig. 5.5C).


97

C

NaCl(mM)

0100

200300

400500

600700

Sho

otet

hano

l-ins

olub

le d

ry m

ass

(g)

0

4

6

8

B

Leaf

are

a(c

m2 )

0200

300

400

500

A

Sho

ot e

xten

sion

(m

m d

-1)

0

4

6

8

10 D

F

NaCl(mM)

0100

200300

400500

600700

E*

**

* ****

**

R2 = 0.8366

R2 = 0.8993

R2 = 0.9233

Figure 5.1. Responses of shoot growth parameters of Atriplex nummularia to uniform ( ) or non-uniform ( ) NaCl treatments: (A) shoot extension, (B) leaf area, and (C) shoot ethanol-insoluble dry mass. For non-uniform treatments, these shoot growth parameters are also plotted against the lowest salinity ( ) and the mean ( ) salinity of the root-zone: (D) shoot extension, (E) leaf area, and (F) shoot ethanol-insoluble dry mass. In D, E and F, the arrows indicate the displacement of the non-uniform values from when plotted against the lowest salinity to when plotted against the mean salinity of the root-zone. Regression curves (cubic relationships) for D, E and F are for the combination of uniform data and non-uniform data plotted against the mean salinity of the root-zone. R2 values are indicated for each curve and are significant (P ≤ 0.05). Plants were grown for 21 days with a split-root system. In uniform treatments both root halves were exposed to 10, 120, 230, 450 or 670 mM NaCl. In non-uniform treatments one root half was exposed to 10, 120, 230 or 450 mM NaCl (indicated on the x-axis) and the other root half was exposed to 670 mM NaCl. The shoot ethanol-insoluble dry mass (g) before treatments were imposed was 1.80 ± 0.22. Plants were sampled at midday. Values are means (n= 5) ± S.E. Asterisks indicate significant differences between means, * (P ≤ 0.05); ** (P ≤ 0.01); *** (P ≤ 0.001).

NaCl (mM) NaCl (mM)


98

B

NaCl(mM)

0 100 200 300 400 500 600 7000

1

2

A

0

1

2

3

4

* ** **

Roo

tet

hano

l-ins

olub

le d

ry m

ass

(g)

Roo

ts

in e

ach

side

etha

nol-i

nsol

uble

dry

mas

s(g

)

High salinity side

Low salinity side

* **

Figure 5.2. Responses of root ethanol-insoluble dry mass of Atriplex nummularia to uniform ( ) or non-uniform ( ) NaCl treatments: (A) entire root system, and (B) roots in each side of the non-uniform treatments. Plants were grown for 21 days with a split-root system. In uniform treatments both root halves were exposed to 10, 120, 230, 450 or 670 mM NaCl. In non-uniform treatments one root half was exposed to 10, 120, 230 or 450 mM NaCl (indicated on the x-axis) and the other root half was exposed to 670 mM NaCl. For uniform treatments the ratios of root/shoot were; 0.42 ± 0.01 (10/10); 0.47 ± 0.02 (120/120); 0.52 ± 0.05 (230/230); 0.54 ± 0.04 (450/450); 0.40 ± 0.08 (670/670). In non-uniform treatments the ratios of root/shoot were; 0.32 ± 0.03 (10/670); 0.35 ± 0.02 (120/670); 0.31 ± 0.02 (230/670); 0.35 ± 0.04 (450/670). The root ethanol-insoluble dry mass (g) before treatments were imposed was 0.42 ± 0.04. Plants were harvested at midday. Values are means (n= 5) ± S.E. Asterisks indicate significant differences between means, * (P ≤ 0.05); ** (P ≤ 0.01).


99

Table 5.1. R2 and P values of regression lines and curves fitted to the entire uniform and non-uniform data set, with non-uniform data either plotted against the lowest salinity in the root-zone or against the mean salinity in the root-zone. n.a. = not available

Figure Figure

R2 P R2 P

Shoot elongation 0.8366 < 0.05 5.1D 0.4302 0.3825 n.a.Leaf area 0.8993 < 0.05 5.1E 0.6623 0.1175 n.a.Shoot ethanol-insoluble dry mass 0.9233 < 0.05 5.1F 0.6900 0.0961 n.a.

Net photosynthetic rate 0.9055 < 0.05 5.2C 0.7976 < 0.05 n.a.Stomatal conductance 0.9340 < 0.05 5.2D 0.6498 0.1279 n.a.

Midday shoot water potential 0.9176 < 0.05 n.a. 0.9801 < 0.05 5.5DLeaf sap osmotic potential 0.9701 < 0.05 5.5E 0.7801 < 0.05 n.a.Leaf water content 0.7577 0.0535 n.a. 0.9701 < 0.05 5.5F

Leaf Na+ concentration 0.9200 < 0.05 5.6D 0.8125 < 0.05 n.a.Leaf K+ concentration 0.7069 < 0.05 5.6E 0.6927 < 0.05 n.a.Leaf Cl- concentration 0.9563 < 0.05 5.6F 0.7744 < 0.05 n.a.

Mean salinity in

root-zone

Lowest salinity in

root-zoneParameter


100

Regression analyses on the combined uniform and non-uniform data sets gave curves of

best fit for shoot water potential (quadratic relationship, Fig. 5.5D) and leaf water

content (cubic relationship, Fig. 5.5F) when plotted against the lowest salinity of the

root-zone. On the other hand, with leaf osmotic potential regression analysis gave a

curve of best fit (quadratic relationship, Fig. 5.5E) when plotted against the mean

salinity of the root-zone (see Table 5.1 for R2 and P values for the regression analyses).

The osmotic potentials of the xylem sap were rather low (highly negative), but the

validity of these measurements can be questioned. Although the initial sap collected was

discarded, the collected sap could still have been contaminated with cytosolic solutes.

Xylem osmotic potentials for uniform treatments (MPa) were: -1.17 ± 0.23 (10/10), -

1.21 ± 0.13 (120/120), -1.41 ± 0.21 (230/230), -1.40 ± 0.19 (450/450) and -2.22 ± 0.28

(670/670). For non-uniform treatments, xylem sap osmotic potentials (MPa) were: -1.17

± 0.29 (10/670), -0.91 ± 0.29 (120/670), -1.32 ± 0.28 (230/670) and -1.41± 0.17

(450/670).

Tissue ion concentrations

In uniform treatments the concentration of Na+ in the young fully expanded leaves

increased with the external NaCl concentration (Fig. 5.6A), and leaf Na+ concentrations

were 70% higher for plants grown with 670 mM than 10 mM NaCl. There were similar

trends for leaf Cl- concentrations in uniform treatments (Fig. 5.6C). Leaf K+

concentrations decreased at concentrations ≥ 120 mM NaCl, with the lowest leaf K+

concentration of 103 mM occurring in plants grown in uniform 450 mM NaCl (Fig.

5.6B).


101

B

NaCl(mM)

0100

200300

400500

600700

Sto

mat

al c

ondu

ctan

ce(m

mol

m-2

s-1

)

050

100

150

200

250

AN

et p

hoto

synt

hetic

rat

e (

mol

m-2

s-1

)

0

5

10

15

20µµ µµ

*** *** **

**

D

NaCl(mM)

0100

200300

400500

600700

C R2 = 0.9055

R2 = 0.9340

Figure 5.3. Responses of leaf gas exchange parameters of Atriplex nummularia to uniform ( ) or non-uniform ( ) NaCl treatments: (A) net photosynthetic rate, and (B) stomatal conductance, both for young fully expanded leaves. For non-uniform treatments, these parameters are also plotted against the lowest salinity ( ) and the mean ( ) salinity of the root-zone: (C) net photosynthetic rate, and (D) stomatal conductance. In C and D, the arrows indicate the displacement of the non-uniform values from when plotted against the lowest salinity to when plotted against the mean salinity of the root-zone. Regression curves (quadratic relationships) for C and D are for the combination of uniform data and non-uniform data plotted against the mean salinity of the root-zone. R2 values are indicated for each curve and are significant (P ≤ 0.05). In uniform treatments both root halves were exposed to 10, 120, 230, 450 or 670 mM NaCl. In non-uniform treatments one root half was exposed to 10, 120, 230 or 450 mM NaCl (indicated on the x-axis) and the other root half was exposed to 670 mM NaCl. Measurements were taken 19 days after imposing treatments, between 1100 and 1300 hours, with PAR of 1500 µmol m-2 s-1, ambient relative humidity of 50–60%, reference CO2 of 380 µmol mol-1. Values are means (n = 4) ± SE, with each replicate under non-uniform treatment being the mean of two measurements per plant of opposing leaves (i.e. one leaf from above each split-root side). Asterisks indicate significant differences between means, * (P ≤ 0.05); ** (P ≤ 0.01); *** (P ≤ 0.001).

NaCl (mM)


102

NaCl(mM)

0 100 200 300 400 500 600 7000

20

30

40

50

Hex

ose

equi

vale

nts

(mM

)

Figure 5.4. Concentration of total soluble sugars (tissue water basis) in young fully expanded leaves of Atriplex nummularia exposed to uniform ( ) or non-uniform ( ) NaCl treatments. Plants were grown for 21 days with a split-root system. In uniform treatments both root halves were exposed to 10, 120, 230, 450 or 670 mM NaCl. In non-uniform treatments one root half was exposed to 10, 120, 230 or 450 mM NaCl (indicated on the x-axis) and the other root half was exposed to 670 mM NaCl. Leaf tissues were sampled at 1100 hours. Values are means (n = 5) ± S.E. with each replicate under non-uniform treatment being the mean of two measurements per plant of opposing leaves (i.e. one leaf from above each split-root side). No significant differences were found between treatments (P ≤ 0.05).


103

C

NaCl (mM)

010

020

030

040

050

060

070

0

Leaf

wat

er c

onte

nt(m

L g

-1 d

ry m

ass)

0

4

5

B

Leaf

sap

os

mot

ic p

oten

tial

(MP

a)

-5

-4

-3

-2

-1

0

A

Mid

day

shoo

t wat

er p

oten

tial

(MP

a)

-5

-4

-3

-2

-1

0

*

F

NaCl (mM)

010

020

030

040

050

060

070

0

E

D R2 = 0.9801

R2 = 0.9701

R2 = 0.9333

Figure 5.5. Responses of shoot water relations parameters in Atriplex nummularia to uniform ( ) or non-uniform ( ) NaCl treatments: (A) midday shoot water potential, (B) osmotic potential of expressed leaf sap including salt bladders, and (C) leaf water content. For non-uniform treatments, these parameters are also plotted against the lowest salinity ( ) and mean ( ) salinity of the root-zone: (D) midday shoot water potential, (E) osmotic potential of expressed leaf sap including salt bladders, and (F) leaf water content. In D, E and F, the arrows indicate the displacement of the non-uniform values from when plotted against the lowest salinity to when plotted against the mean salinity of the root-zone. The regression curves (quadratic relationships for D, E and cubic relationship for F) are fitted to the combined uniform and non-uniform data set. The curves of best fit in D and F were when the non-uniform data were plotted against the lowest salinity in the root-zone, whereas the curve of best fit in E was when the non-uniform data were plotted against the mean salinity of the root-zone. R2 values for these curves are significant (P ≤ 0.05). In uniform treatments both root halves were exposed to 10, 120, 230, 450 or 670 mM NaCl. In non-uniform treatments one root half was exposed to 10, 120, 230 or 450 mM NaCl (indicated on the x-axis) and the other root half was exposed to 670 mM NaCl. Midday water potential and osmotic potentials were determined 21 days after imposing treatments between 1200 and 1300 hours. Values are means (n = 5) ± S.E. In non-uniform treatments, measurements were made from shoot segments and leaves from both sides of the shoot, directly above the low and high-salt side. As there were no differences in the readings between the sides, each replicate is the mean of two measurements per plant from opposing leaves. Asterisks indicate significant differences between means: * (P ≤ 0.05).

