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COMPARING CULTIVATED SUNFLOWER (HELIANTHUS) TO TWO WILD SPECIES FOR TRAITS PUTATIVELY ASSOCIATED WITH DROUGHT RESISTANCE by ETHAN FOSTER MILTON (Under the Direction of Lisa A. Donovan) ABSTRACT Drought is arguably the single greatest abiotic factor limiting plant productivity worldwide. Improving drought resistance of crops can counteract some of its detrimental effects. Identifying and incorporating variation from wild drought resistant congeners into crops is one avenue for improvement. We investigated two wild species of sunflower, Helianthus argophyllus (ARG) and H. niveus spp. tephrodes, (TEPH) hypothesized to be drought resistant and compared them to cultivated H. annuus (ANN) for drought resistance traits related to rooting, leaf and germination characteristics at various ontogenetic stages. Contrary to expectation, wild sunflowers did not outperform ANN for germination and rooting traits putatively associated with drought resistance, but TEPH exhibits leaf traits potentially useful in reducing heat load and water loss. Wild sunflowers do possess some traits that may potentially be useful for improving drought resistance in cultivated sunflower. INDEX WORDS: Drought, Sunflower, Ecophysiology, Germination

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COMPARING CULTIVATED SUNFLOWER (HELIANTHUS) TO TWO WILD SPECIES FOR

TRAITS PUTATIVELY ASSOCIATED WITH DROUGHT RESISTANCE

by

ETHAN FOSTER MILTON

(Under the Direction of Lisa A. Donovan)

ABSTRACT

Drought is arguably the single greatest abiotic factor limiting plant productivity worldwide. Improving

drought resistance of crops can counteract some of its detrimental effects. Identifying and incorporating

variation from wild drought resistant congeners into crops is one avenue for improvement. We

investigated two wild species of sunflower, Helianthus argophyllus (ARG) and H. niveus spp. tephrodes,

(TEPH) hypothesized to be drought resistant and compared them to cultivated H. annuus (ANN) for

drought resistance traits related to rooting, leaf and germination characteristics at various ontogenetic

stages. Contrary to expectation, wild sunflowers did not outperform ANN for germination and rooting

traits putatively associated with drought resistance, but TEPH exhibits leaf traits potentially useful in

reducing heat load and water loss. Wild sunflowers do possess some traits that may potentially be useful

for improving drought resistance in cultivated sunflower.

INDEX WORDS: Drought, Sunflower, Ecophysiology, Germination

COMPARING CULTIVATED SUNFLOWER (HELIANTHUS) TO TWO WILD SPECIES FOR

TRAITS PUTATIVELY ASSOCIATED WITH DROUGHT RESISTANCE

by

ETHAN FOSTER MILTON

B.S., Florida State University, Tallahassee, 2007

A Thesis Submitted to the Graduate Faculty of the University of Georgia in Partial Fulfillment

of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2013

© 2013

Ethan Foster Milton

All Rights Reserved

COMPARING CULTIVATED SUNFLOWER (HELIANTHUS) TO TWO WILD SPECIES FOR

TRAITS PUTATIVELY ASSOCIATED WITH DROUGHT RESISTANCE

by

ETHAN FOSTER MILTON

Major Professor: Lisa A. Donovan

Committee: John M. Burke

Marc van Iersel

Electronic Version Approved:

Maureen Grasso

Dean of the Graduate School

The University of Georgia

May 2013

iv

TABLE OF CONTENTS

Page

LIST OF TABLES………………………………………………………………………………….……...vi

LIST OF FIGURES……………………………………………………………………………………….vii

CHAPTER

1 INTRODUCTIONAND LITERATURE REVIEW………………………………………1

References…………………………………………………………………..…….6

2 COMPARISON OF DESERT ADAPTED HELIANTHUS NIVEUS SSP. TEPHRODES

TO CULTIVATED H. ANNUUS FOR PUTAITVE DROUGHT RESISTANCE TRAITS

AT TWO ONTOGENETIC STAGES…………………………………………………….9

Abstract………………………………………………………………………….10

Introduction……………………………………………………………………...11

Materials and Methods…………………………………………………………..14

Results…………………………………………………………………………...18

Discussion……………………………………………………………………….19

References……………………………………………………………………….25

3 CULTIVATED HELIANTHUS ANNUUS AND WILD RELATIVES (H.

ARGOPHYLLUS AND H. NIVEUS SSP. TEPHRODES) DIFFER IN GERMINATION

RESPONSE TO SIMULATED DROUGHT STRESS………………………………….46

Abstract………………………………………………………………………….47

Introduction……………………………………………………………………...48

Materials and Methods…………………………………………………………..51

Results…………………………………………………………………………...53

Discussion……………………………………………………………………….54

v

Page

References……………………………………………………………………….59

4 CONCLUSIONS …………………………………………………………………...…...72

vi

LIST OF TABLES

Page

Table 2.1: Distribution of seedling means (± SE) for two species of Helianthus (sunflower),

H. annuus and H. niveus ssp. tephrodes for measured traits.……………………………………………..33

Table 2.2: Distribution of mature means (± SE) for two species of Helianthus (sunflower),

H. annuus and H. niveus ssp. tephrodes for measured traits.……………...……………………………...34

Appendix Table 2.1: Distribution of means for normalized difference vegetative index (NDVI)

components R680 and R800 with associated standard error between two species of Helianthus

(sunflowers)………………………………………………………………………………...……………..45

Table 3.1: Percent germination (% Germ) and mean germination time (MGT) (means + SE) for

three species of Helianthus (sunflowers), H. annuus (ANN), H. argophyllus (ARG) and H. niveus

ssp. tephrodes (TEPH) for polyethylene glycol – 6000 induced water stress treatments..…………..........66

Table 3.2: Likelihood ratio contrasts of standardized species Helianthus annuus (ANN),

H. argophyllus (ARG) and H. niveus ssp. tephrodes (TEPH) germination time regressions for

polyethylene glycol – 6000 induced water stress treatments……………………………………..……....67

vii

LIST OF FIGURES

Page

Figure 2.1: Comparison of Helianthus annuus (ANN) and H. niveus ssp. tephrodes (TEPH) at the

seedling stage for measured traits……………………………………………………………….………...29

Figure 2.2: Comparison of Helianthus annuus (ANN) and H. niveus ssp. tephrodes (TEPH) at

the mature stage for measured traits.……………………………………………………………………...31

Appendix Figure 2.1: Comparison of Helianthus annuus seedling accession means for measured

traits..……………………………………………………………………………………………...….........35

Appendix Figure 2.2: Comparison of Helianthus niveus ssp. tephrodes seedling accession means

for measured traits. …………………………………………………………………………….…….........37

Appendix Figure 2.3: Comparison of Helianthus annuus mature accession means for traits

measured.……………………………………………….……………………………………………........39

Appendix Figure 2.4: Species means for normalized difference vegetative index (NDVI) components

at the seedling stage.………………………………………………………………………………...……41

Appendix Figure 2.5: Species means for normalized difference vegetative index (NDVI) components

at the mature stage.…………………………………………………………………………………...……42

Appendix Figure 2.6: Comparison of Helianthus niveus ssp. tephrodes mature accession means

for traits measured.……………………………………………………………………………..…….........43

Figure 3.1: Percent germination after seven days for three species of Helianthus (sunflower)

H. annuus (ANN), H. argophyllus (ARG) and H. niveus ssp. tephrodes (TEPH) for polyethylene

glycol – 6000 induced drought stress treatments………………………………………………………….63

Figure 3.2: Mean germination time after seven days for three species of Helianthus (sunflower)

H. annuus (ANN), H. argophyllus (ARG) and H. niveus ssp. tephrodes (TEPH) for polyethylene

glycol – 6000 induced water stress treatments…………………………………………………………….64

viii

Page

Figure 3.3: Probability density functions for observed germination frequency by day for three

species of Helianthus (sunflower) H. annuus (ANN), H. argophyllus (ARG) and H. niveus ssp.

tephrodes (TEPH) for polyethylene glycol – 6000 induced water stress treatments………………..…….65

Appendix Figure 3.1: Percent germination after seven days for accessions of three species of

Helianthus (sunflower) H. annuus (ANN), H. argophyllus (ARG) and H. niveus ssp. tephrodes

(TEPH) for polyethylene glycol – 6000 induced drought stress treatments.……………...…………........68

Appendix Figure 3.2: Mean germination time after seven days for accessions of three species of

Helianthus (sunflower) H. annuus (ANN), H. argophyllus (ARG) and H. niveus ssp. tephrodes

(TEPH) for polyethylene glycol – 6000 induced drought stress treatments...…………………….……....70

1

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

The availability of water is arguably the single largest abiotic factor that affects productivity in

both natural and agronomic systems (Boyer 1982; Blum 1996; Passioura 1996; Verslues, Agarwal et al.

2006). Abiotic stress, specifically drought, is topic of great concern given the expectation that under

global warming many areas will experience a change in precipitation patterns and an increase in extreme

temperatures. These meteorological changes will bring with them an increase in duration, frequency and

severity of drought (Houghton, Ding et al. 2001; IPCC 2007). Thus there exists an emerging need to

make crops more resistant to the detrimental effects of drought. Crop species and their wild congeners

have long been a focus of study in applied biology. The identification of traits useful for resource uptake,

allocation, reproduction as well as resistance to biotic and abiotic stress has provided insights into the

potential for improving commercial crops (Ashraf and O'Leary 1996). A thorough understanding of

physiological traits associated with drought resistance and improved water use efficiency is crucial for

crop improvement (Ashraf and O'Leary 1996; Passioura 2006; Richards 2006). One potential source for

crop improvement in terms of drought resistance is to identify putative drought resistance traits and

incorporate wild germplasm from species adapted to water stressed environments.

