ecophysiology of the tree fern species dicksonia ......ecophysiology of the tree fern species...

Ecophysiology of the tree fern species

Dicksonia antarctica Labill

and Cyathea australis (R. Br.) Domin

Liubov Vladimirovna Volkova

Submitted in total fulfilment of the requirements

of the degree of Doctor of Philosophy

October 2009

Department of Forest and Ecosystem Science

Melbourne School of Land and Environment

The University of Melbourne

Produced on archival quality paper

-i-

Abstract

Predictions of global warming and associated climate change indicate widespread in-

creases in light intensities, temperatures, and the frequency and severity of droughts in

south-eastern Australia. Understanding the ability of plants to respond and acclimate to

these events is essential to predict species survival and potential impacts on biodiver-

sity.

This study focuses on two tree fern species – Dicksonia antarctica and Cyathea aus-

tralis – two iconic understorey species of south-east Australian forests. These tree ferns

belong to different families and are of contrasting origins, yet often grow together in

south-eastern Australia, typically in shade, often along waterways. Their ecological im-

portance is evident in the high epiphytic diversity on their trunks (ferns, mosses, bryo-

phytes, liverwort etc), and the provision of nursery sites for many tree and shrub spe-

cies. Both species are decreased by timber harvesting practices such as clearcut logging,

with deaths continuing for up to five years in the post-harvest environment. Understand-

ing the relative roles of changing light, water, and temperature in these ongoing declines

is essential to conserving both tree fern populations and their dependent biota.

The Thesis encompasses three controlled experiments and a field study. In the con-

trolled experiments, the tree ferns were acclimated to contrasting growth light environ-

ments (shade or moderate light) and then exposed to an environmental stress (i.e. light,

heat, water deficit). The field study examined relationships between environmental

variables (i.e. light, temperature, plant water status) and photosynthetic capacity pa-

rameters of the tree ferns in their natural environment. Stress responses and acclimation

potential of photosynthetic traits, water relation parameters, and frond traits of the tree

ferns were studied using infra-red gas analysis, pigment determination techniques, and

stable isotope methods.

It was hypothesised that, consistent with their contrasting origins and micro-site prefer-

ences, the two tree fern species would possess different physiological characteristics

-ii-

and therefore respond differently to environmental stresses. It was also hypothesised

that plants grown under contrasting light environments would have different reactions to

and recoveries from environmental stresses.

Overall, plants were able to sustain and recover from high light stress, while interactive

effects of high light and heat were most detrimental to tree fern performance. Both spe-

cies were susceptible to water stress, either alone or in combination with high light. The

hypothesised different responses of the two species (associated with their different ori-

gins) were not confirmed, and reaction to and recovery from stress was mainly unaf-

fected by growth light environment. Both species had low acclimation potential to any

of the applied environmental stresses. Overall, findings from this study indicate that

combined effects of high light and heat most likely cause ongoing decline of tree ferns

in post-harvest environments, and that the distribution of tree ferns will most likely con-

tract under future climate scenarios of higher light, increased temperatures, and de-

creased water availability.

-iii-

Declaration

This is to certify that:

the thesis comprises only my original work towards the PhD except where indicated

in the Preface,

due acknowledgement has been made in the text to all other material used,

the thesis is less than 100,000 words in length, exclusive of tables, maps, bibliogra-

phies and appendices

Liubov Volkova

-iv-

Preface

The climate chamber experiment, Chapter 3, was undertaken in Champenoux, France

using facilities of the Institut National Reserche Agronomique (INRA). I planned, con-

ducted the research, evaluated and presented the data. The results of the study were pre-

sented at the International Eco-Fizz conference, 2007 (a poster) and published in the

scientific journal Functional Plant Biology (Volkova L, Tausz M, Bennett LT, Dreyer

E, 2009. „Interactive effects of high irradiance and moderate heat on photosynthesis,

pigments, and tocopherol in the tree-fern Dicksonia antarctica’). M. Tausz and L.T.

Bennett are the supervisors of my PhD work. Erwin Dreyer, the fourth co-author of the

publication was a hosting party in INRA and supervised my activities. Professor Dreyer

is also an honorary staff member of the Department of Forest and Ecosystem Science at

The University of Melbourne.

Chapter 4 (high light and water stress experiment) has been submitted for publication in

co-authorship with my supervisors and Dr. Andrew Merchant (the University of Syd-

ney). I declare that the execution of the experiment, data evaluation and presentation

were solely my own work, and that A. Merchant gave useful tips on the experimental

design and helped to organise the isotopic analysis of my samples.

Chapters 2 and 5 are written in co-authorships with my supervisors M. Tausz and L.T.

Bennett who helped with usual supervisory roles.

-v-

Acknowledgements

Personal financial support for this study was provided by a Melbourne Research Schol-

arship. Expenses related to research activities (i.e. field study and construction of the

controlled experiments) were partly covered by a research agreement with the Victorian

Department of Sustainability and Environment (TA30874).

I am personally grateful to my University supervisors, Ass. Prof. Michael Tausz and Dr

Lauren Bennett, for their patience, ongoing support and encouragement during my

study. I admire Michael for his ability to think globally and to give me confidence that

everything is possible. I admire Ren for her strong personality, always prompt re-

sponses; ability to carefully examine every detail; her great friendship and care when I

needed it. She was (and is) the Woman, who made me deeply respect women in science.

I am indebted to Erwin Dreyer (INRA, France) for his great deal of support and advice

during my candidature; his personal friendship is very precious to me. I would like to

thank Andrew Merchant for his advice and support throughout my study. My thanks to

Chris Western for his patience, always good advice and for being my personal Counsel-

lor at difficult times.

I acknowledge staff and students at Creswick campus for their support. Thanks particu-

larly to Thomas Wright for his friendship and ongoing help, and Raymond Dempsey for

his help in the field. Thanks also to Matt Lee and Najib Ahmady for always providing

reliable and timely results, and my thanks to all others.

I am grateful to my family: husband, Fedor Torgovnikov, for his patience, support and

help during my study. His ability to fix equipment and build constructions for my ex-

periments was priceless. His patience with my often bad moods due to problems with

experiments and understanding my difficulties made me able to finish this study. I thank

my daughter, Katerina Torgovnikova, for her help with watering and re-potting plants

and her patience with “always busy mum”. I am thankful to my parents-in-law, and

most of all, I want to thank my mum, Svetlana Volkova, for teaching me to never give

up and always reach my targets.

-vi-

Table of Contents

Abstract ...................................................................................................................... i

Declaration .............................................................................................................. iii

Preface ..................................................................................................................... iv

Acknowledgements .................................................................................................... v

Table of Contents ..................................................................................................... vi

List of Figures .......................................................................................................... xi

Chapter 1. Introduction .......................................................................................... 1

1.1. Environmental stresses: light, temperature and water deficit ...................... 1

1.2. Fundamental effects of high irradiance in interaction with high

temperature or drought on plants ............................................................................. 1

1.3. Two tree ferns of contrasting origin ............................................................. 3

1.4. The tree ferns in mountain ash forests of south-eastern Australia ............... 5

1.5. Current knowledge of tree fern ecophysiology ............................................. 6

1.6. Thesis aims and outline ................................................................................. 8

Chapter 2. Effects of sudden exposure to high light on two tree fern species

Dicksonia antarctica, and Cyathea australis, acclimated to different light

intensities. ............................................................................................................ 11

(i) Abstract ....................................................................................................... 11

2.1. Introduction ................................................................................................ 11

2.2. Materials and methods ................................................................................ 13

2.2.1. Plant material ......................................................................................... 13

2.2.2. Experimental design ............................................................................... 14

2.2.3. Light environment of measured frond ..................................................... 15

2.2.4. Maximal quantum yield of photochemistry (Fv/Fm) ................................ 16

2.2.5. Gas exchange measurements .................................................................. 16

2.2.6. Plant water status ................................................................................... 17

2.2.7. Frond traits ............................................................................................. 17

2.2.8. Artificial sunfleck experiment ................................................................. 18

2.2.9. Statistical analyses .................................................................................. 19

-vii-

2.3. Results ......................................................................................................... 19

2.3.1. Light environment of the tree ferns ......................................................... 19

Maximum quantum yield of photochemistry (Fv/Fm) .......................................... 20

2.3.2. Photosynthetic capacity parameters ....................................................... 20

2.3.3. Plant water status ................................................................................... 26

2.3.4. Frond traits ............................................................................................. 26

2.3.5. Artificial sunfleck experiment ................................................................. 29

2.4. Discussion ................................................................................................... 31

2.4.1. Species overview ..................................................................................... 31

2.4.2. Acclimation to growth light environment ............................................... 31

2.4.3. High light stress ...................................................................................... 33

2.4.4. Acclimation to new light environment .................................................... 33

2.5. Summary ..................................................................................................... 34

Chapter 3. Interactive effects of high irradiance and moderate heat on

photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. .. 35

(ii) Abstract ....................................................................................................... 35

3.1. Introduction ................................................................................................ 35

3.2. Material and methods ................................................................................. 39

3.2.1. Plant material ......................................................................................... 39

3.2.2. Climate chamber conditions and experimental design ........................... 39

3.2.3. Frond temperature (Tfrond) ...................................................................... 40



3.2.6. Frond nitrogen and chlorophyll content ................................................. 42

3.2.7. Critical temperature (Tc) ........................................................................ 42

3.2.8. Total tissue osmolality ............................................................................ 43

3.2.9. Pigments and tocopherol determination ................................................. 43

3.2.10. Statistical analysis .............................................................................. 44

3.3. Results ......................................................................................................... 45

3.3.1. Frond temperature (Tfrond) ...................................................................... 45

-viii-

3.3.2. Maximum quantum yield of PS II (Fv/Fm) and photosynthetic capacity

parameters .......................................................................................................... 46

3.3.3. Critical temperature (Tc) ........................................................................ 52

3.3.5. Carotenoids and α-tocopherol ................................................................ 53

3.3.6. Correlations between Tc and biochemical parameters ........................... 57

3.4. Discussion ................................................................................................... 58

3.4.1. Effect of high irradiance, high temperature and their interaction on

photosynthetic capacity parameters of D. antarctica ......................................... 58

3.4.2. Membrane stability of D. antarctica measured via critical temperature 60

3.4.3. Xanthophyll cycle carotenoids, pigments and α-tocopherol ................... 61

3.5. Summary ..................................................................................................... 63

Chapter 4. Interactive effects of high light and water deficit on the tree fern

species Dicksonia antarctica and Cyathea australis .............................................. 65

(iii) Abstract ................................................................................................... 65

4.1. Introduction ................................................................................................ 65


4.2.1. Plant material ......................................................................................... 68

4.2.2. Experimental design ............................................................................... 68

4.2.3. Maximum quantum yield of PSII (Fv/Fm) ................................................ 70

4.2.4. Photosynthetic capacity .......................................................................... 70

4.2.5. Frond water relations ............................................................................. 72

4.2.6. Stable isotope analysis ............................................................................ 72

4.2.7. Relative extractable soil water, REW ..................................................... 72

4.2.8. Statistical analysis .................................................................................. 73

4.3. Results ......................................................................................................... 74

4.3.1. Maximum quantum yield of PS II (Fv/Fm) ............................................... 74

4.3.2. Photosynthetic capacity .......................................................................... 74

4.3.3. Frond survival ......................................................................................... 79

4.3.4. Time course of stomatal conductance during 5 days without water ....... 79

4.3.5. Frond water relations ............................................................................. 81

-ix-

4.3.6. Intrinsic water use efficiency (calculated as Amax/gs, WUEi) and stable

carbon isotope composition (δ13

C) ..................................................................... 83

4.4. Discussion ................................................................................................... 85

4.4.1. Pre-treatment period – species differences and effect of light ............... 85

4.4.2. Water deficit and light interactions ........................................................ 86

4.4.3. Rewatering period ................................................................................... 88

4.5. Summary ..................................................................................................... 89

Chapter 5. Seasonal variations in photosynthesis of the tree ferns Dicksonia

antarctica and Cyathea australis in wet sclerophyll forests of Australia .............. 91

(iv) Abstract ................................................................................................... 91

5.1. Introduction ................................................................................................ 91


5.2.1. Study site and sampling design ............................................................... 94

5.2.2. Tree fern measurement schedule ............................................................ 95

5.2.3. Mean irradiance on measured fronds ..................................................... 96



5.2.6. Frond water potential ............................................................................. 97

5.2.7. Frond traits ............................................................................................. 97

5.2.8. Statistical analysis .................................................................................. 98

5.3. Results ......................................................................................................... 99

5.3.1. Relationships between photosynthesis, growth irradiance and

temperature ......................................................................................................... 99

5.3.2. Water status parameters ....................................................................... 103

5.3.3. Diurnal measurements .......................................................................... 103

5.3.4. Stomatal density .................................................................................... 106

5.4. Discussion ................................................................................................. 106

5.4.1. Comparisons between the two tree fern species ................................... 106

5.4.2. Light as a limiting factor to tree fern photosynthetic performance ...... 107

5.4.3. Temperature as a limiting factor to tree fern photosynthetic performance

.............................................................................................................. 109

-x-

5.4.4. Effects of plant water status and water relation parameters on tree fern

photosynthetic performance .............................................................................. 110

5.4.5. Stomatal density .................................................................................... 111

5.5. Summary ................................................................................................... 113

Chapter 6. Ecophysiology of two tree fern species and implications for their

future management. General discussion and conclusions .................................... 115

6.1. Species overview ....................................................................................... 115

6.2. Overview of light, temperature, and water availability as stresses on tree

fern physiology ...................................................................................................... 117

6.3. Practical implications and future directions ............................................ 119

REFERENCES ...................................................................................................... 123

-xi-

List of Figures

Figure 2.1 Photosynthetic capacity parameters of the tree ferns D. antarctica and C.

australis grown under variable light and shade during the before exposure period, and

then exposed to high light, and measured after two weeks (short-term exposure) and

three months (long-term exposure). ...........................................................................

Figure 2.2 Mesophyll capacity parameters of the tree ferns D. antarctica and C.

australis grown under variable light and shade during the before exposure period, and

then exposed to high light, and measured after two weeks (short-term exposure) and tree

months (long-term exposure) ................................................................................. 24

Figure 3.1. Time course of maximum quantum efficiency of PSII and chlorophyll

content of high irradiance and shaded D. antarctica during three successive temperature

treatments ............................................................................................................... 47

Figure 3.2 Stomatal conductance versus light-saturated rate of net photosynthesis for

high irradiance and shaded D. antarctica .............................................................. 51

Figure 3.3 Time course of critical temperature Tc D. antarctica across the experiment

............................................................................................................................... 52

Figure 3.4 α -Tocopherol content of high irradiance and shaded D. antarctica under

three temperature ................................................................................................... 56

Figure 3.5 Critical temperature versus xanthophyll zeaxanthin of high irradiance and

shaded D. antarctica during three temperature treatments .................................... 57

Figure 4.1 Weather conditions during the experiment .......................................... 73

Figure 4.2 Light saturated net photosynthesis and stomatal conductance of water deficit

and control D. antarctica and C. australis under high and moderate light in three

successive experimental periods (pre-treatment, water deficit and rewatering) .... 78

Figure 4.3. Time course of stomatal conductance of water deficit D. antarctica and C.

australis grown under high and moderate light with decreasing relative extractable soil

water and increasing number of days without water ............................................. 80

-xii-

Figure 4.4 Stable isotope composition and intrinsic water use efficiency and of the tree

ferns D. antarctica and C. australis under high and moderate light in three successive

experimental periods .............................................................................................. 84

Figure 5.1 Location of the tree ferns at the study area .......................................... 95

Figure 5.2 Relationships between photosynthetic capacity parameters and frond traits of

the tree ferns D. antarctica and C. australis and environmental variables ......... 101

Figure 5.3 Climate conditions during diurnal course measurements in summer and

winter ................................................................................................................... 104

Figure 5.4 Relationships between photosynthesis, stomatal conductance and water

pressure deficit based on leaf temperature ........................................................... 105

Figure 5.5 Light response curves of the tree ferns D. antarctica and C. australis in

summer and winter ............................................................................................... 106

Figure 5.6 Stomatal density of the tree ferns D. antarctica and C. australis from light-

exposed and shaded habitats. ............................................................................... 112

-xiii-

List of Tables

Table 2.1 Relative irradiance, Isum (i.e. the fraction of penetrating irradiance in the

photosynthetically active spectral region) of the tree ferns growing under variable light

and shade during the before exposure period, and then exposed to high light, and

measured after two weeks (short-term exposure) and three months (long-term exposure)

.................................................................................................................................... 20

Table 2.2 Photosynthetic capacity parameters of the of the tree ferns D. antarctica and

C. australis grown under variable light and shade during the before exposure period,

and then exposed to high light, and measured after two weeks (short-term exposure) and

three months (long-term exposure) ........................................................................ 23

Table 2.3 Predawn frond water potentials and frond traits of the tree ferns D. antarctica

and C. australis grown under variable light and shade during the before exposure

period, and then exposed to high light, and measured after two weeks (short-term

exposure) and three months (long-term exposure) ................................................ 27

Table 2.4 Dynamic responses of photosynthesis to an artificial sunfleck ............ 30

Table 3.1 Temperature of D. antarctica fronds exposed to high irradiance and under

shade ...................................................................................................................... 60

Table 3.2 Photosynthesis and frond traits of D. antarctica exposed to high irradiance

and under shade before and during three successive temperature treatments ....... 49

Table 3.3 Pigment content and osmolality of D. antarctica fronds exposed to high

irradiance and under shade during three successive temperature treatments ........ 54

Table 4.1 Chlorophyll fluorescence and photosynthetic capacity variables of water

deficit and control tree ferns (D. antarctica and C. australis) grown under high and

moderate light during three successive experimental periods ............................... 76

Table 4.2 Frond water relations of water deficit and control tree ferns (D. antarctica

and C. australis) grown under high and moderate light during three successive

experimental periods ............................................................................................ 100

-xiv-

Table 5.1 Significance of the effect of fixed factors (species and season) and of

covariates (ANCOVA) on photosynthetic capacity parameters and frond traits of the

tree ferns D. antarctica and C. australis .............................................................. 100

Table 5.2 Photosynthetic capacity and water relation parameters of the tree ferns D.

antarctica and C. australis in summer and winter............................................... 102

-1-

Chapter 1. Introduction

1.1. Environmental stresses: light, temperature and water deficit

Predictions of global warming and associated climate change indicate widespread in-

creases in light intensities, temperatures, and the frequency and severity of droughts in

south-eastern Australia (Hennessy et al. 2007). Understanding likely responses of plants

to future threats is critical to land management, and some of the main challenges for

conservational biology will be to anticipate environmental change and to adjust man-

agement approaches accordingly (Rossetto 2008). To achieve this aim, it is crucial to

understand the mechanisms of plants to cope with such changes in environmental fac-

tors, and to understand the limitations of their coping capacity.

1.2. Fundamental effects of high irradiance in interaction with high temperature

or drought on plants

Exposure of plants to high levels of irradiance often leads to photoinhibition and photo-

oxidative stress. Photoinhibition is a decline in the quantum yield of photosynthesis.

The primary sites of light damage are associated with components located in the thyla-

koid membranes of chloroplasts (Havaux et al. 1996). Primary damage occurs within

the reaction centre of photosystem II (PSII), with associated photoinhibition effects

such as decreases in photosynthetic yield, bulk pigment loss with photo-oxidation, loss

of enzyme activity (including Rubisco), and, eventually, even cell death (Long et al.

1994). Photo-oxidative stress is caused by the toxic effects of reactive oxygen species

(ROS) produced in the photosynthetic apparatus under high irradiance (Niyogi 2000).

Plants have developed a number of adaptive mechanisms that allow the photochemical

apparatus to cope with rapid changes in light. For instance, when leaves are exposed to

strong light that is saturating for photosynthesis, the xanthophyll zeaxanthin is rapidly

and reversibly formed by violaxanthin de-opoxidation in bright light via the intermedi-

ate antheraxanthin (e.g. Demmig-Adams and Adams 2006).

-2-

Photoinhibition alone is rarely responsible for plant mortality and the plant may recover

and become fully acclimated (Lovelock et al. 1994). However, when, in addition to high

irradiance, leaves are exposed to other environmental stress factors such as high tem-

perature or drought, there can be sustained reductions in the efficiency of photosynthetic

energy conversion and inhibition of repairs to photodamaged PSII (Murata et al. 2007).

Interactive effects of high light and increased temperature are widely discussed in plant

physiology literature (e.g. Havaux et al. 1991, Kirchgeßner et al. 2003, Dieleman and

Meinen 2007). While some authors suggest that these effects are detrimental for plants

because photosynthesis is particularly sensitive to inhibition by heat stress due to labile

components in the photosynthetic apparatus (Salvucci and Crafts-Brandner 2004), oth-

ers insist that high light alleviates negative effects of high temperatures on plants (Ha-

vaux et al. 1991).

Water supply is among the most important factors limiting plant species distribution

(Howard 1973). The primary effects of water stress on photosynthesis have been com-

prehensively discussed (e.g. Flexas et al. 1998), with stomatal conductance among the

earliest responses that protect plants from extreme water loss. Decreases in intercellular

CO2 concentrations (Ci) after stomatal closure during water stress may induce down-

regulation of photosynthetic apparatus to match available carbon substrate and de-

creased growth (Chaves et al. 2003). A number of drought effects are mediated by an

excess of absorbed light energy in the photosynthetic apparatus, leading to an imbalance

between electron transport and electron consumption and causing photoinhibition and

photo-oxidative stress (Flexas et al. 1999). Hence, an interactive effect of high light and

drought can be fatal to plants (Levitt 1980, Lovelock et al. 1994).

Effects of high light, heat and water deficit on plant performance have been extensively

studied over recent decades, yet these studies have been mainly focused on productive,

overstorey tree species (e.g. oak, Eucalyptus species, Acacia species etc). Understorey

species, with low commercial value, have received much less attention, in spite of their

-3-

specific situation, growing under relatively low irradiance, but experiencing occasional

exposure to high irradiance through sunflecks or removal of overstorey (e.g. Pearcy

1988, Durand and Goldstein 2001). Hence, results on overstorey trees cannot easily be

generalised for these species. Growing concern about biodiversity protection in produc-

tive forests and increasing commitment to sustainable forest management is contribut-

ing to rising interest in the understorey component of forests.

1.3. Two tree ferns of contrasting origin

Ferns, or pteridophytes, are the largest and most complex group of flowerless plants that

reproduce by spores developed in sporangia on the underside of leaves or fronds (Large

and Braggins 2004). Some ferns have adopted the tree growth form and are thus called

tree ferns. Most tree ferns belong to the families Dicksoniaceae and Cyatheaceae (Large

and Braggins 2004). Members of the family Cyatheaceae are the most widespread tree

ferns, with many species showing high degrees of local endemism. Centres of diversity

include the Great Antilles, Central America, the Andes, Madagascar, Malesia (i.e. in-

cluding Indonesia, Philippines and New Guinea). The family Dicksoniaceae has high

diversity in Indonesia and New Guinea, with some species found in isolated pockets in-

cluding St Helena Island and the Fernandez Islands off the coast of Chile (Large and

Braggins 2004).

The tree ferns Dicksonia antarctica Labill. and Cyathea australis (R. Br.) Domin are

iconic and ecologically important understorey plants of Australian forests. Observations

by Ashton (2000) indicated that trunks of tree ferns were favourable sites for the estab-

lishment of most woody species in wet sclerophyll forest dominated by Eucalyptus reg-

nans F. Muell. Tree ferns, particularly D. antarctica, formed an impressive understorey

and were associated with numerous species of ground and epiphytic ferns. Studies by

Lindenmayer et al. (1994) found abundance of the mountain brushtail possum Tricho-

surus caninus Ogilby increased with numbers of C. australis and D. antarctica. The

dead fronds of C. australis were favorite sites for Exoneura bicolor bees (Blows and

Schwarz 1991). Crimson Rosella (Platycercus elegans) birds value sori of D. antarctica

-4-

as an energy rich food, and sori account for 20-30% of the birds‟ diet in autumn and

winter (Magrath and Lill 1983). Both tree fern species are also popular horticulture

commodities for domestic and international markets; for example, in 2003/2004 more

than 50,000 trunks of D. antarctica were exported from Tasmania (Davies 2005).

Both D. antarctica and C. australis are widespread in the temperate zones of Australia

(McCarthy 1998). The most significant habitats for tree ferns are rainforest (cool and

warm temperate) and wet sclerophyll forests, particularly in the deepest, least disturbed

sheltered gullies (Department of Natural Resources and Environment 2002). D. antarc-

tica is common in wet forest and often dominates moist, shady gullies, where it fre-

quently grows in extensive stands. C. australis‟ s habitat ranges from dark gullies to dry

forest fringes and creek banks in quite open areas (McCarthy 1998). Observations indi-

cate that the two tree fern species have overlapping but divergent micro-site prefer-

ences. For example, a study in south-east Australian wet sclerophyll forest found that

tree ferns were more likely to be C. australis than D. antarctica with increasing distance

from a stream (Dignan and Bren 2003).

D. antarctica and C. australis belong to contrasting floristic elements of the Australian

vegetation. While D. antarctica is believed to be endemic to Australia and derived di-

rectly from the original Gondwanan flora, C. australis is considered to be an intrusive

species of the Indo-Malayan flora (Barlow 1994). These different origins combined

with indications of different micro-site preferences suggest the two tree ferns would

have different physiological adaptations to environmental stresses.

During their lifetime, tree ferns can be periodically exposed to the harsh conditions of

post-wildfire environments, which are characterised by increased light intensities and

leaf temperatures, and consequently increased evapotranspiration and water loss. Effects

of these conditions on tree fern physiology have not been studied, but are indicated by

poor survival and ongoing decline of both D. antarctica and C. australis after clearcut

logging in mountain ash (E. regnans) forest (Ough and Murphy 2004). It was found that

only 11-17 % of D. antarctica and C. australis survived one year after clearcut logging,

-5-

and of those remaining, up to 40% of D. antarctica and 65% of C. australis were not

expected to survive another five years (Ough and Murphy 2004). In contrast, much

higher rates of regeneration and survival of the tree ferns were recorded after wildfires

(Ought 2001).

1.4. The tree ferns in mountain ash forests of south-eastern Australia

Mountain ash forest of south-eastern Australia is a unique wet sclerophyll ecosystem

that typically forms an interface between two broad vegetation types, rainforest and dry

sclerophyll forest (Campbell and Clarke 2006). These forests are highly prized as water

catchments for the Melbourne region, and for flora and fauna conservation and recrea-

tion purposes (Attiwill and Fewings 2001). E. regnans, itself, is a valuable timber spe-

cies, and about 40% of these forests are available for timber harvesting (Bennett and

Adams 2004).

The dominant harvesting practice in mountain ash forests includes clearcut, slash burn-

ing of debris and remaining vegetation, and seeding with E. regnans seeds (Bennett and

Adams 2004). Such harvesting practices cause major disturbance, including physical

damage to resprouting plants, changes in soil physical and chemical properties, distur-

bance to soil stored plant propagules, and sudden exposure of understorey plants to full

sunlight (Ough and Murphy 1996).

Reasons for steady declines in tree fern numbers in post harvest environments remain

uncertain. Soil disturbance was suggested as a likely major contributor to poor regenera-

tion of tree ferns a decade after clear-felling compared with wildfire regeneration (Ough

2001). Greater survival of tree ferns was recorded in understorey islands (i.e. areas

within a coupe where trees can be felled but disturbance to understorey species and soil

is minimized) than in logged coupes, but mortality also occurred in understorey islands

across all size classes of tree ferns (Ough and Murphy 1998). Apart from soil distur-

bance, there are other obvious differences between post-wildfire and post-harvesting

environments: a fire-killed forest provides much more shade and many more micro-

-6-

habitats than the relatively uniform ash-bed created by high intensity regeneration burns

after logging (Hickey 1994). Logging also results in sharp edges in the boundary zone,

increasing light penetration up to 100% (Dignan and Bren 2003), which can create high

light stress for vegetation remaining in buffer zones, including understorey islands.

High light stress gives rise to two other stress factors – heat and drought. Heat, because

direct irradiance will also increase leaf temperatures, and drought, because greater leaf

temperatures will lead to a greater evaporative demand. As discussed above, interactive

effects of these three stresses can be fatal for a plant (Levitt 1980). Thus, it is possible

that sudden changes in light intensity, water availability and temperature contribute to

D. antarctica and C. australis mortality in the post-harvest environment.

1.5. Current knowledge of tree fern ecophysiology

Little is known about the physiology of tree ferns. It is obvious that tree ferns tolerate a

broad range of environmental conditions throughout their life cycle. Periodically dis-

turbed by wildfires, they have evolved under a regime of variable light levels from high

(immediately after fires) to low or moderate after canopy re-establishment (Hunt et al.

2002).

Certainly, other studies indicate potential for fern acclimation to different light regimes.

For example, New Zealand ferns from contrasting habitats displayed contrasting charac-

teristics in terms of photosynthetic light compensation point, which were tightly corre-

lated with specific frond area (Bannister and Wildish 1982). Frond characteristics (frond

surface area, epidermis thickness, palisade/ spongy mesophyll ratio, blade size, petiole

length) of a South American Cyathea species were also correlated with the irradiance

regime at its local micro-habitat (Arens 1997). During the course of forest ecosystem

dynamics including gap formation, bushfires, or forest harvesting, tree ferns may be

suddenly exposed to full sunlight. Studies in Hawaii indicated limited capacity of shade-

acclimated tree ferns to quickly and efficiently adjust to sudden increase in irradiance

-7-

due to gap formation (Durand and Goldstein 2001). However, further studies of high

light stress on tree ferns and their rate of recovery are currently lacking.