NaCl (mM)


104

Under non-uniform salinities, leaf Na+ and Cl- concentrations also increased with

increasing salinities in the low-salt side. Under non-uniform conditions with 10–230

mM NaCl in the low-salt side, leaf Na+ and Cl- concentrations increased by 10–50%

compared with the corresponding uniform treatment, but these concentrations did not

differ between the uniform 450 mM NaCl treatments and the non-uniform treatment

with 450 mM NaCl in the low-salt side. Under non-uniform salinities K+ concentrations

were 53–68% of that of the uniform 10 mM NaCl treatment, but only 10/670 had a

lower K+ concentration than its corresponding uniform treatment (i.e. uniform 10 mM

NaCl). Regression analysis performed on the combined uniform and non-uniform data

set showed best fits for leaf Na+ and Cl- (linear relationships, Fig. 5.7D, 5.7F) and leaf

K+ (quadratic relationship, Fig. 5.7E) when plotted against the mean salinity of the root-

zone (see Table 5.1 for R2 and P values for the regression analyses).

Water use

After 19 days, under non-uniform treatment, whole plant water use decreased with

increasing salinity in the low-salt side (Fig. 5.7A). At 450/670 mM NaCl, plant water

use was similar to that measured with uniform 670 mM NaCl, and was ~60% of the use

by plants in the 10/670 mM treatment. This reduction with 450/670 mM NaCl was due

in part to a 20% decline in water uptake rate (root surface area basis – Fig. 5.7B), but

was also driven by the above mentioned decrease in root dry mass allocation to the low-

salt side (Fig. 5.2B). In all non-uniform treatments, water uptake from the high-salt side

was maintained at rates ≤ 0.1 mmol m-2 s-1.

K+ depletion rates from the low- and high-salt sides

To assess whether K+ was taken up mostly from the low-salt side compared with the

high salt-side, a small experiment was conducted in which K+ depletion was measured

in the solution on each side. Only the following treatments were considered: 10/10,

10/450 and 450/450. Uptake rates under uniform salinities declined with 450 mM NaCl,

with K+ net uptake rate 11% of that in plants with uniform 10 mM NaCl (Table 5.2).

Under non-uniform salinity, most of the K+ (72%) was taken up from the low-salt side.

Rates of uptake from each side were related to the NaCl concentrations of the solution

and were similar to those measured from roots in the corresponding uniform salinities.


105

C

NaCl(mM)

0100

200300

400500

600700

0

400

600

800

1000

0

50

100

150

200

250

A

0

400

600

800

1000

B

**

**

*

***

Cl-

(m

M)

K+

(mM

)N

a+

(mM

)

*** ** **

F

NaCl(mM)

0100

200300

400500

600700

D R2 = 0.9200

R2 = 0.7069

R2 = 0.9563

E

Figure 5.6. Ion concentrations (tissue water basis) in young fully expanded leaves of Atriplex nummularia exposed to uniform ( ) or non-uniform ( ) NaCl treatments: (A) Na+, (B) K+, and (C) Cl-

. For non-uniform treatments, these parameters are also plotted against the lowest salinity ( ) and mean ( ) salinity of the root-zone: (D) Na+, (E) K+, and (F) Cl-. In D, E and F, the arrows indicate the displacement of the non-uniform values from when plotted against the lowest salinity to when plotted against the mean salinity of the root-zone. The regression lines (for D, E) and curve (quadratic relationship for F) are fitted to the combined uniform and non-uniform data set. The lines and curve of best fit were when the non-uniform data were plotted against the mean salinity of the root-zone. R2 values for these lines are significant (P ≤ 0.05). In uniform treatments both root halves were exposed to 10, 120, 230, 450 or 670 mM NaCl. In non-uniform treatments one root half was exposed to 10, 120, 230 or 450 mM NaCl (indicated on the x-axis) and the other root half was exposed to 670 mM NaCl. Leaf tissues were sampled at 1100 hours, 21 days after imposing treatments. Values are means (n= 5) ± S.E. In non-uniform treatments, leaves were measured from both sides of the shoot, directly above the low and high-salt side. As there were no differences between the results from each side, each replicate under non-uniform treatments is the mean of two measurements per plant from opposing leaves. Asterisks indicate significant differences between means: * (P ≤ 0.05); ** (P ≤ 0.01); *** (P ≤ 0.001). Note different vertical scale for (B).

NaCl (mM)


106

0 100 200 300 400 500 600 700

Wat

er u

ptak

e ra

te

on a

roo

t sur

face

are

a ba

sis

( m

mol

m-2

s-1

)

0.0

0.1

0.2

0.3

A

NaCl (mM)

Tot

al w

ater

upt

ake

(mL)

0

5

10

15

20

25

B

High salinity side

Low salinity side

Figure 5.7. Responses of plant water uptake by Atriplex nummularia to uniform 670 mM NaCl ( ) or non-uniform ( ) NaCl treatments in the root-zone: (A) whole-plant water uptake, and (B) water uptake rate on a root surface area basis from the low and high-salt sides. In non-uniform treatments one root half was exposed to 10,120, 230 or 450 mM NaCl (indicated on x-axis), and the other root half was exposed to 670 mM NaCl. Water uptake measurements were taken from 0600 to 1800 hours, 20 days after imposing treatments. Values are means (n= 4) ± SE. Root surface area in each side for the uniform 670 mM NaCl treatment was (m2): 0.067 ± 0.003. Root surface areas in the low-salt side for the non-uniform treatments were (m2): 0.123 ± 0.003 (10/670), 0.127 ± 0.007 (120/670), 0.106 ± 0.005 (230/670) and 0.096 ± 0.009 (450/670). Root surface areas in the high-salt side for the non-uniform treatments were (m2): 0.061 ± 0.007 (10/670), 0.070 ± 0.004 (120/670), 0.063 ± 0.005 (230/670) and 0.067 ± 0.007 (450/670). Data for water uptake rates in each side under uniform 670 mM NaCl were averaged, as no differences were found between means in each side.


107

Table 5.2. Depletion rates of K+ in the treatment solutions for the period 8 to 15 days in Atriplex nummularia grown under uniform or non-uniform salinities.

Low salinity side High salinity side

10/10

10/450 28.0 ± 5.0a 10.8 ±5.8b

450/450

NaCl (mM)

K+ uptake rates

(µmol g-1 root fresh mass d-1)

39.7 ± 3.4a*

4.2 ± 3.0b*

Atriplex nummularia was grown for 15 days with a split-root system with uniform or non-uniform salinities in the root-zone. In uniform treatments both root halves were exposed to 10 or 450 mM NaCl, whereas in the non-uniform treatments one root half was exposed to 10 and the other root half to 450 mM NaCl. Depletion rates were determined 7 days after changing solution (from day 8 to 15) and uptake rates were estimated using the uptake rate formula from Williams (1948). Values are means (n = 5) ± SE. Different lower case letters within each column indicate significant differences (P ≤ 0.05). Root fresh masses on each side after 15 days were (g): 4.09 ± 0.30 (10/10, data for the two sides averaged for each replicate); 5.61 ± 0.51 (low salinity side, 10/450); 2.56 ± 0.27 (high salinity side, 10/450); 3.14 ± 0.34 (450/450, data for the two sides were averaged for each replicate). * Data for the two sides were averaged as under uniform 10 and 450 mM NaCl no differences were found between uptake rates in each side.


108

5.5 Discussion

In saline landscapes, the spatial heterogeneity in the salt concentrations experienced by

individual plants is likely to be quite large (see Table 2.1, Chapter 2). When salinity is

heterogeneously distributed in the root-zone, it has been suggested that plants respond

mainly to either: (1) the mean salinity, or (2) the lowest salinity in the root-zone (see

Introduction). As far as I am aware, this is the first time that research into non-uniform

salinity in split-root studies has focused on the use of substantial (moderate to high)

NaCl concentrations in the low-salt side: all other experiments in the literature have used

“NaCl-free” media (e.g. Flores et al., 2002; Hamed et al., 2008 Kirkham et al., 1969,

1972; Messedi et al., 2004; Zekri and Parsons, 1990). Results from this chapter in

which Atriplex nummularia was exposed to laterally non-uniform salinity indicate that if

one half of the root system is at high salinity, then increasing NaCl concentrations on the

low-salt side becomes progressively more damaging to most of the measured plant

parameters. Shoot growth parameters (shoot ethanol-insoluble dry mass, shoot extension

and leaf area), leaf gas exchange, leaf osmotic potential and leaf ion concentrations

(Na+, K+ and Cl-) all responded to the mean salinity of the root-zone (Table 5.1).

Therefore, for such parameters, plants ‘integrate’ the two salinities of the root-zone. For

these parameters, with high salinity already around half the roots, increasing the NaCl

concentration around the other half of the roots brought the mean salinity in the root-

zone out of the “optimum range” for A. nummularia. On the other hand, midday shoot

water potential and leaf water content responded more closely to the lowest salinity of

the root-zone (Table 5.1). As most of the water comes from the low-salt side under non-

uniform salinity it is likely that midday shoot water potential and leaf water content are

influenced mainly by the water potential of that part of the root-zone from where most

water was taken up.

If reductions in shoot growth are related to the mean salinity of the root-zone and to the

salinity tolerance of the species, then the results of earlier studies (Messedi et al., 2004;

Hamed et al., 2008) reporting growth enhancements for the halophytes Sesuvium

portulacastrum and Batis maritima under non-uniform salinities (0 and 800 mM NaCl)

can be explained. With the halophyte Sesuvium portulacastrum when both root halves

where exposed to 800 mM NaCl, shoot biomass declined by 69%, however when one

root half was exposed to 0 mM NaCl and the other root half was exposed to 800 mM


109

NaCl, shoot biomass increased by 20% compared to “control” plants with both root

halves at 0 mM NaCl (Messedi et al., 2004). This enhanced growth in the 0/800 mM

treatment can now be explained as the mean salinity in the root-zone was 400 mM NaCl,

and for this dicotyledonous halophyte, optimal growth occurs in the range 100–400 mM

NaCl (Messedi et al., 2004). Moreover as a salt-free solution was used by Messedi et al.

(2004) as the uniform low-salt “control”, the reduced growth in the control plants

compared to the non-uniform treatments was likely due to a deficiency in the ions

required by these dicotyledonous halophytes for maximal growth (Yeo and Flowers,

1980).