Drought is defined as a prolonged period that is characterized by the absence of precipitation or

supplemental water supply and results in declining soil water availability (Boyer 1982). Broadly defined,

drought resistance is the ability of plants to deal with decreasing soil water availability (lower or more

negative soil water potential) either through internal or external mechanisms. Plants have three strategies

to cope with or “resist” declining soil water availability (Kramer and Boyer 1995): escape, avoidance and

tolerance. This thesis focuses on drought avoidance and drought tolerance, using the terminology most

consistent with Levitt (Levitt 1980) and Verslues (Verslues, Agarwal et al. 2006).

2

Drought avoidance is a plant’s ability to avoid lower internal water potentials even though soil

water potential is declining, by maintaining a favorable balance of water uptake to water loss (Verslues,

Agarwal et al. 2006). This can be achieved with both inherent traits already expressed under well watered

conditions, and the ability to alter traits in response to drought. At the broadest scale, avoidance is

achieved via traits that decrease transpirational water loss, such as stomatal regulation and increased leaf

level water-use efficiency (WUE), and traits that allow plants to take up more water, such greater

allocation of biomass to roots, and deeper rooting (Turner and Begg 1981). Drought tolerance is defined

as the ability of plants to survive and continue metabolic function under substantially reduced internal

water potentials (Verslues, Agarwal et al. 2006). Drought tolerance is generally thought to be achieved by

accumulation of compatible solutes such as glycine betaine and the stiffening of cell walls, both of which

may result in decreased growth and yield. Drought tolerant plants additionally have the capacity to avoid

internal cellular damage by producing protective solutes that minimize the damage due to reactive oxygen

species.

This thesis investigates several aspects of drought avoidance and tolerance in the sunflower

(Helianthus) genus. It is a genus of 49 species composed of both annuals and perennials and contains two

crops native to North America, cultivated sunflower derived from H. annuus and Jerusalem Artichoke H.

tuberosus (Heiser and Smith 1969; Schilling and Heiser 1981). The genus as a whole displays a wide

geographic distribution, occupies an array of diverse habitats, and possess an abundance of variability for

agronomically important traits from yield to abiotic and biotic stress resistance (Heiser and Smith 1969).

Two examples of the variability within this genus are silver leaf sunflower, H. argophyllus and the dunes

sunflower, H. niveus ssp. tephrodes. Both of these species are hypothesized to be drought resistant based

on their native arid habitats and the presence of leaf characteristics associated with species adapted to

water limited environments, such as dense white leaf pubescence (Baldini and Vannozzi 1998; Murillo‐

Amador, López‐Aguilar et al. 2002; Seiler, Gulya et al. 2006; Seiler, Gulya et al. 2006).

Compared to cultivated Helianthus annuus, H. argophyllus and H. niveus ssp. tephrodes grow in

more water limited environments; however these environments are very different in terms of water

3

limitation. Helianthus argophyllus grows along the coastal Texas plain, from saline dunes along the coast

to semi-arid sandy regions further inland, while H. niveus ssp. tephrodes can be found in the extremely

hot and dry Algodones dunes region of the Sonoran Desert. A few studies using inbred lines derived from

H. argophyllus × H. annuus have provided evidence of its usefulness as a source of variation for

performance under drought conditions (Baldini and Vannozzi 1998; Baldini and Vannozzi 1999). The

results of these studies indicate one H. argophyllus × H. annuus line had a higher water use efficiency and

harvest index under drought conditions compared to cultivated sunflower, although the hybrid line

performed more poorly under well watered conditions (Baldini and Vannozzi 1998; Baldini and Vannozzi

1999). Another study evaluated lines of H. argophyllus × H. annuus hybrids for root and shoot growth

under short-term water stress conditions induced by polyethylene glycol (PEG) and found that H.

argophyllus derived lines performed similarly to those derived from H. annuus and wild H. debilis

(Khodarahmpour 2011). More studies of H. argophyllus drought resistant traits are warranted.

Additionally, very little is known about the drought resistance of H. niveus ssp. tephrodes.

When considering applications to agriculture, many studies focus primarily on traits which

convey drought avoidance rather than drought escape or tolerance because the latter strategies typically

associated with a reduced total yield or biomass and are highly specific to a given environment. Chapter

two focuses on leaf and growth traits that can be screened in individuals relatively quickly in order to

evaluate the capacity for novel wild germplasm to be drought avoidant.

Leaf traits that are thought to be associated with reduced water loss and increased leaf level WUE

(photosynthetic carbon gain divided by transpirational water loss) have received attention (Farquhar and

Sharkey 1982; Kramer and Boyer 1995). Greater stomatal sensitivity to water deficits can reduce

transpiration and thus increase leaf level WUE, which can be estimated from leaf carbon isotope ratio

(Farquhar, Ehleringer et al. 1989; Ehleringer, Phillips et al. 1992; Donovan and Ehleringer 1994). Smaller

leaf size and greater leaf reflectance due to pubescence are also expected to enhance leaf level WUE

measurements by keeping leaves cooler (Ehleringer 1980; Turner and Begg 1981). Leaf reflectance can

be used to calculate the Normalized Difference Vegetative Index (NDVI) which is basically an index

4

associated with green biomass, and can be affected by a variety of stress such as N and water deficiency

(Penuelas, Filella et al. 1995). Leaves that are good at harvesting the photosynthetically active

wavelengths while reflecting near infrared wavelengths that heat up leaves have NDVI values tending

towards 1. In addition, water stress typically results in decreased NDVI (Tucker 1979). Greater leaf

toughness, lower specific leaf area (thicker leaves) and low N leaves are often associated with species

adapted to drier habitats (Gummuluru, Jana et al. 1989; Valladares, Sanchez-Gomez et al. 2006).

At the whole plant level, traits such as deeper rooting and greater root to total biomass ratio (root

mass ratio) are also associated with drought avoidance (Verslues, Agarwal et al. 2006). They delay the

onset of internal water deficit, and provide plants with sufficient water to continue growth and survival

once mild internal water deficits occur. Taken together, root traits, leaf traits, and whole plant WUE can

result in a more favorable plant water status under water limited conditions. In chapter 2, we compare

cultivated H. annuus to wild H. niveus ssp. tephrodes to test the following hypothesis under well watered

conditions: 1) Wild sunflower Helianthus niveus ssp. tephrodes seedlings will exhibit rooting

characteristics more consistent with drought resistance than cultivated H. annuus. 2) Wild sunflower H.

niveus ssp. tephrodes adult plants will have a greater water use efficiency and photosynthetic capacity

than cultivated H. annuus. 3) Wild sunflower H. niveus ssp. tephrodes seedlings and adult plants will

have leaf characteristics more consistent with drought resistance than cultivated H. annuus.

Drought also has detrimental effects on stand establishment and uniformity. The timing and onset

of drought can affect final yield in differing degrees of severity depending on the ontogenetic stage in

which the crop is being impacted (Passioura 1996). Passioura (1996) argues that phenology, or the

developmental timing of a crop as it relates to water availability, is an important consideration for

determining drought resistance. In many crop species it has been shown that the two most susceptible

times for crop failure due to drought are the germination to seedling stage and flowering (Ahmad, Ahmad

et al. 2009). Identifying species that confer an advantage or increased percent and uniformity of

germination is one avenue that could be used to improve crop production and stability of yield in

marginal cropping fields. In chapter three, we compare cultivated H. annuus to wild H. niveus ssp.

5

tephrodes and wild H. argophyllus to test the following hypothesis under well watered conditions: 1)

Wild sunflower species H. argophyllus and H. niveus ssp. tephrodes will have a greater percent

germination than cultivated H. annuus under simulated drought stress. 2) Wild sunflower species H.

argophyllus and H. niveus ssp. tephrodes will have a lower mean germination time than cultivated H.

annuus under simulated drought stress.

Taken as a whole, this thesis aims to contribute to the characterization of drought resistance and

related traits in two species of wild sunflower (Helianthus), at various ontogenetic stages. Specifically we

aimed to assess whether putative drought resistance traits in wild sunflower offer a potential avenue for

improving cultivated sunflower.

6

References

Ahmad, S., R. Ahmad, et al. (2009). "Sunflower (Helianthus annuus L.) response to drought stress at

germination and seedling growth stages." Pakistan Journal of Botany 41(2): 647-654.

Ashraf, M. and J. W. OLeary (1996). "Effect of drought stress on growth, water relations, and gas

exchange of two lines of sunflower differing in degree of salt tolerance." International Journal of

Plant Sciences 157(6): 729-732.

Baldini, M. and G. Vannozzi (1998). "Agronomic and physiological assessment of genotypic variation for

drought tolerance in sunflower genotypes obtained from a cross between H. annuus and H.

argophyllus." Agricoltura Mediterranea 128(3): 232-240.

Baldini, M. and G. P. Vannozzi (1999). "Yield relationships under drought in sunflower genotypes

obtained from a wild population and cultivated sunflowers in rain-out shelter in large pots and

field experiments." Helia 22(30): 81-96

Blum, A. (1996). "Crop responses to drought and the interpretation of adaptation." Plant Growth

Regulation 20(2): 135-148.