Effects of high temperature, either alone or with high light, on the physiological per-

formance of tree ferns have also been poorly studied. Tingey et al. (1987) found that

photosynthesis of D. antarctica was particularly susceptible to inhibition with increas-

ing temperature and high light; and Nobel et al. (1984) also mentioned negative effects

of high temperature on gas exchange of ferns. Moreover, there are indications that tree

ferns are very susceptible to temperature increases due to their reticulated vascular sys-

tem (White and Weidlich 1995), which might not be as efficient in delivering water to

fronds as the vascular system of angiosperms (Brodribb et al. 2005). However, more

detailed studies, examining acclimation potential of photosynthetic apparatus of tree

ferns to temperature increases and its reversibility are lacking, despite the obvious im-

portance of this knowledge to predicting species‟ acclimation potential and survival in

the future.

Adequate water supply as an important element for tree ferns can be suggested from

their distributional patterns in the forests (mostly along waterways), and is also men-

tioned in the horticultural literature (e.g. Jones and Clemesha 1993, Large and Braggins

2004). Observational studies indicate that tree ferns can sustain periods of drought if

they are protected by canopy. Ashton (2000) observed that despite infrequent but severe

drought events, tree fern numbers increased by 80% in the lower strata of wet sclero-

phyll forests over 48 years. Hunt et al. (2002) also reported that D. antarctica can main-

tain favourable water relations during short periods of drought if its habitat is limited to

sheltered sites. However, these field observations involve potentially confounding ef-

fects of shade, temperature and (soil and air) humidity, because more shaded sites are

also cooler and moister. Thus, it often remains unresolved whether alleviation of

drought stress is a direct effect of lower irradiance – e. g. shading ameliorates drought-

related photoinhibition and photo-oxidative stress – or an indirect effect of greater water

availability and less evaporative demand in shade.

-8-

1.6. Thesis aims and outline

The principal research objectives of this thesis are the characterisation of physiological

responses of D. antarctica and C. australis to varying light conditions, temperature re-

gimes and water availability. There have been no prior studies of the comparative

physiology of D. antarctica and C. australis, and there has been little previous examina-

tion of the interactive effects of light, temperature and water deficit on tree fern physi-

ology.

In my first experimental study (Chapter 2) I examine effects of high light on photosyn-

thetic capacity parameters of D. antarctica and C. australis in a controlled glasshouse

experiment.

In my second experimental Chapter (Chapter 3), I report effects of high light and light

by temperature interactions on photosynthetic performance of D. antarctica in a con-

trolled climate chamber experiment. This experiment was based in France, which meant

that C. australis could not be included because a European source of this species could

not be found.

In my third experimental Chapter (Chapter 4), I examine effects of water deficit either

alone or in interaction with high light on the photosynthetic capacity of D. antarctica

and C. australis in a semi-controlled, open-air experiment.

In my fourth and final experimental Chapter (Chapter 5) I examine the ecophysiology of

both tree fern species under field conditions in the buffer zones surrounding a clearcut

mountain ash forest of central Victoria, Australia. Here, I examine relationships of

growth irradiance, leaf/air temperatures, plant water status with photosynthesis, frond

traits, and water relation parameters of mature tree ferns over two consecutive years.

In my final Chapter 6, I provide an overall discussion of the results and indicate possi-

ble implications of my findings.

-9-

Each experimental chapter was written as a stand-alone paper for journal submission.

Thus, some repetitions of citations and of text from this Introductory Chapter were in-

evitable.

This Chapter is published

Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species

Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light

intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

-11-

Chapter 2. Effects of sudden exposure to high light on two tree fern

species Dicksonia antarctica, and Cyathea australis, acclimated to

different light intensities.

(i) Abstract

We examined the responses of two tree fern species (Dicksonia antarctica and Cyathea

australis) growing under shade or variable light (intermittent shade) to sudden exposure

to high light. Steady-state gas exchange as well as dynamic responses of plants to artifi-

cial sunflecks indicated that difference in growth light environment had very little effect

on the tree ferns‟ capacity to utilise and acclimate to prevailing light conditions. Two

weeks of exposure to high light (short-term acclimation) led to decreases in all photo-

synthetic parameters and more negative predawn frond water potentials, mostly irre-

spective of previous growth light environment. After three months in high light (long-

term acclimation), D. antarctica fully recovered while C. australis previously grown

under variable light recovered only partially, suggesting high light stress effects under

the variable light environment for this species.

2.1. Introduction

The light environment in the understorey of closed forests is often characterized as a

low level of diffuse light punctuated by intense sunflecks resulting from direct-beam

solar radiation through holes in the canopy (Pearcy 1988). However, through forest eco-

system dynamics including gap formation and bushfires, or anthropogenic management

such as forest harvesting, understorey species can suddenly be exposed to prolonged full

sunlight, a stress factor that can contribute to decline in their photosynthetic perform-

ance (Levitt 1980). Rapid physiological adjustment to unfavourable levels of irradiance

(i.e. acclimation, Lambers et al. 2008) is then required for understorey species survival.





-12-

The tree ferns Dicksonia antarctica (Labill.) and Cyathea australis (R.Br.) Domin are

characteristic and ecologically important understorey plants of south-eastern Australia

(Large and Braggins 2004). Even though both species prefer high rainfall wet sclero-

phyll forests (Jones and Clemesha 1993), they have different micro-site preferences: D.

antarctica is common in wet, shady gullies, whereas the often co-occurring species C.

australis seems to preferentially grow within the forest or even along forest margins

(McCarthy 1998). An observational study confirmed that the greater the distance to the

stream the more likely it‟s to encounter C. australis rather than D. antarctica (Dignan

and Bren 2003), suggesting a greater dependence of D. antarctica on water availability

and shade protection. D. antarctica and C. australis belong to contrasting floristic ele-

ments of the Australian vegetation (Gondwanan vs. Intrusive, Tropical; Barlow 1994),

which suggests different physiological adaptation potential and supports their distribu-

tion patterns within forests.

Periodically disturbed by wildfires, D. antarctica and C. australis have evolved under a

regime of variable light levels from high (immediately after fires) to low or moderate

after canopy re-establishment (Hunt et al. 2002). Evidence of plasticity in frond mor-

phology and anatomy in response to different levels of irradiance was found in a study

on South American Cyathea species (Arens 1997). Hunt et al. (2002) also suggested

that during the period of regeneration of woody species following fire, D. antarctica

may experience prolonged periods of exposure to high light intensities and dry atmos-

pheric conditions. Potential of D. antarctica and C. australis to tolerate a broad range of

light conditions is also indicated in horticultural publications (Jones and Clemesha

1993; Large and Braggins 2004).

Despite their apparent longer term acclimation potential to variable light conditions, tree

ferns seem particularly vulnerable after the formation of large gaps. Studies in Hawaii

found that shade-adapted tree ferns were damaged in disturbed areas and forest gaps,

because they are unable to adjust quickly or efficiently to high light environments (Du-

rand and Goldstein 2001). Moreover, Ough and Murphy (2004) found that only about

11 – 17 % of D. antarctica and C. australis survived one year after clearcut logging in





-13-

the mountain ash (Eucalyptus regnans F. Muell) forests of the Victorian Central High-

lands. Of those remaining, up to 40 % of D. antarctica and 65 % of C. australis would

not survive another five years (Ough and Murphy 2004).

The objectives of this study were to elucidate acclimation potential and vulnerability of

D. antarctica and C. australis to sudden increases in light. The two species were grown

under either full or intermittent shade („variable light‟) in a glasshouse and, after pro-

longed acclimation, were suddenly exposed to high irradiance. We measured steady-

state and dynamic (i.e. sunflecks) photosynthetic responses of the tree ferns acclimated

to each light environment in order to test the following hypotheses:

Responses to light would be different between the two species, with D. antarctica

performing better in full shade and being more prone to high light-induced dam-

age;

Sudden exposure to high light would cause limitations in gas exchange (e.g.

photoinhibition) in both species in the short term (two weeks), with those accli-

mated to full shade most strongly affected;

Both tree fern species would have limited capacity to acclimate to high light even

in the longer term (three months).

2.2. Materials and methods

2.2.1. Plant material

Ten sporophytes of D. antarctica and ten of C. australis (Fern Acres nursery, King

Lake West, Australia) were transplanted into 25-l pots. Potting mix contained (% vol-

ume) composted pine bark (30), gravel (45), coarse fern mulch (5), composted mulch

(14.5), fine fern mulch (5), „Dynamic lifter‟ (0.16: Yates, Padstow, NSW, Australia),

and two types of slow-release fertiliser (0.17 each; Osmocote, Baulkham Hills, NSW,

Australia). Before the experiment, tree ferns were propagated from spores and grown in

an open-air nursery under a dense canopy that provided ca 70% shade. All plants were

about six months old and 20-25 cm tall at the start of the experiment.





-14-

2.2.2. Experimental design

The experiment ran from December 2005 to July 2006 in a glasshouse at the site of the

University of Melbourne‟s Creswick campus, in south-eastern Australia (143º53‟E,

37º25‟S; elevation 392 m above sea level). After the first two weeks, old and fully de-

veloped fronds were cut off and only new fronds, which developed under a designated

growth light environment, were measured during the experiment.

Plants were randomly assigned to two growth light environments in a fully randomised

block design with five replicates (i.e. two species within two treatments per each of five

blocks). The two growth light environments – „shade‟ and „variable light‟ – were ap-

plied using wavelength neutral shadecloth. Shade allowed ca 20% uniform light pene-

tration; whereas the variable light simulated sunflecks – the shadecloth was cut into 12

cm-wide stripes, and these were alternated with uncovered gaps of the same width (the

12 cm width was based on the 20 min movement of the sun at its zenith). Under direct

sun in the glasshouse, the maximum recorded photosynthetic photon flux density

(PPFD) was 1900 µmol photons m-2

s-1

at plant height (PAR range, 400-700 nm, meas-

ured with a Li-Cor quantum sensor).

Plants were watered twice per day to maintain soils at field capacity throughout the ex-

periment. Relative humidity and air temperature in the glasshouse were maintained us-

ing a Humidex I greenhouse climate control system (Nelan Industries Pty. Ltd., Mel-

bourne, Australia). Mean conditions throughout the experiment were: 8ºC minimum

temperature, 24ºC maximum temperature, and > 60 % relative humidity.

Plants were measured at the end of three periods:

1) „Before exposure‟ (early April 2006): measurement of new fully-developed

fronds after four months of growth under the designated light environment

(shade or variable light);





-15-

2) „Short-term exposure‟ (late April 2006): measurements of the same cohort of

fronds after two weeks of shade removal (indication of short-term acclimation

potential);

3) „Long-term exposure‟ (late July 2006): measurements of a new fully developed

cohort of fronds after three months of shade removal (indication of long-term

acclimation potential).

In addition, an artificial sunfleck experiment was conducted for two days in late March

2006 (i.e. before exposure and just after full expansion of new fronds). See below for

details.

Chlorophyll a fluorescence, predawn water potential, and gas exchange parameters

were measured at the end of each of the three experimental periods. Samples for nitro-

gen and chlorophyll were collected at the same time. All measurements were made on

the mid-third of the youngest fully expanded fronds.

2.2.3. Light environment of measured frond

The growth light environment for each plant was calculated from hemispherical photo-

graphs. These were taken at the level of each measured frond using a fish-eye lens

(Nikon, F- 601, Japan). Black and white negatives were scanned and evaluated using

Winphot software (ter Steege 1996). Relative irradiance at the measurement location

(Isum) was calculated according to Niinemets et al. (1998):

Isum = pdif Idif + (1- pdif) Idir Eqn. (1)

Where pdif is the ratio of diffuse irradiance to total irradiance in the photosynthetically

active spectral region (400-700nm) above the plant; and Idif and Idir are the factors of

diffuse and direct radiation that will penetrate to the measured location relative to the





-16-

total irradiance above the plant (ter Steege 1996). All parameters were calculated for

each day of the experiment, taking into account amount of sunshine hours in Victoria

during each month of the experiment (data from the Australian Bureau of Meteorology,

http://www.bom.gov.au/jsp/ncc/climate _averages/sunshine-hours/index.jsp, verified 1

September 2009).

2.2.4. Maximal quantum yield of photochemistry (Fv/Fm)

Predawn chlorophyll a fluorescence was measured on overnight dark-adapted leaves

with a pulse modulated fluorometer (OS-30p, Opti-Sciences, Hudson, USA). Ground

fluorescence (Fo) was obtained with a low intensity modulated light (600 Hz, 650nm,

photosynthetic photon flux density PPFD <1 µmol m-2

s-1

). Maximum fluorescence (Fm)

was induced by a saturating flash. Maximum efficiency of PSII was estimated as Fv/Fm

= (Fm - Fo)/Fm, after Maxwell and Johnson (2000).

2.2.5. Gas exchange measurements

Gas exchange parameters were measured using a Li-Cor 6400 gas exchange system,

equipped with a 2x3 cm broadleaf chamber (Li-Cor, Lincoln, Nebraska, USA).

A light response curve was generated for each plant at CO2 concentration of 400 µmol

mol-1

, block temperature 25ºC, air flow rate 400 µmol air s-1

, and relative humidity

>60%. PPFD was increased stepwise from 0 to 2000 µmol m-2

s-1

. Fronds were induced

in the dark for approximately 10 min and the rate of dark respiration was recorded when

stability was reached. PPFD was then increased in 11 successive steps to 2000 µmol m-

2s

-1 with two measurements per PPFD level. Measurements were recorded once rates of

gas exchange were stable. Apparent quantum yield (ф) and maximum photosynthetic

rate Amax were calculated according to Lambers et al. (2008).

An A-Ci curve was generated according to Long and Bernacchi (2003) with some modi-

fications: PPFD 1000 µmol m-2

s-1

, block temperature 25ºC, air flow rate 400 µmol air s-

http://www.bom.gov.au/jsp/ncc/climate%20_averages/sunshine-hours/index.jsp





-17-

1, and relative humidity > 60%. Reference CO2 concentration was increased from 75 to

2200 µmol mol-1

in 13 successive steps with two measurements per CO2 concentration.

Measurements were recorded once gas exchange parameters were stabilised, which on

average took at least 5 min. After finishing the A-Ci curve, illumination in the leaf

chamber was turned off, CO2 concentration was decreased to ambient and respiration

due to oxidative phosphorylation was recorded after 5 min in the dark.

Using the Farquhar model (Farquhar et al. 1980), maximum carboxylation rate (Vcmax),

and maximum electron transport rate (Jmax) were evaluated by fitting A- Ci curves to

non-rectangular hyperbolas (as described in Dreyer et al. 2001 and Montpied et al.

2009). Triose phosphate use (TPU) limitation was not included in the model, and corre-

sponding points with decreased Amax at elevated Ci were disregarded (Long and Bernac-

chi 2003). The set of primary parameters of Rubisco kinetic properties used here

(Kc=327µmol mol-1

, Ko=282600 µmol mol-1

, Γ*=43.7 µmol mol-1

) are from von

Caemmerer et al. (1994).

The frond area enclosed in the chamber for light response and A- Ci curves was marked,

detached, scanned and calculated using imaging software (UTHSCSA Image Tool Ver-

sion 3, University of Texas, USA). All gas exchange measurements were recalculated

on a frond-area basis.

2.2.6. Plant water status

Predawn frond water potential (Ψpredawn) of each plant was measured at the end of each

period using a pressure chamber (PMS Corvallis, OR, USA).

2.2.7. Frond traits

Specific leaf area (SLA), needed for calculation of nitrogen and chlorophylls on a

frond-area basis, was calculated as the ratio of frond area over frond dry weight (m2 kg

-1

dry weight). Fresh frond samples were collected, the frond area scanned and calculated





-18-

using Scion Image software (Scion Corporation 2000-2001, USA), and frond material

then dried at 60ºC for 48 h for dry weight.

Frond samples were analysed for total nitrogen and carbon content using an elemental

analyser (LECO CHN-1000, Michigan, USA). Frond samples were dried as described

above and ground to a fine powder. Photosynthetic nitrogen use efficiency (PNUE) was

calculated as Amax divided by frond nitrogen content (on a frond-area basis).

For measurements of frond chlorophyll content, four frond discs (each diameter 3.75

mm) were collected, immediately immersed in liquid nitrogen, and stored at -80ºC until

extraction. Chlorophyll a and b were extracted using 1.8 ml of 100% dimethyl sulphox-

ide (DMSO). Extracts were heated for 30 min at 65ºC in a dry block heater Termoline

L+M (Northgate, Queensland, Australia). The supernatant was then transferred to a

spectrophotometer Carry 300 (Varian, The Netherlands). A blank of pure DMSO was

used to calibrate the spectrophotometer at zero absorbance. Chlorophyll a, b and total

concentrations were calculated according to Wellburn (1994).

2.2.8. Artificial sunfleck experiment

Predawn Fv/Fm was recorded for each dark-adapted plant. A frond was then enclosed in

the Li-Cor chamber at PAR 20 µmol m-2

s-1

and photosynthesis rate (A20) and gs20 were

recorded once the readings were stable (after at least 5 min). PPFD was then increased

to 2000 µmol m-2

s-1

in one step, and gas exchange parameters recorded every 10 sec-

onds for 20 min. Fv/Fm immediately and 30 min after the sunfleck were recorded using a

pulse modulated fluorometer (as above). Experimental conditions were: chamber rela-

tive humidity 75-80%, reference CO2 concentration 400 µmol m-2

s-1

and block tempera-

ture 25±1ºC.

The following parameters were calculated to characterise the dynamic response of net

photosynthesis to a sudden increase in PAR from 20 to 2000 µmol m-2

s-1

: maximal pho-





-19-

tosynthesis rate (Amax_ind, calculated from the induction curve) and time to reach 63% of

change in photosynthesis (t63%), nomenclature after Tausz et al. (2005).

2.2.9. Statistical analyses

Repeated-measures models of SPSS 15 (SPSS Inc. Chicago, USA) were used for statis-

tical analyses, with light and species as the between-subject factors and period as the

within-subject factor (all fixed). Effects of period (before exposure; short-term expo-

sure; long-term exposure), growth light environment (variable light; shade) and species

(D. antarctica; C. australis), and period by light by species interactions on each de-

pendent variable were tested. Data for statistical analyses were the values per individual

plant at the end of each period. Significant differences between periods were examined

by using the repeated contrast function (SPSS 15).

A two-way general linear model (SPSS 15) with growth light environment and species

as fixed factors was used to analyse the artificial sunfleck data.

Each dependent variable was checked for normality using the Shapiro-Wilk test and log

transformed if assumptions of normality were not satisfied. Data were checked for ho-

mogeneity of variance using Cochrane‟s test, and it was ensured by visual examination

of scatter plots that means and variances were not correlated across experimental

groups.

2.3. Results

2.3.1. Light environment of the tree ferns

Relative irradiance, Isum, did not differ between species within growth light environment

(P=0.4, data not shown), confirming randomised block design for the two species. Yet,

Isum in the variable light was 2.5 times greater than Isum of shade (Table 2.1). Shade re-

moval increased growth light intensity almost two-fold for variable light plants, and





-20-

more than four-fold for shaded plants (Table 2.1). These relatively high levels of irradi-

ance remained until the end of the experiment.

Table 2.1. Relative irradiance, Isum (i.e. the fraction of penetrating irradiance in the pho-

tosynthetically active spectral region) of the tree ferns growing under variable light and

shade during the before exposure period, and then exposed to high light, and measured

after two weeks (short-term exposure) and three months (long-term exposure). Values

are means (n = 5) ± s.e. Affect abbreviations: P, Period; L, Growth light environment.

Differences in Isum between growth light environments for each period were determined

using one-way ANOVAs‟. Significance level:***P<0.001.

Isum Growth light environment Effects

Variable light Shade L S

Before exposure 0.344±0.02 0.134±0.01 *** n.s. (0.4)

Short-term exposure 0.656±0.03 0.575±0.04 n.s. (0.2) n.s. (0.1)

Long-term exposure 0.617±0.04 0.676±0.05 n.s. (0.9) n.s. (0.4)

Maximum quantum yield of photochemistry (Fv/Fm)

Maximum quantum yield of photochemistry (Fv/Fm) was similar between species and

tended to be lower under variable light than shade in the before exposure period (mean

across species of 0.76 versus 0.82; Table 2.2). Short-term (two week) exposure to high

light led to significant decreases in Fv/Fm of both species irrespective of the growth light

environment (to ca 0.70; Table 2.2). After three months of exposure to high light, Fv/Fm

partially recovered in previously shaded plants but remained low in plants previously

grown under variable light (Period x Light, P<0.001, Table 2.2).

2.3.2. Photosynthetic capacity parameters

Light-saturated rate of net photosynthesis (Amax) and stomatal conductance (gs) at Amax

were similar across species and growth light environments in the before exposure period





-21-

(Figs. 2.1 a, 2.1 b). Both parameters decreased after two weeks of exposure to high light

in both species irrespective of the growth light environment, and both parameters recov-

ered after three months of high light exposure, with the exception of Amax in C. aus-

tralis, which remained low (Figs. 2.1 a, 2.1 b).

Respiration rate in the dark (Rd) was not significantly affected by the growth light envi-

ronment, species or sudden exposure to high light (Fig. 2.1 c). However, three months

after exposure, Rd increased significantly in C. australis previously grown under vari-

able light (Fig. 2.1 c).

Apparent maximum quantum yield (ф) was significantly greater in shade than in vari-

able light plants in the before exposure period, then decreased in previously shaded

plants but increased in variable light plants after two weeks of exposure to high light

(Table 2.2). After three months of exposure to high light, ф increased to near or greater

than the before exposure values in all but previously shade-grown C. australis plants

(Table 2.2). Effect of species on ф was insignificant in all periods.

Light compensation point (LCP) was similar across species and growth light environ-

ments in the before exposure period (Table 2.2). Two weeks of exposure to high light

led to significant increases of LCP in both species with greater increased in previously

shaded than in variable light plants. LCP continued to increase after three months of ex-

posure to high light in all but previously shade-grown D. antarctica (Table 2.2).

The maximal carboxylation rate (Vcmax) as well as the maximal light driven electron flux

(Jmax) did not differ between growth light environments and species in the before expo-

sure period (Figs. 2.2 a, 2.2 b). Two weeks of exposure to high light led to significant

decreases in Vcmax and Jmax, irrespective of species and growth light environments. Vcmax

remained low even after three months of exposure in all plants and only marginally re-

covered in D. antarctica previously grown in variable light (P=0.06). In contrast, recov-

ery of Jmax was observed in all plants with exception for C. australis previously grown

in variable light (Fig. 2.2 b).





-22-

Photosynthetic nitrogen use efficiency (PNUE) was not affected by the growth light en-

vironment but was significantly higher in C. australis in the before exposure period

(Table 2.2). Short-term (two week) exposure to high light did not affect PNUE of vari-

able light plants contrasting with a decrease in shade-grown plants. PNUE significantly

increased after three months of exposure in all plants, with greater rises in plants previ-

ously grown in variable light (Light x Period, P=0.02; Table 2.2).


Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyat-

heaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

-23-

Table 2.2. Photosynthetic capacity parameters of the of the tree ferns D. antarctica and C. australis grown under variable light and shade during

the before exposure period, and then exposed to high light, and measured after two weeks (short-term exposure) and three months (long-term

exposure).

Values are means (n = 5) ± s.e. of: Fv/Fm, maximal quantum yield of photochemistry; ф, the apparent maximum quantum yield; LCP, light com-

pensation point; PNUE, photosynthetic nitrogen use efficiency (Amax/nitrogen content). Effect abbreviations: P, Period; L, Growth light envi-

ronment; S, species. Significance levels:*P≤0.05; **P<0.01; ***P<0.001; x, interactions. Only significant effects and interactions are presented.

Parameters Species Growth light environment

Effects Variable light Shade

Before ex-

posure

Short-term

exposure

Long-term

exposure

Before ex-

posure

Short-term

exposure

Long-term ex-

posure

Fv/Fm D. antarctica 0.78±0.02 0.72±0.03 0.69±0.02 0.83±0.01 0.68±0.02 0.71±0.02 P ***

L x P ** C. australis 0.75±0.02 0.68±0.03 0.68±0.01 0.82±0.01 0.67±0.02 0.73±0.01

ф (mol CO2

mol-1

quanta)

D. antarctica 0.068±0.005 0.079±0.009 0.096±0.031 0.085±0.008 0.059±0.008 0.047±0.002 L*; L x P *

C. australis 0.069±0.009 0.112±0.028 0.106±0.008 0.081±0.012 0.065±0.013 0.080±0.012

LCP (µmol m-

2s

-1)

D. antarctica 9±0 12±3 15±4 12±2 26±6 14±3 P***

C. australis 11±2 12±1 17±3 7±2 15±3 19±5

PNUE (µmol

CO2 molN-1

s-

1)

D. antarctica 25±4 30±1 59±11 33±3 25±3 48±4 P ***; S***

L x P *

L x S *

C. australis 32±5 33±3 70±5 66±13 23±4 56±8





-24-

Am

ax (

mol C

O2 m

-2s-1

)

0

2

4

6

8

Shadegs a

t A

max (

mm

ol H

2O

m-2

s-1

)

0

20

40

60

80

100

120

140

Variable light

Before_exposure

Short_term

Long_term

Rd (

mol C

O2 m

-2s-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Period

Before_exposure

Short_term

Long_term

P-value of effects

P ***

P-value of effects

P ***P x L x S *

P-value of effects

P x L x S*

a

b

c

Fig. 2.1. a) Stomatal conductance under saturating irradiance; b) maximum photosyn-

thetic rate and c) mitochondrial respiration in the dark of the tree ferns D. antarctica

( ) and C. australis ( ) grown under variable light and shade during the before

exposure period, and then exposed to high light, and measured after two weeks (short-

term exposure) and three months (long-term exposure). Values are means (n=5) ± s.e. Ef-

fect abbreviations: S, Species; L, Growth light environment; P, Period. Significance lev-

els:*P≤0.05; ***P<0.001; x, interactions. Only significant effects and interactions are

presented.





-25-

Variable light

Before_exposure

Short_term

Long_term

ShadeV

cm

ax (

mo

l C

O2 m

-2s-1

)

0

10

20

30

40

Period

Before_exposure

Short_term

Long_term

Jm

ax (

mo

l C

O2 m

-2s-1

)

0

20

40

60

80

100

P-value of effects

P*

P-value of effects

P**S**P x S *

a

b

Fig. 2.2. a) Maximal carboxylation rate of Rubisco and b) maximal light driven electron

flux of the tree ferns D. antarctica ( ) and C. australis ( ) grown under vari-

able light and shade during the before exposure period, and then exposed to high light,

and measured after two weeks (short-term exposure) and tree months (long-term expo-

sure). Values are means (n=5) ± s.e. Effect abbreviations: S, Species; P, Period. Signifi-

cance levels:*P≤0.05; **P<0.01; x, interactions. Only significant effects and interactions

are presented.





-26-

2.3.3. Plant water status

Predawn frond water potentials (Ψ predawn) were similar across growth light environ-

ments and species in the before exposure period (-0.3 to -0.4 MPa; Table 2.3). Two

weeks of exposure to high light led to small although significant decreases in Ψ predawn in

both species and both growth light environments (-0.6 to -1.1 MPa), with full recovery

after three months in high light (Table 2.3).

2.3.4. Frond traits

Nitrogen (NA) and carbon (CA) content per frond area were significantly greater in

plants grown under variable light than in shade, and in D. antarctica than in C. australis

(Table 2.3). Two weeks of exposure to high light stimulated increases in CA in shade-

grown plants and decreases in NA in variable light plants of both species. After three

months in high light, NA remained relatively unchanged in shade-grown plants, but de-

creased significantly in plants previously grown in variable light. In contras, CA was

comparable with pre-exposure levels in variable light plants while continued to increase

in previously shade-grown plants (Table 2.3).

Total chlorophyll (a + b) and chlorophyll a content per frond area were significantly

greater in shade-grown than in variable light plants, and in D. antarctica than in C. aus-

tralis (Table 2.3). Sudden exposure to high light had no immediate effect on total chlo-

rophyll, which decreased only after three months of high light exposure (Table 2.3).

Chlorophyll a/b ratio was significantly greater in D. antarctica for much of the experi-

ment, whereas growth light environment and short-term (two week) exposure to high

light had no effect on chlorophyll a/b ratio (Table 2.3).


Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis

(Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

-27-

Table 2.3. Predawn frond water potentials and frond traits of the tree ferns D. antarctica and C. australis grown under variable light and

shade during the before exposure period, and then exposed to high light, and measured after two weeks (short-term exposure) and three

months (long-term exposure). Values are means (n = 5) ± s.e. of: Ψ predawn, predawn frond water potential; NA, nitrogen content on a frond

area basis; CA, carbon content on a frond area basis; Chl total, total chlorophyll content on a frond area basis; Chl a, chlorophyll a content

on a frond area basis; Chl a/b, chlorophyll a/b ratio. Effect abbreviations: P, Period; L, Growth light environment; S, Species. Significance

levels:*P≤0.05; **P<0.01; ***P<0.001; n.s., non significant; x, interactions. Only significant interactions are presented.