The principle that plants respond to the mean salinity of the root-zone may also explain

the discrepancies in the literature concerning non-halophyte growth responses with non-

uniform salinities in the root-zone (see Introduction). Maas and Hoffman (1977)

proposed that for non-halophytes growth in response to salinity can be represented by a

two piece linear relationship, in which yield is initially independent of the salinity of the

root-zone, but above “threshold salinity” yield reduces with increasing salinity in the

root-zone. Therefore for non-halophytes exposed to non-uniform salinity in the root-

zone, if the mean salinity in the root-zone is below the “threshold” then no growth

reduction will be observed. However, yields can be expected to progressively decline as

the mean salinity increases above the threshold. Let us see how this interpretation can be

applied to the example of the non-halophyte Citrus aurantium (Zekri and Parsons,

1990). Exposure of this plant to a non-uniform salinity (NaCl free solutions with an EC

of 1.1 dS m-1 in one root half and a saline solution with EC 8.8 dS m-1 around the other

root half) caused a 21% decline in shoot dry mass compared to control plants with both

root halves in the 1.1 dS m-1 solution (Zekri and Parsons, 1990). However, shoot growth

of plants with both root halves in the 8.8 dS m-1 solution declined by 81%. Furthermore,

in the above non-uniform treatment 75% of the roots were exposed to 1.1 dS m-1 and the

remaining 25% of the roots were exposed to 8.8 dS m-1; thus the “root-weighted mean”

salinity of the total root-zone would have been 3 dS m-1. Therefore using a threshold

salinity of 1.2 dS m-1 and yield reduction rates of 13.5% per dS above threshold

(Bielorai et al., 1978), a salinity of 3 dS m-1 would be expected to cause a yield

reduction of 24%, a value close to the 21% reduction actually observed for shoot dry

mass by Zekri and Parsons (1990) for their non-uniform salinity treatment. Therefore

also for non-halophytes it appears that, under non-uniform salinities, growth responses


110

are dictated by the “root-weighted mean” salinity in the root-zone and the species

specific salinity tolerance.

In the present study non-uniform salinities were associated with increases in leaf Na+

and Cl- concentrations. Similar increases in Na+ and Cl- concentrations in shoot tissues

have been observed in both halophytes and non-halophytes with non-uniform salinities

in the root-zone (non-halophytes: Hajji et al., 2001; halophytes: Hamed et al., 2008,

Messedi et al., 2004). It is, however, not clear whether these increases were caused or

not by the ion uptake from the high-salt side. Although most Na+ and Cl- ions are

excluded at the root surface, there will be some uptake of those ions from the external

solution during water uptake from the high-salt side, and these ions will then move via

bulk flow in the xylem vessels to the shoot (Flowers and Yeo, 2007). It is well

documented for halophytes that growth decreases as NaCl increases to severe and

extreme salinities, however the reasons for such impacts are yet not clear (reviewed

recently by Flowers and Colmer, 2008). In the present study, using the combined data

set from the uniform and non-uniform treatments, there was a significant negative

relationship between leaf Na+ and shoot ethanol-insoluble dry mass (R2 = 0.8076). It is,

however, not possible to conclude unequivocally that these increases in Na+ (and also

Cl-) were the main cause of the reduced growth under non-uniform salinities, because

the salt contents of the bladder cells on the surfaces of the leaves were also included in

the measurement of leaf ions6. In Atriplex amnicola bladder cells accounted for 81–86%

of the leaf Na+ in expanding leaves but ≤ 10% of leaf Na+ in older leaves (Aslam et al.,

1986).

Under non-uniform salinities A. nummularia was able to take up most of its water and

K+ from the low-salt side. Most of the water used by A. nummularia exposed to non-

uniform salinities during the light period came from the root portion with the lowest

salinity (% total water uptake: 85-88% with 10–120 mM in the low-salt side; 77-78%

with 230–450 mM NaCl in the low-salt side). In regard to K+ uptake, data from the

second experiment show that most of K+ depletion (72%) occurred in the low-salt

solution under high non-uniform salinity (Table 5.2). The fact that most K+ came from

the low salt-side was presumably because on the high-salt side Na+ would have

competed with K+ for the same entry pathways (c.f. Maathuis and Amtmann, 1999).

6 Attempts to remove the bladders using the method of Aslam et al. (1986) were not successful.


111

Another experiment supporting the view that nutrients are taken up from the low-salt

side with non-uniform salinities comes from a study of Capsicum annuum (Lycoskoufis

et al., 2005). In this experiment, there was severely reduced growth (i.e. 42% decrease

in shoot dry mass compared with plants exposed uniformly to a NaCl-free solution)

when the roots of the NaCl-free side were exposed to water instead of the standard

nutrient solution. These data support the hypothesis that the non-saline side (or low-salt

side) is important for providing to the plant not only water but also essential nutrients. It

is therefore possible to conclude that under non-uniform salinities, if the low-salt side is

provided with sufficient nutrients, growth will be primarily limited by either elevated

concentrations of Na+ and Cl- in shoot tissues or limited water availability from the low-

salt side rather than the nutritional disturbances usually associated with uniform

salinities in the root-zone (Hajji et al., 2001; Hamed et al., 2008).

One parameter that did not respond to the mean salinity of the root-zone in the present

study was shoot water potential. In contrast to most other parameters (e.g. growth or leaf

gas exchange), under non-uniform conditions midday shoot water potentials resembled

those of plants grown under uniform conditions at the lower salinity level and were 0.9

to 1.6 MPa higher than in plants with the uniform 670 mM NaCl treatment. As most of

the water was taken up from the low-salt side, it would therefore appear that midday

shoot water potentials under non-uniform conditions reflect the water potential of the

root-zone from where the plants are taking up most of the water. Similarly the shoot

water potential of the non-halophytes Phaseolus vulgaris and Hordeum vulgare exposed

to non-uniform salinity were found to be similar to those in plants exposed uniformly to

the NaCl-free medium, whereas stomatal conductances were intermediate between

values in plants uniformly exposed to the NaCl-free medium and salinity (0.4 MPa or

ca. 80 mM NaCl for Phaseolus vulgaris and 1.5 MPa or ca. 315 mM NaCl for Hordeum

vulgare, Kirkham et al., 1972). For example, after 19 days of treatments, shoot water

potential in P. vulgaris exposed to non-uniform salinity only declined by 0.1 MPa

compared to control plants (uniform NaCl-free medium), whereas it declined by -1.93

MPa when both root halves were exposed to ca. 80 mM NaCl (Kirkham et al., 1972).

Therefore, as more than 70% of the whole plant water uptake was taken from the NaCl-

free side (Kirkham et al., 1969), for the non-halophyte P. vulgaris shoot water potentials

under non-uniform conditions also appear to reflect the water potential in the root-zone

where plants are taking up most of the water.


112

In the present study under non-uniform salinities small amounts of water uptake

occurred from high-salt side (% total water uptake: 12-15% with 10–120 mM in the

low-salt side; 22-23% with 230–450 mM NaCl in the low-salt side). This occurred

despite the fact that midday shoot water potentials reflected the water potential in the

root-zone where plants were taking up most water, i.e. the low-salt side. Therefore, in a

similar manner to Chapter 3, there was a gradient between the shoot water potential at

midday (-2 MPa, Fig. 5.5A) and the osmotic potential of the external solution in the

high-salt side (-3 MPa) that might suggest that water should be lost to the solution rather

than taken up from the high-salt side. This apparent anomalous water uptake can be

explained with a simple water uptake model.

Water uptake can be considered to be a two step process: movement across the roots

into the xylem and then up the xylem in response to a tension gradient (Taiz and Zeiger,

2006; Maurel et al., 2010). For the argument here, only the first step, the water

movement across the root, will be discussed. Conceptually water movement (Jv) from

the medium to the xylem can occur through two main pathways (Javot and Maurel,

2002; Knipfer and Frickle, 2010): (i) the cell-to-cell (CTC) pathway, where water

moves across the plasmamembranes (transcellular path) or moves through the

plasmodesmata (symplastic path); and (ii) the apoplastic pathway, where water moves

though the cell walls but is blocked by the casparian strip, unless in some regions of the

root there is a disruption of the continuity of the casparian strip (also called bypass flow,

e.g. Oryza sativa, Faiyue et al., 2010). It is important to stress that there have not yet

been any studies that report on the extent of bypass flow in halophytes. However, it is

expected that, to regulate ion transport to the shoot, such flow must be small (Flowers et

al., 1986).

In general as water flow is proportional to the water potential gradient, water flow can

be defined as (Nobel, 2009; see also Fig. 5.8):

Jv = Lp*(∆P - σ*∆π) (Nobel, 2009) (1)

where Jv is the water flow; Lp is the hydraulic conductivity coefficient; ∆P is the

difference in hydrostatic pressure between the external solution and the xylem; σ is the

reflection coefficient for solutes; ∆π is the difference in osmotic potential between the

external solution and the xylem. From the above formula, it is clear that both changes in


113

xylem π and σ will affect the rate of flow expected in response to a gradient in osmotic

potential. Membranes have different resistances to the passage of solutes and water, and

the term σ describes the relative ease with which solutes cross a membrane (Nobel,

2009). The maximum obtainable value for σ is 1, which indicates that the solute is not

able to penetrate the membrane, i.e. all molecules are reflected, whereas if σ = 0 all

solutes cross the membrane. Hence considering that functional membranes are crossed

in the CTC pathway, generally the CTC pathway will have a σ close to the unity. On the

other hand, in an apoplastic pathway, where no membranes are crossed, σ will be 0. In

summary (see also Fig. 5.8):

Flow in the apoplastic pathway will be: Jv = Lp*∆P (2)

Flow in the CTC pathway will be: Jv = Lp*(∆P - σ*∆π) (3)

Summarising the total water flow that includes both pathways could therefore be given

by:

Jvtotal = Lp*(∆P - σroot*∆π) (4)

and therefore

Jvtotal/ Lp= ∆P - σroot*∆π (5)

where σroot (the reflection coefficient of the roots) is an integrated value that takes into

account the different proportion of apoplastic (or bypass flow) and the CTC pathways

(Zhu and Steudle, 1991).

Using equation 5, the direction of water flow (Jvtotal/ Lp) under non-uniform conditions

can be estimated (Fig. 5.9). Where Jvtotal/ Lp is negative, the root loses water to the

external medium, and where this ratio is positive, there is a net water uptake from the

external medium. Calculation is made here using the water potential in the shoot at

midday for treatment 10/670. The midday shoot water potential measured with the

pressure bomb represents the xylem tension as the osmotic potential component is not

taken into account when measuring water potentials with the Scholander pressure bomb.


114

Hence as the measured xylem tension under non-uniform salinity was -2 MPa and

assuming that there was no resistance in the xylem pathway between the shoot and the

roots, it can be assumed that root xylem tension was also -2 MPa. With this tension in

the root xylem, and an external osmotic potential at 670 mM NaCl of -3.1 MPa (see

Chapter 3), the ratio Jvtotal/ Lp was calculated for a range of osmotic potential in the

xylem (between – 0.1 and -1.1 MPa; Fig. 5.9) and σroot. These calculations show that a

net movement of water from the external medium to the xylem (i.e. Jv/Lp positive)

would occur with a xylem osmotic potential of -0.1 MPa only if σroot ≤ 0.66. On the

other hand, with σroot = 1, water uptake would only occur if xylem osmotic potential ≥ -

1.1 MPa. Therefore, as bypass flow in halophytes is likely to be quite small, it is also

likely that the net water uptake occurs in response to a decline in xylem osmotic

potential, most likely caused by the uptake of ions.

In conclusion, plant responses to non-uniform salinities are an integrated response to the

two salinities of the root-zone and the intrinsic salinity tolerance of the species. The

results presented here are only applicable to the commercial clone of A. nummularia,

“Eyres Green”, and should be confirmed for other ecotypes of A. nummularia and other

dicotyledonous halophytes. Nevertheless these results would suggest that

dicotyledonous halophytes, which generally have an optimum growth around 100–400

mM NaCl, will have an advantage under non-uniform salinities compared with non-

halophytes, as they have the potential to withstand severe/extreme salinities in part of

the root system as long as there are other root portions exposed to lower salinity and the

mean salinity of the root-zone is within the “optimal range”. In regards to water uptake

under non-uniform salinities, declines in xylem osmotic potentials could explain the

water uptake from the external solution to the xylem of the roots in the high-salt side.