Boyer, J. S. (1982). "Plant productivity and environment." Science 218(4571): 443-448.

Donovan, L. A. and J. R. Ehleringer (1994). "Potential for selection on plants for water-use efficiency as

estimated by carbon isotope discrimination." American Journal of Botany 81(7): 927-935.

Ehleringer, J. (1980). Leaf morphology and reflectance in relation to water and temperature stress.

Adaptations of Plants to Water and High Temperature Stress. N. Turner and P. Kramer. New

York, N.Y., Wiley-Interscience: 295-308.

Ehleringer, J. R., S. L. Phillips, et al. (1992). "Seasonal variation in the carbon isotopic composition of

desert plants." Functional Ecology 6(4): 396-404.

Farquhar, G. D., J. R. Ehleringer, et al. (1989). "Carbon isotope discrimination and photosynthesis."

Annual Review of Plant Physiology and Plant Molecular Biology 40: 503-537.

Farquhar, G. D. and T. D. Sharkey (1982). "Stomatal conductance and photosynthesis." Annual Review

of Plant Physiology and Plant Molecular Biology 33: 317-345.

7

Gummuluru, S., S. Jana, et al. (1989). "Genotypic variability in physiological characters and its

relationship to drought tolerance in durum wheat." Canadian Journal of Plant Science 69(3): 703-

711.

Heiser, C. B. and D. M. Smith (1969). The North American sunflowers (Helianthus), Seeman Printery.

Houghton, J. T., Y. Ding, et al. (2001). Climate change 2001: the scientific basis, Cambridge University

Press Cambridge.

IPCC (2007). Climate change 2007: The physical science basis. Contribution of working group I to the

fourth assessment report on the intergovernmental panel on climate change. Solomon, S., Qin, D.,

Manning, M. R., Marquis, M., Averyt, K. B. Tignor, M., Miller, H. and Chen, Z.

Khodarahmpour, Z. (2011). "Effect of drought stress induced by polyethylene glycol (PEG) on

germination indices in corn (Zea mays L.) hybrids." African Journal of Biotechnology 10(79):

18222-18227.

Kramer, P. J. and J. S. Boyer (1995). Water relations of plants and soils, Academic Press.

Lambers, H., F. S. Chapin, et al. (2008). Plant physiological ecology, Springer.

Levitt, J. (1980). Responses of plants to environmental stresses: water, radiation, salt, and other stresses,

Academic Press.

Murillo‐Amador, B., R. López‐Aguilar, et al. (2002). "Comparative effects of NaCl and polyethylene

glycol on germination, emergence and seedling growth of cowpea." Journal of Agronomy and

Crop Science 188(4): 235-247.

Passioura, J. (1996). "Drought and drought tolerance." Plant Growth Regulation 20(2): 79-83.

Passioura, J. (2006). "Increasing crop productivity when water is scarce—from breeding to field

management." Agricultural Water Management 80(1): 176-196.

Penuelas, J., I. Filella, et al. (1995). "Assessment of photosynthetic radiation-use efficiency with spectral

reflectance." New Phytologist 131(3): 291-296.

Richards, R. A. (2006). "Physiological traits used in the breeding of new cultivars for water-scarce

environments." Agricultural Water Management 80(1–3): 197-211.

8

Schilling, E. E. and C. B. Heiser (1981). "Infrageneric classification of Helianthus (Compositae)." Taxon

30(2): 393-403.

Seiler, G., T. Gulya, et al. (2006). "Exploration for wild Helianthus species from the desert Southwestern

USA for potential drought tolerance." Helia 29(45): 1-10.

Seiler, G. J., T. Gulya, et al. (2006). Plant exploration to collect wild Helianthus niveus subspecies for

sunflower improvement. Proceedings Sunflower Research Workshop.

Tucker, C. J. (1979). "Red and photographic infrared linear combinations for monitoring vegetation."

Remote Sensing of Environment 8(2): 127-150.

Turner, N. and J. Begg (1981). "Plant-water relations and adaptation to stress." Plant and Soil 58(1): 97-

131.

Valladares, F., D. Sanchez-Gomez, et al. (2006). "Quantitative estimation of phenotypic plasticity:

bridging the gap between the evolutionary concept and its ecological applications." Journal of

Ecology 94(6): 1103-1116.

Verslues, P. E., M. Agarwal, et al. (2006). "Methods and concepts in quantifying resistance to drought,

salt and freezing, abiotic stresses that affect plant water status." The Plant Journal 45(4): 523-539.

9

CHAPTER 2

COMPARISON OF DESERT ADAPTED HELIANTHUS NIVEUS SSP. TEPHRODES TO

CULTIVATED H. ANNUUS FOR PUTAITVE DROUGHT RESISTANCE TRAITS AT TWO

ONTOGENETIC STAGES1

__________________________ 1Milton, E.F. and L.A. Donovan, to be submitted to Crop Science

10

Abstract

Water availability is a major factor limiting plant productivity in both natural and agronomic

systems. Thus, improving crop species for potentially greater drought resistance has become a major

focus for the scientific community. Identifying traits conferring drought resistance in both existing

cultivars and wild relatives may prove useful for improving crops grown under water limiting conditions.

We tested the expectation that a wild sunflower species, the desert dwelling Algodones Dunes sunflower,

Helianthus niveus ssp. tephrodes (TEPH) would exhibit root and leaf traits consistent with greater

drought resistance as compared to cultivated sunflower H. annuus (ANN) in comparative greenhouse

studies under well-watered conditions. When comparing seedlings for root traits, ANN reached a rooting

depth of 30 cm (DTB) faster than TEPH and achieved a greater belowground biomass with a shorter

height at harvest. However, there were no differences for aboveground biomass and root mass ratio

(RMR), likely due to a two-fold higher DTB for TEPH. Contrary our expectation, TEPH achieves deeper

rooting depth more slowly than ANN, which is likely to be undesirable for environments with limited

surface soil moisture. Comparing mature plants for gas exchange traits, TEPH had a higher instantaneous

water use efficiency (WUE) and photosynthesis on an area basis, though differences may have been due

to ANN leaves being at a later ontogenetic stage when sampled. However, ANN did have a greater

photosynthetic rate on a mass basis, likely due to TEPH having thicker, heavier leaves. ANN and TEPH

did not differ for stomatal conductance, integrated WUE as measured by carbon isotope ratio (13

C) and

leaf nitrogen content. Comparing both seedling and mature plants for individual leaf and canopy traits,

TEPH had thicker and smaller leaves and reduced normalized difference vegetative index (NDVI) likely

due to the presence of dense pubescence. Additionally, TEPH seedlings had tougher leaves, a smaller

canopy surface area and a greater total number of leaves. TEPH leaf and canopy traits could be useful to

reduce heat load and transpirational water loss commonly associated with drought prone environments.

Thus, TEPH deserves further study for leaf traits of potential value for developing drought resistant

cultivated sunflower.

INDEX WORDS: Sunflower, Drought, NDVI, Roots, Pubescence, WUE

11

Introduction

Crop species and their wild relatives have long been a focus of study for the applied side of

biology. Identifying useful traits for resource uptake, resource allocation, reproduction as well as

resistance to biotic and abiotic stress has provided insights into the potential for improving commercial

crops (Ashraf and O'Leary 1996). Drought is a major stress that limits plant productivity in many

agricultural areas. Given the expectation that temperature and precipitation patterns will change under

global warming, the duration, frequency and severity of drought is expected to increase in some regions

(Houghton, Ding et al. 2001; Sheffield and Wood 2008). Thus, a thorough understanding of physiological

traits associated with drought resistance and improved water use efficiency (WUE) has become a primary

focus for crop improvement (Ashraf and O'Leary 1996; Passioura 2006; Richards 2006). Wild congeners

native to water limited environments are a potential source of drought resistance traits for improvements

in crop productivity (Thompson, Zimmerman et al. 1981; Shimshi, Mayoral et al. 1982; Jackson and

Koch 1997; Seiler, Gulya et al. 2006; Hajjar and Hodgkin 2007).

Drought is typically defined as a prolonged absence of precipitation or supplemental water supply

that results in declining soil water availability (Boyer 1982). Plants can resist drought through either

tolerance or avoidance mechanisms. Drought tolerance mechanisms allow for continued metabolic

function under substantially reduced internal water potentials generated by the inability to maintain a

balance between water uptake and loss (Verslues, Agarwal et al. 2006). Drought tolerance can be achieved

by osmotic adjustment and cell wall stiffening, both of which are metabolically expensive and may

decrease productivity (Kramer and Boyer 1995; Verslues, Agarwal et al. 2006). Drought avoidance is the

avoidance or delay of lower internal water potentials under declining soil moisture by maintaining a

favorable balance of water uptake to water loss (Verslues, Agarwal et al. 2006).