Parameters Species Variable light Shade Effects

Before

exposure

Short-term

exposure

Long-term

exposure

Before

exposure

Short-term

exposure

Long-term

exposure

Ψ predawn

(MPa)

D. antarctica -0.4±0.1 -1.1±0.2 -0.5±0.1 -0.3±0.1 -0.9±0.2 -0.4±0.0 P ***

C. australis -0.3±0.1 -0.7±0.1 -0.5±0.1 -0.4±0.1 -0.6±0.1 -0.3±0.2

NA (g m-2

) D. antarctica 4.0±0.3 3.2±0.1 2.0±0.3 2.7±0.1 2.6±0.1 2.1±0.2 P ***; L ***; S ***;

L x P ** C. australis 3.4±0.5 1.9±0.2 1.5±0.1 1.8±0.2 1.8±0.1 1.5±0.1

CA (g m-2

) D. antarctica 74±5 71±4 72±9 48±5 58±5 75±5 P *; L ***; S *

C. australis 62±8 64±6 69±1 39±5 45±5 57±6

Chl total

(µmol m-2

)

D. antarctica 592±71 581±80 465±83 605±65 740±86 493±34 P *; L *; S **

C. australis 433±78 352±52 367±66 567±65 554±103 427±60


Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis

(Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

-28-

Parameters Species Variable light Shade Effects

Before

exposure

Short-term

exposure

Long-term

exposure

Before

exposure

Short-term

exposure

Long-term

exposure

Chl a (µmol

m-2

)

D. antarctica 454±51 442±64 337±63 454±46 538±60 394±24 P*; L*; S***

C. australis 321±57 264±40 267±55 422±48 400±72 312±45

Chl a/b D. antarctica 3.3±0.2 3.4±0.2 1.3±0.2 3.1±0.1 2.6±0.1 1.5±0.1 S***; L x P *

C. australis 2.6±0.3 3.0±0.3 2.4±0.2 2.9±0.1 2.7±0.2 2.7±0.3





-29-

2.3.5. Artificial sunfleck experiment

Predawn Fv/Fm was significantly lower in variable light than shade-grown plants (never-

theless near 0.80), with the lowest Fv/Fm recorded for C. australis (0.78). Immediately

after a sunfleck, Fv/Fm decreased to near 0.50 for both species irrespective of the growth

light environment, but partly recovered within 30 min, although recovery of variable

light C. australis was least (0.70; Table 2.4). Photosynthesis rate in low light (A20) as

well as maximal photosynthesis rate calculated from the induction curve Amax_ind did not

differ between species and growth light environments (Table 2.4). Increase in stomatal

conductance (∆gs) in response to a sunfleck was insignificant for both species irrespec-

tive of the growth light environment (Table 2.4). Time to reach 63% of change in pho-

tosynthesis (t63%) did not differ significantly between species and growth light environ-

ments (Table 2.4).





-30-

Table 2.4. Dynamic responses of photosynthesis to an artificial sunfleck. Values are

means (n = 5) ± s.e. of: Fv/Fm _pr, Fv/Fm predawn; Fv/Fm _ immed, Fv/Fm immediately after

the sunfleck; Fv/Fm _30 min, Fv/Fm 30 min after sunfleck; A20, maximal photosynthesis

rate at PAR 20 µmol m-2

s-1

; Amax _ind, maximal photosynthesis rate calculated from the

induction curve; ∆ gs (gsmax–gs20), difference between stomatal conductance at the end of

the experiment (gsmax) and at PAR 20 µmol m-2

s-1

(gs20); t 63%, time to reach 63% of

change in photosynthesis. Effect abbreviations: S, Species; L, Growth light environ-

ment. Significance levels:*P≤0.05; n.s.,-non significant. Only significant interactions

are presented.

Parameter Species Light regime P-value of effect

Variable Shade

Fv/Fm _pr D. antarctica 0.81±0.01 0.81±0.01 L *, L x S *

C. australis 0.78±0.01 0.82±0.00

Fv/Fm _ immed D. antarctica 0.53±0.04 0.49±0.03 n.s.

C. australis 0.53±0.04 0.53±0.03

Fv/Fm _30 min D. antarctica 0.76±0.01 0.71±0.02 L x S *

C. australis 0.70±0.02 0.72±0.01

A20

(µmol m-2

s-1

)

D. antarctica 0.51±0.12 0.15±0.18 n.s.

C. australis 0.42±0.11 0.54±0.13

Amax _ind

(µmol m-2

s-1

)

D. antarctica 6.25±0.29 3.94±0.68 n.s.

C. australis 5.46±0.89 5.86±0.59

∆ gs

(mmol m-2

s-1

)

D. antarctica 26±7 51±13 n.s.

C. australis 35±16 29±26

t 63% (s) D. antarctica 27±4 82±25 n.s.

C. australis 49±18 22±4





-31-

2.4. Discussion

2.4.1. Species overview

D. antarctica and C. australis had comparable maximum photosynthetic rates, Amax and

mesophyll capacity parameters (i.e. Vcmax and Jmax). Photosynthetic rates were within the

range previously reported in the literature for D. antarctica (Amax of 6 – 11 µmol m-2

s-1

(Hunt et al. 2002; Volkova et. al 2009), while Vcmax and Jmax were within the lowest range

of values, measured under similar light intensities and leaf temperatures, among a large

number of species reviewed by Wullschleger (1993).

N content per frond area was significantly greater in D. antarctica than in C. australis.

Values of up to 4 g m-2

in D. antarctica are beyond the range previously recorded for tree

fern species (e.g. Cibotium menziesii (Hook) max 2.1 g m-2

, Durand and Goldstein 2001).

D. antarctica also had significantly greater chlorophyll a/b ratio, due to its greater chloro-

phyll a content, which is a characteristic of sun acclimated plants (Arens 1997). This re-

sult was at odds with this species‟ putative preference for well-shaded microhabitats

(Dignan and Bren 2003). However, potential confounding effects of tree fern age should

be considered given that our study involved young sporophytes rather than field-grown

adults (Herbinger et al. 2007). Although D. antarctica seems to show attributes of a more

light-adapted plant at the leaf scale, differences at the whole-tree scale, such as less hy-

draulic conductance (typical for trees with small rooting volumes), may contribute to re-

stricting this species to wetter and shadier sites (see, for instance, McDowell et al. 2008).

In addition, results of a field study on mature tree ferns indicated more similar values in

chlorophyll a/b ratios between the two species (Volkova et al, in preparation).

2.4.2. Acclimation to growth light environment

We observed plasticity of frond traits in response to the growth light environment in both

species (except for frond shape, which did not differ between the two growth light envi-

ronments). More carbon per frond area, CA, in plants grown under variable light, indicated

greater carbon gain under increased light availability (Oikawa et al. 2006). Shade-grown

plants had more total chlorophyll (a + b) per frond area, indicating an enhanced invest-





-32-

ment of resources to improve light harvesting in low irradiance (Niinemets et al. 1998).

Although shade leaves commonly have lower chlorophyll a/b ratio, the growth light envi-

ronment did not affect chlorophyll a/b ratio in our tree ferns, possibly because plasticity

of this parameter is limited in ferns. Due to a lack of data on acclimation of chlorophyll

a/b ratio in other tree fern species, we compared our tree ferns with shade adapted

Trichomanes ferns, where the chlorophyll a/b ratio did not change after plants were trans-

ferred to high light (Johnson et al. 2000). We believe that such a comparison with non-

arboreal ferns is appropriate as according to Large and Braggins (2004) „tree fern‟ is an

arbitrary term, applied to any ferns with large erect rhizomes.

In terms of photosynthetic capacity parameters both species displayed limited acclimation

potential to the growth light environment (i.e. effect of light was significant only for the

maximum apparent quantum yield, ф), especially given that all measurements were made

on fronds that developed under those environments. Greater ф of shade-acclimated plants

probably allowed plants to photosynthesise more efficiently under low light (Seidlova et

al. 2009).

Acclimation of plants to variable light did not lead to more efficient use of sunflecks –

none of the measured parameters during sunflecks differed significantly from shaded

plants. These results are in agreement with findings for the fern Polypodium virginianum

L. by Gildner and Larson (1992) and confirm our suggestion of low acclimation potential

of the tree fern photosynthetic apparatus to changing light conditions.

Significant decreases in Fv/Fm after the sunfleck treatment indicated engagement of non-

photochemical quenching, which was in agreement with other studies (e.g. Watling et al.

1997). There was no significant stomatal response to the artificial sunfleck, suggesting

that stomata were not controlling dynamic photosynthetic response in the tree ferns, as

was previously found in a co-occurring tree species Nothofagus cunninghamii Oerst.

(Tausz et al. 2005).





-33-

2.4.3. High light stress

Irrespective of previous growth light environment, sudden exposure to high light resulted

in significant decreases in a number of parameters, namely Fv/Fm, Amax, gs, Vcmax, Jmax and

Ψpredawn in both species. Decreases in photosynthetic capacity parameters (i.e. Amax, Vcmax,

Jmax) are consistent with effects of high light stress (Larcher 2003). The apparent quantum

yield of photosynthesis, ф, was the only parameter more strongly affected in shade-

acclimated plants than in those grown under variable light, probably indicating that excess

irradiance reduced the quantum yield of photosynthesis via inactivation or down-

regulation of PSII (Montgomery et al. 2008) – greater decrease in Fv/Fm in shade-grown

plants is in agreement with this statement.

Significant decreases in Ψ predawn were not associated with changes in water availability as

plants were watered to field capacity of the potting mix at all times and may point towards

the effect of increased transpiration due to increase in leaf temperature trigged by sudden

exposure to high light (Levitt 1980). Decreases in Ψ predawn in our plants (to -1.1 MPa)

possibly indicated hydraulic stress. Ferns are known to have dichotomous branching veins

which are not very efficient in delivering water to fronds. Furthermore, vein density is

very low in these species (Brodribb et al. 2007).

2.4.4. Acclimation to new light environment

Non-recovery of Fv/Fm after three months of exposure indicated mild, albeit significant,

photoinhibition in all plants, consistent with findings of Guo et al. (2006), who observed

non-recovery in Fv/Fm of tropical rainforest sub-canopy species after transfer to high light.

However, mild photoinhibition would have very little effect on Amax measured under satu-

rated light (Zhu et al. 2004) and therefore would not contribute markedly to longer-term

persistence of the tree ferns under high light conditions.

Photosynthesis (Amax) of D. antarctica but not C. australis recovered after three months

exposure to high light. This result is consistent with field observations by Ough and Mur-





-34-

phy (2004) of greater decline in C. australis than D. antarctica within five years of clear-

cut logging.

While Vcmax remained low even after three months of exposure to high light in all plants,

full recovery (and even further increase of Jmax in D. antarctica) suggest that electron

transport and RuBP regeneration capacity, rather than Rubisco activity, were limiting fac-

tors to tree fern photosynthesis, as suggested by Wise et al. (2004).

Increases in Rd with prolonged exposure to high light of C. australis pre-acclimated to

variable light suggests greater energy loss by this species (Seidlova et al. 2009) and may

contribute to the lack of recovery of Amax. These results are consistent with our earlier

suggestion and findings by Ough and Murphy (2004) that C. australis appeared to be

more vulnerable to high light stress.

2.5. Summary

Regardless of putative differences in origin and observed differences in micro-site prefer-

ences, both tree fern species had comparable photosynthetic capacity parameters. Steady-

state gas exchange as well as dynamic responses of plants to an artificial sunfleck indi-

cated that difference in the growth light environment had very little effect on the tree

ferns‟ capacity to utilise and acclimate to prevailing light conditions. Sudden exposure to

high light led to decreases in all photosynthetic parameters and more negative predawn

frond water potentials, indicating high light stress responses that were mostly irrespective

of the growth light environment. Despite its field preference for shadier and wetter sites,

D. antarctica showed greater acclimation capacity to sustained high light stress than C.

australis under glasshouse conditions. After three months in high light, D. antarctica fully

recovered while C. australis previously grown under variable light recovered only par-

tially, indicating limited capacity of these plants to acclimate to high light, or perhaps

suggesting some previous light stress under the variable light environment. Results of this

study can be used in planning of forest management practises for better protection of bio-

diversity.


Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate

heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant

Biology In press.

-35-

Chapter 3. Interactive effects of high irradiance and moderate heat on

photosynthesis, pigments, and tocopherol in the tree fern Dicksonia

antarctica.

(ii) Abstract

Effects of high irradiance and moderate heat on photosynthesis of the tree fern Dick-

sonia antarctica were examined in a climate chamber under two contrasting irradiance

regimes (900 and 170 µmol photons m-2

s-1

) and three sequential temperature treatments

(15ºC; 35ºC; back to 15ºC). High irradiance led to decline in predawn quantum yield of

photochemistry, Fv/Fm (0.73), maximal Rubisco activity (Vcmax; from 37 to 29 µmol m-2

s-1

), and electron transport capacity (Jmax; from 115 to 67 µmol m-2

s-1

). Temperature

increase to 35ºC resulted in further decreases in Fv/Fm (0.45) and in chlorophyll bleach-

ing of high irradiance plants, while Vcmax and Jmax were not affected. Critical tempera-

ture for thylakoid stability (Tc) of D. antarctica was comparable with other higher plants

(ca 47ºC), and increases of Tc with air temperature were greater in high irradiance

plants. Increased Tc was not associated with accumulation of osmotica or zeaxanthin

formation. High irradiance increased the xanthophyll cycle pigment pool (V+A+Z, 91

vs. 48 mmol mol-1

chlorophyll-1

), de-epoxidation state (56% vs. 4%), and α-tocopherol.

Temperature increase to 35ºC had no effect on V+A+Z and de-epoxidation state in both

light regimes, while lutein, β-carotene and α-tocopherols increased, potentially contrib-

uting to increased membrane stability under high irradiance.

3.1. Introduction

While understorey species of evergreen forests often experience high intensity sun-

flecks, they are not usually exposed to prolonged periods of high irradiance (Lovelock

et al. 1998; Tausz et al. 2005). A protective canopy usually creates a favourable micro-

climate with more moderate temperature fluctuations and greater air humidity than in

the above-canopy atmosphere. However, during the course of forest ecosystem dynam-




Biology In press.

-36-

ics including gap formation, bushfires, or anthropogenic management such as forest

harvesting, understorey species may be suddenly exposed to full sunlight and high tem-

peratures, stress factors that can contribute to temporary decline of these species. Ac-

cording to climate-change projections, these factors are likely to become even more sig-

nificant in the future, as temperatures are predicted to increase, and disturbances in for-

est canopies may become more frequent (Hennessy et al. 2007).

In the short term, exposure of shade-acclimated plants to high levels of irradiance often

leads to photoinhibition and photo-oxidative stress. Photoinhibition alone is rarely re-

sponsible for plant mortality and the plant may recover and become fully acclimated.

Photo-oxidative stress is caused by the toxic effects of reactive oxygen species (ROS)

produced in the photosynthetic apparatus under high irradiance when carbon assimila-

tion is light-saturated (Niyogi 2000). Many plants can, to a certain extent, acclimate to

increased irradiance through enhanced dissipation of absorbed light energy in the thyla-

koids, a process related to the conversion of the light harvesting xanthophyll violaxan-

thin to the energy quenching zeaxanthin (Demmig-Adams and Adams 2006). Protection

against high irradiance can also involve the accumulation of tocopherol (Munné-Bosch

2005), an antioxidant that scavenges toxic ROS and contributes to thylakoid membrane

stability.

When, in addition to high irradiance, leaves are exposed to other environmental stress

factors such as high temperature, there can be sustained reductions in the efficiency of

photosynthetic energy conversion and inhibition of repairs to photodamaged photosys-

tem II (PSII; Murata et al. 2007). Photosynthesis is particularly sensitive to inhibition

by heat stress due to labile components in the photosynthetic apparatus (Salvucci and

Crafts-Brandner 2004). The thylakoid membrane is one of the main temperature stress

targets and changes during acclimation occur at that level (Ducruet et al. 2007). The de-

gree of thermostability of the thylakoids can be estimated by the critical temperature Tc

– the temperature threshold above which irreversible damage occurs to PSII (Schreiber

and Berry 1977). Tc changes with growing conditions, reflecting thermal acclimation of

the photosynthetic apparatus (Ducruet et al. 2007). Chlorophyll fluorescence yield is a




Biology In press.

-37-

sensitive indicator of the state of thylakoids, and can be used to assess Tc in plants as the

temperature threshold above which ground level fluorescence (F0) increases (e.g. Froux

et al. 2004). The mechanisms underlying acclimatory changes in Tc are still poorly un-

derstood, although some results point towards stabilising effects of protective com-

pounds on thylakoids. For example, the xanthophyll zeaxanthin (Havaux and Gruszecki

1993; Havaux and Tardy 1996), as well as increased soluble sugar concentration (Hüve

et al. 2006), are believed to have a stabilising effect and shift Tc towards higher tem-

peratures.

Our model understorey species, the tree fern Dicksonia antarctica (Labill., Dick-

soniaceae), is known to decline after clearcut logging in Victoria, Australia (Ough and

Murphy 2004). These tree ferns are iconic and ecologically significant understorey spe-

cies in many humid forest types in the Southern Hemisphere, including Australian tem-

perate rain forests and wet sclerophyll (eucalypt) forests (Large and Braggins 2004).

They support a large epiphytic diversity on their trunks and provide nursery sites for

many tree and shrub species as well as nesting and feeding sites for marsupials, insects

and birds (Lindenmayer et al. 1994; Roberts et al. 2005). Decline in D. antarctica num-

bers is expected to negatively impact on many dependent species and thus maintenance

of tree ferns is often an objective of forest management plans (Department of Natural

Resources and Environment 2002).

The reasons for poor survival and ongoing decline of D. antarctica after logging remain

uncertain (Ough and Murphy 2004), but exposure to high irradiance, combined with in-

creased air and frond temperatures, could be contributing factors. Periodically disturbed

by wildfires in their natural habitat, D. antarctica are exposed to a broad range of irradi-

ance during their lifetime (Hunt et al. 2002), which suggests that this species is able to

at least partly acclimate to different levels of irradiance. Certainly, other studies indicate

potential for fern acclimation to different light regimes. For example, New Zealand

ferns from contrasting habitats displayed contrasting characteristics in terms of photo-

synthetic light compensation point, which were tightly correlated with specific frond

area (Bannister and Wildish 1982). Frond characteristics (frond surface area, epidermis




Biology In press.

-38-

thickness, palisade/spongy mesophyll ratio, blade size, petiole length) of a South

American Cyathea species (another important tree fern genus) were also correlated with

its local irradiance (Arens 1997). However, other studies suggest limited capacity of

shade-acclimated tree ferns to efficiently adjust to increased irradiance (Durand and

Goldstein 2001). To our knowledge, only a few studies have examined effects of high

temperature, either alone or with high irradiance on the physiological performance of

tree ferns: Tingey et al. (1987) found that photosynthesis of D. antarctica was particu-

larly susceptible to inhibition with increasing temperature and high light; and Nobel et

al. (1984) also mentioned negative effects of high temperature on gas exchange of ferns.

D. antarctica’ s natural distribution in Australia is limited to the temperate zone

(McCarthy 1998), characterised by cool to warm conditions (Köppen classification Aus-

tralian Bureau of Meteorology, http://www.bom.gov.au/iwk/climate_zones, verified 13

August 2009), perhaps indicating that the species has limited potential for acclimation

to temperature increases (such as after clearcut logging, but potentially also due to cli-

mate change), making it susceptible to ongoing decline.

In this study, we investigated the responses of D. antarctica to high irradiance, moder-

ately high temperature (+35ºC), and a combination of both under fully controlled cli-

mate chamber conditions. Measuring Tc together with a number of variables related to

photosynthesis, chlorophyll fluorescence, and chloroplast pigments, we addressed the

following specific questions:

Are photosynthetic parameters of D. antarctica adversely affected by a) high ir-

radiance; b) high temperatures; and c) their interactions?

Does membrane stability (measured via critical temperature, Tc,) increase in D.

antarctica fronds with increased temperature (indicative of an acclimation to high

temperature), and if yes, are Tc changes associated with accumulation of osmotica

or zeaxanthin formation?

Do other potentially protective thylakoid compounds, such as carotenoids and to-

copherol, change in relation to high irradiance and high temperature?

Are effects of high temperature on the above parameters reversible?




Biology In press.

-39-

3.2. Material and methods


One-year-old sporophytes of Dicksonia antarctica Labill (HSK Gardening and Leisure

Avon Dassett, UK) were transplanted into 10-l pots, containing a mixture of sand and

peat (50/50 v/v) and 40g slow release fertiliser (Nutricote 100, Chisso-Asahi Fertilizer

Co. Ltd, Tokyo, Japan, N/P/K, %, 13/13/13) per pot. The plants were grown under uni-

form sunlit conditions in a naturally illuminated glasshouse at INRA, Champenoux,

France (48º44‟N, 6º14‟E) for two months in spring 2007. At the end of this period, the

plants were transferred to a climate chamber (Chambre Phytotronique STRADER, An-

gers, France) at Champenoux.

3.2.2. Climate chamber conditions and experimental design

Irradiance in the climate chamber was provided by two types of 400 W lamps (HQI

Philips (mercury halide) and SONT Philips (sodium halide) Koninklijke Philips Elec-

tronics N.V., Eindhoven, The Netherlands) and resulted in a photosynthetic photon flux

density (PPFD) of 900 µmol photons m-2

s-1

at plant height (PAR range, 400-700 nm,

measured with a Li-Cor quantum sensor). Relative humidity was 70-80%, air tempera-

ture was controlled to ± 0.5ºC (see temperature treatments below), and photoperiod was

16 h day-1

.

The tree ferns were randomly assigned to two experimental groups (n = 7 in each). One

group was shielded by a wavelength-neutral shade mesh (17% light transmission,

PPFD: 170 µmol photons m-2

s-1

) – „shade‟, the other exposed to full light (900 µmol

photons m-2

s-1

) – „high irradiance‟. Such levels are representative of irradiance condi-

tions in the open on overcast winter and clear summer days respectively, at typical D.

antarctica field sites in mountain ash forests of Victoria, Australia. Plants were kept

well-watered at all times and were rotated daily at random within their designated ir-

radiance regime.




Biology In press.

-40-

A sequence of temperature treatments was applied as follows:

(1) 10 days at 15ºC day and night;

(1a) 3 days at 25°C day and night (to avoid heat shock);

(2) 12 days at 35°C/25°C day/night (typical hot summer days for D. antarctica in

the field);

(3) 10 days at 15°C day and night (to check reversibility of temperature effects).

Chlorophyll a fluorescence and critical frond temperature were measured for each indi-

vidual every 1 to 2 days throughout the experiment. Photosynthesis was recorded from

net CO2 uptake (A) versus intercellular CO2 concentration (A-Ci curves) on the first day

of the experiment and at the end of each temperature treatment. Samples for nitrogen

(N) content, xanthophyll analyses, and osmolality were taken at the end of each of the

three temperature treatments. All measurements were made on the mid-third of the

youngest fully-expanded fronds that were of healthy appearance (i.e. not discoloured).

Total chlorophyll content was measured for each individual every 1 to 2 days through-

out the experiment; measurements were made over the entire plant irrespective of frond

condition.

3.2.3. Frond temperature (Tfrond)

Frond temperature (as 10 random points across the entire plant) was measured twice

during the 35ºC temperature treatment (beginning/end) at predawn and midday, and

once at the end of the experiment (during the second 15ºC temperature treatment), using

an IR laser thermometer (Raynger PM, Raytech Inc, Santa Cruz, CA, USA).


Fv/Fm was derived from chlorophyll a fluorescence measured on dark-acclimated fronds

(at the end of the „night‟ period) with a modulated fluorometer (PAM 2000, Heinz Walz




Biology In press.

-41-

GmbH, Effeltrich, Germany). Maximum quantum yield of PSII was estimated as Fv/Fm

= (Fm–F0)/Fm, after Maxwell and Johnson (2000).


Gas exchange parameters were measured using a Li-Cor 6400 gas exchange system,

equipped with a 2x3 cm broadleaf chamber (Li-Cor, Lincoln, Nebraska, USA). All gas

exchange measurements were conducted at the reference frond temperature of 25ºC. For

each plant, an A-Ci curve was generated at PPFD 1000 µmol m-2

s-1

, frond temperature

25ºC, air flow rate 400 µmol air s-1

, and relative humidity (RH)>60%. Photosynthesis

was induced at a CO2 mole fraction of 50 µmol mol-1

for 15-20 min prior to measure-

ments to ensure maximal stomatal opening and maximal activity of Calvin cycle en-

zymes. CO2 mole fraction was then increased in 13 successive steps to 2200 µmol mol-1

with two measurements at each step. After finishing the A-Ci curve, illumination in the

leaf chamber was turned off, CO2 mole fraction was decreased to 400 µmol mol-1

and

respiration rate was recorded after 5 min in the dark. The frond area enclosed in the Li-

Cor chamber was marked, photographed and calculated using imaging software

(UTHSCSA Image Tool Version 3, University of Texas, USA). Values for Amax and gs

at ambient CO2 (400 µmol mol-1

) were derived from these curves.

Using a biochemical photosynthetic model Farquhar (Farqhuar et al. 1980), apparent

(i.e. assuming that mesophyll conductance to CO2, gi, is infinite) maximum carboxyla-

tion rate (apparent Vcmax), and the maximum apparent rate of electron transport (appar-

ent Jmax) were estimated by fitting the A-Ci curves to the model as described in Mont-

pied et al. (2009). Triose phosphate use (TPU) limitation was not included in the model;

points with decreasing A at high CO2 mole fractions were disregarded. A set of primary

parameters of Rubisco kinetic properties used herein, Kc= 327 µmol mol-1

, Ko = 282600

µmol mol-1

, Γ* = 43.7 µmol mol-1

, were taken from von Caemmerer et al. (1994).

Then, gi was estimated with the curve fitting approach introduced by Ethier and

Livingston (2004) and described by Montpied et al. (2009) and real Vcmax and Jmax were




Biology In press.

-42-

computed based on the A-Cc (chloroplastic CO2 mole fraction); therefore, only corrected

Vcmax and Jmax (i.e., under the hypothesis of finite gi) are given and discussed in this

study.

3.2.6. Frond nitrogen and chlorophyll content

Frond samples were analysed for nitrogen (N) content using an elemental analyser

(NCS 2500, CE instrument Thermo Quest, Milano, Italy). Samples were dried at 60ºC

for 48 h (to determine dry weight) and then ground to a fine powder. Frond area of fresh

samples was scanned and calculated using Scion Image software (Scion Corporation

2000-2001, USA), and these data used to calculate N content on a frond area basis.

Frond chlorophyll content was estimated from transmittance values measured with the

Minolta SPAD-502 Chlorophyll meter (Minolta, Illinois, USA; hereafter „SPAD‟). Val-

ues were the mean of two to three separate pinnules per plant (randomly selected irre-

spective of frond colour).

Total chlorophyll (a + b) was also measured by HPLC (see Pigments and tocopherol

determination).

3.2.7. Critical temperature (Tc)

Critical temperature was estimated in vivo from the sharp rise of basal chlorophyll a

fluorescence under increasing temperature (Schreiber and Berry 1977). Disks of tree

fern pinnules were placed into a temperature-controlled aluminium body, with the fibre-

optics of the fluorometer (PAM 2000, Walz, Effelrich, Germany) pointing at the sam-

ple. Ground fluorescence (F0) was induced with a red diode at low PPFD of about 1

µmol m-2

s-1

. Temperature of the aluminium body was increased gradually (1°C min-1

)

from 20°C to 60°C. F0 was continuously recorded and critical temperature (Tc) was es-

timated graphically at the beginning of the heat-induced fluorescence rise (Froux et al.

2004).




Biology In press.

-43-

3.2.8. Total tissue osmolality

Total tissue osmolality was measured using freeze-point depression from hot water ex-

tracts of dried frond tissue (Callister et al. 2006). Approximately 60 mg of dried ground

frond tissue (as prepared for N analysis) was placed in a 2-ml polypropylene vial to

which 1.6 ml of deionised water was added. The samples were placed in a water-bath at

90ºC for 60 min. The samples were left to cool to room temperature, centrifuged at

10,000 x g for 2 min and 1 ml of the supernatant was transferred to a 1.7-ml polypro-

pylene vial. Osmolality of the solution was measured using an OSMOMAT 030 cryos-

copic osmometer (Gonotec, Berlin, Germany).

3.2.9. Pigments and tocopherol determination

Frond discs (3.75 mm diameter) were collected at midday at the end of each of the three

temperature treatments and immediately frozen in liquid nitrogen. Samples were freeze-

dried, sealed with silicagel in airtight plastic bags, and kept at -20ºC until analysis.

Four discs per plant were ground in a Matrix Mill (Retsch MM301, Germany) at the

temperature of liquid nitrogen. To avoid the presence of traces of acid in the acetone

used for the extraction, 0.5 g l-1

of calcium carbonate were added to samples prior to

grinding (García-Plazaola and Becerril 1999). The resulting powder was extracted with

0.5 ml of ice-cold acetone, homogenised and centrifuged at 4ºC for 1 min at 15000 x g.

The pellets were re-extracted as described above to a combined sample volume of 1 ml.

Extracts were stored in sealed vials at -20ºC until analysis. Prior to injection, samples

were centrifuged at 4ºC for 20 min at 15000 x g and the clean supernatant was trans-

ferred into HPLC vials.