115

Figure 5.8. Schematic diagram of the pathway of water flow from the external medium to the xylem across a plant root. Water flow from the external medium to the xylem is directly proportional to the hydraulic conductivity of the pathway (Lp) and is driven by a water potential gradient, that has a tension (P) and an osmotic (π) component, between the medium and the root xylem. Water movement (Jv) from the medium to the root xylem can occur through two main pathways: (i) the cell-to-cell (CTC) pathway, where water moves across a plasmamembranes (transcellular path) or moves through plasmodesmata (symplastic path); (ii) the apoplastic pathway (APO), where water moves though the cell walls but is blocked by the casparian strip, unless in some regions there is a disruption of the continuity of the casparian strip which allows water flow (bypass flow). Each pathway will have a different reflection coefficient (σ). In general the CTC pathway will have a σ close to the unity whereas the apoplastic pathway will have σ = 0 (Knipfer and Frickle, 2010). The reflection coefficient measured for the root (σroot) might be defined as an integrated values that takes into account the different proportions of the apoplastic and the cell-to-cell pathways (Zhu and Steudle, 1991). In regard to bypass flow (question mark in diagram) there are no studies in the literature that have been conducted to determine the extent of bypass flow in halophytes. However, in order to regulate ion transport to the shoot it is expected that this flow must be small in order (Flowers et al., 1986).

?

CTC: Jv = Lp*( ∆∆∆∆P - σσσσ*∆∆∆∆ππππ) APO: Jv = Lp*( ∆∆∆∆P)

Jv total = Lp*(∆∆∆∆P - σσσσroot *∆∆∆∆ππππ)


116

Figure 5.9. Relationship between Jvtotal/Lp (Jvtotal = water flow; Lp= hydraulic conductance coefficient) and root reflection coefficients (σroot) and xylem osmotic potential (πxylem, MPa). For Jvtotal/Lp, positive values indicate water uptake from the external solution and negative values indicate water loss from the roots. In plants exposed to 10/670 mM NaCl, Jvtotal/Lp values were estimated assuming no resistance in the shoot/root pathway and that the pressure in the xylem of the roots was therefore equivalent to the water potential of the shoots measured with a pressure bomb (– 2 MPa). On the high salt side, the external solution had an osmotic potential (πmedium) of -3.1 MPa and no pressure component (Pmedium= 0). .

σσσσ0.0 0.2 0.4 0.6 0.8 1.0

J wat

er*L

p

(m s

-1)

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

1.10.90.70.50.30.1

Jv*L

p

(m s

-1)

πmedium = - 3.1 MPaPxylem = - 2.0 MPa

πxylem(MPa)

B

root

ππππxylem

(- MPa)

Jv

tota

l/Lp

Chapter 6 Concluding Discussion

117

Chapter 6

Concluding Discussion

6. Chapter 6: Concluding Discussion


118

6.1 Summary of key findings

6.1.1. Plant responses to non-uniform salinity in the root-zone

The results described in this thesis enable discussion of the general principles that

governed the responses of Atriplex nummularia to non-uniform salinities. In Chapters 3

and 5, most of the measured physiological parameters (e.g. shoot growth or leaf gas

exchange) responded more closely to the average salinity of the root-zone, rather than

being driven by the salinity of the low-salt side. The only exceptions were shoot water

potentials and leaf water contents which responded more closely to the lowest salinity in

the root-zone. Results in Chapter 4 in which plants were grown under non-uniform

salinity with low (10 mM NaCl) and extreme salinity (1500 mM NaCl) in each root half,

show that calculation of the mean salinity of the root-zone needs to be made as a “root-

weighted” mean salinity. In Chapters 3 and 5, the root ethanol-insoluble dry mass

allocation between the low and the high-salt sides were relatively similar, and if the

mean salinity were to be corrected by the amount of roots in each side, the “root-

weighted mean” salinity would decline by ca. 10 to 50 mM NaCl for treatments 10/670,

120/670, and 230/670 and would be similar to the “non-root-weighted mean” for

450/670. However, if the dry mass allocation between the low and the high-salt side

were to be significantly affected (e.g. Chapter 4 where 79% of the root ethanol-insoluble

dry mass was in the low-salt side), then the mean salinity of the root-zone would differ

quite significantly from the “root-weighted mean” salinity. Hence for A. nummularia

exposed simultaneously to 10 and 1500 mM NaCl, while the mean salinity of the root-

zone was 755 mM NaCl, the “root-weighted mean” salinity was 316 mM NaCl (Chapter

4). Drawing this together, Fig. 6.1 presents all the data from all experimental chapters

for whole plant ethanol insoluble dry mass and stomatal conductance of young fully

expanded leaves (as a percent of the uniform 10 mM treatment), and has related these to

either the lowest salinity of the root-zone (Fig. 6.1A, D), the mean salinity of the root-

zone (Fig. 6.1B, E), or the “root-weighted mean” salinity of the root-zone (Fig. 6.1C, F).

Correlations of these plant parameters were sharpest (i.e. R2 values highest) when

related to the “root-weighted mean” salinity of the root-zone. Moreover, if whole plant

dry mass from Chapter 3, that were likely to be exposed to a higher relative humidity,

and had different growth responses to increasing NaCl in the root-zone compared to

plants in Chapters 4 and 5 that were exposed to relative humidity of 55-56% (see

Section 6.1.4), are taken out, R2 values for whole plant ethanol-insoluble dry mass


119

decline further when data are plotted against either the lowest or the mean salinity in the

root-zone but increases when data are plotted against the “root-weighted mean” salinity

in the root-zone. R2 values for whole ethanol-insoluble plant dry mass without data from

Chapter 3 are; 0.5420 (plotted against the lowest salinity in the root-zone); 0.4931

(plotted against the mean salinity in the root-zone); 0.7210 (plotted against the “root-

weighted mean” salinity in the root-zone). It is clear that under non-uniform salinities

plants respond more closely to the “root-weighted mean” salinity, than the lowest

salinity or the mean salinity of the root-zone (Fig. 6.1). With extreme non-uniform

salinity, compensatory root growth in the low-salt side shifted the mean salinity in the

root-zone towards the low-salt side; this explains why plants exposed to extreme non-

uniform salinity had a similar ethanol-insoluble dry mass to plants grown with uniform

10 mM NaCl (Fig. 6.1). Therefore, compensatory root growth in the low-salt side

observed with extreme salinity in one root portion contributed to the plant responses to

non-uniform salinities by shifting the mean salinity of the root-zone towards the low-salt

side.

6.1.2. Ion relations under non-uniform salinities

Non-uniform salinities were associated with increases in leaf Na+ and Cl- concentrations

compared to those in plants with the uniform low salt treatments. As dicotyledonous

halophytes, such as A. nummularia, are able to use the accumulation and sequestration

of inorganic ions to adjust their osmotic potential (e.g. Atriplex hymenelytra, Bennert

and Schmidt, 1984; reviewed extensively for other halophytes in Flowers and Colmer,

2008), it is most likely that part of these ions were used for osmotic adjustment. With

non-uniform salinity, where a plant has a portion of its roots in a solution with low

salinity, and most of the water comes from that root portion, it is conceivable that

growth restriction could be related to limited water availability rather than to toxic

concentrations of ions in plant tissues, as water uptake from the high-salt side is limited.

For example in Chapters 3 and 4, whole plant water uptake under non-uniform salinities

declined to 61–73% of that for plants exposed to uniform 10 mM NaCl. Thus, it might

be possible that the increases in ions in the shoot tissues under non-uniform salinities

could improve plant performance, as Na+ and Cl- can act as “cheap” osmolytes for the

plants to maintain low water potentials (Maathuis, 2007).


120

The data presented in this thesis suggests that under non-uniform salinities, A.

nummularia takes up most K+ from the low-salt side. In Chapter 5, with high non-

uniform salinity (10/450), 72% of the K+ was taken up from the low salt solution (Table

5.2). It is presumed that this occurred because K+ and Na+ compete for the same entry

pathways (Maathuis and Amtmann, 1999), and most K+ uptake under non-uniform

salinities must therefore come from the low-salt side. Independently of a uniform or

non-uniform salinity in the root-zone, with increasing NaCl concentrations in the root-

zone leaf K+ declined compared to plants exposed to uniform 10 mM NaCl. For plants

grown with 10/1500 mM NaCl, there was a 47% decline in leaf K+ concentrations

without adverse effects on growth; this might indicate that for plants with uniform 10

mM NaCl in the root-zone, K+ uptake is generally in excess of that required for

maximum growth.

Under non-uniform salinities shoot ion concentrations increased compared with the

plants grown under uniform low salinity. For example, with one root half exposed to 10

mM NaCl and the other exposed to 1500 mM NaCl, leaf Na+ and Cl- concentrations

increased to 1.7–2.4 times the concentrations found in plants grown with 10 mM NaCl

in both root halves. Due to these increases in shoot Na+ and Cl- concentrations, under

non-uniform salinities there were generally sharp declines in the shoot K+:Na+ ratio (e.g.

1.12:1 with 10/10 to 0.1:1 in 10/1500, Chapter 4) compared with plants in uniform 10

mM NaCl. Nevertheless, these low K+:Na+ ratios under non-uniform salinities were

likely to be adequate owing to substantial compartmentation of Na+ in the vacuoles

(Short and Colmer, 1999) and possible substitution of K+ by Na+ as an osmoticum in the

vacuoles to maintain turgor, while still maintaining adequate K+ for the cytoplasm

(Flowers and Läuchli, 1983; Wyn Jones and Gorham, 2002; Subbaroa et al., 2003;

Karrenberg et al., 2006).


121

NaCl(mM)

010

020

030

040

050

060

070

080

00

40

60

80

100

120

NaCl(mM)

010

020

030

040

050

060

070

080

0

Eth

anol

-inso

lubl

e dr

y m

ass

% c

ontr

ol (

10/1

0)

0

40

60

80

100

120Lo

w s

alin

ity0

40

60

80

100

120

Mea

n sa

linity

R2 = 0.6529

R2 = 0.4796

R2 = 0.7141

R2 = 0.7384

R2 = 0.6976

Wei

ghte

d av

erag

e sa

linity

Whole plant ethanol-insoluble dry mass Stomatal conductance

R2 = 0.8550

A

B

C

D

E

F

Figure 6.1. Responses to different methods of expressing salinity in the root-zone of: (A,B,C) whole plant ethanol-insoluble dry mass and (D,E,F) stomatal conductance expressed as % of the uniform 10 mM NaCl treatment in Atriplex nummularia exposed to uniform (Chapter 3 , Chapter 4 , Chapter 5 ) or non-uniform (Chapter 3 , Chapter 4 , Chapter 5) NaCl treatments. Values have been plotted in relation to (A, D) the lowest salinity of the root-zone, (B, E) the mean salinity of the root-zone, or (C, F) the “root-weighted mean” salinity (mean NaCl concentration ‘root-weighted’ for root ethanol-insoluble dry mass in the low and high-salt sides) of the root-zone. Regression curves (quadratic relationships for B and E and cubic relationships for the remaining) were fitted to the combined uniform and non-uniform data sets. The regression curve of best fit occurred when the data were plotted against the ‘root-weighted average’ salinity of the root-zone. R2 values are shown and were significant (P ≤ 0.05). In uniform treatments, Atriplex nummularia was exposed to 10, 120, 230, 450, 500, 670 mM NaCl and 1500 mM NaCl. Averages for the uniform 1500 mM NaCl are not shown as the plants were moribund; for the record, whole plant ethanol-insoluble dry mass and stomatal conductance at 1500 mM were 38% and 11% of those of control plants (exposed to uniform 10 mM NaCl) respectively. If data from Chapter 3, that were likely to be exposed to a higher relative humidity and had different growth responses to increasing NaCl in the root-zone (see Section 6.1.4) are taken out, R2 values for whole plant ethanol-insoluble dry mass are: 0.5420 (when data are plotted against the lowest salinity in the root-zone), 0.4931 (when data are plotted against the mean salinity in the root-zone) and 0.7210 (when data are plotted against the root-weighted salinity in the root-zone). In non-uniform treatments the plants were exposed to: 10, 120, 230 or 450 mM NaCl in one root half and 10 mM in the other root half (Chapter 3), to 10, 120, 230 or 450 mM NaCl in one root half and 670 mM NaCl in the other root half (Chapter 5), and to 500 or 1500 mM NaCl in one root half and 10 mM NaCl in the other root half (Chapter 4).