At the broadest scale, avoidance is achieved through traits that decrease transpirational

water loss, such as stomatal regulation and increased leaf level water-use efficiency (WUE,

photosynthetic carbon gain divided by transpirational water loss), and traits that allow plants to

take up more water, such as greater allocation of biomass to roots and deeper rooting (Turner and

12

Begg 1981; Verslues, Agarwal et al. 2006). Drought avoidance mechanisms can also be thought of as

reducing water loss by minimizing transpiration and heat load. Plants that avoid drought often show an

increased investment to rooting structures, either through deeper rooting or a greater ratio of root biomass

to total biomass, known as root mass ratio (RMR) (Verslues, Agarwal et al. 2006). Deeper rooting can

allow for continued water uptake under drought conditions, because deeper soil layers tend to hold greater

reserves of water than shallower soil horizons (Passioura 2006). Thus, allocation of root biomass to

deeper rooting, particularly in early growth stages, would be beneficial for cropping systems in drought

prone environments (Blum 1996). Previous work in sunflower has shown variation among species in

primary root elongation rates. A study comparing six wild species of Helianthus and two cultivated

varieties showed potential for greater primary root elongation in both the wild H. petiolaris and the H.

annuus based dwarf cultivated line 471D, with H. petiolaris showing the greatest rate of primary root

elongation under well-watered conditions among the eight lines tested (Seiler 1994). Thus, the ability to

quickly develop a deep rooting system, and the capacity to allocate more resources to root development is

a primary area of focus for increased drought resistance through a greater capacity to extract available soil

moisture.

Leaves also play an important role in drought resistance by reducing transpirational water loss

and heat load. Leaf traits associated with reduced water loss and increased leaf-level WUE have received

a lot of attention (Farquhar and Sharkey 1982; Kramer and Boyer 1995). The control of gas exchange via

stomatal regulation (i.e. stomatal conductance) coupled with the maintenance of adequate photosynthetic

rate on an area basis can reduce water loss due to transpiration and thus increase WUE. WUE is measured

in two different ways; instantaneous WUE and integrated WUE. Instantaneous WUE can be thought of as

a plant’s ability to regulate stomatal conductance at the scale of minutes or seconds, while integrated

WUE, estimated from leaf carbon isotope ratio (13

C), can be used to estimate WUE over a leaf’s lifetime

(Farquhar, Ehleringer et al. 1989; Donovan and Ehleringer 1994).

Leaf characteristics that reduce heat load, such as increased leaf pubescence, and smaller

individual leaf surface area, can help to minimize transpirational water loss. Smaller, thicker leaves or a

13

reduction in specific leaf area (SLA, leaf area divided by leaf dry weight) contribute to leaf water

conservation by minimizing the surface area to volume ratio (Lopez, Chauhan et al. 1997). Smaller,

thicker leaves also concentrate photosynthetic machinery, which coupled with reduced water loss, can

facilitate greater instantaneous WUE (Craufurd, Wheeler et al. 1999). Leaf pubescence, a trait often

associated with desert species, can serve to increase leaf reflectance and is generally thought to help

plants regulate water use. Ecological studies have shown that the occurrence and extent of leaf

pubescence increases with habitat aridity (Sandquist and Ehleringer 1997). The increased pubescence

reflects excess radiation and is thought to help reduce heat load and reduce transpiration by increasing the

leaf boundary layer (the area of still air around a leaf) which serves to trap moisture and reduce water loss

(Ehleringer 1984). The leaf reflectance parameter normalized difference vegetative index (NDVI) is a

common index calculated from reflectance values in the red (660 nm) and infrared spectrum (800 nm) and

has been used at the canopy level to measure green biomass and is interpreted as a measure of canopy

health (Tucker 1979; Penuelas, Filella et al. 1995). Assessing the two components of NDVI can provide

insight into how a species is dealing with excess radiation driving photosynthesis (680nm) and radiation

responsible for increasing heat load (800nm). To our knowledge NDVI specifically has not be used as a

measure for individual leaf pubescence estimates, however previous studies have shown differences in the

density of leaf pubescence using a greater range of wavelengths (Gausman, Menges et al. 1977).

We evaluated putative drought resistance traits under well-watered conditions in two species of

annual sunflower, cultivated Helianthus annuus (hereafter ANN) and its wild congener H. niveus ssp.

tephrodes (here after TEPH). TEPH has been hypothesized to be drought resistance based on habitat and

dense white leaf pubescence (Heiser and Smith 1969; Seiler 1992). We compared these species for

putative drought resistance under well-watered conditions at two ontogenetic stages, seedling and mature.

Evaluation of drought resistance focused on three trait categories: root establishment (seedling), leaf

strategies to minimize water loss and moderate leaf temperature (seedling and mature), and instantaneous

gas exchange parameters (mature). Specifically we addressed the following hypotheses under well-

watered conditions: 1) TEPH seedlings will have a more rapid proliferation of roots, taking fewer days to

14

reach a rooting depth of 30cm (DTB) and allocate more resources to their root development than ANN

(fewer DTB, larger RMR and belowground biomass); 2) Mature TEPH plants will have greater

photosynthetic capacity and WUE than ANN (greater photosynthetic rate on an area and mass basis,

greater leaf nitrogen content, lower stomatal conductance, greater instantaneous and integrated WUE);

3) TEPH seedlings and mature plants will have leaf characteristics more consistent with drought

resistance than ANN (decreased SLA, increased leaf toughness, decreased individual leaf area, decreased

canopy area with a greater number of leaves, decreased NDVI).

Materials and Methods

Study Species

Both ANN and TEPH are members of the section Helianthus within the genus Helianthus which

is comprised of thirteen species of annual diploids (n = 17) (Schilling and Heiser 1981). Species from

Section Helianthus are predominantly found in the southwestern United States and are thought to be well

adapted to sandy soils and arid condition (Seiler and Rieseberg 1997). Wild ANN, common sunflower, is

the most widespread of all the sunflower species, with a range from southern Canada to northern Mexico,

although predominantly found in the western United States. Wild ANN is described as a weedy species,

having the greatest diversity of habitat and morphology of all the sunflowers. Wild individuals typically

grow 1.0 - 4.0 m tall and are usually branched (Seiler and Rieseberg 1997). Wild ANN is the progenitor

of cultivated sunflower, one of a few crop species native to North America. Cultivated ANN is grown

primarily for oil and confectionary purposes. It is the fifth largest oilseed crop in the world, and

production in the United States accounts for approximately 5% of production worldwide (Seiler and Jan

2010). The Algodones Dunes sunflower, TEPH a wild relative of Common Sunflower (ANN), grows in

the sandy arid habitat of the Algodones Dunes (annual precipitation 62 mm) in the Sonoran desert and

typically grows to a height of 0.5-1.5 m (Barbour and Billings 1988; Jepson and Hickman 1993; Seiler

and Rieseberg 1997). The habitat is characterized by an average temperature of 11.4˚ C in January and

32.7˚C in July. TEPH has been hypothesized to be drought resistant and likely useful as germplasm

resource for sunflower crop improvement primarily based on its arid desert habitat. This hypothesis is

15

further substantiated by the presence of dense leaf pubescence, low SLA and reduced leaf size, all of

which are characteristic of drought resistance (Turner and Begg 1981; Chaves, Maroco et al. 2003).

Experimental Design

The seedling study was conducted in the University of Georgia Plant Biology greenhouses in

spring 2012. The experimental design was a randomized complete block design, with 2 species, 4

accessions per species and 12 plants for each line divided into 4 blocks, for a total of 96 measured plants.

Plants for each accession within each block were averaged to a single data point to avoid

pseudoreplication, giving a total analyzed sample size of n = 32. All seed was obtained from the USDA

National Plant Germplasm System and accession names for each of the following species represent

USDA National Genetic Resources Program (GRIN, http://www.ars-grin.gov/). For ANN the accession

identifiers are PI-642777 (HA-412-HO), PI-560141 (RHA-373), PI-578872 (HA-383) and PI-607506

(RHA 415). For TEPH the accession identifiers are PI-664653 (AMES-27850), PI-613758 (AMES-6852),

PI-650018 (AMES-27422), and PI-664643 (AMES-27421). Seeds were scarified by excising the blunt

end of each achene and then germinated on filter paper in petri dishes. On 4 February 2012, germinated

seedling were transferred to 2.5 cm plugs containing Fafard 3B potting mix (Conrad Fafard Inc.,

Agawam, MA) in a growth chamber (12/12 light cycle under 1000 mol at 22°C). On 11 February,

seedlings were transplanted into 2 liter tree pots measuring 30 cm deep, and filled to the top with Fafard

3B soil mixture (Conrad Fafard, Inc. Agawam, MA). Plants were watered daily and fertilized 3 times

weekly with a 30ppm (based on nitrogen) Jack’s Professional 20-20-20 fertilizer solution (J.R. Peters,

Inc. Allentown, PA).

The mature plant study was conducted in the University of Georgia Plant Biology greenhouse in

summer of 2010. The initial experimental design was a randomized complete block design, with 2

species, 10 accessions of TEPH (6 replicates each) and 6 accessions of ANN (3 replicates), divided into 3

blocks for a total n= 234. All seed was obtained from the USDA National Plant Germplasm System and

accession names for each of the following species represent USDA GRIN identifiers. For ANN the

accession identifiers are PI-642777 (HA-412-HO), PI-560141 (RHA-373), PI-578872 (HA-383) and PI-

16

607506 (RHA-415). For TEPH the accession identifiers are PI-664653 (AMES-27850), PI-613758

(AMES-6852), PI-650018 (AMES-27422), PI-664643 (AMES-27421), PI-650017(AMES-27420), PI-

650019 (AMES-27830), PI-650020 (AMES-27831), PI-650021 (AMES-27832), PI-664651 (AMES-

27847) and PI-664654 (AMES-27851). However, two ANN accessions did not have adequate

germination PI-494567 (ANN1811) and PI-659440 (ANN1238) and one accession of TEPH, PI-664651

(AMES-27847) and were excluded from this study. We sampled a subset of 2-3 individuals per block for

each accession, and replicates for each accession within each block were averaged to a single data point to

avoid pseudoreplication, giving a total analyzed sample size of n = 39. Germinated seedlings of TEPH

were transferred to 2.5 cm plugs containing Fafard 3B potting mix (Conrad Fafard, Inc. Agawam, MA)

and grown for approximately one week to develop roots before being transplanted to 4 liter pots filled to

the top with Fafard 3B potting mix (Conrad Fafard, Inc. Agawam, MA). All ANN accessions were direct

sown into pots. Plants were given 30 g of Osmocote ® 14-14-14 slow release fertilizer (Everris

International B.V.) and kept well watered with drip irrigation.