HPLC separation of chloroplast pigments and tocopherols was according to the methods

given in Tausz et al. (2003). Chromatographic conditions were:




Biology In press.

-44-

Pigments: 25 x 4.6 mm Spherisorb ODS 25 µm column. Gradient: solvent A: ace-

tonitrile: methanol: water = 100:5:10 (v/v/v), solvent B: ethylacetate: acetone = 1:2

(v/v), 10% B to 70% B in 17 min, hold at 70% B for 5 min, return to 10% B in 5

min. Flow rate 1 ml min-1

. The injection volume was 20 µl, photometric detection at

440 nm.

α-Tocopherol: 25 x 4.6 mm Spherisorb ODS 25 µm column. Solvent 100% metha-

nol isocratic. Flow rate 1 ml min-1

. Injection volume was 20 µl. Fluorescence detec-

tion excitation 295 nm, emission 325 nm.

Acetone, acetonitrile, methanol and ethyl acetate were of HPLC grade and water was

deionized. A standard of α-tocopherol was purchased from Sigma (Sigma-Aldrich, Cas-

tlehill, NSW, Australia); standards for carotenoids and chlorophyll a and b were pre-

pared as follows: several generic extracts were prepared in 100% acetone and measured

at three wavelengths in the spectrophotometer (Varian UV/V 300, USA). Using the

equations of Lichtenthaler (1987), chlorophyll a, b and total carotenoid concentrations

were calculated at a spectrophotometer resolution range of 1 – 4 nm. The same extracts

were then re-run in the HPLC and conversion factors for chlorophyll a, b and total caro-

tenoids were calculated, disregarding the minor differences in carotenoid absorption co-

efficients at the wavelength in question.

3.2.10. Statistical analysis

Repeated measures models of SPSS 15 (SPSS Inc. Chicago, USA) were used for statis-

tical analysis, with irradiance as the between-subject factor and temperature as the

within-subject factor (both fixed). Effects of irradiance (high irradiance, shade), and 3

levels of temperature (15ºC, 35°C, back to 15ºC), and irradiance by temperature interac-

tions on each dependent variable were analysed. Data for statistical analyses were the

values per individual plant at the end of each temperature treatment. Photosynthetic pa-

rameters, measured before the start of the experiment were not used in the model.




Biology In press.

-45-

3.3. Results

3.3.1. Frond temperature (Tfrond)

Tfrond did not differ among plants at the beginning of the experiment (data not shown).

After the temperature increased to 35ºC, Tfrond was similar in shaded and high irradiance

plants at predawn (below 25°C) but differed on average by 1.5ºC at midday (around

35°C vs. 33.5°C, Table 3.1). By the end of the treatment at 35ºC, Tfrond of shaded plants

was on average 0.7ºC cooler at predawn, and 3.3ºC cooler at midday (Table 3.1).

Table 3.1. Temperature of Dicksonia antarctica fronds (Tfrond) exposed to high irradi-

ance and under shade. Values are means ± s.e. (n = 7 plants, ten measurements per

plant) at predawn and midday on the first (1) and last (12) days of two temperature

treatments (35ºC and back to 15ºC). P values indicate significance of difference be-

tween high irradiance and shaded plants within temperature treatments (Student‟s t-test)

35ºC

P

Back to 15ºC

P High

irradi-

ance

Shaded

Differ-

ence

High

irradi-

ance

Shaded Differ-

ence

Pre-

dawn

Day 1 24.3±0.1 24.2±0.1 0.1 0.30 15.3±0.0 16.0±0.0 -0.7 <0.001

Day 12 23.0±0.2 22.3±0.2 0.7 0.02

Midday

Day 1 34.9±0.6 33.4±0.3 1.5 0.02 19.7±0.4 17.2±0.4 2.5 <0.001

Day 12 34.6±0.2 31.3±0.5 3.3 0.002




Biology In press.

-46-

3.3.2. Maximum quantum yield of PS II (Fv/Fm) and photosynthetic capacity

parameters

Predawn Fv/Fm remained close to the optimum value of 0.83 in shaded plants across all

temperature treatments. In contrast, Fv/Fm declined after the first day of exposure to high

irradiance (Fig. 3.1 a). The 35ºC treatment resulted in further decreases of Fv/Fm (Fig.

3.1 a). After return to 15°C, a partial recovery of Fv/Fm was detected.




Biology In press.

-47-

Fig. 3.1. a) Time course of maximum quantum efficiency of PSII and b) chlorophyll

content of high irradiance (open symbols) and shaded (closed symbols)D. antarctica

during three successive temperature treatments(delineated by dotted lines). Values are

means of n = 7 (± s.e.). P-values indicate significance of effects of irradiance (I), tem-

perature (T), and irradiance by temperature interaction (I x T).




Biology In press.

-48-

Photosynthetic parameters were comparable among all plants at the start of the experi-

ment (Table 3.2, „before‟). Ten days of high irradiance (at 15ºC) resulted in decreases in

both light-saturated rate of net photosynthesis at a reference temperature of 25°C,

Amax25

, and corresponding stomatal conductance gs (Table 3.2; Fig. 3.2). Increasing the

temperature to 35ºC induced stomatal opening in all plants (Table 3.2). However, while

Amax of all shaded plants also increased (Table 3.2. Fig. 3.2), Amax was less responsive in

high irradiance plants (Fig. 3.2). Nonetheless, all changes in gs and Amax were fully re-

versible upon return to 15ºC (Table 3.2).

When measured at the reference temperature of 25°C, maximal carboxylation rate,

Vcmax, and maximal light-driven electron flux Jmax decreased in response to increased

irradiance. Both were insensitive to temperature treatments regardless of irradiance re-

gime (Table 3.2). Mesophyll conductance to CO2, gi, was highly variable, probably ow-

ing to the low accuracy of the fitting procedure used. As a result, no significant effects

of irradiance and temperature could be detected (Table 3.2).

Nitrogen content per frond area (g m-2

) was not affected by either irradiance or tempera-

ture (Table 3.2). Photosynthetic nitrogen use efficiency (PNUE), calculated as Vcmax/N,

was lower under high irradiance than in shade and remained such until the end of the

experiment. Changes in temperature did not affect PNUE (Table 3.2).

Total chlorophyll content (in SPAD units) decreased at 35ºC under high irradiance and

remained low thereafter (Fig. 3.1 b). A similar albeit not significant effect was detected

for chlorophyll a + b in HPLC extracts (Table 3.2). It should be noted that visually

damaged fronds were avoided for HPLC analyses, while SPAD measurements were

made across entire fronds. Chlorophyll a/b ratios were similar between irradiance re-

gimes during the 15ºC temperature treatment. With temperature increase to 35ºC, chlo-

rophyll a/b ratios decreased in high irradiance plants in contrast to a significant increase

in shaded plants (Table 3.2). With temperature return to 15ºC, chlorophyll a/b ratios of

high irradiance plants recovered to the initial values.


Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern

Dicksonia antarctica. Functional Plant Biology In press.

-49-

Table 3.2. Photosynthesis and frond traits of D. antarctica exposed to high irradiance and under shade before and during three successive

temperature treatments. Values are means ± s.e. (n = 7) of Amax25

, light-saturated net CO2 assimilation rate at 25°C; gs, stomatal conduc-

tance under saturating irradiance at 25°C; NA, nitrogen content on a frond area basis; PNUE, photosynthetic nitrogen use efficiency

(Vcmax25

/NA) at 25°C; Chl total, total chlorophyll content in SPAD units and on a frond area basis; Chl a/b, chlorophyll a/b ratio; Vcmax25

,

maximal carboxylation rate of Rubisco at 25°C; Jmax25

, maximal light driven electron flux at 25°C; gi, mesophyll conductance to CO2

measured at 25°C; Effect abbreviations: I, Irradiance; T, temperature; I x T, irradiance by temperature interaction; Significance lev-

els:*P≤0.05; **P<0.01; ***P<0.001; n.s., non significant; n.d., no data

Parameter

Irradiance regime

Temperature treatments Significance of

effects (P)

Before 15ºC 35ºC Back to 15ºCº

Amax25

(µmol CO2 m-2

s-1

) High irradiance 6.0±0.5 3.7±0.5 5.1±0.6 3.9±0.4

I***; T* Shade 6.4±0.4 6.4±0.8 7.6±0.3 5.6±0.5

gs at Amax25

(mmol H2O m-2

s-1

)

High irradiance 82±11 57±6 108±7 50±6 I*; T***

Shade 80±7 80±14 152±12 70±5

Vcmax25

(µmol CO2 m-2

s-1

) High irradiance 37.0±2.0 29.3±2.7 23.2±2.8 29.1±3.3 I***

Shade 36.0±1.0 37.1±2.9 40.6±3.8 34.5±4.2

Jmax 25

(µmol CO2 m-2

s-1

) High irradiance 115±6 68±9 50±7 85±11 I**

Shade 105±6 85±10 86±12 98±10




-50-

Parameter

Irradiance regime

Temperature treatments Significance of

effects (P)

Before 15ºC 35ºC Back to 15ºCº

gi (mmol CO2 m-2

s-1

)

High irradiance 155±64 141±53 143±115 115±25 n.s.

Shade 115±35 222±63 294±195 259±112

NA (g m-2

)

High irradiance n.d. 16.9±1.0 15.9±0.6 17.4±0.7

n.s. Shade n.d. 16.5±0.7 16.0±0.8 15.5±2.1

PNUE (Vcmax25

/N),(µmol

mol-1

N-1

m-2

)

High irradiance n.d. 204±21 201±24 175±13 I**

Shade n.d. 278±18 311±30 300±41

Chl total (SPAD units) High irradiance n.d. 49.1±1.7 39.3±2.3 34.1±2.1

I**; T***;

I x T*** Shade n.d. 48.9±0.4 47.1±0.5 47.3±1.0

Chl total (a + b)

(µmol m-2

)

High irradiance n.d. 782±142 721±111 675±90 n.s.

Shade n.d. 810±63 914±127 843±81

Chl a/b

High irradiance n.d. 2.36±0.05 2.29±0.05 2.41±0.07 I**; T**;

I x T*** Shade n.d. 2.46±0.05 2.71±0.05 2.66±0.07




-51-

Fig. 3.2. Stomatal conductance, gs versus light-saturated rate of net photosynthesis Amax for high irradiance (on the right) and shaded (on

the left) D. antarctica measured at the standardised temperature of 25ºC during three temperature treatments: 15ºC (open circle), 35ºC

(closed diamond) and back to 15ºC (open triangle). Values are means of n = 6 (high irradiance) and n = 7 (shaded).




Biology In press.

-52-

3.3.3. Critical temperature (Tc)

During the 15°C treatment, critical temperature for photochemistry (Tc) was similar un-

der shade and high irradiance (means of 47.5ºC and 47.2ºC respectively; Fig. 3.3). In-

crease in temperature resulted in significant rises of Tc that were greatest under high ir-

radiance. Tc started to decrease after return to 15°C, although pre-treatment values were

not reached by the end of the experiment (Fig. 3.3).

Fig. 3.3. Time course of critical temperature, Tc of high irradiance (open symbols) and

shaded (closed symbols) D. antarctica across the experiment. Values are means of n =

7 (± s.e.). P-values indicate significance of effects of irradiance (I), temperature (T), and

irradiance by temperature interaction (I x T).




Biology In press.

-53-

3.3.4. Total tissue osmolality

Total tissue osmolality was not affected by irradiance but decreased significantly during

the 35ºC treatment (Table 3.3). It increased to close to the original values after return to

15°C.

3.3.5. Carotenoids and α-tocopherol

Neoxanthin and lutein contents (per mol total chlorophyll) were significantly greater

under high irradiance, whereas α- and β-carotene contents remained comparable to

shaded plants (Table 3.3). Temperature increase to 35ºC led to significant increase in β-

carotene and lutein in high irradiance plants. These pigments tended to remain high on

return to 15ºC, although α-carotene significantly decreased under high irradiance (Table

3.3).

The xanthophyll cycle pigment pool (i.e., Violaxanthin, Antheraxanthin, and Zeaxan-

thin, V+A+Z) was significantly greater under high irradiance for the whole experiment.

Changes in temperature did not affect V+A+Z under either irradiance regime (Table

3.3). The de-epoxidation state of xanthophylls (expressed as (0.5A+Z)/(V+A+Z)) was

greater under high irradiance than shade (on average 44% vs. 3%; Table 3.3). It de-

creased by ca 32% under high irradiance upon return to 15°C, but was insensitive to

temperature under shade. Small amounts of lutein-epoxide were detected (Table 3.3),

but they were not affected by light, temperature and their interactions.

Content of α-tocopherol was significantly greater under high irradiance than shade (Fig.

3.4). Whereas α-tocopherol was invariant to temperature changes in shaded plants, α-

tocopherol increased in high irradiance plants with temperature increase to 35ºC, and

remained high after the temperature returned to 15ºC; due to the lack of a high light-low

temperature control, we cannot clearly separate the high irradiance and high temperature

effects on these changes.




-54-

Table 3.3. Pigment content and osmolality of D. antarctica fronds exposed to high irradiance and under shade during three successive

temperature treatments. Values are means ± s.e. (n = 7) of: osmolality; carotenoids (on a chlorophyll basis): lutein, neoxanthin and xantho-

phyll pool (i.e. Violanxanthin, Antheraxanthin, Zeaxanthin, V+A+Z), α- and β-carotene, lutein-epoxide and the de-epoxidation state of

violaxanthin = (0.5A+Z)/(V+A+Z). Effect abbreviations: I, Irradiance; T, temperature; I x T, irradiance by temperature interaction; Signifi-

cance levels:*P≤0.05; **P<0.01; ***P<0.001; n.s. –not significant

Irradiance regime

Temperature treatments Significance of effects

(P) 15ºC 35ºC Back to 15ºC

Osmolality (mosmol g d.w.-1

) High irradiance 1.56±0.06 1.33±0.04 1.45±0.05

T*** Shade 1.55±0.02 1.40±0.03 1.51±0.01

V+A+Z (mmol mol-1

chl-1

) High irradiance 91.9±9.2 118±15.0 113.7±15.9

I*** Shade 47.8±1.7 45.8±1.2 45.5±1.2

De-epoxidation (%) High irradiance 56.6±3.4 53.1±7.7 21.4±4.7

I***; T***; I x T*** Shade 4.4±1.1 2.3±0.8 1.6±0.6

Lutein (mmol mol-1

chl-1

) High irradiance 263±9 339±25 388±29

I***; T**; I x T** Shade 185±4 186±3 198±6




-55-

Irradiance regime

Temperature treatments Significance of effects

(P) 15ºC 35ºC Back to 15ºC

Neoxanthin (mmol mol-1

chl-1

) High irradiance 61.3±2.6 65.9±3.8 69.3±3.6

I*** Shade 45.2±1.9 46.2±1.1 48.4±2.0

α-carotene (mmol mol-1

chl-1

) High irradiance 11.1±2.3 24.2±3.4 5.8±1.5

I*; T***; I x T** Shade 16.1±2.6 24.1±2.4 20.9±1.8

β-carotene (mmol mol-1

chl-1

) High irradiance 62.2±7.8 94.6±8.7 89.3±7.0

I***; T** Shade 44.7±5.7 59.4±5.3 59.3±3.3

Lutein-epoxide

(mmol mol-1

chl-1

)

High irradiance 5.2±1.7 6.3±1.4 6.2±2.2

n.s.

Shade 3.4±0.4 3.9±0.4 4.3±0.3




Biology In press.

-56-

Fig. 3.4 α - Tocopherol content of high irradiance (open bars) and shaded (closed bars)

D. antarctica under three temperature treatments. Values are means of n = 7 (± s.e.). P-

values indicate significance of effects of irradiance (I), temperature (T), and irradiance

by temperature interaction (I x T).




Biology In press.

-57-

3.3.6. Correlations between Tc and biochemical parameters

No significant correlation was detected between Tc and osmolality, tocopherol, and α-

and β-carotenes (correlations not shown). Correlations between Tc and zeaxanthin, pre-

sented as Z/(Z+V) (after Havaux and Gruszecki 1993) were also not significant, either

for each irradiance treatment separately or for the combined data (Fig. 3.5).

Fig. 3.5. Critical temperature, Tc versus xanthophyll zeaxanthin (expressed as Z/(Z+V);

after Havaux and Gruszecki, 1993) of high irradiance (open symbols) and shaded

(closed symbols) D. antarctica during three temperature treatments: 15ºC (circle), 35ºC

(diamond) and back to 15ºC (triangle). Each point represents an individual measure-

ment. Relationships were also non-significant when data were separated by irradiance

treatment. Z, zeaxanthin; V, violaxanthin.




Biology In press.

-58-

3.4. Discussion

3.4.1. Effect of high irradiance, high temperature and their interaction on pho-

tosynthetic capacity parameters of D. antarctica

Photosynthetic capacity of D. antarctica in this study was within ranges reported in the

literature. Maximum light saturated rates of net photosynthesis (Amax25

) were compara-

ble with values reported for the same species: 6 – 10.8 µmol m-2

s-1

(Nobel et al. 1984)

and 8.3 µmol m-2

s-1

(Hunt et al. 2002). Maximum carboxylation rates, Vcmax25

(the

maximal in vivo Rubisco activity), and the maximum rate of electron transport, Jmax25

, at

the reference temperature of 25°C were comparable to some shade tolerant tree species,

such as silver fir (Abies alba Mill, Robakowski et al. 2002), and mesophyll conduc-

tance, gi, corresponded to typical values of evergreen trees among the species reviewed

by Ethier and Livingston (2004).

Photosynthetic capacity of D. antarctica was adversely affected by high irradiance.

Jmax25

decreased with respect to shaded plants, and this decrease was paralleled by a de-

crease of maximum quantum yield of PSII, Fv/Fm, indicating a moderate but chronic

photoinhibition (Table 3.2; Tallon and Quiles 2007). High irradiance also led to de-

creases in Amax25

, due to both reduced stomatal conductance gs and decreased photosyn-

thetic capacity, i.e., Rubisco activity and Jmax25

. A number of processes may result in

deactivation of Rubisco – for example, remobilisation and export of nitrogen from the

leaves, interruption in the electron transport chain, or the presence of reactive oxygen

species (ROS). In our case, deactivation of Rubisco in high irradiance plants was not

related to a remobilisation of frond nitrogen – the N content per frond area was not af-

fected by irradiance. This can be seen also through the decline of photosynthetic nitro-

gen use efficiency (PNUE). However, high irradiance alone did not affect total chloro-

phyll content and chlorophyll a/b ratios, indicating that photoprotective mechanisms

were efficient enough to avoid chlorophyll degradation.

High temperature had no negative effects on photosynthetic capacity of D. antarctica

under shade. Increasing temperature to 35ºC even stimulated Amax25

. Increases in Amax25




Biology In press.

-59-

were in this case solely due to increases in gs, as we found no effects of temperature on

photosynthetic capacity (Vcmax25

, Jmax25

). The increase in chlorophyll a/b ratio in shaded

plants with increasing temperature perhaps indicates temperature-stimulated resynthesis

of photosynthetic reaction centres relative to light-harvesting antenna complexes, which

commonly coincides with other changes in pigment composition, e.g. lutein, β-carotene

(Haldimann 1999).

The interactive effect of high irradiance and high temperature led to severe photoinhibi-

tion in agreement with earlier findings (Berry and Björkman 1980). Temperature in-

crease stimulated gs yet without commensurate increases in Amax25

, indicating that meta-

bolic limitations (e. g. Rubisco activity) governed Amax25

(consistent with the findings of

e.g. Law and Crafts-Brandner 1999 on Rubisco activation). Many studies have shown a

negative effect of moderate heat and photoinhibition on the activation of Rubisco medi-

ated by an activase (for details see Salvucci and Crafts-Brandner 2004). In our study,

we did not measure the activity of Rubisco-activase, but found no increase in photosyn-

thetic capacity to increased temperatures. Prolonged photoinhibition and heat interac-

tion resulted in decreases in chlorophylls as in numerous other studies (e.g. Lambers et

al. 2008). Decreases in chlorophyll a/b ratio indicated that under prolonged light stress,

chlorophylls were destabilised with chlorophyll a being more sensitive than chlorophyll

b (Yamamoto et al. 2008).

A return of temperature to 15ºC induced stomatal closure thus reversing Amax25

to the

initial values at 15°C, as found by Ghouil et al. (2003). The only partial recovery of

Fv/Fm in high irradiance plants demonstrated detrimental interactive effects of high ir-

radiance and temperature on Fv/Fm, suggesting that degradation processes (i.e. bleaching

of chlorophylls or photo-degradation of thylakoid complexes) did not allow rapid re-

covery of Fv/Fm (Ottander et al. 1995). The fact that Vcmax and Jmax remained non-

responsive possibly underlined the high sensitivity of Rubisco to high irradiance in D.

antarctica.




Biology In press.

-60-

In summary, photosynthetic capacity and photosynthetic nitrogen use efficiency were

rapidly affected by exposure to high irradiance under 15°C, while chlorophylls and pre-

dawn Fv/Fm declined further under the combination of high irradiance and high tempera-

ture.

3.4.2. Membrane stability of D. antarctica measured via critical temperature

Critical temperature, Tc, recorded in D. antarctica was ca 47ºC, comparable with a

range of overstorey species, such as Quercus petraea (46.7ºC; Dreyer et al. 2001).

Comparable data for other tree fern species are currently lacking.

An increase in air temperature induced an increase in Tc, as found by many authors (e.g.

Dreyer et al. 2001). This increase in Tc was larger under high irradiance than shade, co-

inciding with a 3.3°C higher midday frond temperature under high irradiance. Previ-

ously published data suggest that an increase in Tc by 1ºC (as found in our study) re-

quires an increase in ambient temperatures of about 10ºC (Froux et al. 2004). We there-

fore believe that the larger rise in Tc in high irradiance plants was not solely caused by

the difference in frond temperature, but directly related to high irradiance effects, which

resulted in enhanced thermostability of thylakoid membranes.

Contrary to findings by Hüve et al. (2006), increased thermostability of the thylakoid

membranes was not associated with the accumulation of osmotically active substances.

In our study, osmotically active solutes even decreased when temperature increased. It

may be argued that the hot water extract method results in artefacts, because some cell

wall or other material can be brought into solution as a result of the grinding and extrac-

tion procedures (Callister et al. 2006). Yet, this method is widely used (e.g. Merchant et

al. 2006) and moreover, a tight correlation was found among methods even though the

absolute results were different (Callister et al. 2006). Our observation therefore seems

reliable.




Biology In press.

-61-

With subsequent temperature decrease to 15ºC, we found a tendency for Tc to return to

the initial values, even though this return was not complete. The observed lag confirms

the hysteresis found by Froux et al. (2004), when the increase in Tc with increasing

temperature is faster than relaxation from this effect after temperature decrease.

3.4.3. Xanthophyll cycle carotenoids, pigments and α-tocopherol

Exposure of D. antarctica to high irradiance alone resulted in almost two-fold increase

in xanthophyll cycle carotenoids (V+A+Z), both on a chlorophyll as well as on a frond

area basis. A high de-epoxidation state was also recorded (50% vs. 2-4% in the shade).

Values for V+A+Z were within the range of values presented for other species (Thayer

and Björkman 1990). The maximum de-epoxidation state of the xanthophyll cycle was

lower than maximum values measured in epiphytic ferns (Tausz et al. 2001). An in-

crease was also recorded in pools of other carotenoids such as neoxanthin and lutein in

response to high irradiance. Values were within the range reported for other ferns, e.g. a

range of epiphytic species (Tausz et al. 2001) or the tree fern Cyathea microdonta (Ma-

tsubara et al. 2009). These carotenoids (e.g. neoxanthin) may preserve PSII from photo-

inactivation and protect membrane lipids from photo-oxidation by ROS (North et al.

2007). The observed increase in lutein (located primarily in both the proximal and distal

light harvesting centres of PSI and PSII) is probably associated with the acclimation of

antennae to increasing irradiance (Senger et al. 1993). As antenna size is usually re-

duced in response to increasing irradiance, it is also likely that an increasing fraction of

these carotenoids is not bound to antenna proteins. High irradiance also induced in-

creases in α–tocopherols, known for their protective function in thylakoids, consistent

with findings elsewhere (e.g. García-Plazaola and Becerril 1999; Munné-Bosch 2005).

Increased temperature stimulated an increase in Tc in shaded plants but without simulta-

neous accumulation of zeaxanthin, which is contrary to observations by Havaux and

Gruszecki (1993) and Havaux and Tardy (1996). Irrespective of an increased Tc, high

temperature had no effect on concentration of α-tocopherol in shaded plants. According

to our results, it is therefore unlikely that increased membrane stability as measured by




Biology In press.

-62-

Tc is directly and generally dependent on zeaxanthin or α-tocopherol. There was some

coincidence of increased Tc with increases in α- and β-carotenes in shaded plants, which

may have reflected changes in carotenoid synthesis rates. Carotenes may also contribute

to an overall increase in membrane stability, as proposed by Tausz et al. (2001).

Interactions between irradiance and temperature had no additional effect on the xantho-

phyll cycle pool and de-epoxidation state of high irradiance plants. In contrast to

V+A+Z, the combination of high irradiance with high temperature led to further in-

creases in the amount of lutein and β-carotene, which remained greater until the end of

experiment. It is not clear from our experiment whether sustained concentrations of ca-

rotenoids indicated their role as a last resort in membrane photoprotection under ex-

treme stress, or simply reflected their superior stability under such conditions. In con-

trast to shaded plants, temperature increase stimulated an almost two-fold increase in α-

tocopherol in high irradiance plants. Although these results for high irradiance plants

appear to support findings of Llusià et al. (2005) – who suggested that increased toler-

ance to high temperatures might be at least partly due to an increase in α-tocopherol –

our results for shaded plants indicate that changes in Tc can occur independently of

changes in α-tocopherol.

Temperature return to 15°C did not affect total V+A+Z pool of high irradiance plants,

but the de-epoxidation state decreased by 30% to remain significantly higher than in

shaded plants. De-epoxidation of the xanthophyll cycle is driven by an acidic thylakoid

lumen, which can be the consequence of an imbalance between electron transport and

electron consumption (Demmig-Adams and Adams 2006). It seems that heat related

changes in the photosynthetic apparatus lead to a relaxation of the pH gradient upon

temperature decrease, despite the continuation of high irradiance. This may be related to

a sustained decrease in light use efficiency as suggested by persistently low Fv/Fm val-

ues, and other, as yet unexplained, changes in the photosynthetic membrane. Such fur-

ther changes were also expressed in the observed change in Tc, possibly in combination

with an increased electron consumption rate upon relaxation of the high temperature.

Temperature decrease did not affect concentration of carotenoids except for α-carotene.




Biology In press.

-63-

Significant decreases in α-carotene in high irradiance plants can be explained in terms

of its ease of oxidation to lutein under conditions of oxidative stress (Senger et al.

1993), or its conversion to β-carotene to increase scavenging of free radicals in core

complexes under conditions of stress (Kirchgeßner et al. 2003).

In summary, increased thylakoid stability in D. antarctica observed during our experi-

ment could not be explained by any of the measured changes in pigments or α-

tocopherol, although they may all play partial roles. Alternative or additional explana-

tions may involve presence of certain heat shock proteins or changes in the composition

of membrane lipids, as suggested by Sinsawat et al. (2004). Discrepancies with earlier

literature may be related to the fact that pigment changes are fast responses to tempera-

ture increases (in the order of one day; as e. g. in Havaux and Tardy 1996), while we

investigated longer term acclimation (12 days) to high temperature. Longer term accli-

mation of plants to rising temperatures is also related to the appearance of polar lipids

with saturated fatty acids causing a decrease in membrane fluidity (Zsófi et al. 2009). In

parallel with an increased threshold temperature for thermal inactivation of PSII (Down-

ton et al. 1984) this can increase thylakoid stability. The difference between the rate of

acclimation and de-acclimation supports this hypothesis: the fast rise may be initially

due to rapid changes in pigments that are later completed by slower changes in lipid

composition. The reversal of these changes may be slower, which would be the cause

for a slow return to initial levels of stability. However, our data do not allow confirma-

tion of this speculation.

3.5. Summary

High irradiance caused chronic photoinhibition (measured as sustained decrease in

maximum PSII quantum efficiency), and decreases in all photosynthetic capacity pa-

rameters of D. antarctica. Whilst we observed some acclimation in terms of increases in

protective carotenoids, which may have sufficed to avoid chlorophyll degradation, simi-

lar or even decreasing chlorophyll a/b ratios indicated limited short-term acclimation

potential of D. antarctica fronds to high irradiance. Temperature alone appeared to have




Biology In press.

-64-

no negative effect on photosynthesis of D. antarctica, possibly suggesting that this spe-

cies can thrive under moderately high temperature as long as it is shaded. However,

there was ample evidence of severe damage to the photosynthetic apparatus when high

irradiance was combined with moderately high temperatures. Therefore we can specu-

late that future scenarios predicting higher temperatures and more frequent disturbances

may decrease the competitiveness of D. antarctica.