NaCl (mM)


122

In conclusion under non-uniform salinities there is an increase Na+ and Cl-

concentrations whereas K+ concentrations decline in shoot tissues. Therefore it is likely

that the degree to which ion uptake will affect plant growth will probably depend on: (i)

the rate of Na+ and Cl- uptake from the high-salt side; (ii) uptake from the low-salt side

of water and the most important essential nutrient (i.e. K+ and Ca2+), for which uptake

will be inhibited on the high-salt side (Hajji et al., 2001); (iii) the rates of accumulation

of Na+ and Cl- in the transpiring shoot tissues and (iv) the plant’s ability to tolerate high

Na+ and Cl- concentrations in the shoot tissues.

6.1.3. Water uptake and potential gradients under non-uniform salinity

Shoot water potential and leaf water content were the only two parameters that

responded more closely to the lowest salinity of the root-zone, than to the “root-

weighted mean” salinity of the root-zone. Under non-uniform salinity, with ≥ 500 mM

NaCl in the high-salt side, plants took up water mostly from the low-salt side (76–89%

of total water uptake, all experimental chapters). Thus midday shoot water potential and

leaf water content under non-uniform salinity, that are similar to those under uniform 10

mM NaCl, indicate that these parameters were influenced mainly by the water potential

in that part of the root-zone from where most water was taken up. As a direct

consequence, there was a water potential gradient on the high-salt side in the direction of

water movement from the roots towards the external solution. Such a water loss to the

external solution could have then resulted in the movement of water from the shoot or

from the roots of the low-salt side to the roots in the high-salt side (also called hydraulic

redistribution, Caldwell et al., 1998). However, in all experiments, the opposite

occurred; there was always some water uptake, albeit reduced, from the solution of the

high-salt side (% total water uptake, Chapters 3 and 5: 10-15% with 10–120 mM in the

low-salt side; 22-23% with 230–450 mM NaCl in the low-salt side). It is important to

stress that shoot water potentials were measured with a Scholander pressure bomb,

therefore the value obtained represents only the xylem tensions as xylem osmotic

potentials were not measured. With a simple water uptake model, it was shown that

water uptake from the high-salt side could occur if xylem osmotic potential declined,

most likely through the uptake of ions into the xylem from the solution of the high-salt

side (Chapter 5).


123

Changes in xylem osmotic potential under non-uniform salinities are also likely to affect

other processes driven by water potential gradients such as hydraulic redistribution.

Hydraulic redistribution is the movement of water via a plant’s root system that is driven

by tension gradients within the root system and generally occurs when there is a reduced

transpiration or when stomata are closed (Caldwell et al., 1998). By extension of the

principle that hydraulic redistribution is driven by xylem tension gradients between roots

in different parts of the soil profile, it is possible that this process also occurs in saline

environments, which are often heterogeneous (see Chapter 2). In saline conditions,

however, declines in xylem osmotic potentials and low root hydraulic conductivity

might reduce tension gradients within the root system and thus diminish hydraulic

redistribution.

Uptake of Na+ and Cl- from saline solutions (e.g. Suaeda maritima, Clipson and

Flowers, 1987; Lotus spp. Teakle et al., 2007; Sade et al., 2010) is likely to cause a

decline in xylem osmotic potential in roots exposed to high salinities. Moreover, due to

the reduced xylem sap flow rates, declines in xylem osmotic potentials are expected to

be exacerbated in periods of low transpirational demands, the same period when the

process of hydraulic redistribution is most likely to occur. For example in the halophyte

Suaeda maritima, it was found that with 200 and 400 mM NaCl in the root-zone Na+

concentrations in the xylem at night increased to values 2 to 3 times those measured

during the day, due to the lower rate of xylem sap flow at night compared with the day

(Clipson and Flowers, 1987). Similar increases in xylem sap Na+ concentrations or

osmolality were found with declining xylem sap flow rate in Zea mays, Hordeum

vulgare and Lycopersicon esculentum (Lopez et al., 2003; Munns, 1985; Jackson et al.,

1996). Therefore, if xylem osmotic potentials were to decline in the roots of the high-

salt side, this would reduce or even abolish completely the water potential gradients

between the roots and the external solution (as shown for water uptake during light

periods, Chapter 5).

As described in Chapter 5, water movement between the medium and the xylem can

occur through 2 pathways, the cell-to-cell and apoplastic pathways, and volume flows

are directly proportional to root hydraulic conductivity (Knipfer and Frickle, 2010;

Nobel, 2009). However, as purely apoplastic flow to the xylem is expected to be small

(Flowers et al., 1986; Knipfer and Frickle, 2010), the cell-to-cell pathway will play the

major role in plant radial water movement. The cell-to-cell pathway is affected by the


124

activity of water channels, proteins named aquaporins in the membranes, and changes in

channel activities are thought to mediate increases or declines in root hydraulic

conductivity, viz. permeability to water (Javot and Maurel, 2002). Declines in root

hydraulic conductivity observed at night and with salinity stress were associated with

declines in the activity of water channels (Boursiac et al., 2005; Clarkson et al., 2000;

Henzler et al., 1999; Martinez-Ballesta et al., 2003; Sade et al., 2010), and such declines

in water channels activity would also increase the resistance to the outflow of water

from the roots of the high-salt side at night.

Concurrent declines in root hydraulic conductivity and xylem osmotic potentials will

therefore reduce the tension gradient in the xylem in the root system under non-uniform

salinities. Due to declines in root hydraulic conductivity and xylem osmotic potentials

water loss to the external medium would be either reduce or would completely stop.

Without this water loss to the external solution, root xylem tension will not increase on

the high-salt side and the tension gradient in the xylem, that drives hydraulic

redistribution, will not be created. A data set that would support such view is given in a

study where hydraulic redistribution in the halophyte Sarcobatus vermiculatus was

found to occur on sites with low salinity but was found to decline on a site that had

higher salinities in the soil solution (salinity in the soil solutions were not indicated,

Richards and Donovan, 2005). Similarly there was no evidence that suggested that

hydraulic redistribution occur in A. nummularia (Appendix). In conclusion, it would

appear that the importance and occurrence of hydraulic redistribution in saline

environments could be more limited compared to that in non-saline environments.

Large predawn disequilibrium between plant water potential and nutrient solution water

potentials was found under uniform salinities and non-uniform salinities (Chapter 3).

Under non-uniform salinities the disequilibrium was found when comparing plant water

potential with the water potential of the nutrient solution in the 10 mM NaCl side. This

predawn disequilibrium in Chapter 3, also reported for other halophytes, could have

been caused by: nighttime transpiration; low plant hydraulic conductance and/or

accumulation of solutes in the root apoplast (see Chapter 3; Donovan et al., 1999;

Donovan et al., 2003; James et al., 2006). Solutes can accumulate in root and shoot

tissue, and apoplastic accumulation of solutes in leaf tissue has been found to play an

important role in maintaining cell turgor pressures at predawn in the halophyte

Sarcobatus vermiculatus (James et al., 2006). High concentrations of apoplastic solutes


125

in leaves of S. vermiculatus, calculated to range between -0.75 and -2.12 MPa, have

been identified as one of the main mechanism causing the decline in leaf water potential

(more negative) at predawn, which prevented the equilibration with the higher soil water

potentials around the active roots. Considering that halophytes generally accumulate

high quantities of osmotica (Flowers and Colmer, 2008), lower leaf water potentials

might prevent excessive cell turgor pressures in cells (James et al., 2006). From the data

in Chapter 3, assuming that leaf water potential had equilibrated with the water potential

of the 10 mM NaCl medium, with the measured leaf osmotic potentials, cell turgor

would have ranged between 1.7 and 2 MPa in uniform 10 mM NaCl and non-uniform

treatments. It is however important to recall that the measured leaf osmotic potentials

included bladders and apoplastic solutes; nevertheless, even taking into account that,

these values are well above the low turgor pressures reported for other halophytes (0.05

– 0.3 MPa over a range of salinities; see James et al., 2006 and Clipson et al., 1985).

Under heterogeneous salinities, mostly in a scenario where salinities in the soil solution

vary with time and space, and the plants switch between water sources of different

salinities, the regulation of leaf water potential via the apoplast might play an important

role. The regulation of solute accumulation in the apoplast might be an efficient

mechanism to quickly regulate cell turgor pressure as water content in the apoplast is 5

to 40% of that in the symplast (Kramer, 1983; James et al., 2006; Sattelmacher, 2000).

6.1.4. Growth under uniform salinities

In Chapters 3 and 5, it was noted that there were two different growth responses of A.

nummularia to increasing uniform NaCl concentrations in the root-zone (Fig. 6.2). In

Chapter 5 there was an enhancement in whole plant ethanol-insoluble dry mass with 120

and 230 mM NaCl in the root-zone. In contrast in Chapter 3, maximum whole plant

ethanol-insoluble dry mass occurred with 10 mM NaCl and at higher NaCl

concentrations ethanol-insoluble dry mass declined (Fig. 6.2). Similar differences in

growth with increasing uniform salinities in the root-zone have been previously

observed for Atriplex halimus exposed to high and a low relative humidity (Gale et al.,

1970). With plants exposed to high (69%) relative humidity, highest growth occurred

with a NaCl-free medium and total dry mass declined with increasing salinities, but

when relative humidity was lower (27%), highest total dry mass was found at 120 mM


126

NaCl. In the present thesis the two different growth curves are likely to be caused by the

fact that the first experiment in Chapter 3 was conducted in a different environment

compared to the experiment in Chapter 5. Experiment 1 in Chapter 3 was conducted in a

naturally-lit phytotron with day/night temperature of 20/15°C and an unknown but

presumably high a relative humidity. On the other hand, the experiment in Chapter 5

was conducted in controlled-environment room with day/night temperature of 20/15°C

and an average relative humidity of 55%. However, as relative humidity was not

monitored in the experiment in Chapter 3, it is not possible to conclude that these

different growth response curves were unequivocally related to the different relative

humidities in the two experiments. Nevertheless it is possible that the lower relative

humidity increased the evapotranspirational demands in Chapter 5 which increased ion

delivery to the shoots. This increased ion delivery to the shoots would in turn have

enhanced plant growth at low-moderate salinities. In support of this contention,

compared to Na+ concentrations in leaves in Chapter 3, Na+ concentrations in Chapter 5

were 2.2–2.8 times and 1.7 times higher, for plants exposed to 10 and 230 mM NaCl,

respectively. It could then be argued that the increases in Na+ and Cl- concentrations in

the tissues in Chapter 5 resulted in increased turgor pressure in elongating cells, and this

would in turn have lead to higher elongation rates (Yeo and Flowers, 1980).