Traits Measured

The seedlings were assessed for root growth rate based on days to reach a 30cm rooting depth, i.e.

the bottom of the pot. Plants were visually scored by checking for emerged root tips at a hole in the center

and the four corners of the pot which had been cut ~ 1cm at each corner side to form a flap that could be

pulled down for inspection. When a plant root reached a 30cm rooting depth the plant was then harvested.

Harvested plants were scored for number of true leaves, total leaf area (LI 3100, LiCor Instruments,

Lincoln, NE), the leaf area of the pair of the most recent fully expanded leaves, stem height, and the leaf

reflectance parameter NDVI (Unispec Spectral Analysis System PP Systems Inc., Amesbury, MA)

averaged for the pair of the most recent fully expanded leaves. NDVI was calculated from the reflectance

(R) of near infrared (NIR, 800 nm) and red (R, 680 nm) wavelengths following the equation (R800-

R680)/( R800+R680) (Tucker 1979). Roots were harvested after washing off the substrate. All biomass was

dried at 60⁰C for at least 48 hours before weighing. Biomass was separated into aboveground biomass

(leaves and stems) and belowground biomass (roots). Additionally, we calculated specific leaf area (SLA)

17

as leaf area divided by leaf mass, and root mass ratio (RMR) as root biomass divided by total biomass.

In the mature study, plants were assessed for NDVI and SLA as they were in the seedling study,

except that they were measured or calculated on a single leaf. Additionally we measured instantaneous

gas exchange (LI 6400, LiCor Instruments, Lincoln, NE) over three days, with a single block being

measured on each day. Gas exchange measurements were conducted within a growth chamber to ensure

uniformity of ambient environmental conditions. Each plant was moved to the growth chamber and

allowed to adjust to ambient conditions in the growth chamber (photosynthetic photon flux of 1000

mol, air temperature of 30˚ C and vapor pressure deficit of 2.0 kPa), for at least 20 minutes before being

measured. From instantaneous gas exchange measurements we obtained photosynthetic rate on an area

basis, stomatal conductance, and instantaneous photosynthetic WUE. Photosynthesis on a mass basis was

calculated as photosynthesis on an area basis divided by SLA. It should be noted that the majority of

ANN plants had set bud or were beginning to flower, approximately reproductive stage (R) R1/2 during

gas exchange measurements (Schneiter and Miller 1981). Following the completion of gas exchange

measurements, plants were harvested. Leaves used for gas exchange were excised, scanned using a

flatbed scanner, measured for leaf reflectance values, and tested for leaf toughness as the average of 8

measurements using a Chatillon force gauge model DFE (Ametek Inc., Largo, FL). Leaves were dried at

60⁰C for at least 48 hours before weighing. Scanned leaf images were used to calculate area with image

analysis software ImageJ (U.S. National Institute of Health, Bethesda, MD) (Schneider et al. 2012). From

leaf chemistry analysis we obtained leaf N and leaf carbon isotopic ratio (leaf 13

C, measured on a

continuous flow mass spectrometer, University of Georgia Soils Ecology Lab, Athens, GA). Leaf 13

C is

a proxy for plant integrated WUE, a measure that incorporates the concentration of intercellular CO2 over

the entire lifespan of a leaf. The concentration of intercellular CO2 can be used as an estimate of

integrated WUE provided that leaf temperatures had been similar (Farquhar et al. 1989; Ehleringer et al.

1992; Donovan and Ehleringer 1994). A higher leaf 13

C (less negative) indicates higher WUE.

18

Statistical Analysis:

All analyses were carried out using a general linear model (PROC GLM) in the statistical

software package SAS® v. 9.3 (SAS Institute Inc. 2011). Main effects comparisons for species were

analyzed using the analysis of variance (ANOVA, PROC GLM in SAS V 9.3) and a significance of

P<0.05. Accessions comparisons within species were also analyzed using ANOVA and compared by least

squares means using a significance of P<0.05.

Results

At the seedling stage, ANN and TEPH differed for DTB. ANN had a smaller DTB, reaching a

rooting depth of 30cm much more quickly than TEPH (Figure 2.1, Table 2.1). However, ANN and TEPH

did not differ for RMR at time of harvest, but did differ in root biomass (Figure 2.1, Table 2.1). The lack

of species difference in RMR is likely due to a longer growing period for TEPH, because it took longer to

reach a rooting depth of 30 cm. A within-species analysis was also conducted to examine variation among

accessions. For ANN, accessions differed for DTB and belowground biomass, but not RMR (Table 2.1,

Appendix Figure 2.1). For TEPH, accessions differed for DTB but not RMR and root biomass (Table 2.1,

Appendix Figure 2.2).

At the mature stage, species differed for photosynthetic rate on an area basis, photosynthetic rate

on a mass basis and instantaneous WUE, but not integrated WUE, leaf nitrogen, or stomatal conductance

(Figure 2.2, Table 2.2). TEPH exhibited a higher photosynthetic rate on a mass basis and greater

instantaneous WUE. However ANN expressed a greater photosynthetic rate on a mass basis (Figure 2.2,

Table 2.2). Within-species accession variation was also examined (Table 2.1, Appendix Figure 2.3). ANN

accessions differed for photosynthetic rate on a mass basis and leaf nitrogen content, but not

photosynthetic rate on an area basis, instantaneous WUE or integrated WUE (Table 2.2, Appendix Figure

2.3). TEPH accession did not differ for traits related to photosynthetic capacity and WUE.

At the seedling stage, ANN and TEPH differed for leaf area of the most recent fully expanded

leaf, NDVI, SLA, total leaf area, and number of leaves (Figure 2.1, Table 2.1). TEPH had a smaller,

thicker leaves (lower leaf area and SLA) with a reduced NDVI which comprised a smaller canopy from a

19

greater total number of leaves as compared to ANN (Figure 2.1, Table 2.1). Components of NDVI were

also assessed individually and species differed for R680. TEPH reflected a higher percentage of incident

radiation at 680 nm than ANN, exhibiting a 1% increase in reflectance (Appendix Figure 2.4, Appendix

Table 2.1). ANN and TEPH did not differ in reflectance at R800 (Appendix Figure 2.4, Appendix Table

2.1). ANN accessions differed for total canopy leaf area, individual leaf area, and SLA, but not NDVI or

number of leaves (Table 2.1, Appendix Figure 2.1). TEPH accessions varied for SLA and number of

leaves, but not for NDVI, canopy leaf area or individual leaf area (Table 2.1, Appendix Figure 2.2). For

the seedling stage TEPH and ANN were also assessed for the productivity correlates aboveground

biomass and stem height. Species did not differ for aboveground biomass, but TEPH was taller at time of

harvest (Figure 2.1, Table 2.1). These results are likely due to the fact that TEPH took nearly twice as

long as ANN to reach a rooting depth of 30cm and subsequently undergo harvest.

At the mature stage, ANN and TEPH followed similar patterns for leaf traits at the seedling stage.

Species differed for leaf area of the most recent fully expanded leaf, NDVI, SLA as well as leaf toughness

(Figure 2.2, Table 2.2). Compared to ANN, TEPH leaves at the mature stage were smaller, thicker and

tougher (lower individual leaf area and SLA and greater leaf toughness) and had a reduced NDVI (Figure

2.2, Table 2.2). Components of NDVI were also assessed individually and species differed for R680.

TEPH reflected a higher percentage of incident radiation at 680 nm than ANN, exhibiting a 6% increase

in reflectance (Appendix Figure 2.5, Appendix Table 2.1). ANN and TEPH did not differ for R800

(Appendix Figure 2.5, Appendix Table 2.1). ANN accessions differed for individual leaf area, SLA and

leaf toughness, but not NDVI (Table 2.2, Appendix Figure 2.3). TEPH accession differed for SLA and

leaf toughness, but not individual leaf area or NDVI (Table 2.2, Appendix Figure 2.4).

Discussion

The aim of this study was to compare cultivated ANN and its wild relative TEPH for rooting and

canopy traits in order to identify novel trait characteristics conferring putative drought avoidance.

Because root development and architecture plays a critical role in supplying crops with adequate water,

one focus centered around seedling root elongation rate and RMR. Given previous findings concerning

20

the increased root elongation rate at the seedling stage in other wild sunflower species as compared to

cultivated varieties, TEPH was compared to ANN primarily based on its desert habitat and leaf

morphology. Because TEPH is generally described as exhibiting leaf morphology associated with

transpiration and heat load reduction, we also evaluated a suite of leaf traits in both species. Generally

speaking, the smaller, thicker and more pubescent leaves of TEPH are thought to reduce transpirational

water loss and heat load to aid in drought avoidance and may be desirable traits when breeding for

improved drought resistance in cultivated sunflower for drought resistance. Likewise, both species were

evaluated at the mature stage to assess the maintenance of adequate photosynthetic rates in light of

drought avoidance characteristics because avoidant plants may exhibit decreased photosynthetic rates that

could lead to an overall reduction in yield under non-water limiting conditions.