This Chapter is submitted for publication

Volkova L, Bennett LT, Merchant A and Tausz M “Shade does not ameliorate drought effects on the

tree fern species Dicksonia antarctica and Cyathea australis"

-65-

Chapter 4. Interactive effects of high light and water deficit on the

tree fern species Dicksonia antarctica and Cyathea australis

(iii) Abstract

We examined the responses of two tree fern species (Dicksonia antarctica and Cyathea

australis) growing under moderate and high light regimes to short-term water deficit

followed by rewatering. Under adequate water supply, morphological and photosyn-

thetic characteristics differed between species. D. antarctica, although putatively the

more shade and less drought adapted species, had greater chlorophyll a/b ratio, and

greater water use efficiency and less negative δ13

C. Both species were susceptible to

water deficit regardless of the light regime showing significant decreases in photosyn-

thetic parameters (Amax, Vcmax, Jmax) and stomatal conductance (gs) in conjunction with

decreased relative frond water content (RWC) and predawn frond water potential (Ψ

predawn). Stomatal conductance under moderate light responded later and at lower soil

water content. More shaded D. antarctica seemed to be most vulnerable to drought as

evidenced by greatest decreases in Ψ predawn, and lowest stomatal conductance and pho-

tosynthetic rates. Both tree fern species were able to recover after short but severe water

stress.

4.1. Introduction

Drought is considered to be one of the most important factors limiting plant perform-

ance (e. g. Ribas-Carbo et al. 2005). Shade grown plants are potentially less tolerant to

reduced soil moisture than light grown plants because survival in light limiting envi-

ronments generally requires a large leaf area, which can only be supported under moist

conditions (Smith and Huston 1989). This is supported by a number of studies e. g. Val-

ladares and Pearcy (2002), Valladares and Niinemets (2008), which have found that

shade and drought tolerance cause conflicting requirements for biomass investment.




-66-

Shade tolerance favours foliage for efficient light capture, whereas drought tolerance

requires predominant investment in roots for efficient water uptake.

During periods of insufficient water supply, plants may be exposed to light conditions

in excess of their ability to use in photosynthetic fixation. The resulting imbalance be-

tween electron transport and consumption leads to photoinhibition and photo-oxidative

stress (Flexas et al. 1999). Thus, an interactive effect of high light and water deficit can

be more detrimental than water deficit alone (Levitt 1980, Lovelock et al. 1994). Val-

ladares and Pearcy (2002) observed that the capacity to withstand severe drought was

not enhanced in the shade but was decreased due to increased below-ground competi-

tion for water with established trees. Furthermore, photoinhibition becomes relatively

more important for carbon gain in shade than in sun due to the relatively more important

effect of low photochemical efficiency under low light following sunflecks (Valladares

and Pearcy 2002).

The tree ferns Dicksonia antarctica (Labill.) and Cyathea australis (R.Br.) Domin are

ecologically important (Lindenmayer et al. 1994, Roberts et al. 2005) understorey spe-

cies of south-eastern Australian forest ecosystems, including wet sclerophyll forests and

cool temperate rainforests characterised by mild to warm summers with occasionally

short periods of droughts and high temperatures (Australian Bureau of Meteorology,

2006). D. antarctica and C. australis, are believed to have different micro-habitat pref-

erences as D. antarctica typically dominates wet, shady gullies while C. australis is

more common along forest margins (McCarthy 1998). An observational study con-

firmed that the greater the distance to the stream the more likely it‟s to encounter C.

australis than D. antarctica (Dignan and Bren 2003). D. antarctica and C. australis

belong to contrasting floristic elements of the Australian vegetation (Gondwanan vs.

Tropical, Groves 1994) thus physiological adaptation to micro-habitats (if they exist)

are likely to have arisen during the contrasting phytogeographical history of these spe-

cies.




-67-

During their lifetime, tree ferns can be periodically exposed to harsh conditions of post-

wildfire environments, characterised by increased light intensities and leaf temperatures,

and consequently increased evapotranspiration and water loss. Direct effects of these

conditions on tree fern physiology have not been studied, but are indicated by poor sur-

vival and ongoing decline of both D. antarctica and C. australis after clearcut logging

(Ough 2001). Understanding negative effects of excess light and water deficit on tree

fern persistence is of increasing importance given predictions of more frequent drought

and fire events in climate change scenarios relevant to south-eastern Australia

(Hennessy et al. 2007).

Both D. antarctica and C. australis are able to tolerate short periods of drought if some

shade is available. Despite infrequent but severe drought events, tree fern numbers in-

creased by 80% in the lower strata of wet sclerophyll forests over 48 years (Ashton

2000). Hunt et al. (2002) also reported that D. antarctica can maintain favourable water

relations during short periods of drought, if its habitat is limited to sheltered sites. This

apparent ameliorating effect of shade on drought might simply be due to reduced water

loss from soil and plants, rather than a direct effect of light intensity. Observational field

studies usually have to accept such confounding effects between shade, temperature and

(soil and air) humidity, because more shaded sites are also cooler and moister. Thus, it

often remains unresolved whether alleviation of drought stress is a direct effect of lower

irradiance – e. g. shading ameliorates drought-related photoinhibition and photo-

oxidative stress – or an indirect effect of greater water availability.

In this study, we compared interactive effects of short-term water deficit and high light

on the physiological performance of two tree ferns – one (D. antarctica) putatively less

drought/light tolerant than the other (C. australis) – by applying short-term but severe

water deficit treatment to potted individuals under semi-controlled conditions. We ana-

lysed key variables relevant to photosynthetic capacity (gas exchange, chlorophyll a

fluorescence), and plant water relations (water potential, osmolality, carbon isotope dis-

crimination) during a drought-rewatering cycle. Our research hypotheses were:




-68-

The tree fern species will display contrasting physiological characteristics, owing

their different origins and microclimate preferences, where D. antarctica will be

more susceptible to water deficit;

Water deficit will be detrimental for high light exposed tree ferns, while shade

will ameliorate the negative effects of water deficit;

The tree ferns will be able to recover after short but severe water stress, with those

in shade recovering faster.



Twenty sporophytes of Dicksonia antarctica (Labill.) and twenty of Cyathea australis

(R.Br. Domin) (Fern Acres nursery, King Lake West, central Victoria, Australia) were

transplanted two months before the experiment into spacious 25-l pots, containing (%

volume) composted pine bark (30), gravel (45), coarse fern mulch (5), composted mulch

(14.5), fine fern mulch (5), „Dynamic lifter‟ (0.16, Yates, Padstow, NSW, Australia),

and two types of slow-release fertiliser (0.17 each) Osmocote Baulkham Hills, NSW,

Australia (18+4.8+8.3 mg) and (16+3.5+10+1.2 mg). Before the experiment plants grew

in an open-air nursery under the dense canopy, providing ca 70% of shade. All plants

were two and a half years-old at the start of the experiment.

4.2.2. Experimental design

The experiment ran from February to April 2008 under the prevailing summer to au-

tumn weather at Creswick Campus of the Melbourne School of Land and Environment,

The University of Melbourne, Victoria, Australia (143º 53‟ E, 37º 25‟ S; 392 m above

sea-level).

Ten plants of each species were randomly assigned to two light regimes – “moderate

light” (35% of ambient light intensity by a wavelength neutral shade-cloth) and “high

light” (70% of ambient light intensity). We choose 35% of ambient light as most repre-




-69-

sentative for field conditions of south-eastern Australia (personal observations), while

70% of ambient light was the required minimum (derived from previous experiments,

unpublished) to protect plants from high light stress. Maximum photosynthetic photon

flux density (PPFD, 400 – 700 nm, measured with a Li-Cor PAR sensor, Li-Cor, USA)

at noon on clear sunny days was on average 600 µmol photons m-2

s-1

in moderate light

and 1000 µmol m-2

s-1

in high light at frond height, (ambient maximum PPFD ca

1800 µmol m-2

s-1

). Measurements with a Solarmeter (Ultraviolet Radiometer 5.0, So-

lartech Inc.) confirmed that UV-A and UV-B filtration was similar to that in the PAR

range (30% and 60% filtration, respectively). Weather conditions were recorded by a

weather station (Tain™ Electronics, Melbourne, Australia).

Within each light regime, 5 plants per species were randomly assigned to two water

treatments: - watered daily to field capacity for the entire experiment (control) and wa-

ter-deficit (deficit). Plants were divided into five blocks, each containing one plant per

species and four treatments (i.e. all combinations of “control” and “deficit” with “mod-

erate light” and “high light”). All measurements (as below) within one block were made

on the same day, with a one day lag in the application of water deficit treatments for

consecutive blocks. Plants were rotated randomly within their designated light regime

three times per week to minimise differences in light and temperature conditions.

Plants were measured:

(1) After 20 days of acclimation to designated light regimes, all plants regularly wa-

tered to field capacity – “pre-treatment” period;

(2) After 10 days of treatment – “water deficit” period (deficit plants not watered for

5 days then maintained at 10% of field capacity for next 5 days);

(3) Twenty days of watering to field capacity – “rewatering” period;

Additional shade cloth (to reach 50% of ambient light intensity) was placed over

high light plants to avoid damage in extremely hot weather (>35°C) during the

first 10 days of the rewatering period (Fig. 4.1).




-70-

Each plant was measured for chlorophyll a fluorescence, predawn water potential and

gas exchange at the end of each of the three experimental periods. In addition, stomatal

conductance (gs) was measured daily for the first five days of water deficit period from

8 to 11.30 a.m. Samples for chlorophyll content, osmolality and stable isotopes were

taken in the morning at the end of each of the three periods. All measurements were

made on the mid-third of the youngest fully expanded fronds.

4.2.3. Maximum quantum yield of PSII (Fv/Fm)

Maximum quantum yield of PSII (Fv/Fm) was measured at predawn with a pulse modu-

lated fluorometer (OS-30p, Opti-Sciences, Hudson, USA). Ground fluorescence (F0)

was obtained with a low intensity modulated light (600 Hz, 650nm, PPFD<1 µmol m-2

s-

1). Maximum fluorescence (Fm) was induced by a saturating flash. Maximum quantum

yield of PSII was estimated as Fv/Fm = (Fm–F0)/Fm, nomenclature after Maxwell and

Johnson (2000).

4.2.4. Photosynthetic capacity

Gas exchange was measured with a Li-Cor 6400 portable photosynthesis measurement

device, equipped with a 2x3 cm broadleaf chamber with red-blue LEDs (Li-Cor, Lin-

coln, Nebraska, USA). All gas exchange measurements were conducted at the reference

frond temperature of 25ºC. For each plant, an A-Ci curve was generated after Long and

Bernacchi (2003) with some modifications: PPFD was 1000 µmol m-2

s-1

, frond tem-

perature was 25ºC, air flow rate was 400 µmol air s-1

, and relative humidity (RH)>60%.

CO2 mole fraction was increased in 13 successive steps from 50 to 2200 µmol mol s-1

with two measurements at each step. After finishing the A-Ci curve, illumination in the

leaf chamber was turned off, CO2 mole fraction was decreased to 400 µmol mol-1

and

respiration rate was recorded after 5 min in the dark. The frond area enclosed in the

chamber was marked, detached, scanned and calculated using Scion Image programme

(Scion Corporation 2000-2001, USA). Gas exchange measurements were then recalcu-

lated for real frond area.




-71-

Intrinsic water use efficiency (WUEi) was calculated as Amax/gs; where Amax and gs were

measured at the conditions mentioned above and reference CO2 of 397±1 µmol mol s-1

.

Using the Farquhar model (Farqhuar et al. 1980), maximum carboxylation rate (Vcmax),

and maximum electron transport rate (Jmax) were evaluated by fitting A-Ci curves to the

model, as described in Dreyer et al. (2001). Triose phosphate use (TPU) limitation was

not included in the model, and corresponding points with decreased A at elevated Ci

were disregarded. The set of primary parameters of Rubisco kinetic properties used

herein (Kc=327µmol mol-1

, Ko=282.6 mmol mol-1

, Γ*=43.7 µmol mol-1

) are from von

Caemmerer et al. (1994).

A time course of gs versus days of water withheld was measured using the Li-Cor 6400

gas exchange system, with chamber conditions as above and a reference CO2 concentra-

tion of 397±1 µmol mol-1

.

For measurements of frond chlorophyll content, four frond discs (3.75 mm diameter)

were immediately immersed in liquid nitrogen and then stored at -80ºC until extraction.

Chlorophyll a and b were extracted using 1.8 ml of 100% dimethyl sulphoxide

(DMSO). Extracts were heated for 30 min at 65ºC in a dry block heater (Termoline

L+M, Northgate, Queensland, Australia). The supernatant was then transferred to a mi-

croplate reader (Tecan GmbH, Austria). Blank microplates were scanned and their ab-

sorbance was deducted from final measurements. Chlorophyll a, b and total were calcu-

lated according to Wellburn (1994). The absorbance of 200 μl of sample in a microplate

was converted into a 1 cm pathlength, and then corrected using correction coefficients

(Warren 2008). Correction coefficients for chlorophyll a and b were calculated from 20

samples using regressions of initial measurement against repeated measurement in a

spectrophotometer (Carry 300, Varian, The Netherlands) at the same wavelengths (all

regressions were highly significant P<0.001).

Leaf mass per area (LMA), needed to calculate total chlorophyll on a frond area, was

calculated as dry weight/frond area (g m-2

).




-72-

4.2.5. Frond water relations

Predawn frond water potential (Ψ predawn) of each tree fern was measured using a pres-

sure chamber (PMS Corvallis, OR, USA) at the end of each of the three periods.

Concentrations of osmotically active solutes (osmolality) were measured via freeze-

point depression from fresh sap extracts using an OSMOMAT 030 cryoscopic osmome-

ter (Gonotec, Berlin, Germany), nomenclature by De Costa et al. (2007).

Relative water content (RWC) was determined as follows: pinnae were detached and

weighed (fresh weight), then floated in water for ca 5 h in the dark to reach full hydra-

tion. Pinnae were blotted dry with tissue paper and weighed (saturated weight), then

dried at 60ºC for 48 h and weighed again (dry weight). RWC (%) was calculated as:

(fresh weight – dry weight)/(saturated weight – dry weight) x 100.

4.2.6. Stable isotope analysis

Due to rapid turnover of the soluble carbon pool, we tracked differences in stable iso-

tope composition (δ13

C) of a hot water extract (e.g. Warren et al. 2007). Hot water ex-

tracts were prepared according to Callister et al. (2006). Extract sub-samples (100 µl)

were dried in tin capsules at 50ºC for 48 h, and δ13

C determined by IRMS (EuroVector,

IsoPrime Mass Spectometer, Manchester, UK) with Dumas flash combustion. Analysis

of δ13

C (in ‰ units) was against a tertiary standard (Acetanilide: δ13

C =33.44‰ and

C=71.09%), which was calibrated against a PDB (Pee Dee Belemnite) standard.

4.2.7. Relative extractable soil water, REW

Relative extractable soil water (REW), was calculated as the soil moisture content at

each day of water deficit divided by soil moisture content at field capacity minus soil

moisture content at permanent wilting point (after Bogeat-Triboulot et al. 2007). For




-73-

this, each pot of water deficit treated plants was weighed at field capacity and at the end

of each day of water withheld. Potential differences in evaporation from the pot surface

between light regimes was taken into account by placing a plant-free pot in each of the

light regimes and weighed at the same time as treated plants (Merchant et al. 2006). Soil

moisture content at field capacity was determined gravimetrically by oven drying soils

at 105ºC for 24 h.


Effects of species (D. antarctica, C. australis), light (moderate, high), water treatment

(control, deficit), and their interactions on dependent variables were analysed using the

general linear models of SPSS (SPSS Inc. Chicago, USA). Variables were graphically

checked for deviations from normality, homogeneity of variances was tested using

Levene‟s test, and means and variances were not correlated across treatments. Each pe-

riod (pre-treatment, water deficit, rewatering) was analysed separately.

DOY

42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96

Ma

xim

al d

aily

air t

em

pe

ratu

re,o

C

0

5

10

15

20

25

30

35

40

45

50

55

Da

ily R

H a

t 9

a.m

., %

0

20

40

60

80

100Pre-treatment Drought RewateringRewatering

(extra shadeon high light plants)

(shade removal)

Fig. 4.1 Weather conditions during the experiment. Dotted grey line is maximum daily

air temperature (ºC), black line - daily relative humidity (RH, %) at 9 a.m. Vertical lines

indicate stages of the experiment for Block 1.




-74-

4.3. Results

4.3.1. Maximum quantum yield of PS II (Fv/Fm)

Even during the pre-treatment period, both species had lower Fv/Fm than what is consid-

ered an optimum value for healthy fronds (0.83, Maxwell and Johnson 2000, Table 4.1).

Fv/Fm did not differ between species but was significantly higher (P<0.001) in moderate

light plants during the pre-treatment and the water deficit periods (Table 4.1). Fv/Fm was

not affected by water treatment in the water deficit period regardless of the light regime,

but was significantly greater in control than deficit plants in the rewatering period.

Drought stressed D. antarctica in moderate light had significantly lower Fv/Fm in the

rewatering period (Table 4.1).

4.3.2. Photosynthetic capacity

Light saturated net photosynthesis (Amax) and stomatal conductance (gs) at Amax were

significantly greater in C. australis than D. antarctica in the pre-treatment and the water

deficit periods, but not the rewatering period (Fig. 4.2). Water deficit resulted in a sig-

nificant reduction in Amax and gs in both species (water treatment P<0.001), with mar-

ginally higher, albeit non-significant, values under high light. Amax and gs recovered

with rewatering in both species, and under both light regimes (Fig. 4.2).

The maximum carboxylation rate, Vcmax, and the maximum rate of electron transport,

Jmax, did not differ significantly between species and light regimes in the pre-treatment

period (Table 4.1). However, water deficit caused significant decreases in both variables

for both species – an effect that may have been most pronounced for D. antarctica un-

der moderate light, although increasingly inaccurate Ci estimates at very low gs made

impossible to construct meaningful A-Ci curves (i.e. missing estimates for Vcmax Jmax;

Table 4.1). Vcmax and Jmax recovered to near control values with rewatering for both spe-

cies, although Jmax was significantly lower in C. australis in this period (Table 4.1).




-75-

Total chlorophyll content per frond area (µmol m-2

) did not significantly differ between

species, light regimes, and water treatments across all experimental periods (Table 4.1).

Chlorophyll a/b ratios were significantly greater in D. antarctica than C. australis in the

pre-treatment period (P=0.01, Table 4.1). However, this difference was not detected for

the remainder of the experiment, with chlorophyll a/b ratios of both species largely in-

variant to light regimes and water treatments (Table 4.1).


Volkova L, Bennett LT, Merchant A and Tausz M “Shade does not ameliorate drought effects on the tree fern species Dicksonia antarctica and Cyathea australis"

-76-

Table 4.1 Chlorophyll fluorescence and photosynthetic capacity variables of water deficit and control tree ferns (D. antarctica and C. aus-

tralis) grown under high and moderate light during three successive experimental periods.

Values are means (n=5) ± s.e. of: Fv/Fm, maximum quantum yield of PSII, Vcmax, maximal carboxylation rate of Rubisco (µmol m-2

s-1

);

Jmax, maximal light driven electron flux (µmol m-2

s-1

); Chl total (µmol m-2

), total chlorophyll content on a frond area basis, Chl a/b, chloro-

phyll a/b ratio. Effect abbreviations: L, Light regime; W, Water treatment; S, Species; x, interaction. Significance levels:* P<0.05; **

P<0.01; *** P<0.001; n.s., non significant, n/d, none detected.

Variable Species

High light Moderate light Significance of effects, P

Pre-

treatment

Water deficit Rewatering Pre-

treatment


treat-

ment

Water

defi-

cit

Rewa-

tering control deficit control deficit

control deficit control deficit

Fv/Fm

D. ant-

arctica 0.72±0.0 0.76±0.0 0.73±0.0 0.78±0.0 0.73±0.0 0.78±0.0 0.81±0.0 0.78±0.0 0.77±0.01 0.64±0.0

L*** L*** W***

SxL** C. aus-

tralis 0.73±0.0 0.75±0.0 0.75±0.0 0.74±0.0 0.68±0.0 0.75±0.0 0.78±0.0 0.78±0.0 0.79±0.01 0.75±0.0

Vcmax

D. ant-

arctica 36.4±5 30.4±4 16.9±3 39.8±4 37.6±6 37.9±3 26.4±5 n/d 40.0±1.8 37.3±4

n.s. W** n.s.

C. aus-

tralis 33.3±4 38.0±3 21.0±4 34.3±2 36.5±4 50.5±8 33.0±3 26.0±3 45.7±3 38.2±3

Jmax

D. ant-

arctica 106±13 141±35 98±13 154±19 141±35 111±8 88±20 n/d 145±12 139±25

n.s. W** S***

C. aus-

tralis 91±11 96±8 53±10 101±7 90±6 144±27 83±6 69±10 109±7 103±12



-77-

Variable Species


Pre-

treatment


treatment


treat-

ment

Water

defi-

cit

Rewa-

tering control deficit control deficit


Chl total

D. ant-

arctica 382±30 277±14 218±12 288±12 219±13 372±31 275±40 253±31 251±36 268±25

n.s. n.s. n.s. C. aus-

tralis 349±15 285±66 242±18 252±14 222±23 354±23 256±13 219±18 328±65 261±25

Chl a/b

D. ant-

arctica 4.0±0.1 3.5±0.1 3.4±0.2 3.5±0.1 3.5±0.2 4.1±0.1 3.4±0.2 3.4±0.2 3.4±0.1 3.5±0.2

S** n.s. n.s. C. aus-

tralis 3.9±0.1 2.8±0.3 3.4±0.1 3.4±0.1 3.0±0.4 3.8±0.1 3.4±0.1 3.2±0.2 3.0±0.3 3.5±0.1



-78-

Period

pre-treatment water deficit rewatering

Am

ax (

mol m

-2s-1

)

0

2

4

6

8

10

12

pre-treatment water deficit rewatering

High lightgs (

mm

ol m

-2s-1

)

0

50

100

150

200

250

300Moderate light

Period

S* S***W**

n.s.

Pre-treatment

Waterdeficit

Re-watering

S* S**W**

n.s.

Pre-treatment

Waterdeficit

Re-watering

Fig. 4.2. Stomatal conductance and light saturated net photosynthesis of water deficit (hatch) and control (solid) D. antarctica (white bars)

and C. australis (grey bars) under high and moderate light in three successive experimental periods (pre-treatment, water deficit and rewa-

tering). Values are means n=5 (± s.e.). Effect abbreviations: S, Species; W, Water treatment; none of interactions were significant. Signifi-

cance levels:* P<0.05; ** P<0.01; *** P<0.001; n.s., non significant




-79-

4.3.3. Frond survival

Approximately 1/3 of fronds of water deficit plants survived the water deficit period – a

proportion that did not significantly differ between species and light regimes (high light:

33±7% for D. antarctica and 24±4% for C. australis; moderate light: 27±4% for D.

antarctica and 35±6% for C. australis).

4.3.4. Time course of stomatal conductance during 5 days without water

Stomatal response to withholding water did not differ between species but was affected

by light regime (Fig. 4.3). Water deficit plants under high light decreased gs by almost

50% relative to controls after one day without water, at relative extractable soil water

(REW) of ca 80% (Fig. 4.3). In contrast, plants under moderate light maintained initial

stomatal conductance for 1 day longer and decreased gs at lower REW (ca 65%;

Fig.4.3).




-80-

REW, %

204060801000

20

40

60

80

100

Days without water

day 1day 2

day 3day 4

day 5

High light

gs (

% o

f contr

ols

)

0

20

40

60

80

100

Moderate light

5 days of water withheld

REW,%

20406080100

Days without water

day 1day 2

day 3day 4

day 5

C. australis C. australis

D. antarctica D. antarctica

gs control gs control

gs controlgs control

Fig. 4.3. Time course of stomatal conductance of water deficit D. antarctica and C. aus-

tralis grown under high and moderate light during first five days of water deficit. Where

gs, stomatal conductance, and REW, relative extractable soil water. Values are means

(n=5) ± s.e.




-81-

4.3.5. Frond water relations

Significant differences in frond water relations were detected between species in the

pre-treatment period. D. antarctica had significantly lower Ψ predawn, RWC and signifi-

cantly greater osmolality (Table 4.2). With the exception of Ψ predawn, these species dif-

ferences were not detected during the water deficit period, although the difference in

osmolality was reinstated after rewatering.

Frond water relations of both species were significantly affected by water treatments.

Water deficit significantly decreased Ψ predawn and RWC of deficit relative to control

plants of both species under both light regimes (Table 4.2). These effects were reversed

in the rewatering period. Osmolality was not affected by water deficit; however, a sig-

nificant species by water treatment interaction was detected in the rewatering period

(i.e. increased osmolality of control D. antarctica relative to water deficit plants, and the

opposite trend in C. australis; Table 4.2).

Light regimes did not significantly affect frond water relations of either species, apart

from incomplete Ψ predawn recovery of deficit D. antarctica under moderate light in the

rewatering period (Light x Water; P=0.04; Table 4.2).



-82-

Table 4.2. Frond water relations of water deficit and control tree ferns (D. antarctica and C. australis) grown under high and moderate

light during three successive experimental periods. Values are means (n=5) ± s.e. of: Ψ predawn, predawn frond water potential (MPa); osmo-

lality (osmol kg-1

); RWC, relative water content (%). Effect abbreviations: L, Light regime; W, Water treatment; S, Species; x, interaction.

Significance levels:* P<0.05; ** P<0.01; *** P<0.001; n.s., non significant.

Variable Species


Pre-

treatment

Water deficit Rewatering

Pre-

treatment


treat-

ment

Wa-

ter

defi

cit

Re-

watering



Ψ predawn

D. antarc-

tica -0.3±0.0 -0.7±0.2 -1.3±0.2 -0.3±0.1 -0.3±0.1 -0.3±0.0 -0.5±0.1 -2.5±0.3 -0.2±0.1 -0.4±0.1

S ** S *

W*

**

LxW*

C.

australis -0.2±0.0 -0.5±0.0 -1.2±0.4 -0.4±0.1 -0.3±0.0 -0.2±0.0 -0.5±0.0 -1.3±0.4 -0.2±0.0 -0.3±0.1

Osmolality

D. antarc-

tica 0.69±0.0 0.62±0.1 0.61±0.0 0.79±0.1 0.68±0.0 0.71±0.0 0.60±0.0 0.68±0.1 0.79±0.0 0.72±0.0

S** n.s. S***

SxW**

C.

australis 0.64±0.0 0.63±0.0 0.60±0.0 0.56±0.0 0.66±0.0 0.62±0.0 0.58±0.0 0.62±0.0 0.63±0.0 0.65±0.0

RWC

D.

antarctica 95±0 94±1 92±2 97±0 96±1 94±1 95±2 93±1 96±0 96±1

S* W* n.s.

C. aus-

tralis 97±1 97±1 89±3 97±0 97±1 97±0 94±1 90±5 97±1 94±1




-83-

4.3.6. Intrinsic water use efficiency (calculated as Amax/gs, WUEi) and stable

carbon isotope composition (δ13

C)

During the pre-treatment period, WUEi was significantly greater in D. antarctica and

was not affected by light regime. Water deficit treatment significantly increased WUEi

of deficit relative to control plants irrespective of the light regime and species. With re-

watering, WUEi was greater in C. australis than D. antarctica (Fig. 4.4).

δ13

C was significantly more negative in C. australis across the experiment (Fig. 4.4).

During water stress period, δ13

C of deficit plants under high light were comparable with

pre-treatment values, but δ13

C became more negative in control plants under moderate

light, particularly in D. antarctica (Light x Water interaction, P=0.02; Fig. 4.4). This

effect was not detected in the rewatering period, when δ13

C within species was compa-

rable across water treatments and light regimes (Fig. 4.4).


Volkova L, Bennett LT, Merchant A and Tausz M “Shade does not ameliorate drought effects on the tree fern species Dicksonia antarctica and Cyathea

australis"

-84-

Moderate light

Period

Pre-treatment water deficit rewatering

WU

Ei (

mol m

ol-1

)

0

20

40

60

80

100

Period

Pre-treatment water deficit rewatering

High light

13C

(m

mol m

-2s-1

)

-30

-25

-20

-15

-10

-5

S * W ** S *

Pre-treatment

Waterdeficit

Re-watering

S *** S ***W **L x W *

S*

Pre-treatment

Waterdeficit

Re-watering

Fig. 4.4. Stable isotope composition, δ13

C and intrinsic water use efficiency, WUEi and of water deficit (hatch) and control (solid) D.

antarctica (white bars) and C. australis (grey bars). Values are means (n=5); ± s.e. Effect abbreviations: S, Species; W, Water treat-

ment; L, Light regime; L x W, light by water treatment interaction. Significance levels:* P<0.05; ** P<0.01; *** P<0.001




-85-

4.4. Discussion

4.4.1. Pre-treatment period – species differences and effect of light

Photosynthetic capacity of both species were within range for tree fern species reported

in the literature: 6 – 10.8 µmol m-2

s-1

(Nobel et al. 1984) and 8.3 µmol m-2

s-1

(Hunt et

al. 2002) for D. antarctica, or even similar to some middle storey canopy species in

these types of forests, e.g. Nothofagus cunninghamii (Tausz et al. 2005). Maximum car-

boxylation rates, Vcmax (the maximal in vivo Rubisco activity), and the maximum rate of

electron transport, Jmax at the reference temperature of 25°C were within the lowest val-

ues among the large number of species reviewed by Wullschleger (1993).

D. antarctica had greater WUEi that together with its less negative δ13

C in the pre-

treatment period suggests that photo-assimilation may be comparatively less susceptible

to changes in water status.