127

NaCl(mM)

0 100 200 300 400 500 600 700

Who

le p

lant

etha

nol-i

nsol

uble

dry

mas

s%

con

trol

(10

/10)

20

40

60

80

100

120

140

160

Figure 6.2. Growth response curve (whole plant ethanol-insoluble expressed as % of uniform 10 mM NaCl) for Atriplex nummularia exposed to uniform (Chapter 3 , Chapter 5 ) NaCl treatments. Experiments in these two chapters were conducted in two different environments: in experiment 1 of Chapter 3 plants were grown in a naturally-lit phytotron with day/night temperature of 20/15 °C and an unknown, but presumably high relative humidity. In contrast in Chapter 5 plants were grown in a controlled-environment room with day/night temperature of 20/15 °C and a relative humidity of 55%. The average whole plant dry mass of the uniform 10 mM NaCl treatments in Chapters 3 and 5 were (g): 4.2 (Chapter 3) and 8.0 (Chapter 5).


128

6.2 Implications of the thesis for saltland capability assessment

Saltland capability assessment is a tool that helps in the selection of species for saline

landscapes (Bennett and Barrett-Lennard, 2008). It has been suggested that the

capability of saltland sites for agriculture may be diagnosed on the basis of average ECe

values in the subsoil and on the depth and salinity of the groundwater (Barrett-Lennard

et al., 2008). However, considering that saline sites typically are a mosaic of different

salinities (Bennett et al., 2009), the number of sampling points and where they should be

taken are key aspects for a correct land capability assessment. For example, for a plant

such as A. nummularia that has an extensive root system (Jones and Hodgkinson, 1969)

that is likely to encounter a range of different salinities, measuring salinity at just one (or

few) points and over shallow depths could give quite a misleading estimation of the

mean salinity of the plants’ root-zone. In a natural community of A. nummularia, the

root systems of individual plants were found to extend horizontally for more than 10 m

and for more than 3.5 m below the soil surface (Jones and Hodgkinson, 1969). Therefore

for a correct assessment of the salinity the plant is exposed to, salinity around species

with large root systems should be measured at numerous points in a large radius around

the plant, perhaps with instruments such the EM38, an electromagnetic conductivity

meter that can allow a quick measurement of the bulk soil salinity. Although in this

thesis I have only focused on the the commercial clone of A. nummularia, “Eyres

Green”, and responses of other halophytes should also be investigated, the results of the

thesis further emphasise the need for rigorous land capability protocols for the

assessment of perennial plants for saline lands (cf. Bennett et al., 2009).

This thesis is the first to conduct a comprehensive evaluation of the physiology of a

halophyte under non-uniform salinity using the dicotyledonous halophyte Atriplex

nummularia. Conventional studies of salinity tolerance have considered the responses of

plants to uniform salinity in the root-zone. The naturally inherent variability of soil

salinity, however, means that understanding responses to non-uniform salinities is

essential. In the present thesis, A. nummularia was able to maintain growth with one

root half exposed to 1500 mM NaCl, a level that completely inhibited root and shoot

growth when uniformly applied to the root-zone. The fact that dicotyledonous

halophytes have a high Na+ and Cl- tissue tolerance and have an optimal growth in the

range of 10–400 mM NaCl, would suggest that dicotyledonous halophytes will have a

greater ability to tolerate non-uniform distribution of salts in the root-zone and that


129

investigating their salinity tolerance under uniform conditions may have underestimated

their potential performance in the field.

6.3 Limitations and future studies

Prior to this work, there have been only two studies on halophyte physiology under non-

uniform salinities (Hamed et al., 2008; Messedi et al., 2004), and this thesis has opened

up a number of areas that require further research. The key areas arising from this work

that still require attention are:

1. Assess more in detail the uptake patterns of K+, Na+, and Cl- under non-uniform

salinities. Evidence presented in Chapter 5 showed a greater depletion in K+ from

the solution of the low-salt side and lead to the conclusion that most, if not all of

the K+ (e.g. treatment 10/1500), under high and extreme non-uniform salinities

comes from the low-salt side. One limitation of the results presented in this thesis

was that, as 10 mM NaCl was used in the low-salt side, higher leveles of Na+ and

Cl- in leaves could also have occurred through the uptake of these ions from the

low-salt side. However, increased shoot Na+ and Cl- under non-uniform salinities

in some non-halophyte (Hajji et al., 2001) and halophytes (Messedi et al., 2004;

Hamed et al., 2008) exposed to non-uniform salinities suggest that the bulk of net

Na+ and Cl- uptake occurred from the high-salt side as NaCl-free solution were

used in the low-salt side. Experiments to support (or otherwise) the hypothesis that

K+ ions are mainly taken up from the low-salt side Na+ and Cl- ions from the high-

salt side could be done by examining the uptake of labelled 22Na, 42K, 36Cl and by

measuring K+ depletion rates in the nutrient solution in each side. Sequential

harvests in time should also be taken to measure changes in plant ion contents.

Depletion rates of other nutrients such as NO3- and Ca2+ could also be monitored.

In addition, experiments could be conducted by removing one by one essential

nutrients from the solution of the low-salt side, as done in Messedi et al. (2004).

2. Assess the role of K+ recycling under non-uniform salinities and its impact in

maintaining functional meristematic root tips on the high-salt side. Recovery test

conducted in Chapter 4 showed that, even after 20 days of exposure to 1500 mM

NaCl on the high-salt side of the extreme non-uniform treatment, there was new

root formation 24 to 48 h after stepping down the solution to 10 mM NaCl.


130

Experiments could be conducted by using 42K in the low-salt side and by

monitoring its movement from the roots in the low-salt side to the shoot and then

back to the roots of the high-salt side. Cryo-analytical scanning electron

microscopy could also be used to determine K+ and Na+ concentrations in the

cytoplasm and vacuoles in different parts of the growing root tip (e.g. Läuchli et

al., 2008).

3. Determine whether declines in shoot growth under non-uniform salinities are

caused by increases in Na+ and Cl- in shoot tissues. One limitation of the results

presented in this thesis was that salt bladders were included when measuring leaf

ions; attempts were made to remove bladders but these were not successful.

Measuring ions in leaf tissues without salt bladders would give an indication

whether under non-uniform salinities declines in growth are associated, or not,

with increases in leaf mesophyll Na+ and Cl- concentrations. Moreover,

quantitative cryo-analytical scanning electron microscopy could be used to

determine ion concentrations in specific leaf cells (e.g. Läuchli et al., 2008)

4. Establish whether the roots in the high-salt side do cause declines in stomatal

conductance under non-uniform salinities. In all experimental chapters, non-

uniform salinities resulted in reduced stomatal conductances. However, the critical

experiment to determine whether under non-uniform salinity stomatal conductance

is controlled by non-hydraulic signals deriving from the high-salt side was not

conducted. As done with partial root drying experiments (e.g. Saab and Sharp,

1989; Gowing et al., 1990), it would be possible to have an indication of whether

there are positive signals from the root of the high-salt side that trigger stomatal

closure by excising the roots in the high-salt side and monitoring changes with

time in leaf stomatal conductance or elongation rates.

5. Measure and determine the changes in xylem osmotic potential with uniform and

non-uniform increasing salinities in the root-zone. The model presented in Chapter

5 suggested that changes in xylem osmotic potential can affect both water uptake

and hydraulic redistribution under non-uniform salinities. Attempts were made to

measure xylem osmotic potential in Chapter 5 but osmotic potentials obtained for

the xylem sap were rather large (ca. -1 MPa in all treatments) and therefore need

confirmation. Xylem sap was collected with the pressure bomb, and despite


131

discarding the initial sap collected, the collected fluid could have been

contaminated with cytosolic solutes. Moreover in the literature there is very little

information on xylem osmotic potentials in halophytes. Long-term experiments to

estimate xylem sap concentration could be done by measuring plant water uptake

and having sequential harvests to measure increases in plant ion contents in time;

in this kind of experiment the concentration of ions in the xylem sap can be

calculated as the ratio of the increase in ion content between harvests and the

water used between those harvests (Flowers, 1985). Alternatively for diurnal

changes in xylem sap concentration, experiments could be conducted using

spittlebugs (e.g. Teakle et al., 2007) or root pressurization (e.g. Munns, 1985) to

collect xylem sap. The spittlebug method, however, might be more appropriate as

it would allow having non-uniform treatments whereas with the current root

pressurization method only experiments with uniform salinities in the root-zone

could be conducted. It is not known, however, if spittlebugs will feed on

halophytes. Moreover experiments using 22Na coupled with water uptake

measurements on each side would also enable the estimation of Na+

concentrations in the xylem in each root side.

6. Assess whether apoplastic bypass flow occurs in Atriplex nummularia. It has been

hypothesized that, to regulate ion transport to the shoot, bypass flow must be small

in halophytes (Flowers et al., 1986), but it is important to confirm it

experimentally as bypass flow could greatly influence the xylem osmotic potential

and therefore all processes affected by water potential gradients. Experiments

could be conducted using the apoplastic dye PTS coupled with water use

measurements (cf. Yeo et al., 1987).

7. Experimentally confirm that hydraulic redistribution is more limited in saline

landscapes due to ion uptake from the high-salt side. During my PhD project,

attempts were made to measure hydraulic redistribution in response to a water

potential gradient but these experiments were not successful and have not been

included in the main body of the thesis (results in the Appendix). To my

knowledge there are only 3 reports in the literature where hydraulic redistribution

has been investigated in saline systems (Armas et al., 2010; Hao et al., 2009;

Richards and Donovan, 2005). It should be verified whether hydraulic

redistribution is limited in saline conditions due to ion uptake by using a similar


132

split-root system as the one used in this thesis and measuring water transfer (using

enriched water, D2O, in one side of the split-root system) from one root half to the

other overnight. In these experiments, comparisons could be made between non-

halophytic species, with different abilities to exclude ions at the root surface, and

halophytes that can actively accumulate ions. Moreover, it should also be verified

whether this process can occur in the field with the use of deuterium enriched

water (e.g. Caldwell and Richards, 1989; Smart et al., 2005) and heat pulse

sensors (Burgess et al., 2001; Hao et al., 2009).

6.4 Conclusion

This study adds to the knowledge of halophyte physiology, which has previously

generally been studied over a range of uniform salinities in the root-zone. Atriplex

nummularia was able to maintain growth with one root half exposed to low salinity,

even when the other half was exposed to salinities as high as 1500 mM NaCl, a level

that inhibited growth and damaged tissues when at uniform concentrations in the root-

zone. Like most dicotyledonous halophytes, A. nummularia showed optimal growth in

the range 10–400 mM NaCl; so if the “root-weighted mean” salinity in the root-zone

was within this range, plants expressed optimal growth even with extreme salinity in

one root half. Given the intrinsic heterogeneity of saline landscapes, the preferential root

growth and water uptake in least saline areas combined with the high salinity tolerance

of dicotyledonous halophytes supports the hypothesis that these species have the

potential to withstand large variations in salinities in the root-zone. This ability to

exploit local areas of lower salinity might explain the presence of halophytic vegetation

in heterogeneous saline landscapes that include soil with high to extreme salinity.