Rooting characteristics: seedling

Under well-watered conditions, seedlings of ANN and TEPH differed for the number of days to

reach a rooting depth of 30 cm but did not differ for RMR. In this study, the number of days to reach a

rooting depth of 30 cm is used as a proxy for root growth rate. Contrary to expectations, ANN had a

greater root growth rate under non-water limiting conditions at the seedling stage despite speculation

surrounding the desert dwelling species TEPH. Quicker time to deep rooting could prove to be beneficial

in cultivated sunflower (Seiler 1994; Seiler 1998). Previous work in primary and lateral root elongation

demonstrated that a hybrid line developed from a cross between cultivated H. annuus and the wild H.

petiolaris, a species inhabiting dry prairies (hereafter PET), had longer primary roots than a standard

height cultivated ANN genotype (9 and 5.2 cm respectively) at 10 days after planting (Seiler 1994).

However it should be noted that a dwarf variety of cultivated ANN, 471D, had a primary root length of

8.9 cm 10 days after planting and was nearly identical to PET (Seiler 1994). Although TEPH and PET

both grow in dry environments, TEPH did not exhibit the same rooting strategy in this study. This may be

due to the non-water limiting conditions of this experiment, typical of a well irrigated crop field, where

ANN likely was able to better use resources given the intense selection for increased growth rate that

cultivated varieties have undergone during domestication. Differences in root allocation have been noted

21

in coffee between drought sensitive and resistant lines under well-watered conditions, with drought

resistant lines showing greater final rooting depth at time of harvest (Pinheiro, DaMatta et al. 2005).

Though TEPH had a slower root growth rate when compared to ANN in well watered conditions, it might

be worthwhile to evaluate TEPH and ANN under drought stress and test for differences in rooting

allocation.

Given the difference in root growth rate between TEPH and ANN, it is surprising that there was

no difference for RMR and aboveground biomass, though belowground biomass was greater for ANN

than for TEPH. Because RMR is a ratio dependent upon growth allocation (belowground biomass to total

biomass) and TEPH was slower to reach a rooting depth of 30 cm, the lack of difference between the two

species is likely due to the greater number of growing days (nearly 2 fold greater than ANN) for TEPH.

Because TEPH is a desert species, the general expectation is that allocation of total biomass to roots is

higher compared to species found in more mesic habitats (Barbour 1973), however root mass accounted

for ~8% of total biomass in ANN and ~7% in TEPH.

Leaf characteristics: seedling and mature

TEPH possesses favorable leaf morphology to offset canopy heat load at both the seedling and

mature stages. When comparing seedlings, TEPH produced smaller leaves with a lower SLA, or a thicker

and/or denser leaf than ANN. Mature TEPH also produced smaller leaves with reduced SLA, indicating

that the relative species difference holds through ontogenetic stages. It has been shown that leaf size and

SLA are positively correlated with moisture availability, and specifically a lower SLA is thought to

contribute to protection from desiccation (Mooney and Dunn 1970). Under conditions of high radiation

and low soil moisture availability, reduced SLA and accompanied reduction in leaf size can help prevent

desiccation by reducing transpirational surface area (Mooney and Dunn 1970; Ackerly, Knight et al.

2002). Smaller, thicker leaves are also associated with reduced boundary layer resistance which

contributes to increased leaf cooling (Mooney and Dunn 1970; McDonald, Fonseca et al. 2003). Larger

leaves, such as those found in ANN at both the seedling and adult stages, shed absorbed heat more slowly

than the smaller leaves found in TEPH and can actually exceed ambient air temperatures during sunny

22

days. Under mesic conditions heat load is not as detrimental to the metabolic function of the plant because

transpiration serves to cool the plant, however under decreasing available soil moisture, plants cannot use

water as freely for heat dissipation and thus smaller leaves serve to reduce damage by heat load

(McDonald, Fonseca et al. 2003). Crop size also plays an important role in total water consumption and is

affected primarily by leaf area index, or the total amount of leaf area given the total ground surface

occupied by the plant canopy. At the whole plant level, an increase in total leaf area increases potential

transpiration and can accelerate water consumption and decrease water use efficiency. Thus, by reducing

total leaf area, plants are able to reduce their total water consumption (i.e. less transpiration) (Blum 1996).

Because TEPH exhibits these leaf characteristics throughout development, it could prove to be a source of

beneficial alleles for improved drought resistance for sunflower crops grown in drought prone

environments.

TEPH also exhibited lower values of NDVI at both the seedling and adult stage. Lower values

typically indicate “less green” biomass on a canopy wide scale, which could be an indication of TEPH’s

dense leaf pubescence. In order to determine what was driving TEPH to be lower than ANN for NDVI we

compared the individual components of NDVI, R680 and R800 at both the adult and mature stages. We

found that TEPH had lower values because it was reflecting a higher percentage of incident radiation at

680 nm than ANN at both the seedling (1% increase in reflectance) and mature (6% increase in

reflectance) stages. We observed no difference in R800 between TEPH and ANN at either stage. This

resulted in a lower NDVI for TEPH. It has been shown that pubescence increases as aridity levels

increases (Ehleringer 1984). A thick covering of leaf hairs under high light environments could prove to

be advantageous in reflecting excess radiation and thus might be worthwhile for breeding purposes

specifically for environments with a large heat load (Ehleringer 1984). However, it should be noted that

reflectance is typically across the entire spectrum, which includes photosynthetically active radiation

(Ehleringer 1984). When considering breeding for increased pubescence, it may only be desirable for

environments with large amounts of radiation, i.e. near the equator or marginal desert cropping lands.

23

Photosynthesis, WUE and leaf chemistry: mature

Maintaining adequate photosynthetic carbon gain under well-watered conditions is crucial for

generating acceptable yields in cropping systems. Though plants may be drought resistant, if lines

displaying characteristics associated with drought avoidance also show decreased photosynthetic rates

under well-watered conditions they may not be favorable targets for breeding. Though ANN and TEPH

differed for photosynthetic rate on an area basis at the mature stage, it should be noted, that ANN’s

photosynthetic rate on an area basis was lower than expected based on previous measurements (Sobrado

and Turner 1986). Sobrado and Turner (1986) report a maximum photosynthetic rate of approximately 40

µmol m2 s

-1 for cultivated ANN compared to our rate of 36.34 ± 1.26 µmol m

2 s

-1 (Table 2.2). We suggest

that our rates were lower than previously reported values due to differences in sampled leaf age. We

sampled ANN plants during bud set or approximately reproductive stage R1/2 from Schneiter and Miller

(Schneiter and Miller 1981). A previous glasshouse study done on cultivated sunflower found

photosynthetic rate to decrease by nearly half within 15 days of recording the maximum photosynthetic

rate and the authors concluded that leaf age is a major determining factor controlling photosynthetic

output (Rawson and Constable 1980). Although TEPH had a greater photosynthetic rate on an area basis,

ANN was found to have a greater photosynthetic rate on a mass basis as compared to TEPH (Figure 2.2),

due to a greater SLA for ANN. Photosynthetic rate on a mass basis has been shown to be positively

correlated with both SLA and nitrogen content on a mass basis and is generally associated with an

increased relative growth rate(Wright, Reich et al. 2004). Our data for ANN and TEPH concerning

photosynthetic rate on a mass basis and SLA are consistent with the trend outlined above and suggest

TEPH leaf thickness may not be conducive for the potential of high productivity as inferred by

photosynthetic rate on a mass basis under optimal conditions.

We observed lower stomatal conductance for TEPH as compared to ANN. TEPH was able to

maintain a comparable photosynthetic rate on an area basis under decreased stomatal conductance which

is reflected in a higher instantaneous WUE. However, instantaneous WUE is a measurement that changes

over the course of minutes given changes in environmental conditions and may not be as robust as

24

measuring WUE over longer time courses. In order to get a more comprehensive measurement of water

use efficiency we also evaluated plants for integrated WUE estimated from leaf 13

C. We found no

difference in integrated WUE for ANN and TEPH under well-watered conditions. However, integrated

WUE is a rather plastic trait and change in environmental conditions can have significant effects on this

measurement (Richards 1996). Therefore even though differences were not detectable in this study, it

should not be ruled out that TEPH could have a higher integrated WUE under drought stress and thus

may provide an advantage in crop yield in agricultural areas with limited rainfall.

Conclusion

This study suggests that under well watered conditions, ANN roots grow more quickly at the

seedling stage. At the leaf level for both seedling and mature plants, TEPH had smaller, thicker more

pubescent leaves which are congruent with decreased heat load under water limited and high light

conditions. At the mature stage TEPH had a lower photosynthetic rate on a mass basis suggesting there is

a tradeoff between leaf traits that reduce heat load and WUE and photosynthetic rate on a mass basis.

While it may not be desirable to use TEPH in breeding for root growth rate, it could be useful for

desirable leaf level heat load reducing traits. These traits need to be further explored under water limited

conditions.