Chlorophyll a/b ratios were also significantly higher in D. antarctica, a result inconsis-

tent with this species‟ putative preference for well shaded microhabitats. Normally,

chlorophyll a/b ratios are lower in shade acclimated than sun foliage because of greater

chlorophyll b contents (Hoober et al. 2007). For D. antarctica, we observed greater

chlorophyll a content while b was comparable between species (data not shown). How-

ever, our study involved young sporophytes and it must be noted that these species dif-

ferences might change at later stages in the tree fern life cycle.

Both species displayed low acclimation potential to changes in light regime - neither

photosynthetic capacity (except Fv/Fm) nor water relation variables were affected by a

nearly two-fold difference in maximum irradiance. Reduction in Fv/Fm usually indicates

down-regulation of PSII (i.e. photoinhibition, Savitch et al. 2000), yet because Fv/Fm

was measured at predawn, the down-regulation should be relaxed, indicating either

damage of PSII or sustained down-regulation of some type. Down-regulation or damage

of PSII in high light plants may affect maximum light use efficiency of photosynthesis,

and likely depress photosynthetic performance - yet effects of light regimes on Amax

were non significant, suggesting small differences in Fv/Fm under moderate and high




-86-

light had minimal effect on Amax. These differences would become more relevant for

photosynthesis during low light periods and therefore integrated carbon gain (Zhu et al.

2004) – an issue not addressed in this study.

4.4.2. Water deficit and light interactions

Both species were susceptible to short-term water stress. Deeper shade did not amelio-

rate the negative effects of water deficit, and there is no evidence to suggest that water

deficit in combination with high light was more detrimental to plant function. The ab-

sence of significant water treatment by species interactions indicates that both species

responded to water deficit in a similar way. The only parameter insensitive to water

deficit was Fv/Fm - while this is in agreement with some studies (e.g. Epron and Dreyer

1992), others showed that drought reduced predawn Fv/Fm more in shade than in sun

(Valladares and Pearcy 2002). In our study, plants under moderate light had consistently

higher predawn Fv/Fm than high light plants even under water deficit, indicating full re-

covery of PSII overnight.

We detected contrasting stomatal behaviour under the different light regimes - plants

growing under moderate light closed stomata later and at lower relative extractable soil

water content. This indicated slower stomatal response under lower light, a result con-

sistent with the findings of Roberts et al. (1984) where stomata of bracken fern from the

most shaded understorey level were less sensitive to soil moisture treatments than those

from more irradiated levels. Surprisingly, even at REW of 30%, stomatal conductance

was not close to zero, possibly indicating limited stomatal control or high residual cu-

ticular conductance (which was not distinguished from stomatal conductance in our

measurements).

Decreases in gs by ca 70%, relatively to the pre-treatment values indicated that stress

was rather severe – the result we aimed at. However, a straightforward definition of

drought stress severity as given by Flexas and Medrano (2002) – that is, moderate stress

intensity at gs <150 and severe at <50 mmol H2O m-2

s-1

– appeared to be unsuitable in

our study. Ferns seem to have generally low stomatal conductance (Doi et al. 2006,

Hunt et al. 2002) and in our study, gs of D. antarctica rarely exceeded 150 mmol H2O




-87-

m-2

s-1

even with adequate watering. The severity of drought stress was also confirmed

by the decrease in mesophyll capacity parameters of photosynthesis - both Vcmax and

Jmax decreased significantly after ten days of drought, confirming findings by Bota et al.

(2004) that impaired Rubisco activity and/or RuBP regeneration do not limit photosyn-

thesis until drought is severe and A and gs are strongly depressed.

Reduced values of gs and Amax in D. antarctica under moderate light resulted in in-

creased error in the computation of Ci, therefore it was not possible to accurately con-

struct A-Ci curves for these plants and measure Vcmax and Jmax values. This precluded a

formal significance test of the result. D. antarctica under moderate light also had greater

decreases in Ψ predawn which most probably reflect conditions close to the point of turgor

loss in some fronds, and it can be speculated that D. antarctica under moderate light

was most severely affected by water deficit treatment, possibly as a consequence of

greater water loss due to the slower stomatal closure under moderate light.

RWC can affect Rubisco activity and photochemistry of plants in response to drought.

According to Flexas et al. (2006) Rubisco activity remains essentially unaffected by wa-

ter stress until gs drops below 50 mmol H2O m-2

s-1

regardless of species. While our re-

sults on D. antarctica support this suggestion, gs of C. australis varied from less than 50

to above 100 mmol H2O m-2

s-1

, yet Rubisco activity (measured via Vcmax) decreased

significantly. Despite a significant decrease in RWC under water deficit, RWC of both

species remained around 90%. This corresponds to ca 30% of the cases reviewed by

Flexas and Medrano (2002), where decrease in Rubisco activity was associated with

high RWC between 90-100%. Possibly, our inability to detect significant osmotic ad-

justment in this study suggests that RWC is maintained in these species by a combina-

tion of alternative factors. High solute levels in frond tissues may provide „pre-emptive‟

protection against rapid changes in water status, a concept that would require additional

investigation given the lack of data on tree fern chemistry and physiology. If water con-

servation measures such as early and rapid stomatal closure fail, fronds may be left

without strong tissue level tolerance mechanisms. In our study, this was indicated by

necroses of two-thirds of fronds by the end of the water deficit period. In agreement




-88-

with their ecological distribution, tree ferns may be unable to tolerate a major drop in

RWC instead relying on an „avoidance‟ strategy of frond death.

We observed significant increases in WUEi under severe water stress in line with the

stomatal responses. This was accompanied by significant reductions in photosynthetic

activity. For carbon isotopic composition, the general lack of significant changes be-

tween treatments may be due to low photosynthetic activity limiting the contribution of

new assimilates to the water extractable pool (Dawson et al. 2002). The observed sig-

nificant δ13

C shift in control plants under moderate light during the rewatering period

(when air temperatures increased for ca 10ºC) could be related to stomatal closure in

response to high air temperature while Rubisco remained active. Indeed, our other study

of heat and light interactions indicate that moderate light may have mediated effects of

heat on control plants (Volkova et al. 2009). An increase in air temperature can also ex-

plain relatively large changes for some variables within a species between pre-treatment

and control (Fv/Fm, Ψ predawn, RWC etc).

Overall, there were some indications that shade exacerbated drought stress effects in D.

antarctica (i.e. mesophyll capacity parameters, Ψ predawn). These findings are consistent

with results elsewhere indicating that shaded plants can be more susceptible to drought,

although the mechanisms explaining such an effect in other studies – such as increased

below-ground competition, greater reduction in predawn Fv/Fm (Valladares and Pearcy

2002) – were apparently not applicable to our study.

4.4.3. Rewatering period

Both species of tree fern recovered from short-term water deficit with rewatering (i.e.

treatment effect was non significant at this stage for all parameters but Fv/Fm). Recovery

of plants was mostly unaffected by light regime, however, due to an unexpected heat

wave we had to put an additional shade protection over high light plants, which resulted

in rather small differences in PPFD between light regimes during first 10 days of rewa-

tering, and probably smoothed potential differences during recovery.




-89-

Decreases in Fv/Fm in the rewatering period were presumably mediated by further in-

crease in ambient air temperatures (Galle et al. 2007). We speculate that the greater de-

crease in Fv/Fm of deficit plants was an after-effect of the treatment, which may have

made them more sensitive to the following conditions.

Species differences in Ψ predawn, RWC, Amax, gs, chlorophyll a/b during the pre-treatment

period were no longer evident in the rewatering period. This could be partly explained

by effects of more extreme temperatures combined with different cohorts of fronds, but

might also reflect some subtly different responses in frond physiology between the two

species triggered by the treatments.

4.5. Summary

D. antarctica and C. australis displayed contrasting physiological characteristics some-

times contrary of what would have be expected from species‟ different origin and mi-

cro-site preferences - greater WUEi, chlorophyll a/b ratios and less negative δ13

C for D.

antarctica would indicate this species as more drought and light tolerant. Results of our

study also suggest that shade does not ameliorate drought effects on C. australis and D.

antarctica. However, both species were resilient to short-term severe water stress, their

ability to restore physiology of surviving fronds and re-sprout after considerable loss of

fronds undoubtedly plays a role for both species exposed to more extreme changes in

environmental conditions. These findings are consistent with their distribution in tem-

perate forest systems that are subject to seasonal droughts and rapid changes in forest

canopy structure.


Volkova L, Bennett LT and Tausz M “Seasonal variations in photosynthesis of the tree ferns Dick-

sonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) in wet sclerophyll forests of

Australia

-91-

Chapter 5. Seasonal variations in photosynthesis of the tree ferns

Dicksonia antarctica and Cyathea australis in wet sclerophyll forests

of Australia

(iv) Abstract

Steady state and dynamic responses of two tree fern species of contrasting origins,

Dicksonia antarctica (Gondwanan) and Cyathea australis (Pan-tropical), were studied

over two consecutive years under field conditions in wet sclerophyll forest of south-

eastern Australia. Irrespective of their different origins and micro-site preferences, there

were no significant differences in photosynthetic performance between the two species.

Growth irradiance (from open sites to dense canopy cover) had very little effect on pho-

tosynthetic rates of the tree ferns. Both species performed better in winter than in sum-

mer, when photosynthetic rates reached higher values under similar irradiance and leaf

temperatures. However, at the same leaf temperature, Fv/Fm was significantly lower in

winter than in summer, suggesting some cold-induced limitation in PSII efficiency indi-

cating persistent photoinhibition associated with cold winter mornings. Both species

displayed seasonal acclimation in a number of measured photosynthetic parameters and

frond traits. Acclimation of stomatal density to spatial variation in growth irradiance

seemed limited in both species, although stomatal pattern differed between species. Be-

cause there were no significant differences between the two species in photosynthetic

parameters, both species could be described by the same carbon gain and water use

models at the leaf-scale.

5.1. Introduction

The tree ferns Dicksonia antarctica (Labill) and Cyathea australis (R. Br.) Domin are

well known Australian representatives of the fern genera Cyathea and Dicksonia, which

include most tree fern species worldwide. Cyathea has a broad global distribution,




Australia

-92-

whereas Dicksonia is diverse throughout Indonesia and New Guinea, and can be found

in isolated pockets including off the coast of Chile (Large and Braggins 2004).

Little is known about the photosynthesis and stomatal conductance of D. antarctica and

C. australis in their natural habitats. This knowledge gap effectively excludes tree ferns

from long-term carbon uptake and energy flux calculations, and limits the validity of

predictions of their survival under climate change. Yet, these tree ferns often form the

dominant understorey component of wet sclerophyll forests in south-eastern Australia

(Ough and Murphy 1996). These forests support as much as 1,053 tonnes carbon ha-1

in

above-ground living biomass (Keith et al. 2009), although the contribution of tree ferns

remains unknown. Tree ferns of the genus Cyathea account for 33% of above-ground

biomass in dwarf forests of Puerto Rico (Weaver 2008). Understorey species of ever-

green forests world wide contribute an average of 49% to ecosystem respiration (Mis-

son et al. 2007).

D. antarctica and C. australis are species of great ecological importance (Lindenmayer

et al. 1994, Roberts et al. 2005). Both species are broadly distributed in the wetter parts

of south-eastern Australia, with D. antarctica dominating wet, shady gullies, while C.

australis can also extend to forest margins (McCarthy 1998). Although D. antarctica

and C. australis can grow together in wet sclerophyll forests, previous studies have in-

dicated they have different micro-habitat preferences. Dignan and Bred (2003) found

greater probability of a tree fern being C. australis than D. antarctica with increasing

distance from a stream, suggesting greater drought tolerance of C. australis. The postu-

lated difference in origin of the two species – Gondwanan for D. antarctica compared

with pan-tropical for C. australis (Page and Clifford 1981) – also suggests different

physiological adaptation potential consistent with their observed distribution patterns

within forests.

During their lifetime, tree ferns of south-eastern Australia can be periodically exposed

to the harsh conditions of post-wildfire environments, characterised by increased irradi-

ance and leaf temperatures, and consequently stronger evapotranspiration and water




Australia

-93-

loss. Sensitivity to high temperatures, high irradiance and decreased water could poten-

tially limit growth and distribution of tree ferns in the field, although there are few field

data to evaluate this theoretical model.

Ferns, as other plants (angiosperms or gymnosperms), have displayed a capacity to ac-

climate to changing irradiance. For example, leaf characteristics (frond surface area,

epidermis thickness, palisade/spongy mesophyll ratio, blade size, petiole length) of a

South American Cyathea species were found to be correlated with local irradiance

(Arens 1997). However, stomatal density, which might be expected to increase with in-

creasing light intensity (Casson and Gray 2008), was found to have limited plasticity in

the tree fern Cyathea caracasana in response to light environment (Arens 1997).

To evaluate relationships between physiological performance of the tree ferns and pre-

vailing environmental conditions, we analysed diurnal and seasonal trends in gas ex-

change during summer and winter over two consecutive years using pairs of D. antarc-

tica and C. australis at contrasting micro-habitats (shaded creek-side and more exposed

rocky knoll). The objectives of this study were:

To determine if D. antarctica and C. australis showed differences in morphologi-

cal and physiological frond traits among contrasting micro-habitats in the field.

The postulated difference in their origin in particular leads us to hypothesise that

the species would have different seasonal acclimation to temperature, with D.

antarctica having higher photosynthesis in winter, and C. australis favouring

summer;

To determine whether light, temperature and plant water status limited physio-

logical performance in the field;

To examine acclimation of stomatal density in the two tree ferns; that is, would

stomatal density differ between the two species and/or between high light exposed

and shaded habitats?




Australia

-94-


5.2.1. Study site and sampling design

Our study site was established in mountain ash (Eucalyptus regnans F. Muell) forest in

the Victorian Central Highlands (145‟42ºE 37‟35ºS, elevation 450 m). The average an-

nual rainfall of a representative weather station (Healesville, 145,53ºE 37.68ºS, eleva-

tion 131 m) is 1021 mm. October is the wettest (106 mm) and January is the driest (58

mm) month on average. Mean maximum temperature of the hottest month (February) is

26ºC and mean minimum of the coldest month (July) is 4ºC (Australian Bureau of Me-

teorology, 2009 www.bom.gov.au/climate /averages/cdo/about/about-stats.shtml, veri-

fied August 2009).

The experimental design involved tree ferns on the margins of a logging coupe that was

clearcut in late 2003 and early 2004, then slash-burnt in March 2004. The coupe is

bounded by the Acheron River to the south, and a creek to the east. Retained undis-

turbed vegetation buffer zones were maintained along the waterways in accordance with

the Code of Forest Practices (Department of Natural Resources and Environment,

1996), and were 200 m wide along the Acheron River and 20 m along the creek.

We selected eight mature individuals of each of two species, Dicksonia antarctica (La-

bill.) and Cyathea australis (R.Br.) Domin. To cover the full range of local environ-

mental conditions evenly for both species, tree ferns were selected in pairs (one from

each species) growing in close vicinity (Fig. 5.1). Such pairs were selected from the

most exposed sites at the fringe of the clearing on a rocky knoll (elevation up to 490 m

above sea level, proximity to the creek 49 - 100 m) to the most sheltered sites in buffer

zones (average elevation 450 m) near waterways (ca 2 m).

http://www.bom.gov.au/climate%20/averages/cdo/about/about-stats.shtml




Australia

-95-

Fig. 5.1. Location of the tree ferns at the study area. Where D is D. antarctica and C. is

C. australis

5.2.2. Tree fern measurement schedule

Each plant was measured for pre-dawn chlorophyll a fluorescence, predawn/midday

water potential, gas exchange measurements (diurnal and maximal net CO2 assimilation

rate Amax) in winter (August-September) and summer (December-January) over two

consecutive calendar years (2006-2008). Gas exchange measurements were made over

two consecutive days during each season per year. Diurnal measurements were made

once in summer (December 2006) and winter (August-September 2007). Frond samples

for nitrogen and chlorophyll content were collected at the same time as gas exchange

measurements. All measurements were made on the mid-third of the youngest fully ex-

panded fronds of similar north-facing orientation.

Road

Creek

C C

C

C

C; D

C

C C

D D

D

D

D

D

D

Unlogged area




Australia

-96-

Samples for stomatal density determination were collected in January 2007. For this

analysis only, fronds that were most representative of the tree fern‟s light environment

were collected; that is, most light-exposed fronds of tree ferns growing in the open, and

most shaded fronds of shade-grown tree ferns. Orientation (e.g. north facing) was not

taken into account.

5.2.3. Mean irradiance on measured fronds

Mean daily photosynthetic photon flux density (daily PPFD) was estimated from hemi-

spherical photographs (Nikon 601 F-601, Japan, fisheye lens), which were taken from

the position of each measured frond. Black and white negatives were scanned and

evaluated using Winphot software (ter Steege 1996). Calculated irradiance (sum-

mer/winter) ranged from 40/17 mol m-2

d-1

for a tree fern growing in open habitat to

13/3 mol m-2

d-1

for a tree fern growing under dense canopy near the creek.


Maximal quantum yield of photochemistry (Fv/Fm) was measured at predawn (5 a.m. in

summer, 6 a.m. in winter) with a pulse modulated fluorometer (OS-30p, Opti-Sciences,

Hudson, USA). Ground fluorescence (F0) was obtained with a low intensity modulated

light (600 Hz, 650nm, PPFD<1 µmol m-2

s-1

). Maximum fluorescence (Fm) was induced

by a saturating flash. Maximum efficiency of photosystem II (PSII) was estimated as

Fv/Fm = (Fm–F0)/Fm, nomenclature after Maxwell and Johnson (2000).


Light-saturated rates of net photosynthesis (Amax) and gs at Amax were measured using a

Li-Cor 6400 portable photosynthesis measurement system, equipped with a 2x3 cm

broadleaf chamber with red-blue LEDs (Li-Cor, Lincoln, Nebraska, USA). PPFD was

1500 µmol m-2

s-1

(determined as saturating by preliminary light response curves), air

flow rate was 400 µmol air s-1

, reference CO2 concentration 400 µmol mol-1

, and leaf




Australia

-97-

temperature and relative humidity were kept at ambient values. The frond area enclosed

in the Li-Cor chamber was marked, photographed and calculated using imaging soft-

ware (UTHSCSA Image Tool Version 3, University of Texas, USA).

Diurnal courses of gas exchange under ambient conditions were measured using the Li-

Cor transparent chamber. Air/leaf temperature and water pressure deficit based on leaf

temperature (VPD) were also recorded.

5.2.6. Frond water potential

Predawn frond water potential was chosen as a measure of plant water status, because it

is an appropriate estimate for the water availability in the soil reached by the roots

(Jones 1992).

Predawn (Ψ predawn) and midday (Ψ midday) frond water potentials were measured using a

pressure chamber (PMS Corvallis, OR, USA) at predawn 5 a.m./6 a.m. and midday 12

p.m./1 p.m. (summer/winter, respectively).

5.2.7. Frond traits

Specific leaf area (SLA) was calculated as the ratio of frond area over frond dry weight

(m2 kg

-1 dry weight). Fresh frond samples were collected, the frond area scanned and calcu-

lated using Scion Image software (Scion Corporation 2000-2001, USA), and frond ma-

terial was then dried at 60ºC for 48 h for dry weight. Different cohorts of fully-

expanded fronds produced in summer and winter, were used for calculation of SLA.

Frond samples were dried as described above and ground to a fine powder, then ana-

lysed for total nitrogen and carbon content using an elemental analyser (LECO CHN-

1000, Michigan, USA). Photosynthetic nitrogen use efficiency (PNUE) was calculated

as Amax divided by frond nitrogen content (on a frond area basis).




Australia

-98-

For measurements of frond chlorophyll content, four frond discs (diameter of 3.75 mm

each) were collected, immediately immersed in liquid nitrogen, and stored at -80ºC until

extraction. Chlorophyll a and b were extracted using 1.8 ml of 100% dimethyl sulphox-

ide (DMSO). Extracts were heated for 30 min at 65ºC in a dry block heater Termoline

L+M (Northgate, Queensland, Australia). The supernatant was then transferred to a

spectrophotometer Carry 300 (Varian, The Netherlands). A blank of pure DMSO was

used to calibrate the spectrophotometer at zero absorbance. Chlorophyll a, b and total

were calculated according to Wellburn (1994).

Fresh frond material (from 3-4 fronds per tree fern) was analysed for stomatal density

by variable pressure scanning electron microscopy (VP-SEM, model Leo 1450 VP; Leo

Electron Microscopy Inc., NY, USA). A flat cross-section of both sides of each frond

was scanned and stomatal density was calculated for four randomly chosen views (giv-

ing 12 to 16 stomatal density estimates per tree fern).


A general linear model of SPSS 15 (SPSS Inc. Chicago, USA) was used to analyse ef-

fects of species (D. antarctica, C. australis) and season (summer, winter) on each de-

pendent variable using the following covariates: estimated mean daily PPFD, leaf tem-

perature during gas exchange measurement, Tleaf, and predawn frond water potential Ψ

predawn (as a measure of plant water status).

Every variable was checked for normality using the Shapiro-Wilk test, and log trans-

formed if the assumption of normality was not satisfied. Relationships of covariates

with dependent variables were visually checked for linearity and differences in slopes

(see Fig. 5.2). In this analysis, the effect of season would only be significant if seasonal

differences were over and above the variation explained by seasonal differences in

growth irradiance, temperature, and water status as defined by the covariates.




Australia

-99-

For diurnal course measurements we used boundary-line analysis in a scatter- plot of all

data points of each parameter against the environmental variable in question. Functions

for boundary line analysis were chosen according to González-Rodríguez et al. (2001)

and Larcher (2003), with accuracy of fits evaluated using r2 (in all fits, r

2 >0.85).

5.3. Results

5.3.1. Relationships between photosynthesis, growth irradiance and tempera-

ture

Predawn quantum yield of photochemistry Fv/Fm never decreased below 0.7. It was

negatively related to Tleaf (significant covariate effect in Table 5.1), but did not differ

between the two species. It was significantly higher in summer, despite a negative rela-

tionship with Tleaf, reflecting a flatter slope in their relationship in summer than in win-

ter (Fig 5.2).

Tleaf was positively correlated with the light-saturated rate of net photosynthesis Amax

and stomatal conductance gs, while growth irradiance was significantly correlated to

Amax, but not gs (Table 5.1, Fig. 5.2). Amax and gs were similar for both species (Table

5.1), and were significantly higher in winter than in summer (Fig. 5.2).

Nitrogen content per frond area, NA was correlated with growth irradiance and Tleaf (Ta-

ble 5.1). NA was significantly greater in D. antarctica than in C. australis, and in winter

than in summer (Fig. 5.2). Photosynthetic nitrogen use efficiency PNUE (Amax/NA) did

not vary significantly between species and seasons and was not significantly affected by

any of the tested covariates (Table 5.2).

SLA was inversely related to growth irradiance (Table 5.1). In addition, C. australis

displayed significantly greater specific leaf area, and SLA was significantly greater in

summer than in winter (Fig. 5.2; Table 5.1).




Australia

-100-

None of the covariates had a significant effect on total chlorophyll or chlorophyll a/b

(Table 5.2). Total chlorophyll content (on an area basis) was significantly greater in D.

antarctica and in winter, while chlorophyll a/b ratios were comparable between species

and seasons (Table 5.2).

Table 5.1 Significance of the effect of fixed factors (species and season) and of covari-

ates (ANCOVA) on photosynthetic capacity parameters and frond traits of the tree ferns

D. antarctica and C. australis

Where: Fv/Fm, maximal quantum yield of photochemistry; Amax, light saturated rates of

net photosynthesis; gs, stomatal conductance at Amax; SLA, specific leaf area; NA, frond

nitrogen content on an area basis. Significance levels:*P<0.05; **P<0.01; ***P<0.001;

n.s., non significant; (+) positive relationship; (-) inverse relationship. No interactive

effects were significant

Effect and covari-

ates

Fv/Fm Amax gs SLA NA

Effect

Species n.s.(0.1) n.s (0.9) n.s. (0.1) * *

Seasons *** ** * ** **

Covariate

Mean daily PPFD

(mol m-2

d-1

) n.s. (0.08) (+)** n.s. (0.6) (-)* (+)**

Tleaf (ºC) (-)*** (+)** (+)* n.s. (0.2) (+)*

Ψ predawn (MPa) (+)* n.s. (0.4) n.s. (0.8) n.s. (0.08) n.s. (0.5)




Australia

-101-

Tleaf (oC)

0 10 20 30 40

Fv/F

m

0.68

0.70

0.72

0.74

0.76

0.78

0.80

0.82

0.84

0.86

Daily mean PPFD (mol m-2

d-1

)

0 10 20 30 40

SLA

(m

2kg

-1)

0

5

10

15

20

25

Am

ax (

mol m

-2s-1

)

0

2

4

6

8

10

12

14

16A

max (

mol m

-2s-1

)

0

2

4

6

8

10

12

14

16

gs (

mol m

-2s

-1)

0.00

0.05

0.10

0.15

0.20

0.25

NA (

g m

-2)

0

1

2

3

4

5

6

7

Fig. 5.2. Relationships between photosynthetic capacity parameters and frond traits of

the tree ferns D. antarctica and C. australis and environmental variables

Where: Amax - light saturated rates of net photosynthesis; gs, stomatal conductance at

Amax; Fv/Fm, maximum quantum yield of photosystem II; SLA, specific leaf area; NA,

frond nitrogen content on an area basis. D. antarctica (triangle) and C. australis (circle)

in winter (open symbols) and summer (closed symbols). Regressions are indicated by

dashed lines in summer, and solid lines in winter. All regressions were highly signifi-

cant (P<0.01). See Table 5.1 for significance of effects and relationships.


Volkova L, Bennett LT and Tausz M “Seasonal variations in photosynthesis of the tree ferns Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae)

in wet sclerophyll forests of Australia

-102-

Table 5.2. Photosynthetic capacity and water relation parameters of the tree ferns D. antarctica and C. australis in summer and winter. Values

are means (n = 32) ± s.e. of: PNUE, photosynthetic nitrogen use efficiency, Chl total, total chlorophyll content on a frond area basis; Chl a/b,

chlorophyll a/b ratio; WUEi, intrinsic water use efficiency; Ψ predawn and Ψ midday, predawn and midday frond water potentials. Significance lev-

els: **P<0.01; ***P<0.001; n.s., non-significant; n/a, non applicable; (+) positive relationship (covariates tested were mean daily PPFD, Tleaf, Ψ

predawn). No interactive effects were significant.

Parameter Summer Winter Effects

D. antarctica C. australis D. antarctica C. australis Species Seasons Covariates

PNUE

(µmol CO2 mol-1

N-1

s-1

)

39±5 37±7 33±6 51±11

n.s. (0.3) n.s. (0.7) n.s.

Chl total (µmol m-2

) 390±51 279±27 725±88 496±58 ** ** n.s.

Chl a/b 3.0±0.1 3.1±0.2 3.5±0.4 3.1±0.1 n.s. (0.7) n.s. (0.7) n.s.

Ψ predawn (MPa) 1 -0.6±0.1 -0.5±0.1 -0.1±0.0 -0.1±0.0 n.s. (0.5) *** n/a

Ψ midday (MPa) -1.1±0.1 -1.2±0.1 -0.5±0.1 -0.3±0.1 n.s. (0.9) *** n.s.

WUEi

(µmol CO2 mol-1

H2O-1

) 125±16 109±12 62±6 64±12

n.s. (0.2) n.s. (0.8) PPFD* (+)

1-t-test




Australia

-103-

5.3.2. Water status parameters

Predawn frond water potential (Ψ predawn) did not differ between species but was signifi-

cantly more negative in summer (t-test, P<0.001, Table 5.2). Fv/Fm was positively re-

lated to Ψ predawn (Table 5.1). Midday frond water potential (Ψ midday) was not related to

any of the tested covariates. It was similar between species but significantly more nega-

tive in summer than in winter (Table 5.2).

Growth irradiance had a significant positive effect on intrinsic water use efficiency

WUEi (Table 5.2). WUEi did not vary between species and seasons once covariates

were accounted for (Table 5.2).

5.3.3. Diurnal measurements

Instantaneous values of PPFD, leaf/air temperature, and leaf-to-air vapour pressure

deficit (VPD) at the measured fronds did not differ between species within season (t-test

P>0.1, data not shown). These variables differed significantly between seasons, with

mean daily PPFD, air temperature, and VPD in summer double that in winter (Fig.5.3).

Boundary-line analysis showed near-linear relationships between A and gs in summer

for both species, while in winter these relationships approached a saturation curve with

A reaching saturation at gs of ca 0.15 mol m-2

s-1

in both species (Fig. 5.4 a). VPD be-

came limiting to gs only at values greater than 1 kPa, which occurred only in summer

(Fig. 5.4 b). Optimal leaf temperature for photosynthesis in winter was on average 15ºC

for D. antarctica and 20ºC for C. australis, while in summer, A continued to rise at leaf

temperatures above 30ºC in both species (Fig. 5.4 c).

From light response curves fitted to the data of the diurnal measurements (Fig. 5.5),

Amax did not differ significantly between species but was significantly (P<0.05) higher

in summer than in winter (mean 17.4 vs. 8.6 µmol m-2

s-1

respectively). Light saturation

was reached at about 1100 in winter and nearly 1500 µmol photons m-2

s-1

in summer.




Australia

-104-

Time (h)

9:00 11:00 1:00 3:00 5:00

PP

FD

(

mol m

-2s

-1)

0

500

1000

1500

2000

T a

ir (

oC

)

0

10

20

30

9:00 11:00 1:00 3:00 5:00

WinterSummerR

H (

%)

0

20

40

60

80

100

Fig. 5.3. Climate conditions during diurnal course measurements in summer and winter.