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Appendix

Hydraulic redistribution in Atriplex nummularia under non-uniform

salinities

A. Appendix: Hydraulic redistribution in Atriplex nummularia under non-uniform salinities

Appendix

156

A.1 Introduction

When plants are subject to heterogeneous soil water contents in the root-zone, the

transfer of water from zones of high water potential to zones of low water potential has

been observed during periods of low transpiration (Richard and Caldwell, 1987). This

water transfer has been termed “hydraulic redistribution” (Caldwell et al., 1998).

Hydraulic redistribution has been documented for several species, usually with a water

potential gradient within the root-zone, as some root portions were located in moist soil

whereas other were located in drier soils (e.g. Brooks et al., 2006; Burgess et al., 1998,

2000, 2001; Howard et al., 2009; Richards and Caldwell, 1987; Smart et al., 2005;

Yoder and Nowak, 1999). Hydraulic redistribution is driven by water potential gradients

within the root-zone and generally occurs when there is a reduced transpiration or when

stomata are closed (Caldwell et al., 1998; Yoder and Novak, 1999). For example, in C3

and C4 species hydraulic redistribution was detected mainly at night when stomata were

closed whereas in the CAM species Yucca schidigera, that has its stomata open at night,

hydraulic redistribution occurred during the day (Yoder and Novak, 1999). Once shoot

water potential increases, soil water potential of the dry soil regions prevails as the main

source of water potential gradient causing the water to move out from the roots to the

dry soil. As the water moves from the roots to the dry soil, xylem tensions in the “dry

root” increase and a tension gradient is created, causing the water to move from the

roots exposed to wet soil to those exposed to dry soil.

By extension of this general principle that hydraulic redistribution is a function of the

water potential gradient between various points within the plant-soil continuum

(Meinzer et al., 2004; Howard et al., 2009), it would seem reasonable to postulate that

this process may also occur in plants in heterogeneous saline environments. Recently,

reverse xylem sap flow, that has previously been associated with hydraulic

redistribution (i.e. Burgess et al., 1998, 2000, 2001), was detected in dwarf Rhizophora

mangle growing with seasonal and spatial salinity heterogeneity in its root-zone, with

higher salinity in the upper 30 cm of the soil profile declining to – 1.75 MPa (ca. 35 dS

m-1) at the peak of the dry season (Hao et al., 2009). This reverse flow (maximum

reverse sap flow: 2 cm h-1) did not only occur at night, but was also detected on some

occasions during the day. In another study conducted in an arid coastal sand dune

system with a saline groundwater (depth ∼ 3.5 m and ECw 25 dS m-1), there was

evidence that suggested that the deep-rooted Pistacia lentiscus (that accessed the saline

Appendix

157

groundwater) redistributed water to the shallow rooted Juniperus phoenicea, with roots

in a non-saline but dry soil region (Armas et al., 2010). The isotope signature of the

xylem sap of J. phoenicea plants growing with P. lentiscus plants was found to be

similar to that of P. lentiscus, with a signature that was in-between that of the

groundwater and the water 1 m below the dune surface. On the other hand, the isotope

signature of the xylem sap of J. phoenicea plants isolated from P. lentiscus were

different and matched that of the soil 1 m above the water table.

To test whether hydraulic redistribution occurs in Atriplex nummularia under non-

uniform salinities, plants were grown with uniform and non-uniform low to severe

salinities in the root-zone. Using a split-root system as in Chapter 3, plants were exposed

simultaneously to low (10 mM NaCl) and high or severe (335 or 670 mM NaCl) salinity

in the root-zone. In a second experiment to determine whether hydraulic redistribution

occurs in Atriplex nummularia with non-uniform soil water content in the root-zone,

plants were grown in deep soil columns and were watered from the bottom in order to

create a gradient in soil water content, with a wet region at the bottom and a dry soil

region at the top of the soil column.

The following three hypotheses were tested: (i) transfer of water will occur from the

roots of the low-salt side to the roots of the high-salt side during periods of low

transpirational demand, viz. at night; (ii) as hydraulic redistribution is a function of

water potential gradient, then the amount of water moved from the low to the high-salt

side will increase with increasing salinity in the high-salt side; (iii) hydraulic

redistribution will occur in response to differences in soil water content in the root-zone,

and water will move from roots in moist soil (less negative soil water potential) to roots

in drier soils (more negative soil water potential).

A.2 Materials and Methods

Experiment 1 – Split-root experiment in nutrient solution

A commercial clone of Atriplex nummularia, “Eyres Green” (Tamlin’s Nursery, South

Australia), was used. Stem cuttings (10 cm long) with about 5 nodes and leaves on the

Appendix

158

upper two nodes were taken from a mother plant. Cuttings were propagated in a

phytotron with day/night temperatures of 20/15 °C. The stem cuttings were moistened

at the base, dipped in a hormonal rooting powder (‘Richgro Root Strike’, containing 3 g

kg-1 indole-butyric acid) and planted into drained containers filled with washed white

sand. The containers were flushed daily with water until small roots were visible at the

shoot base, and then were irrigated with 0.1-strength nutrient solution for 4 days,

followed by 0.5-strength for 7 days, and thereafter full-strength solution was used. The

full-strength nutrient solution consisted of (mM): 4.7 K2SO4, 9.3 CaCl2, 5.0 Na2SO4, 1.0

MgSO4, 0.7 Ca(NO3)2, 0.3 K2HPO4, 0.2 NH4H2PO4; and (µM): 80 Fe EDDHA

(‘Sequestrene 138’); 23 H3BO3, 2 MnSO4, 2 ZnSO4, 0.5 CuSO4, 0.5 Na2MoO4. The

nutrient solution was adjusted to pH 6, using KOH.

Four weeks later, when roots were about 2 cm long, established cuttings were

transferred to a naturally-sunlit phytotron with day/night temperatures of 20/15 °C.

Cuttings were washed free of sand and transferred to 4.5 L plastic pots containing

aerated full-strength nutrient solution. This solution contained the same nutrient

concentrations reported above, except that 0.1 mM Na2O3Si and 1.0 mM MES were

added; the pH was again adjusted to 6, using KOH. There were 4 plants per 4.5 L pot

and nutrient solutions were topped up with deionized water as required and renewed

weekly. Two weeks after transferring the cuttings to nutrient solution culture, plants

were selected for shoot and root uniformity, and transferred into split-root pots (one

plant per pot, with 1.2 L per side). On the 4th day after transferring the plants to the split-

root pots, 5 treatments were imposed with 3 replications: 10/10D, 10D/335, 10/335D,

10D/670, 10/670D. The first control treatment, with both root halves at 10 mM NaCl had

deuterium enriched nutrient solution (D) added to one randomly chosen side. The other

two non-uniform treatments had deuterium enriched nutrient solution (D) added either to

the 10 mM side or to the 335 and the 670 mM side. Deuterium enriched solution was

prepared by adding 99.8% D2O to the different solutions, with a final average

concentration in the solutions of 4.88 atom% D2O.

Stable isotope application and collection. 12 hours prior to harvest, at 1800 hours,

nutrient solution were changed in all pots and deuterium enriched solutions and non-

enriched solutions were added to all designated pots. All pots were bubbled with water

vapour free air to reduce changes in isotopic signature. The following day, at 0600

hours, nutrient solutions were collected from all pots; 10 mL of the solution was

Appendix

159

collected in air-tight 30 mL vials, filled to the top, and stored at 4°C. The deuterium

signature of the nutrient solution samples was determined by the Universty Western

Australia Stable Isotopes Facility using a High Temperature Conversion Elemental

Analyzer TC/EA (Thermo Finnigan MAT GmbH, Bremen, Germany). For procedures

used to determine deuterium concentrations in water samples refer to Gehre et al.

(2004), and for the normalisation technique see Paul et al. (2007).

Harvest. At the harvest, shoot and root fresh masses were recorded. For all treatments

the two halves of the root system were harvested separately. All shoot and root tissues

were oven dried at 60°C to determine dry mass.

Experiment 2 – Deep pot experiment with soil

Seeds of Atriplex nummularia were surface-sterilized with 0.04% bleach and were

germinated in Petri dishes containing a filter paper moistened with 5 mL of 0.5 mM

CaSO4, in a phytotron with day/night temperatures of 20/15 °C. Five days later,

seedlings were transplanted into washed white sand and flushed daily with 0.1-strength

nutrient solution for 4 days, followed by 0.5-strength for 7 days, and thereafter full-

strength solution was used. The solution used was similar to that used in experiment 1.

Three weeks after transplanting, seedlings were transferred to deep columns (height:

150; diameter: 24 cm) filled with steam sterilized sandy soil (96% sand, 3% clay and 1%

silt; bulk density of 1.3 g/cm3) up to ca. 5 cm from the top of the pots. The base of each

pot contained ~15 cm of crushed granite stones to improve the drainage of water out of

the pots. Plastic beads were used to cover the soil surface to reduce evaporation. Plants

were flushed daily or every 2 days with full-strength nutrient solution for 7 months. One

week prior to treatments and during the experimental period, plants in all treatments

were watered with deionized water. After 5 days plants were harvested (prior

experiment showed that 5 days were sufficient to observe significant drying of the

superficial layer of the soil column). Three treatments were tested with 3 replications:

T1 – plants watered from the top (250 mL deionized water); T2 – plants watered from

the bottom by establishing a watertable using a 30 cm deep container attached to the soil

columns for 2 hours; T3 – plants with roots cut 45 cm from the top and watered from the

bottom by establishing a watertable using a 30 cm deep container attached to the soil

Appendix

160

columns for 2 hours. In T3 roots were severed by sawing through the pots and then

pulling a metallic wire through the soil to excise the roots without removing them from

the soil column.

Diurnal fluctuation in soil water potential. Changes in soil water potentials in the first

7–13 cm of the soil column were monitored from 24 hours before imposing treatments

until the end of the experiment using tensiometers (JetFill tensiometer, ICT International

Pty Ltd, Armadale, Australia). Readings were taken daily at 0600 and 1800 hours and

another 2 to 3 times during each light period.

Shoot water potentials. Predawn and midday shoot water potential were measured on

excised stems on Days 0 and 5 of treatments using a Scholander pressure chamber.

Predawn and midday shoot water potential were taken from 0400 to 0500 hours and

from 1130 to 1230 hours respectively.

Stable isotope application and root collection. Twelve hours prior to harvest, at 1800

hours, plants watered from the top (T1) continued to be watered with deionized water

whereas plants in T2 and T3 were watered from the bottom with deuterium enriched

solutions for 2 hours. The enriched solution was prepared by adding 99.8% D2O to

deionized water, with a final deuterium concentration of 5.78 atom% D2O. The

following day (day of the final harvest), at 0500 hours, beads and tensiometers were

removed and subsamples of the roots with adhering soil in the top 15 cm of the soil

column were sampled. Roots were collected by randomly taking and grouping 3 soil

cores from the top 15 cm of the soil column and then by separating the roots from the

soil. Loosely adhering soil was separated from the roots by lightly shaking the roots.

Roots were then stored in plastic vials sealed with parafilm and immediately stored in

dry ice. Samples were then stored in a cool room at 4°C. Water was extracted from the

samples using azeotropic distillation (Revesz and Woods, 1990; Thorburn et al., 1993b).

The deuterium concentration in the extracted water was determined at the University of

Western Australia’s Stable Isotopes Facility as described above.