25

References

Ackerly, D., C. Knight, et al. (2002). "Leaf size, specific leaf area and microhabitat distribution of

chaparral woody plants: contrasting patterns in species level and community level analyses."

Oecologia 130(3): 449-457.

Ashraf, M. and J. W. O'Leary (1996). "Effect of drought stress on growth, water relations, and gas

exchange of two lines of sunflower differing in degree of salt tolerance." International Journal of

Plant Sciences 157(6): 729-732.

Barbour, M. G. (1973). "Desert dogma reexamined: root/shoot productivity and plant spacing." American

Midland Naturalist 89(1): 41-57.

Barbour, M. G. and W. D. Billings (1988). North American Terrestrial Vegetation. New York, NY,

Cambridge University Press.

Blum, A. (1996). "Crop responses to drought and the interpretation of adaptation." Plant Growth

Regulation 20(2): 135-148.

Boyer, J. S. (1982). "Plant Productivity and Environment." Science 218(4571): 443-448.

Chaves, M. M., J. P. Maroco, et al. (2003). "Understanding plant responses to drought—from genes to the

whole plant." Functional Plant Biology 30(3): 239-264.

Craufurd, P. Q., T. R. Wheeler, et al. (1999). "Effect of temperature and water deficit on water-use

efficiency, carbon isotope discrimination, and specific leaf area in peanut." Crop Science 39(1):

136-142.

Donovan, L. A. and J. R. Ehleringer (1994). "Potential for selection on plants for water-use efficiency as

estimated by carbon isotope discrimination." American Journal of Botany 81(7): 927-935.

Ehleringer, J. (1984). "Ecology and ecophysiology of leaf pubescence in North American desert plants."

Biology and chemistry of plant trichomes: 113-132.

Ehleringer, J. (1984). Ecology and ecophysiology of leaf pubescence in North American desert plants.

Biology and Chemistry of Plant Trichomes. P. L. H. E. Rodriguez, I. Mehta New York, Plenum

Press: 113-132.

26

Farquhar, G. D., J. R. Ehleringer, et al. (1989). "Carbon isotope discrimination and photosynthesis."

Annual Review of Plant Physiology and Plant Molecular Biology 40: 503-537.

Farquhar, G. D. and T. D. Sharkey (1982). "Stomatal conductance and photosynthesis." Annual Review

of Plant Physiology and Plant Molecular Biology 33: 317-345.

Gausman, H. W., R. M. Menges, et al. (1977). "Pubescence affects spectra and imagery of silverleaf

sunflower (Helianthus argophyllus)." Weed Science 25(5): 437-440.

Hajjar, R. and T. Hodgkin (2007). "The use of wild relatives in crop improvement: a survey of

developments over the last 20 years." Euphytica 156(1-2): 1-13.

Heiser, C. B. and D. M. Smith (1969). "The North American sunflowers (Helianthus)." (22).

Houghton, J. T., Y. Ding, et al. (2001). Climate change 2001: the scientific basis, Cambridge University

Press Cambridge.

Jackson, L. E. and G. W. Koch (1997). The ecophysiology of crops and their wild relatives. Ecology in

Agriculture. L. E. Jackson. San Diego Academic Press: 3-37.

Jepson, W. L. and J. C. Hickman (1993). The Jepson Manual: Higher Plants of California, University of

California Press.

Kramer, P. J. and J. S. Boyer (1995). Water Relations of Plants and Soils, Academic Press.

Lopez, F. B., Y. S. Chauhan, et al. (1997). "Effects of timing of drought stress on leaf area development

and canopy light interception of short duration pigeonpea." Journal of Agronomy and Crop

Science 178(1): 1-7.

McDonald, P. G., C. R. Fonseca, et al. (2003). "Leaf-size divergence along rainfall and soil-nutrient

gradients: is the method of size reduction common among clades?" Functional Ecology 17(1): 50-

57.

Mooney, H. A. and E. L. Dunn (1970). "Convergent Evolution of Mediterranean-Climate Evergreen

Sclerophyll Shrubs." Evolution 24(2): 292-303.

Passioura, J. (2006). "Increasing crop productivity when water is scarce—from breeding to field

management." Agricultural Water Management 80(1): 176-196.

27

Penuelas, J., I. Filella, et al. (1995). "Assessment of photosynthetic radiation-use efficiency with spectral

reflectance." New Phytologist 131(3): 291-296.

Pinheiro, H. A., F. M. DaMatta, et al. (2005). "Drought tolerance is associated with rooting depth and

stomatal control of water use in clones of Coffea canephora." Annals of Botany 96(1): 101-108.

Rawson, H. and G. Constable (1980). "Carbon Production of Sunflower Cultivars in Field and Controlled

Environments. I. Photosynthesis and Transpiration of Leaves, Stems and Heads." Functional

Plant Biology 7(5): 555-573.

Richards, R. (1996). "Defining selection criteria to improve yield under drought." Plant Growth

Regulation 20(2): 157-166.

Richards, R. A. (2006). "Physiological traits used in the breeding of new cultivars for water-scarce

environments." Agricultural Water Management 80(1–3): 197-211.

Sandquist, D. R. and J. R. Ehleringer (1997). "Intraspecific variation of leaf pubescence and drought

response in Encelia farinosa associated with contrasting desert environments." New Phytologist

135(4): 635-644.

SAS Institute Inc. 2011. Base SAS® 9.3 Procedures Guide. Cary, NC: SAS Institute

Inc.

Schilling, E. E. and C. B. Heiser (1981). "Infrageneric classification of Helianthus (Compositae)." Taxon

30(2): 393-403.

Schneiter, A. and J. Miller (1981). "Description of sunflower growth stages." Crop Science 21(6): 901-

903.

Seiler, G., T. Gulya, et al. (2006). "Exploration for wild Helianthus species from the desert Southwestern

USA for potential drought tolerance." Helia 29(45): 1-10.

Seiler, G. and C.-C. Jan (2010). Basic information. Genetics, Genomics and Breeding of Sunflower. G.

Seiler, C.-C. Jan, J. Hu and C. Kole. Boca Raton, FL, Science Publishers, Inc: 1-50.

Seiler, G. J. (1992). "Utilization of wild sunflower species for the improvement of cultivated sunflower."

Field Crops Research 30(3): 195-230.

28

Seiler, G. J. (1994). "Primary and lateral root elongation of sunflower seedlings." Environmental and

Experimental Botany 34(4): 409-418.

Seiler, G. J. (1998). "Influence of temperature on primary and lateral root growth of sunflower seedlings."

Environmental and Experimental Botany 40(2): 135-146.

Seiler, G. J. and L. H. Rieseberg (1997). Systematics, origins and germplasm resources of the wild and

domesticated sunflower. Madison, WI, American Society of Agronomy.

Sheffield, J. and E. F. Wood (2008). "Global Trends and Variability in Soil Moisture and Drought

Characteristics, 1950–2000, from Observation-Driven Simulations of the Terrestrial Hydrologic

Cycle." Journal of Climate 21(3): 432-458.

Shimshi, D., M. L. Mayoral, et al. (1982). "Responses to Water Stress in Wheat and Related Wild

Species." Crop Science. 22(1): 123-128.

Sobrado, M. A. and N. C. Turner (1986). "Photosynthesis, dry matter accumulation and distribution in the

wild sunflower helianthus petiolaris and the cultivated sunflower helianthus annuus as Influenced

by water deficits." Oecologia 69(2): 181-187.

Thompson, T. E., D. C. Zimmerman, et al. (1981). "Wild Helianthus as a genetic resource." Field Crops

Research 4(0): 333-343.

Tucker, C. J. (1979). "Red and photographic infrared linear combinations for monitoring vegetation."

Remote Sensing of Environment 8(2): 127-150.

Turner, N. and J. Begg (1981). "Plant-water relations and adaptation to stress." Plant and Soil 58(1): 97-

131.

Verslues, P. E., M. Agarwal, et al. (2006). "Methods and concepts in quantifying resistance to drought,

salt and freezing, abiotic stresses that affect plant water status." The Plant Journal 45(4): 523-539.

Wright, I. J., P. B. Reich, et al. (2004). "The worldwide leaf economics spectrum." Nature 428(6985):

821-827.

29

DT

B

(d

ay

s)

15

30

45

RM

R

(g g

-1)

0.03

0.06

0.09

ND

VI

0.3

0.6

0.9

TO

TA

L

(cm

2)

15

30

45

AR

EA

(cm

2)

15

30

45

HE

IGH

T

(cm

)

4

8

12

LE

AV

ES

(#)

5

10

15

SL

A

(cm

2 g

-1)

150

300

450

ANN TEPH

AG

B

(g)

0.1

0.2

0.3

ANN TEPH

RO

OT

S

(g)

0.0075

0.0150

0.0225

***

* ***

** ***

** ***

**

Figure 2.1

30

Figure 2.1 Comparison of Helianthus annuus (ANN) and H. niveus ssp. tephrodes (TEPH) at the

seedling stage for measured traits. Measured traits include, DTB (in the number of days to reach a rooting

depth of 30 cm), RMR (root mass ratio, root to total plant mass ratio), NDVI (the normalized difference

vegetative index), SLA (specific leaf area), TOTAL (total leaf area per plant), AREA (average leaf area

for a pair of most recent fully expanded leaves), HEIGHT (the stem height measured from the base of the

main stem to growing tip), LEAVES (the number of true leaves), AGB (above ground biomass) and

ROOTS (below ground biomass). P-values for significance are indicated by *, where * P = 0.05, ** P =

0.01 and *** P > 0.001.