Lines indicate means.




Australia

-105-

Tleaf

(oC)

0 10 20 30 40

A (

mo

l m

-2s

-1)

0

2

4

6

8

10

12

14

16

VPD (kPa)

0 1 2 3 4 5 6

gs (

mol m

-2 s

-1)

0.00

0.05

0.10

0.15

0.20

Cyathea australis

gs (mol m-2 s-1)

0.00 0.05 0.10 0.15 0.20

A (

mo

l m

-2s

-1)

0

2

4

6

8

10

12

14

16

a

b

c

Tleaf

,oC

5 10 15 20 25 30 35 40

gs, m

ol m

-2 s

-1

0.00

0.05

0.10

0.15

0.20

5 10 15 20 25 30 35 40

Dicksonia antarctica

0.00 0.05 0.10 0.15 0.20

0 1 2 3 4 5 6

0 10 20 30 40

Fig. 5.4. Relationships between photosynthesis, stomatal conductance and water pres-

sure deficit based on leaf temperature. Where: (a) Photosynthesis (A) versus stomatal

conductance (gs); (b) gs vs. water pressure deficit based on leaf temperature (VPD); (c)

A vs. leaf temperature (Tleaf) of the tree ferns C. australis (on the left) and D. antarctica

(right) in summer (open circles) and winter (closed triangles). Boundary-line fits are

indicated by dashed lines in summer and dotted lines in winter (for a and c only).




Australia

-106-

PAR, mol photons m-2

s-1

0 500 1000 1500 2000 2500

A,

mo

lCO

2 m

-2s

-1

-4

0

4

8

12

16

Cyathea australis

0 500 1000 1500 2000 2500


Fig. 5.5. Light response curves of the tree ferns D. antarctica and C. australis in sum-

mer and winter. Where: open circles – summer; closed circles – winter. Boundary-line

fits are indicated by dashed lines in summer and dotted lines in winter (r2 >0.9 in all

fits).

5.3.4. Stomatal density

Fronds of both species had no stomata on their adaxial (upper) surface. D. antarctica

displayed a more regular stomatal pattern in contrast to the random distribution of sto-

mata in C. australis (Fig. 5.6). Stomatal density did not differ significantly between

species (P=0.08) and was not correlated with growth irradiance (r =-0.007, P>0.9).

Leaf hairs were only observed on fronds of C. australis. Growth irradiance marginally

affected leaf hair density (P=0.06), which varied from 22 leaf hairs mm-2

on high light

exposed fronds to 11 leaf hairs mm-2

on shaded fronds, with an overall average of 13

hairs per mm-2

.

5.4. Discussion

5.4.1. Comparisons between the two tree fern species




Australia

-107-

Regardless of their putatively different origins and distribution patterns within forests,

the majority of photosynthetic parameters did not differ between our two tree fern spe-

cies. Light saturated rates of net photosynthesis (Amax) were similar and within the range

reported previously for D. antarctica (Hunt et al. 2002, Volkova et al. 2009). Specific

leaf areas of D. antarctica and C. australis were in the range of SLAs for humid tem-

perate and tropical forests (Reich et al. 1999).

Both D. antarctica and C. australis performed better in winter, when photosynthetic

rates reached higher values under similar irradiance and leaf temperatures. Both higher

stomatal conductance, through less diffusive resistance to CO2, and greater frond N con-

tent, via increased carboxylation capacity dependent on N-rich proteins such as Rubisco

(Niinemets and Tenhunen 1997), could jointly enable greater assimilation rates.

Chlorophyll a/b ratios did not vary between seasons, suggesting low acclimation of both

tree ferns for this parameter, consistent with previous observations of ferns in the genus

Trichomanes (Johnson et al. 2000).

5.4.2. Light as a limiting factor to tree fern photosynthetic performance

Under our field conditions light-saturation of photosynthesis was recorded at above

1100 µmol m-2

s-1

, considerably higher than previously reported for D. antarctica (600

µmol m-2

s-1

for plants grown under natural illumination in a glasshouse, and 150 µmol

m-2

s-1

for plants grown in a gully, Hunt et al. 2002). This might reflect physiological

acclimation to open canopy conditions created after logging (Kursar and Coley 1999),

or differences between studies related to plant and frond age.

Growth irradiance (measured via mean daily PPFD) had stimulating effects on Amax and

NA confirming results with other tree species (e.g. González-Rodríguez et al. 2001,

Oliveira and Peñuelas 2004, Niinemets 2007). Increase in WUEi (Amax/gs) with increas-

ing growth irradiance possibly reflected the strong relationship between light and Amax.




Australia

-108-

Linear relationships between A and gs, as observed in summer indicate that photosyn-

thesis was limited only by gs (Jones 1992), which, in turn, tended to decrease with in-

creases in VPD. In winter, A reached a plateau at ca gs 0.15 mol m-2

s-1

, indicating addi-

tional non-stomatal limitation of A (such as amount or activity of Rubisco or the rate of

electron transport, Massonnet et al. 2007).

In contrast to results from many other species (e.g. Romero and Botía 2006, Tazoe et al.

2009), gs at Amax (which reflects longer term response of gs to growth irradiance) was

insensitive to mean daily PPFD, indicating a lack of acclimation in gs to observed

growth irradiance for our tree fern species. In addition, there was no significant relation-

ship between instantaneous gs and instantaneous PPFD (as measured during the diurnal

measurements, P=0.9). This is in contrast to common knowledge that increasing light

induces gs until a saturation point is reached (Larcher 2003), a relationship commonly

used in models of stomatal responses. Such results may suggest poor responsiveness of

tree ferns‟ stomata to light and confirm observations of Doi and Shimazaki (2008),

when stomatal conductance of the fern Adiantum capillus-veneris showed much higher

light responsiveness when the light was applied to the lower leaf surface, where stomata

were situated, than when it was applied to the upper surface (as in our case). Similar

non-responsiveness of stomata to instantaneous light was found for Vicia faba (Mott et

al. 2008), leading to the conclusion that mesophyll signals play decisive roles in pre-

conditioning stomatal response to light.

The absence of significant relationship between the growth irradiance and Fv/Fm indi-

cates that PSII of both species fully recovered overnight regardless of received mean

daily PPFD.

Inverse relationship between the growth irradiance and SLA is consistent with the view

that sun leaves are more sclerophyllous (Groom and Lamont 1997, Niinemets 2007).

However, in contrast to the opinion that higher level of sclerophylly (i.e. decreased

SLA) results either from combinations of less water availability and high light intensi-

ties or from nutrient impoverished soils (for details see Groom and Lamont 1997), we




Australia

-109-

found lower SLA in winter‟s than summer‟s frond cohorts, when light intensities were

lower and water availability was greater. In agreement with Turner (1994) we can only

conclude that increase in sclerophylly was not related to increased drought tolerance or

acclimation to drier conditions of the tree ferns.

Overall, the growth irradiance stimulated photosynthetic capacity and nitrogen alloca-

tion in fronds, yet, it had little effect on stomata or PSII in these tree fern species.

5.4.3. Temperature as a limiting factor to tree fern photosynthetic performance

Both tree fern species displayed broad temperature optima for net photosynthesis simi-

lar to a wide range of C3 plants reviewed by Kattge and Knorr (2007). Optimum tem-

peratures were higher in summer, confirming acclimation potential of the tree ferns to

higher growth temperatures, consistent with our previous study (Volkova et al. 2009).

Inverse relationship of Tleaf with Fv/Fm reflects the sensitivity of PSII to high tempera-

tures, which is often cited as the most heat-sensitive component of photosynthesis in

temperate species (e.g. Berry and Björkman 1980). Over and above the effect of sea-

sonal differences in Tleaf, season also had a significant additional effect on Fv/Fm – at the

same Tleaf, Fv/Fm was significantly lower in winter than in summer (Fig. 5.2) – suggest-

ing some cold-induced limitation in PSII efficiency. Reduction of Fv/Fm during winter is

confirmed by numerous other studies (see Wittmann et al. 2007), and may be related to

either chilling-induced photo-degradation of PSII components, or overnight retention of

de-epoxidised xanthophylls (Adams and Demmig- Adams 1994). Furthermore, Larcher

and Nagele (1992) demonstrated that photosynthetic capacity of Fagus sylvatica stems

decreased in winter and even short-term rewarming treatments could not restore it to

summer values. Thus, we may suggest that in winter, the tree ferns may experience per-

sistent photoinhibition of PSII induced by cold morning temperatures that are common

for this study site.




Australia

-110-

Significant positive relations between NA and temperature might reflect changes in soil

nutrient mineralisation and thus N availability with temperature. In addition, a study by

Muller et al. (2009) indicated that temperature rather than irradiance primarily deter-

mined changes in NA in natural conditions for broad-leaved evergreen species.

Evaporative cooling through increased stomatal conductance and associated transpira-

tion implies a negative relationship between Tleaf and gs (see Snider et al. 2009). In con-

trast, we observed a positive relationship of Tleaf with gs, which may indicate that in our

study area gs is affected by low rather than high temperatures. Stomatal closure can also

occur when the water supply from the roots is restricted because of low temperatures in

the root zone (Davies et al. 1982).

5.4.4. Effects of plant water status and water relation parameters on tree fern

photosynthetic performance

Plant water status (measured via Ψ predawn) showed no significant relationship with any

of the measured parameters except Fv/Fm. Apparently, slightly, albeit significantly, more

negative Ψ predawn in summer (-0.05 MPa) relative to winter (-0.01 MPa) was not enough

to impose significant limitations on any of the measured variables. In agreement with

their ecological distribution in moist sites, tree ferns may be unable to tolerate a larger

drop in water potential, instead relying on an „avoidance‟ strategy involving frond loss,

as indicated in another open-air study by the first author (yet unpublished data). Sensi-

tivity of Fv/Fm to plant water status suggests that the efficiency of the photosystem

likely decreased due to reduced efficiency of the light-harvesting and antenna com-

plexes to deliver quanta to reaction centres (Wright et al. 2009).

No effect of season on intrinsic water use efficiency (WUEi) indicates prodigal or water

spending strategy by both species – the strategy, appropriate for plants that are subject

to droughts of short duration (Passioura 1982). Ferns are known for poor efficiency in

water transport through the leaf (Sack and Holbrook 2006), and together with the ob-




Australia

-111-

served low acclimation potential of WUEi to seasonal fluctuations illustrates why many

fern species are confined to moist environments (Franks and Farquhar 1999).

5.4.5. Stomatal density

Stomatal patterns but not density differed between our two tree fern species. In contrast

to many angiosperms, where stomatal density is higher in leaves from sunny habitats

(Larcher 2003), stomatal density of both tree fern species did not correlate with the

growth irradiance. A similar lack of relationship between stomatal density and the

growth irradiance was observed for tree ferns of Cyatheaceae family by Arens (1997),

and for whole fern communities by Kessler et al. (2007). Our results indicate that tree

ferns (Pteridophyte) lack acclimation in terms of changing stomatal density in response

to their light environment.

Leaf hairs, known to reduce leaf temperature due to their reflection function (Lambers

et al. 2008) or to protect against UV-B radiation (Grammatikopoulos et al. 1994), were

found only in C. australis, possibly reflecting its microclimate preferences and assumed

Pan-tropical origin (i.e. adaptation to greater light intensities).




Australia

-112-


High light exposed habitat Shaded habitat

a b

Cyathea australis

High light exposed habitat Shaded habitat

c d

Fig. 5.6. Stomatal density of the tree ferns (a, b) D. antarctica and (c, d) C. australis

from (a, c) light-exposed and (b, d) shaded habitats.




Australia

-113-

5.5. Summary

Regardless of different origins and micro-site habitats, there were no significant differ-

ences in terms of photosynthetic performance between D. antarctica and C. australis,

and both tree fern species had greater photosynthetic capacity in winter. Low tempera-

tures appeared to be most limiting factor on plant performance under field conditions.

Plant water status did not vary markedly and had no effect on any of the measured pa-

rameters. Similar to other plants, both species of tree ferns displayed seasonal acclima-

tion in a number of measured photosynthetic parameters and frond traits (i.e. Fv/Fm,

Amax, gs, NA, chlorophyll total, SLA). Acclimation of stomatal density to spatial varia-

tion in growth irradiance among micro-sites seemed limited in both species, while

stomatal pattern differed between species.

Because there were no significant differences between the two species in photosynthetic

parameters, both species can probably be described by common carbon gain and water

use models at the leaf scale.

-114-

.

-115-

Chapter 6. Ecophysiology of two tree fern species and implications for

their future management. General discussion and conclusions

6.1. Species overview

The maximum amount of carbon fixed per unit leaf area and time or Amax (Taiz and

Zeiger 2002) is believed to be low in ferns compared with many other plants (Nobel et

al. 1984). In fact, both tree fern species in all of my studies had maximal photosynthetic

rates similar to a range of tree species in comparable ecosystems (e.g. middle-storey

canopy species Nothofagus cunninghamii Oerst (Tausz et al. 2005), Canarian laurel for-

est tree species Persea indica (González-Rodríguez et al. 2002), or Laurus azorica

(González-Rodríguez et al. 2001, Chapters 2-5).

According to von Caemmerer and Farquhar (1981), changes in Amax reflect changes in

both stomatal conductance (gs) and mesophyll capacity parameters (i.e. Vcmax and Jmax).

Stomatal conductance was greater in C. australis than D. antarctica at the beginning of

the water stress experiment (Chapter 3), but was comparable between two species in all

other experiments (Chapters 2 and 5, with an average of 100 mmol m-2

s-1

). The maxi-

mum carboxylation rate, Vcmax, or in vivo apparent Rubisco activity, was comparable in

both species across all experiments (Chapters 2, 4), while the maximum rate of electron

transport, Jmax, was consistently higher in D. antarctica than C. australis (Chapters 2,

4). Overall, both species had relatively low values of Vcmax and Jmax comparable with

some shade tolerant tree species, such as silver fir (Abies alba Mill, Robakowski et al.

2002, Chapters 2-5).

Stable isotope composition, δ13

C, is related to the ratio of internal to external CO2 con-

centrations (Ci/Ca), and therefore can be a useful tool in assessing intrinsic water use

efficiency, WUEi (Dawson et al. 2002). Lowest δ13

C assumes greater WUE (Farquhar

et al. 1989). δ13

C values of both tree fern species were within the ranges reported for C3

plants (-20 to -35‰, Dawson et al. 2002). However, despite lower δ13

C in C. australis‟s

-116-

fronds (closer to -30‰, Chapter 4), it was D. antarctica that had significantly greater

instantaneous (i. e. measured by gas exchange) WUEi. Values for WUEi in juveniles of

both species (range of 43-80µmol CO2 mol-1

H2O; Chapter 4) were comparable with

those recorded for mature tree ferns in the field study (Chapter 5), yet they were double

those recorded for an evergreen herbaceous fern of the family Adiantaceae Adiantum

reniforme var. sinensis (Liao et al. 2008).

Specific leaf area (SLA) is an index of sclerophylly (Lamont et al. 2002) and tends to be

higher in tropical and humid temperate forests (Reich et al. 1999). D. antarctica had

consistently lower SLA than C. australis across several experiments (e.g. 8.6 vs. 10.4

m2kg

-1 respectively). However, compared to a broad range of species, both species had

SLA similar to native Hawaiian tree ferns of the genus Cibotium (C. chamissoi), grown

in semi-wet to wet forests (Durand and Goldstein 2001).

Concentrations of chlorophylls were comparable between tree fern species in the first

glasshouse experiment (high light stress), but were greater in D. antarctica than C. aus-

tralis in other experiments involving older plants (Chapters 4, 5). Total chlorophyll

concentrations (per leaf area) of both species were comparable with a wide range of tree

species (e.g. shade grown Fagus sylvatica (Lichtenthaler 2007). Absolute values of total

chlorophyll concentrations only significantly differed in the second experiment, which

examined high light and heat interactions (Chapter 3). This could be due to the different

methods used to determine chlorophyll concentrations (HPLC vs. LECO), and possibly

due to plant age (Louis et al. 2009). Chlorophyll a/b ratio was comparable between the

two species in mature, field plants (Chapter 5), but was consistently higher in D. antarc-

tica than C. australis in juvenile plants (Chapters 2, 4) due to greater chlorophyll a con-

centrations.

Overall, my project found that the photosynthetic characteristics of D. antarctica and C.

australis were comparable and within reported ranges for a number of plant types. Thus,

the different origins of the two species, and their apparent preference for different mi-

-117-

cro-sites, do not appear to have resulted in a divergence of their physiological responses

to a range of environmental stresses.

6.2. Overview of light, temperature, and water availability as stresses on tree fern

physiology

My results in Chapter 2 (high light stress) indicated immediate down-regulation of pho-

tosynthetic capacity parameters by tree ferns in response to high light, but acclimation

to high light after prolonged (three month) exposure. These results are consistent with

the view that high light alone does not have detrimental effect on photosynthesis and

plants can often fully recover (Levitt 1980, Lovelock et al. 1994).

In Chapter 3, I examined interactive effects of high light and moderate heat on photo-

synthetic capacity of D. antarctica. Here, combination of high light and moderate heat

led to negative effects including chlorophyll bleaching and severe photoinhibition

(measured as a decrease in Fv/Fm<0.4). Given the comparable responses to stresses by

both tree fern species in all other studies (see above), it is reasonable to assume that

high light with moderate heat would be equally problematic for C. australis. This result

of detrimental effects of light by temperature interactions supports findings of many

other studies (e.g. Al-Khatib and Paulsen 1999, Montgomery et al. 2008), although it is

in contrast to Havaux et al. (1991), who stated that high light could alleviate negative

effects of high temperatures.

In Chapter 4, I studied interactive effects of high light and water deficit on photosyn-

thetic capacity and water relation parameters of both species D. antarctica and C. aus-

tralis. It appeared that both species were susceptible to water deficit either alone or in

interaction with high light. This funding was in contrast with Levitt (1980) stating that

interactive effect of high light and water deficit can be more detrimental than water

deficit alone. Stomatal response to water withheld was slower in shade-grown than in

high light exposed plants, confirming the findings with bracken fern where stomata

from the most shaded understorey level were less sensitive to soil moisture treatments

-118-

than those from more irradiated levels (Roberts et al. 1984). Probably as a result of it

there were some indications that shade did not ameliorate but rather intensified drought

effects on D. antarctica, the result confirming observation of Valladares et al. (2002)

who stated that the capacity to withstand severe drought was not enhanced in the shade

but decreased due to increased below-ground competition for water with established

trees.

The focus of Chapter 5 was on mature tree ferns growing in their natural environment,

the mountain ash forests of the Victorian Central Highlands, Australia. Measurements

of photosynthetic capacity as well as water relations and frond parameters over two

consecutive years indicated that both species, irrespective of their different origins and

apparent differences in micro-site preferences, performed better in winter than in sum-

mer. Low light and low temperatures were limiting factors for the tree ferns perform-

ance in the field. In contrast to many reports in the literature (e.g. Romero and Botía

2006, Tazoe et al. 2009), stomatal conductance of the tree ferns did not correlate with

growth irradiance but was positively correlated only with temperature. Plant water

status (Ψ predawn) had no effect on any of the measured parameters (except maximal

quantum yield of photochemistry, Fv/Fm), possibly because decreases in Ψ predawn to -

0.05 MPa were not enough to impose limitations. Another interpretation is that tree

ferns lack the ability to adjust to decreased water availability and cannot endure more

pronounced drops in tissue water potentials. This may also explain observations in both,

field and glasshouse, plants, where ferns shed fronds under more severe water deficit.

Both species showed characteristics of a water spending strategy irrespective of season

(summer or winter), i.e. WUEi in summer, when temperature was higher and less water

available, was comparable to WUEi in winter, a season characterised by lower tempera-

tures and greater rainfalls. Such prodigal strategy is common and effective for plants

that only occasionally experience drought (Passioura 1982). Using this interpretation,

results of this field Chapter were consistent with indications of low acclimation of the

tree ferns to water availability found in the controlled experiment involving juvenile

plants (Chapter 4). Tree ferns growing at open sites showed little difference in stomatal

-119-

density from those growing under dense canopy cover, indicating a lack of acclimation

of stomatal density in the tree ferns to growth irradiance. This is in contrast to observa-

tions on many other tree species (Larcher 2003), but consistent with findings of Arens

(1997) on the tree fern Cyathea caracasana.

6.3. Practical implications and future directions

Despite their ecological importance as dominant understorey species in Australia‟s wet

sclerophyll forests, the physiology of D. antarctica and C. australis has previously been

under-examined. Understorey plants like these two species may account for as much as

33% of living above-ground biomass (Weaver 2008), and, in evergreen forests world-

wide, contribute on average 49% of total ecosystem respiration (Misson et al. 2007).

Thus, the paucity of knowledge about tree fern physiology not only limits our ability to

predict potential impacts of climate change on their distribution but also excludes a sig-

nificant component of temperate forests from total carbon balance calculations (i.e. by

excluding a significant portion of potential carbon sinks and sources).

Two experiments and one observational field study (Chapters 2, 4 and 5) indicated that

D. antarctica and C. australis are fairly similar with respect to (eco)physiological and

morphological characteristics. Correspondingly, growth and carbon sequestration mod-

els could use a common set of parameters for both species, which would greatly sim-

plify the task of including tree ferns in process-based carbon accounting or environ-

mental risk assessment models. Moreover, because D. antarctica and C. australis be-

long to the two main tree fern families Dicksoniaceae and Cyatheaceae – arguably the

most important tree fern families worldwide – the knowledge created in this thesis will

be a sound starting point for understanding the ecophysiology of other tree ferns across

a potentially broad range of environmental conditions.

The problem of poor survival and ongoing decline of the tree fern numbers after clear-

cut logging (Ought and Murphy 1998) was one of the main inspirations for this study.

My experimental results, particularly Chapter 3 (high light and high temperature), indi-

-120-

cate detrimental effects of combined high light and moderate temperatures on tree fern

health, which should be considered when designing logging configurations. For exam-

ple, understorey islands should be designed to minimise tree fern numbers near edges

where they will be exposed to greater light intensities and consequently higher tempera-

tures. This could involve consideration of a minimum distance to edge for tree fern

populations of considerable numbers.

The results of Chapter 4 (high light and water deficit) reiterated the importance to tree

ferns of retaining buffer zones along waterways. This experiment also indicated that re-

tention of understorey islands in the middle of logging coupes away from waterways,

might not lead to substantial improvement in the protection of tree ferns in logging

coupes under climate change predictions of increasing drought frequency. Indeed, my

results indicated that shade led to slower stomatal responses in D. antarctica thereby

intensifying drought stress. Thus, design of understorey islands should take tree fern

needs for access to water into consideration, and should thus be placed on lower rather

than upper slopes.

Climate change predictions indicate that the severity and frequency of both droughts

and fire will increase in south-eastern Australia (Hennessy et al. 2007), leading to

changes in species distributions (see Fitzpatrick et al. 2008). Thus, species better

adapted to drought and fire could expand their range, while other elements of native

vegetation might be at risk (Watt et al. 2009). Findings from my study indicate that the

range of tree ferns in south-eastern Australia may contract under a more irradiated (i.e.

due to fire-induced canopy death), warmer, and drier climate. In addition, the current

divergence in micro-site preferences between the two studied species might become less

evident. That is, current localised extensions of C. australis to forest margins might

contract to more shaded, moister sites to be closer to the current distribution of D. ant-

arctica. This emphasises the need for buffer zones along waterways, which under future

climates could be considered refugia for tree ferns – that is, isolated areas of habitat that

retain the environmental conditions that were once widespread, Stewart and Lister

2001).

-121-

While my study provides useful indications of tree fern responses to environmental

stresses, it was limited to photosynthetic capacity parameters at the leaf-level scale. To

increase accuracy of carbon models, future studies should consider dynamics of frond

development, including frond longevity, and the potentially different physiology of

fronds of different age cohorts. In addition, precise micro-climate data will be another

essential component for improved modelling of tree fern ecophysiology and of under-

storey plants in general. Modelling at landscape to regional scale would be of particular

benefit to conservation goals, but scaling errors need to be considered.

Overall, my study revealed that both species of the tree ferns might be vulnerable in the

future because of their low acclimation potential to environmental stresses. These find-

ings highlight the need to develop more flexible conservation policies to maintain tree

fern communities under optimal conditions where they can be most resilient to both

predicted and unexpected future changes.

-123-

REFERENCES

Adams WW, Demmig-Adams B (1994) Carotenoid composition and down-regulation

of photosystem II in 3 conifer species during the winter. Physiologia Plantarum

92, 451-458.

Al-Khatib K, Paulsen GM (1999) High-temperature effects on photosynthetic processes

in temperate and tropical cereals. Crop Science 39, 119-125.

Arens NC (1997) Responses of leaf anatomy to light environment in the tree fern Cyat-

hea caracasana (Cyatheaceae) and its application to some ancient seed ferns.

Palaios 12, 84-94.

Ashton DH (2000) The Big Ash Forest, Wallaby Creek, Victoria - changes during one

lifetime. Australian Journal of Botany 48, 1-26.

Attiwill PM, Fewings JM (2001) 'Sustainable management of the Mountain ash (Euca-

lyptus regnans F. Muell) forests in Central Highlands, Victoria, Australia.'

(Blackwell science)

Bannister P, Wildish KL (1982) Light compensation points and specific leaf areas in

some New-Zealand ferns. New Zealand Journal of Botany 20, 421-424.

Barlow BA (1994) Phytogeography of the Australian region. In 'Australian vegetation'.

(Ed. RH Groves) pp. xvii, 562 p. (Cambridge University Press: Cambridge,

[England]; Oakleigh, Vic.)

Bennett LT, Adams MA (2004) Assessment of ecological effects due to forest harvest-

ing: approaches and statistical issues. Journal of Applied Ecology 41, 585-598.

Berry J, Björkman O (1980) Photosynthetic response and adaptation to temperature in

higher plants. Annual Review of Plant Physiology and Plant Molecular Biology

31, 491-543.

Blows MW, Schwarz MP (1991) Spatial-distribution of a primitively social bee - does

genetic population-structure facilitate altruism. Evolution 45, 680-693.

Bogeat-Triboulot MB, Brosche M, Renaut J, Jouve L, Le Thiec D, Fayyaz P, Vinocur

B, Witters E, Laukens K, Teichmann T, Altman A, Hausman JF, Polle A, Kan-

gasjarvi J, Dreyer E (2007) Gradual soil water depletion results in reversible

changes of gene expression, protein profiles, ecophysiology, and growth per-

formance in Populus euphratica, a poplar growing in arid regions. Plant Physiol-

ogy 143, 876-892.

Bota J, Medrano H, Flexas J (2004) Is photosynthesis limited by decreased Rubisco ac-

tivity and RuBP content under progressive water stress? New Phytologist 162,

671-681.

Brodribb TJ, Feild TS, Jordan GJ (2007) Leaf maximum photosynthetic rate and vena-

tion are linked by hydraulics. Plant Physiology 144, 1890-1898.

Brodribb TJ, Holbrook NM, Zwieniecki MA, Palma B (2005) Leaf hydraulic capacity

in ferns, conifers and angiosperms: impacts on photosynthetic maxima. New

Phytologist 165, 839-846.

Callister AN, Arndt SK, Adams MA (2006) Comparison of four methods for measuring

osmotic potential of tree leaves. Physiologia Plantarum 127, 383-392.

-124-

Campbell ML, Clarke PJ (2006) Response of montane wet sclerophyll forest understo-

rey species to fire: Evidence from high and low intensity fires. Proceedings of

the Linnean Society of New South Wales 127, 63-73.

Casson S, Gray JE (2008) Influence of environmental factors on stomatal development.

New Phytologist 178, 9-23.

Chaves MM, Maroco JP, Pereira JS (2003) Understanding plant responses to drought -

from genes to the whole plant. Functional Plant Biology 30, 239-264.

Davies S (2005) Harvesting of Manferns (Dicksonia antarctica) in Tasmania. Forest

Practices News 6 5-6.

Davies WJ, Rodriguez JL, Fiscus EL (1982) Stomatal behavior and water movement

through roots of wheat plants treated with abscisic acid. Plant Cell and Envi-

ronment 5, 485-493.

Dawson TE, Mambelli S, Plamboeck AH, Templer PH, Tu KP (2002) Stable isotopes in

plant ecology. Annual Review of Ecology and Systematics 33, 507-559.

De Costa W, Zörb C, Hartung W, Schubert S (2007) Salt resistance is determined by

osmotic adjustment and abscisic acid in newly developed maize hybrids in the

first phase of salt stress. Physiologia Plantarum 131, 311-321.

Demmig-Adams B, Adams WW (2006) Photoprotection in an ecological context: the

remarkable complexity of thermal energy dissipation. New Phytologist 172, 11-

21.

Department of Natural Resources and Environment (2002) Victorian tree fern manage-

ment plan. Department of Natural Resources and Environment, East Melbourne,

Australia.

Dieleman JA, Meinen E (2007) Interacting effects of temperature integration and light

intensity on growth and development of single-stemmed cut rose plants. Scientia

Horticulturae 113, 182-187.

Dignan P, Bren L (2003) A study of the effect of logging on the understorey light envi-

ronment in riparian buffer strips in a south-east Australian forest. Forest Ecology

and Management 172, 161-172.