Harvest. At the harvest, after sampling superficial roots, the shoot were removed and

oven dried at 60°C to determine dry mass. To collect soil samples, successive holes

were drilled in the side of all columns at 20, 45, 80 and 110 cm from the surface and soil

Appendix

161

cores were collected from those depths. Each soil core was divided into two subsamples;

one was used to determine soil water content and the other to determine the amount of

roots present. Subsamples of the soil cores previously taken from the first 15 cm of the

soil profile (see above) were also used to determine soil water content. Soil water

content was determined by weighing 22–37 g of soil mass before and after oven drying

the samples at 105°C for 24 hours. For the second subsample, roots were washed free of

soil and then oven dried at 60°C to determine root dry mass per unit volume of soil and

later total root dry mass in the entire soil column was estimated.



used to identify significant differences between treatments, depending on the data set.

Unless otherwise stated, the significance level was P ≤ 0.05.

A.3 Results and Discussion

Experiment 1 – Split-root experiment in nutrient solution

In experiment 1, after 21 days of treatments, there were no significant differences in

shoot and root dry mass amongst treatments (Table A1). Similarly no differences were

found for leaf area amongst treatments. Total root dry mass also did no differ and root

dry mass allocation between the low and high-salt sides did not differ in both non-

uniform treatments.

In order to evaluate whether hydraulic redistribution occurred in A. nummularia in

response to an osmotic gradient, deuterium enriched solution (4.88 atom%) was added

to one side of the split-root and deuterium concentration was measured in the other side.

Water movement was calculated by considering the initial signature of the enriched and

non-enriched solution and the solution signature after 12 hours of treatments (Fig. A1).

No differences were found in the calculated water movement from the side exposed to

deuterium enriched water to the other side where roots where exposed to non-enriched

solution amongst treatments.

Appendix

162

Experiment 2 – Deep pot experiment with soil

After 5 days of treatments, there were no significant differences in shoot and root dry

mass amongst treatments (Table A2). Predawn and midday shoot water relations were

measured after 0 and 5 days of treatments in experiment 2 (Table A3). Similarly to shoot

dry mass, no differences for predawn or midday water potentials were found amongst

treatments.

The water potential in the upper 7–13 cm of soil in the pot declined when plants were

watered from the bottom, with sharper declines occurring in plants with severed roots

(Fig. A2). Compared to the initial values, after 5 days or treatments, soil water potentials

in the upper 7–13 cm of the soil profile were 1.9 times lower when roots were cut

whereas when roots were intact, soil water potential was only 1.3 times lower compared

with pots watered from the soil surface. Similarly to soil water potentials, when plants

were watered from the bottom there was a decline in soil water content in the first 15 cm

of the soil column, with larger declines when the root system was severed (Fig. A3).

When the root system was intact soil water content in the first 15 cm of the soil column

was 72% of that in pots watered from the top, whereas when roots were severed soil

water content declined to 56% of that in pots from the top. These differences between

T2 and T3 are likely to be caused by the fact that plants with their root system severed

half way down the column (T3) had only access to the water available in the top 45 cm

of the soil column whereas in T2, where the root system was intact, plants had access to

water from the entire soil column.

To evaluate whether hydraulic redistribution occurred in A. nummularia with different

soil water content in the root-zone, deuterium enriched water (5.78 atom%) was added

to the bottom of the soil column and the deuterium concentration in root tissues and

closely adhering soil was measured (Fig. A4). In T2, when the root system was intact,

only one of three replicates had a relatively high deuterium concentration (0.00021883

atom%); by contrast the other 2 replicates only had enrichment to the same level as T1

and T2 (0.00016017 and 0.00015643 atom%). Therefore, no statistical difference (P≤

0.05) was found in deuterium concentration in root tissues and closely adhering soil

between the three treatments.

Appendix

163

Table A1. Shoot dry mass, leaf area and root dry mass in each side of the split-root pots of Atriplex nummularia exposed for 21 days to uniform and non-uniform salinities in the root-zone (experiment 1).

TreatmentShoot dry mass

(g)Leaf area

(m2)

Root dry mass low salt side

(g)

Root dry mass high salt side

(g)

10/10 6.23 ± 0.70 3.73 ± 0.2510/335 6.98 ± 0.91 3.96 ± 0.60 1.62 ± 0.12 1.70 ± 0.1910/670 7.17 ± 0.66 4.01 ± 0.36 1.65 ± 0.08 1.67 ± 0.18

1.41 ± 0.08*

In uniform treatments plants had both root halves exposed to 10 mM NaCl. In non-uniform treatments one root half was exposed to 10 mM NaCl and the other root half was exposed to 335 or 670 mM NaCl. Values are means (n= 3) ± SE for uniform 10 mM NaCl and means (n= 6) ± SE for non-uniform treatments. * As under uniform 10 mM NaCl no differences were found between the means of each side, data for the two sides were averaged.

Appendix

164

Treatment(mM NaCl/mMNaCl)

10/10D 10D/335 10D/670 10/335D 10/670DCal

cula

ted

wat

er m

ovem

ent f

rom

enr

iche

d si

de to

non

-enr

iche

d si

de

(mL)

0

2

4

6

8

10

12

14 Control Low salt side enriched

High salt side enriched

Figure A1. Calculated water movement from the side where roots were exposed to deuterium enriched solution to the other sides where roots where exposed to non-enriched solution in Atriplex nummularia grown for 21 days in a split-root system (experiment 1). D indicates enriched side. Solutions were collected after exposing roots for 12 hours, during the night, to a deuterium enriched solution (atom% 4.88). Solutions from the non-enriched side were collected and deuterium concentrations measured. The water movement was calculated by considering the initial signature of the enriched and non-enriched solution and the solution signature after 12 hours of treatments. Values are means (n=3) ± S.E. No significant differences were found amongst treatments (P ≤ 0.05). Control treatment had 10 mM NaCl in both root halves whereas non-uniform treatments had one root half was exposed to 10 mM NaCl and the other half to 335 or 670 mM NaCl.

10/10D 10D/335 10D/670 10/335D

10/670D

Appendix

165

From the above results, it is not clear whether hydraulic redistribution does occur in A.

nummularia. Hydraulic redistribution has been previously found to occur in other

halophytes with heterogeneous soil water content in the root-zone (e.g. Sarcobatus

vermiculatus, Snyder et al., 2008; Ambrosia dumosa, Yoder and Nowak, 1999).

Interestingly hydraulic redistribution in Sarcobatus vermiculatus was found to be more

prevalent at sites with low salinities and deep groundwater (9.4 m) but declined in more

saline sites with a shallower groundwater (Richards and Donovan, 2005). Hydraulic

redistribution is a process driven by a tension gradient in the xylem caused by water loss

to the external medium. However there is a possibility that under heterogeneous saline

conditions, due to declines in xylem osmotic potential caused by ion uptake in the more

saline zone, there could be reduced or no water loss to the external solution and this

could reduce the tension gradient needed to drive hydraulic redistribution or abolish it

completely. This issue has been discussed in more detail in Chapter 6. More studies are

required for a comprehensive understanding of hydraulic redistribution in halophytes

and non-halophytes and whether it is affected by ion uptake under non-uniform

salinities.

Appendix

166

Table A2. Predawn and midday water potentials of Atriplex nummularia after 0 and 5 days of treatment (experiment 2).

Predawn (MPa)

Midday (MPa)

Predawn (MPa)

Midday (MPa)

T1 -2.2 ± 0.1 -2.5 ± 0.1 -2.0 ± 0.2 -2.5 ± 0.1T2 -2.0 ± 0.1 -2.4 ± 0.1 -2.1 ± 0.1 -2.3 ± 0.0T3 -2.1 ± 0.1 -2.9 ± 0.2 -2.4 ± 0.2 -2.7 ± 0.2

D0 D5

Treatment

Atriplex nummularia was grown for 7 months in deep columns and exposed to 3 treatments for 5 days before harvesting the plants. Treatments included a control treatment (pots watered from the top, T1) and 2 treatments where pots were watered from the bottom at 120 cm from the surface (T2 and T3). Plants with a watertable had either an intact root-system (T2) or their roots were cut at 45 cm from the surface. Values are means (n=3) ± S.E. No significant differences were found between predawn or midday water potentials between treatments at both times (P ≤ 0.05). Average shoot and root dry mass across treatments were (g): 107 ± 6 and 46 ± 5, respectively.

Appendix

167

Hours from imposing treatments

-36 -24 -12 0 12 24 36 48 60 72 84 96 108 120

-14

-12

-10

-8

-6

-4

Soi

l wat

er p

oten

tial

(kP

a)

-14

-12

-10

-8

-6

-4

A. T1

-14

-12

-10

-8

-6

-4

B. T2

C. T3

Figure A2. Changes in soil water potential in the upper 7–13 cm of the soil columns with Atriplex nummularia over 5 days of treatments: (A) pots watered from the top – T1, (B) pots watered from the bottom up to 120 cm from the soil surface – T2, and (C) pots watered from the bottom up to 120 cm from the soil surface and with roots cut at 45 cm from soil surface – T3 (experiment 2). Water potentials were measured with tensiometers from 24 hours before imposing the treatments and for the entire 5 days of the experiment. Atriplex nummularia was grown for 7 months in deep columns and treatments were imposed for 5 days before harvesting the plants. The shaded boxes under the x-axis indicate the dark periods. Values are means (n=3) ± S.E. Statistical analysis was conducted only for water potential data after 120 hours from imposing treatments, and only T3 was significantly lower than T1 (P ≤ 0.05). No differences were found between T1 and T2, or T2 and T3.

Appendix

168

C. T3

0 2 4 6 8 10

0

20

40

60

80

100

120

B. T20

20

40

60

80

100

120

Soi

l dep

th(c

m)

0

20

40

60

80

100

120

A. T1

Soil water content(% dry wt)

Figure A3. Soil water content at different depths 5 days after imposing the treatments in Atriplex nummularia: (A) pots watered from the top – T1, (B) pots watered from the bottom up to 120 cm from the soil surface – T2, and (C) pots watered from the bottom up to 120 cm from the soil surface and with the root system cut at 45 cm from soil surface – T3 (experiment 2). Atriplex nummularia were grown for 7 months in deep columns and treatments were imposed for 5 days before harvesting the plants. Values are means (n=3) ± S.E. In the first 15 cm of the soil column, soil water content in T2 and T3 was significantly lower than in T1, and soil water content in T3 was significantly lower than in T2 (P ≤ 0.05).

Appendix

169

*

Treatments

T1 T2 T3

Deu

teriu

m c

once

ntra

tion

in r

oots

and

clo

sely

adh

erin

g so

il(a

tom

%)

0.00010

0.00012

0.00014

0.00016

0.00018

0.00020

0.00022

#

Figure A4. Changes in deuterium concentration (atom%) in roots of Atriplex nummularia and the closely adhering soil in the first 15 cm of the soil column (experiment 2). Pots of Atriplex nummularia were watered from the top with deionized water (T1), watered with deuterium enriched water from the bottom up to 120 cm from the soil surface (T2), or watered with deuterium enriched water from the bottom up to 120 cm from the soil surface and with the root system cut at 45 cm from soil surface (T3). Values are means (n=3) ± S.E. Values are means (n=3) ± S.E. No significant differences were found between deuterium concentrations amongst treatments (P ≤ 0.05). In T2 only one sample had a high deuterium concentration (0.00021883 atom%) while the other 2 replications had enrichment values the same as those in T1 and T2 (i.e. 0.00016017 and 0.00015643 atom%).

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