Doi M, Shimazaki KI (2008) The stomata of the fern Adiantum capillus-veneris do not

respond to CO2 in the dark and open by photosynthesis in guard cells. Plant

Physiology 147, 922-930.

Doi M, Wada M, Shimazaki K (2006) The fern Adiantum capillus-veneris lacks

stomatal responses to blue light. Plant and Cell Physiology 47, 748-755.

Downton WJS, Berry JA, Seemann JR (1984) Tolerance of photosynthesis to high tem-

perature in desert plants. Plant Physiology 74, 786-790.

Dreyer E, Le Roux X, Montpied P, Daudet FA, Masson F (2001) Temperature response

of leaf photosynthetic capacity in seedlings from seven temperate tree species.

Tree Physiology 21, 223-232.

Ducruet JM, Peeva V, Havaux M (2007) Chlorophyll thermofluorescence and ther-

moluminescence as complementary tools for the study of temperature stress in

plants. Photosynthesis Research 93, 159-171.

Durand LZ, Goldstein G (2001) Photosynthesis, photoinhibition, and nitrogen use effi-

ciency in native and invasive tree ferns in Hawaii. Oecologia 126, 345-354.

Epron D, Dreyer E (1992) Effects of severe dehydration on leaf photosynthesis in

Quercus-petraea (Matt) Liebl - photosystem-II efficiency, photochemical and

-125-

nonphotochemical fluorescence quenching and electrolyte leakage. Tree Physi-

ology 10, 273-284.

Ethier GJ, Livingston NJ (2004) On the need to incorporate sensitivity to CO2 transfer

conductance into the Farquhar-von Caemmerer-Berry leaf photosynthesis model.

Plant Cell and Environment 27, 137-153.

Farquhar GD, Ehleringer JR, Hubick KT (1989) Carbon isotope discrimination and pho-

tosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology

40, 503-537.

Farquhar GD, von Caemmerer S, Berry JA (1980) A biochemical model of photosyn-

thetic CO2 assimilation in leaves of C3 species. Planta 149, 78-90.

Fitzpatrick MC, Gove AD, Sanders NJ, Dunn RR (2008) Climate change, plant migra-

tion, and range collapse in a global biodiversity hotspot: the Banksia (Pro-

teaceae) of Western Australia. Global Change Biology 14, 1337-1352.

Flexas J, Escalona JM, Medrano H (1998) Down-regulation of photosynthesis by

drought under field conditions in grapevine leaves. Australian Journal of Plant


Flexas J, Escalona JM, Medrano H (1999) Water stress induces different levels of pho-

tosynthesis and electron transport rate regulation in grapevines. Plant Cell and

Environment 22, 39-48.

Flexas J, Medrano H (2002) Drought-inhibition of photosynthesis in C3 plants: Stomatal

and non-stomatal limitations revisited. Annals of Botany 89, 183-189.

Flexas J, Ribas-Carbó M, Bota J, Galmés J, Henkle M, Martínez-Cañellas S, Medrano

H (2006) Decreased Rubisco activity during water stress is not induced by de-

creased relative water content but related to conditions of low stomatal conduc-

tance and chloroplast CO2 concentration. New Phytologist 172, 73-82.

Franks PJ, Farquhar GD (1999) A relationship between humidity response, growth form

and photosynthetic operating point in C3 plants. Plant Cell and Environment 22,

1337-1349.

Froux F, Ducrey M, Epron D, Dreyer E (2004) Seasonal variations and acclimation po-

tential of the thermostability of photochemistry in four Mediterranean conifers.

Annals of Forest Science 61, 235-241.

Galle A, Haldimann P, Feller U (2007) Photosynthetic performance and water relations

in young pubescent oak (Quercus pubescens) trees during drought stress and re-

covery. New Phytologist 174, 799-810.

García-Plazaola JI, Becerril JM (1999) A rapid high performance liquid chromatogra-

phy method to measure lipophilic antioxidants in stressed plants: Simultaneous

determination of carotenoids and tocopherols. Phytochemical Analysis 10, 307-

313.

Ghouil H, Montpied P, Epron D, Ksontini M, Hanchi B, Dreyer E (2003) Thermal op-

tima of photosynthetic functions and thermostability of photochemistry in cork

oak seedlings. Tree Physiology 23, 1031-1039.

Gildner BS, Larson DW (1992) Photosynthetic response to sunflecks in the desiccation-

tolerant fern Polypodium virginianum. Oecologia 89, 390-396.

González-Rodríguez AM, Morales D, Jiménez MS (2001) Gas exchange characteristics

of a Canarian laurel forest tree species (Laurus azorica) in relation to environ-

mental conditions and leaf canopy position. Tree Physiology 21, 1039-1045.

-126-

González-Rodríguez AM, Morales D, Jiménez MS (2002) Leaf gas exchange character-

istics of a Canarian laurel forest tree species [Persea indica (L.) K. Spreng.] un-

der natural conditions. Journal of Plant Physiology 159, 695-704.

Grammatikopoulos G, Karabourniotis G, Kyparissis A, Petropoulou Y, Manetas Y

(1994) Leaf hairs of olive (Olea europaea) prevent stomatal closure by Ultravio-

let-B radiation. Australian Journal of Plant Physiology 21, 293-301.

Groom PK, Lamont BB (1997) Xerophytic implications of increased sclerophylly: in-

teractions with water and light in Hakea psilorrhyncha seedlings. New Phytolo-

gist 136, 231-237.

Guo XR, Cao KF, Xu ZF (2006) Acclimation to irradiance in seedlings of three tropical

rain forest Garcinia species after simulated gap formation. Photosynthetica 44,

193-201.

Haldimann P (1999) How do changes in temperature during growth affect leaf pigment

composition and photosynthesis in Zea mays genotypes differing in sensitivity to

low temperature? Journal of Experimental Botany 50, 543-550.

Havaux M, Greppin H, Strasser RJ (1991) Functioning of photosystem I and photosys-

tem II in pea leaves exposed to heat-stress in the presence or absence of light -

analysis using in vivo fluorescence, absorbency, oxygen and photoacoustic

measurements. Planta 186, 88-98.

Havaux M, Gruszecki WI (1993) Heat-induced and light-induced chlorophyll a fluores-

cence changes in potato leaves containing high or low levels of the carotenoid

zeaxanthin - indications of a regulatory effect of zeaxanthin on thylakoid mem-

brane fluidity. Photochemistry and Photobiology 58, 607-614.

Havaux M, Tardy F (1996) Temperature-dependent adjustment of the thermal stability

of photosystem II in vivo: Possible involvement of xanthophyll cycle pigments.

Planta 198, 324-333.

Havaux M, Tardy F, Ravenel J, Chanu D, Parot P (1996) Thylakoid membrane stability

to heat stress studied by flash spectroscopic measurements of the electrochromic

shift in intact potato leaves: Influence of the xanthophyll content. Plant Cell and

Environment 19, 1359-1368.

Hennessy KB, Fitzharris BC, Bates N, Harvey SM, Howden L, Hughes J, Salinger and

R. Warrick (2007) Australia and New Zealand. In 'Climate Change 2007: Im-

pacts, Adaptation and Vulnerability. Contribution of Working Group II to the

Fourth Assessment Report of the Intergovernmental Panel on Climate Change'.

(Eds ML Parry, OF Canziani, JP Palutikof, PJ van der Linden and CE Hanson)

pp. 507-540. (Cambridge University Press, Cambridge, UK)

Herbinger K, Then C, Haberer K, Alexou M, Löw M, Remele K, Rennenberg H,

Matyssek R, Grill D, Wieser G, Tausz M (2007) Gas exchange and antioxidative

compounds in young beech trees under free-air ozone exposure and comparisons

to adult trees. Plant Biology 9, 288-297.

Hickey JE (1994) A floristic comparison of vascular species in Tasmanian old growth

mixed forest with regeneration resulting from logging and wildfire. Australian

Journal of Botany 42, 383-404.

Hoober JK, Eggink LL, Chen M (2007) Chlorophylls, ligands and assembly of light-

harvesting complexes in chloroplasts. Photosynthesis Research 94, 387-400.

-127-

Howard TM (1973) Studies in the ecology of Nothofagus Cunninghamii forest. Part 3.

Two limiting factors: light intensity and water stress. Australian Journal of Bot-

any 21, 93-102.

Hunt MA, Davidson NJ, Unwin GL, Close DC (2002) Ecophysiology of the Soft Tree

Fern Dicksonia antarctica Labill. Austral Ecology 27, 360-368.

Hüve K, Bichele I, Tobias M, Niinemets Ü (2006) Heat sensitivity of photosynthetic

electron transport varies during the day due to changes in sugars and osmotic po-

tential. Plant Cell and Environment 29, 212-228.

Johnson GN, Rumsey FJ, Headley AD, Sheffield E (2000) Adaptations to extreme low

light in the fern Trichomanes speciosum. New Phytologist 148, 423-431.

Jones DL, Clemesha SC (1993) 'Australian Ferns and fern allies.' (The Currawong

Press)

Jones HG (1992) 'Plants and microclimate: a quantitative approach to environmental

plant physiology.' (Cambridge University Press: Cambridge [England] ; New

York, NY, USA)

Kattge J, Knorr W (2007) Temperature acclimation in a biochemical model of photo-

synthesis: a reanalysis of data from 36 species. Plant Cell and Environment 30,

1176-1190.

Keith H, Mackey BG, Lindenmayer DB (2009) Re-evaluation of forest biomass carbon

stocks and lessons from the world's most carbon-dense forests. Proceedings of

the National Academy of Sciences 106, 11635-11640.

Kessler M, Siorak Y, Wunderlich M, Wegner C (2007) Patterns of morphological leaf

traits among pteridophytes along humidity and temperature gradients in the Bo-

livian Andes. Functional Plant Biology 34, 963-971.

Kirchgeßner HD, Reichert K, Hauff K, Steinbrecher R, Schnitzler JP, Pfündel EE

(2003) Light and temperature, but not UV radiation, affect chlorophylls and ca-

rotenoids in Norway spruce needles (Picea abies (L.) Karst.). Plant Cell and En-

vironment 26, 1169-1179.

Kursar TA, Coley PD (1999) Contrasting modes of light acclimation in two species of

the rainforest understory. Oecologia 121, 489-498.

Lambers H, Chapin FS, Pons TL (2008) 'Plant physiological ecology.' (Springer: New

York)

Lamont BB, Groom PK, Cowling RM (2002) High leaf mass per area of related species

assemblages may reflect low rainfall and carbon isotope discrimination rather

than low phosphorus and nitrogen concentrations. Functional Ecology 16, 403-

412.

Larcher W (2003) 'Physiological plant ecology: ecophysiology and stress physiology of

functional groups.' (Springer: New York)

Larcher W, Nagele M (1992) Changes in photosynthetic activity of buds and stem tis-

sues of Fagus-Sylvatica during winter. Trees-Structure and Function 6, 91-95.

Large MF, Braggins JE (2004) 'Tree ferns.' (CSIRO publishing: Collingwood, Vic.)

Law RD, Crafts-Brandner SJ (1999) Inhibition and acclimation of photosynthesis to

heat stress is closely correlated with activation of ribulose-1,5-bisphosphate car-

boxylase/oxygenase. Plant Physiology 120, 173-181.

Levitt J (1980) 'Responses of plants to environmental stresses.' (Academic Press: New

York)

-128-

Liao JX, Jiang MX, Huang HD (2008) Effects of soil moisture on ecophysiological

characteristics of Adiantum reniforme var. sinensis, an endangered fern endemic

to the Three Gorges region in China. American Fern Journal 98, 26-32.

Lichtenthaler HK (1987) Chlorophylls and carotenoids - pigments of photosynthetic

biomembranes. Methods in Enzymology 148, 350-382.

Lichtenthaler HK (2007) Biosynthesis, accumulation and emission of carotenoids, al-

pha-tocopherol, plastoquinone, and isoprene in leaves under high photosynthetic

irradiance. Photosynthesis Research 92, 163-179.

Lindenmayer DB, Cunningham RB, Donnelly CF, Triggs BJ, Belvedere M (1994) The

conservation of arboreal marsupials in the montane ash forests of the central

highlands of Victoria, South-Eastern Australia. 5. Patterns of use and the micro-

habitat requirements of the mountain brushtail possum Trichosurus caninus

ogilby in retained linear habitats (wildlife corridors). Biological Conservation

68, 43-51.

Llusià J, Peñuelas J, Munné-Bosch S (2005) Sustained accumulation of methyl salicy-

late alters antioxidant protection and reduces tolerance of holm oak to heat

stress. Physiologia Plantarum 124, 353-361.

Long SP, Bernacchi CJ (2003) Gas exchange measurements, what can they tell us about

the underlying limitations to photosynthesis? Procedures and sources of error.

Journal of Experimental Botany 54, 2393-2401.

Long SP, Humphries S, Falkowski PG (1994) Photoinhibition of photosynthesis in na-

ture. Annual Review of Plant Physiology and Plant Molecular Biology 45, 633-

662.

Louis J, Meyer S, Maunoury-Danger F, Fresneau C, Meudec E, Cerovic ZG (2009)

Seasonal changes in optically assessed epidermal phenolic compounds and chlo-

rophyll contents in leaves of sessile oak (Quercus petraea): towards signatures

of phenological stage. Functional Plant Biology 36, 732-741.

Lovelock CE, Jebb M, Osmond CB (1994) Photoinhibition and recovery in tropical

plant species: response to disturbance. Oecologia 97, 297-307.

Lovelock CE, Kursar TA, Skillman JB, Winter K (1998) Photoinhibition in tropical for-

est understorey species with short- and long-lived leaves. Functional Ecology

12, 553-560.

Magrath R, Lill A (1983) The use of time and energy by the crimson rosella in a tem-

perate wet forest in winter. Australian Journal of Zoology 31, 903-912.

Matsubara S, Krause GH, Aranda J, Virgo A, Beisel KG, Jahns P, Winter K (2009)

Sun-shade patterns of leaf carotenoid composition in 86 species of neotropical

forest plants. Functional Plant Biology 36, 20-36.

Maxwell K, Johnson GN (2000) Chlorophyll fluorescence - a practical guide. Journal of

Experimental Botany 51, 659-668.

McCarthy PM (Ed.) (1998) 'Ferns, gymnosperms and allied groups.' Flora of Australia

(CSIRO Publications: Melbourne)

McDowell N, Pockman WT, Allen CD, Breshears DD, Cobb N, Kolb T, Plaut J, Sperry

J, West A, Williams DG, Yepez EA (2008) Mechanisms of plant survival and

mortality during drought: why do some plants survive while others succumb to

drought? New Phytologist 178, 719-739.

-129-

Merchant A, Tausz M, Arndt SK, Adams MA (2006) Cyclitols and carbohydrates in

leaves and roots of 13 Eucalyptus species suggest contrasting physiological re-

sponses to water deficit. Plant Cell and Environment 29, 2017-2029.

Misson L, Baldocchi DD, Black TA, Blanken PD, Brunet Y, Yuste JC, Dorsey JR, Falk

M, Granier A, Irvine MR, Jarosz N, Lamaud E, Launiainen S, Law BE, Longdoz

B, Loustau D, McKay M, Paw KT, Vesala T, Vickers D, Wilson KB, Goldstein

AH (2007) Partitioning forest carbon fluxes with overstory and understory eddy-

covariance measurements: A synthesis based on FLUXNET data. Agricultural

and Forest Meteorology 144, 14-31.

Montgomery RA, Goldstein G, Givnish TJ (2008) Photoprotection of PSII in Hawaiian

lobeliads from diverse light environments. Functional Plant Biology 35, 595-

605.

Montpied P, Granier A, Dreyer E (2009) Seasonal time-course of gradients of photosyn-

thetic capacity and mesophyll conductance to CO2 across a beech (Fagus sylva-

tica L.) canopy. Journal of Experimental Botany 60, 2407-2418.

Mott KA, Sibbernsen ED, Shope JC (2008) The role of the mesophyll in stomatal re-

sponses to light and CO2. Plant Cell and Environment 31, 1299-1306.

Muller O, Oguchi R, Hirose T, Werger MJA, Hikosaka K (2009) The leaf anatomy of a

broad-leaved evergreen allows an increase in leaf nitrogen content in winter.

Physiologia Plantarum 136, 299-309.

Munné-Bosch S (2005) The role of -tocopherol in plant stress tolerance. Journal of

Plant Physiology 162, 743-748.

Murata N, Takahashi S, Nishiyama Y, Allakhverdiev SI (2007) Photoinhibition of pho-

tosystem II under environmental stress. Biochimica Et Biophysica Acta-

Bioenergetics 1767, 414-421.

Niinemets Ü (2007) Photosynthesis and resource distribution through plant canopies.

Plant Cell and Environment 30, 1052-1071.

Niinemets Ü, Kull O, Tenhunen JD (1998) An analysis of light effects on foliar mor-

phology, physiology, and light interception in temperate deciduous woody spe-

cies of contrasting shade tolerance. Tree Physiology 18, 681-696.

Niinemets Ü, Tenhunen JD (1997) A model separating leaf structural and physiological

effects on carbon gain along light gradients for the shade-tolerant species Acer

saccharum. Plant Cell and Environment 20, 845-866.

Niyogi KK (2000) Safety valves for photosynthesis. Current Opinion in Plant Biology

3, 455-460.

Nobel PS, Calkin HW, Gibson AC (1984) Influences of PAR, temperature and water

vapor concentration on gas exchange by ferns. Physiologia Plantarum 62, 527-

534.

North HM, De Almeida A, Boutin JP, Frey A, To A, Botran L, Sotta B, Marion-Poll A

(2007) The Arabidopsis ABA-deficient mutant aba4 demonstrates that the major

route for stress-induced ABA accumulation is via neoxanthin isomers. Plant

Journal 50, 810-824.

Oikawa S, Hikosaka K, Hirose T (2006) Leaf lifespan and lifetime carbon balance of

individual leaves in a stand of an annual herb, Xanthium canadense. New Phy-

tologist 172, 104-116.

-130-

Oliveira G, Peñuelas J (2004) Effects of winter cold stress on photosynthesis and photo-

chemical efficiency of PSII of the Mediterranean Cistus albidus L. and Quercus

ilex L. Plant Ecology 175, 179-191.

Ottander C, Campbell D, Öquist G (1995) Seasonal changes in photosystem II organiza-

tion and pigment composition in Pinus sylvestris. Planta 197, 176-183.

Ough K (2001) Regeneration of Wet Forest flora a decade after clear-felling or wildfire

- is there a difference? Australian Journal of Botany 49, 645-664.

Ough K, Murphy A (1996) The effect of clearfell logging on tree-ferns in Victorian Wet

Forest. Australian Forestry 59, 178-188.

Ough K, Murphy A (1998) Understorey Island: a method of protecting understorey flora

during clearfelling operations. Department of Natural Resources and Environ-

ment, VSP Internal report # 29, Heidelberg.

Ough K, Murphy A (2004) Decline in tree-fern abundance after clearfell harvesting.

Forest Ecology and Management 199, 153-163.

Page CN, Clifford HT (1981) Ecological biogeography of Australian conifers and ferns.

In 'Ecological biogeography of Australia'. (Ed. A Keast) pp. 471-498. (Dr. W.

Junk bv Publishers: Hague-Boston-London)

Passioura JB (1982) Water in the soil-plant-atmosphere continuum. In 'Physiological

Plant Ecology II. Water Relations and Carbon Assimilation'. (Eds OL Lange, PS

Nobel, CB Osmond and H Ziegler) pp. 5–33. (Springer-Verlag: Berlin, Heidel-

berg, New York)

Pearcy RW (1988) Photosynthetic utilization of lightflecks by understory plants. Aus-

tralian Journal of Plant Physiology 15, 223-238.

Reich PB, Ellsworth DS, Walters MB, Vose JM, Gresham C, Volin JC, Bowman WD

(1999) Generality of leaf trait relationships: A test across six biomes. Ecology

80, 1955-1969.

Ribas-Carbo M, Taylor NL, Giles L, Busquets S, Finnegan PM, Day DA, Lambers H,

Medrano H, Berry JA, Flexas J (2005) Effects of water stress on respiration in

soybean leaves. Plant Physiology 139, 466-473.

Robakowski P, Montpied P, Dreyer E (2002) Temperature response of photosynthesis

of silver fir (Abies alba Mill.) seedlings. Annals of Forest Science 59, 163-170.

Roberts J, Wallace JS, Pitman RM (1984) Factors affecting stomatal conductance of

Bracken below a forest canopy. Journal of Applied Ecology 21, 643-655.

Roberts NR, Dalton PJ, Jordan GJ (2005) Epiphytic ferns and bryophytes of Tasmanian

tree-ferns: A comparison of diversity and composition between two host species.

Austral Ecology 30, 146-154.

Romero P, Botía P (2006) Daily and seasonal patterns of leaf water relations and gas

exchange of regulated deficit-irrigated almond trees under semiarid conditions.

Environmental and Experimental Botany 56, 158-173.

Rossetto M (2008) From populations to communities: understanding changes in rainfor-

est diversity through the integration of molecular, ecological and environmental

data. Telopea 12, 47-58.

Sack L, Holbrook NM (2006) Leaf hydraulics. Annual Review of Plant Biology 57, 361-

381.

Salvucci ME, Crafts-Brandner SJ (2004) Mechanism for deactivation of Rubisco under

moderate heat stress. Physiologia Plantarum 122, 513-519.

-131-

Savitch LV, Massacci A, Gray GR, Huner NPA (2000) Acclimation to low temperature

or high light mitigates sensitivity to photoinhibition: roles of the Calvin cycle

and the Mehler reaction. Australian Journal of Plant Physiology 27, 253-264.

Schreiber U, Berry JA (1977) Heat-induced changes of chlorophyll fluorescence in in-

tact leaves correlated with damage of photosynthetic apparatus. Planta 136, 233-

238.

Seidlova L, Verlinden M, Gloser J, Milbau A, Nijs I (2009) Which plant traits promote

growth in the low-light regimes of vegetation gaps? Plant Ecology 200, 303-318.

Senger H, Wagner C, Hermsmeier D, Hohl N, Urbig T, Bishop NI (1993) The influence

of light intensity and wavelength on the contents of -carotene and -carotene

and their xanthophylls in green algae. Journal of Photochemistry and Photobiol-

ogy B-Biology 18, 273-279.

Sinsawat V, Leipner J, Stamp P, Fracheboud Y (2004) Effect of heat stress on the pho-

tosynthetic apparatus in maize (Zea mays L.) grown at control or high tempera-

ture. Environmental and Experimental Botany 52, 123-129.

Smith T, Huston M (1989) A theory of the spatial and temporal dynamics of plant

communities. Vegetatio 83, 49-69.

Snider JL, Choinski JS, Wise RR (2009) Juvenile Rhus glabra leaves have higher tem-

peratures and lower gas exchange rates than mature leaves when compared in

the field during periods of high irradiance. Journal of Plant Physiology 166,

686-696.

Stewart JR, Lister AM (2001) Cryptic northern refugia and the origins of the modern

biota. Trends in Ecology & Evolution 16, 608-613.

Taiz L, Zeiger E (2002) 'Plant physiology.' (Sinauer Associates, Inc., Publishers: Sun-

derland, Mass.)

Tallon C, Quiles MJ (2007) Acclimation to heat and high light intensity during the de-

velopment of oat leaves increases the NADH DH complex and PTOX levels in

chloroplasts. Plant Science 173, 438-445.

Tausz M, Hietz P, Briones O (2001) The significance of carotenoids and tocopherols in

photoprotection of seven epiphytic fern species of a Mexican cloud forest. Aus-

tralian Journal of Plant Physiology 28, 775-783.

Tausz M, Warren CR, Adams MA (2005) Dynamic light use and protection from excess

light in upper canopy and coppice leaves of Nothofagus cunninghamii in an old

growth, cool temperate rainforest in Victoria, Australia. New Phytologist 165,

143-155.

Tausz M, Wonisch A, Grill D, Morales D, Jimenez MS (2003) Measuring antioxidants

in tree species in the natural environment: from sampling to data evaluation.

Journal of Experimental Botany 54, 1505-1510.

Tazoe Y, von Caemmerer S, Badger MR, Evans JR (2009) Light and CO2 do not affect

the mesophyll conductance to CO2 diffusion in wheat leaves. Journal of Experi-

mental Botany 60, 2291-2301.

ter Steege H (1996) Winphot 5: a Programme to analyze vegetation induces, light, and

light quality from hemispherical photographs. In 'Tropenbos Guyana Reports 95-

2. Tropenbos Guyana Programme'. (Georgetown, Guyana)

Thayer SS, Björkman O (1990) Leaf xanthophyll content and composition in sun and

shade determined by HPLC. Photosynthesis Research 23, 331-343.

-132-

Tingey DT, Evans RC, Bates EH, Gumpertz ML (1987) Isoprene emissions and photo-

synthesis in 3 ferns - the influence of light and temperature. Physiologia Planta-

rum 69, 609-616.

Turner IM (1994a) Sclerophylly: primarily protective. Functional Ecology 8, 669-675.

Valladares F, Niinemets Ü (2008) Shade tolerance, a key plant feature of complex na-

ture and consequences. Annual Review of Ecology, Evolution, and Systematics

39, 237-257.

Valladares F, Pearcy RW (2002) Drought can be more critical in the shade than in the

sun: a field study of carbon gain and photo-inhibition in a Californian shrub dur-

ing a dry El Niño year. Plant Cell and Environment 25, 749-759.

Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance

and moderate heat on photosynthesis, pigments, and tocopherol in the tree-fern


von Caemmerer S, Evans JR, Hudson GS, Andrews TJ (1994) The kinetics of ribulose-

1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of

photosynthesis in leaves of transgenic tobacco. Planta 195, 88-97.

von Caemmerer S, Farquhar GD (1981) Some relationships between the biochemistry

of photosynthesis and the gas exchange of leaves. Planta 153, 376-387.

Warren CR (2008) Rapid measurement of chlorophylls with a microplate reader. Jour-

nal of Plant Nutrition 31, 1321-1332.

Warren CR, Bleby T, Adams MA (2007) Changes in gas exchange versus leaf solutes

as a means to cope with summer drought in Eucalyptus marginata. Oecologia

154, 1-10.

Watling JR, Robinson SA, Woodrow IE, Osmond CB (1997) Responses of rainforest

understorey plants to excess light during sunflecks. Australian Journal of Plant


Watt MS, Kriticos DJ, Manning LK (2009) The current and future potential distribution

of Melaleuca quinquenervia. Weed Research 49, 381-390.

Weaver PRL (2008) Dwarf forest recovery after disturbances in the Luquillo Mountains

of Puerto Rico. Caribbean Journal of Science 44, 150-163.

Wellburn AR (1994) The spectral determination of chlorophyll a and chlorophyll b, as

well as total carotenoids, using various solvents with spectrophotometers of dif-

ferent resolution. Journal of Plant Physiology 144, 307-313.

White RA, Weidlich WH (1995) Organization of the vascular system in the stems of

Diplazium and Blechnum (Filicales). American Journal of Botany 82, 982-991.

Wise RR, Olson AJ, Schrader SM, Sharkey TD (2004) Electron transport is the func-

tional limitation of photosynthesis in field-grown Pima cotton plants at high

temperature. Plant Cell and Environment 27, 717-724.

Wullschleger SD (1993) Biochemical limitations to carbon assimilation in C3 plants - a

retrospective analysis of the A/Ci curves from 109 species. Journal of Experi-

mental Botany 44, 907-920.

Yamamoto Y, Aminaka R, Yoshioka M, Khatoon M, Komayama K, Takenaka D, Ya-

mashita A, Nijo N, Inagawa K, Morita N, Sasaki T, Yamamoto Y (2008) Quality

control of photosystem II: impact of light and heat stresses. Photosynthesis Re-

search 98, 589-608.

-133-

Zhu XG, Ort DR, Whitmarsh J, Long SP (2004) The slow reversibility of photosystem

II thermal energy dissipation on transfer from high to low light may cause large

losses in carbon gain by crop canopies: a theoretical analysis. Journal of Ex-

perimental Botany 55, 1167-1175.

Zsófi Z, Váradi G, Bálo B, Marschall M, Nagy Z, Dulai S (2009) Heat acclimation of

grapevine leaf photosynthesis: mezo- and macroclimatic aspects. Functional

Plant Biology 36, 310-322.

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s:

Volkova, Liubov Vladimirovna

Title:

Ecophysiology of the tree fern species Dicksonia antarctica Labill and Cyathea australis (R.

Br.) Domin

Date:

2009

Citation:

Volkova, L. V. (2009). Ecophysiology of the tree fern species Dicksonia antarctica Labill and

Cyathea australis (R. Br.) Domin. PhD thesis, Dept. of Forest and Ecosystem Science,

Melbourne School of Land and Environment, The University of Melbourne.

Persistent Link:

http://hdl.handle.net/11343/37893

File Description:

Ecophysiology of the tree fern species Dicksonia antarctica Labill and Cyathea australis (R.

Br.) Domin

Terms and Conditions:

Terms and Conditions: Copyright in works deposited in Minerva Access is retained by the

copyright owner. The work may not be altered without permission from the copyright owner.

Readers may only download, print and save electronic copies of whole works for their own

personal non-commercial use. Any use that exceeds these limits requires permission from

the copyright owner. Attribution is essential when quoting or paraphrasing from these works.

ecophysiology of the tree fern species dicksonia ......ecophysiology of the tree fern species...

Documents