31

A

mo

l m

-2 s

-1)

15

30

45

gs

(m

mo

l m

-2 s

-1)

0.4

0.8

1.2

iWU

E

(mm

ol

/ m

ol)

20

40

60

AM

AS

S

(nm

ol

g-1

s-1

)

300

600

900

AR

EA

(cm

2)

75

150

225

ND

VI

0.3

0.6

0.9

1

3C

(‰

)

-30

-20

-10

% N

2

4

6

ANN TEPH

SL

A

(cm

-2 g

-1)

75

150

225

ANN TEPH

TO

UG

H

(N)

0.2

0.4

0.6

*

* ***

*** ***

*** ***

Figure 2.2

32

Figure 2.2 Comparison of Helianthus annuus (ANN) and H. niveus ssp. tephrodes (TEPH) at the mature

stage for measured traits. Measured traits include, A (photosynthesis on an area basis), gs (stomatal

conductance), iWUE (instantaneous water use efficiency), AMASS (photosynthesis on a mass basis),

AREA (area of most recent fully expanded leaf), NDVI (the normalized difference vegetative index),

13

C (integrated water use efficiency), N (percent leaf nitrogen), TOUGH (leaf toughness) and SLA

(specific leaf area). P-values for significance are indicated by *, where * P = 0.05, ** P = 0.01 and *** P

> 0.001.

33

Table 2.1 Distribution of seedling means (± SE) for two species of Helianthus (sunflower), H. annuus and H. niveus ssp. tephrodes for

measured traits. Measured traits include, DTB (in the number of days to reach a rooting depth of 30 cm), RMR (root mass ratio, root to

total plant mass ratio), NDVI (the normalized difference vegetative index), SLA (specific leaf area), TOTAL (total leaf area per plant),

AREA (average leaf area for a pair of most recent fully expanded leaves), HEIGHT (the stem height measured from the base of the main

stem to growing tip), LEAVES (the number of true leaves), AGB (above ground biomass) and ROOTS (below ground biomass). Means

represent the pooled values across accessions for each species.

Species Accession

Trait H. annuus H.niveus ssp.

tephrodes

H. annuus H.niveus ssp.

tephrodes

F df P F df P F df P

DTB 23.06 ± 0.87 38.06 ± 0.94 354.79 1, 31 <0.0001 6.10 3, 3 0.0038 9.22 3, 3 0.0004

RMR 0.08 ± 0.01 0.08 ± 0.01 0.29 1, 31 ns 1.33 3, 3 ns 0.14 3, 3 ns

NDVI 0.79 ± 0.01 0.75 ± 0.01 6.96 1, 31 0.0154 0.05 3, 3 ns 0.27 3, 3 ns

SLA 487.76 ± 6.99 306.34 ± 14.31 1072.11 1, 31 <0.0001 10.27 3, 3 0.0002 62.89 3, 3 <0.0001

LA 45.50 ± 5.15 31.18 ± 3.15 354.79 1, 31 0.0012 14.97 3, 3 <0.0001 2.72 3, 3 ns

MRFEL LA 39.18 ± 4.08 18.76 ± 1.32 115.07 1, 31 <0.0001 41.62 3, 3 <0.0001 1.83 3, 3 ns

HEIGHT 8.31 ± 0.58 10.77 ± 1.10 9.34 1, 31 0.0060 5.05 3, 3 0.0086 11.19 3, 3 0.0001

LEAVES 7.1250 ± 0.23 12.88 ± 0.70 107.10 1, 31 <0.0001 0.25 3, 3 ns 9.02 3, 3 0.0005

AGB 0.2435 ± 0.02 0.23 ± 0.02 0.66 1, 31 ns 13.36 3, 3 <0.0001 4.20 3, 3 0.0178

ROOTS 0.021 ± 0.002 0.016 ± 0.001 8.38 1, 31 0.0087 5.46 3, 3 0.0062 2.82 3, 3 ns

34

Table 2.2 Distribution of mature means (± SE) for two species of Helianthus (sunflower), H. annuus and H. niveus ssp. tephrodes for measured

traits. Measured traits include, A (photosynthesis on an area basis), gs (stomatal conductance), iWUE (instantaneous water use efficiency),

AMASS (photosynthesis on a mass basis), AREA (area of most recent fully expanded leaf), NDVI (the normalized difference vegetative

index), 13

C (integrated water use efficiency), N (percent leaf nitrogen), TOUGH (leaf toughness) and SLA (specific leaf area). Means

represent the pooled values across accessions for each species.

Species Accession

Trait H. annuus H. niveus ssp.

tephrodes

H. annuus H.niveus ssp.

tephrodes

F df P F df P F df P

A 36.34 ± 1.26 41.22 ± 1.17 5.07 1, 37 0.0342 1.29 2, 3 ns 1.56 2, 8 ns

gs 0.91 ± 0.05 0.87 ± 0.04 0.49 1, 37 ns 0.84 2, 3 ns 1.05 2, 8 ns

iWUE 42.91 ± 2.93 49.27 ± 1.38 4.91 1, 37 0.0368 0.81 2, 3 ns 0.84 2, 8 ns

AMASS 708.12 ± 46.02 622.59 ± 16.53 10.28 1, 37 0.0009 11.41 2, 3 <0.0001 2.11 2, 8 ns

AREA 194.81 ± 10.07 30.99 ± 1.83 1338.74 1, 37 <0.0001 18.16 2, 3 <0.0001 1.59 2, 8 ns

NDVI 0.81 ± 0.01 0.69 ± 0.01 61.99 1, 37 <0.0001 0.19 2, 3 ns 1.72 2, 8 ns

13

C -30.62 ± 0.16 -30.66 ± 0.10 0.04 1, 37 ns 0.28 2, 3 ns 1.41 2, 8 ns

%N 4.91 ± 0.27 4.85 ± 0.09 1.25 1, 37 ns 10.49 2, 3 0.0002 1.73 2, 8 ns

TOUGH 0.43 ± 0.04 0.57 ± 0.03 36.40 1, 37 <0.0001 9.96 2, 3 0.0002 7.20 2, 8 0.0002

SLA 253.95 ± 61.81 156.46 ± 7.86 20.58 1, 37 0.0001 5.26 2, 3 0.0065 6.63 2, 8 0.0002

35

RO

OT

S (

g)

0.01

0.02

0.03

AG

B (

g)

0.15

0.30

0.45

LE

AV

ES

(#

)3

6

9

HE

IGH

T (

cm)

4

8

12

AR

EA

(cm

2)

20

40

60

TO

TA

L (

cm2

)

25

50

75

SL

A (

cm2 g

-1)

200

400

600

ND

VI

0.3

0.6

0.9

DT

B (

da

ys)

10

20

30

RM

R (

g g

-1)

0.04

0.08

0.12ns

ns

ns

BB

B

A

B BA A

B

CC

A

A

A

B

B

A

BC

AB

C

A

B

CC

AA

A

B

Appendix Figure 2.1

36

Appendix Figure 2.1 Comparison of Helianthus annuus seedling accession means for measured traits.

Measured traits include, DTB (days to reach 30cm rooting depth), RMR (root mass ratio), NDVI

(normalized difference vegetative index), SLA (specific leaf area), TOTAL (total leaf), AREA (leaf area

of most recent fully expanded leaf), HEIGHT (stem height), LEAVES (number of true leaves), AGB

(above ground biomass) and ROOTS (below ground biomass). Means which share letters do not differ for

LSMeans at P < 0.05. From left to right means represent GRIN accession identifiers PI-642777 (HA-412-

HO), PI-560141 (RHA-373), PI-578872 (HA-383) and PI-607506 (RHA 415).

37

DT

B (

da

ys)

15

30

45

RM

R (

g g

-1)

0.04

0.08

0.12N

DV

I

0.3

0.6

0.9

SL

A (

cm2 g

-1)

150

300

450

LA

(cm

2)

20

40

60

MR

FE

L L

A (

cm2)

10

20

30

HE

IGH

T (

cm)

5

10

15L

EA

VE

S

5

10

15

AG

B (

g)

0.1

0.2

0.3

RO

OT

S (

g)

0.0075

0.0150

0.0225

ns ns

ns

ns

ns

AB AB

A

B

A

A

A

B

A A

BB

A

C

B

C

ABB C

Appendix Figure 2.2

38

Appendix Figure 2.2 Comparison of Helianthus niveus ssp. tephrodes seedling accession means for

measured traits. Measured traits include, DTB (days to reach 30cm rooting depth), RMR (root mass

ratio), NDVI (normalized difference vegetative index), SLA (specific leaf area), TOTAL (total leaf),

AREA (area of most recent fully expanded leaf), HEIGHT (stem height), LEAVES (number of true

leaves), AGB (above ground biomass) and ROOTS (below ground biomass). Means which share letters

do not differ for LSMeans at P < 0.05. From left to right means represent GRIN accession identifiers PI-

664653 (AMES-27850), PI-613758 (AMES-6852), PI-650018 (AMES-27422), and PI-664643 (AMES-

27421).

39

A

( m

ol

m-2

s-1

)

15

30

45

gs

(mm

ol

m-2

s-1)

0.4

0.8

1.2

iWU

E

(mm

ol

/ m

ol

)

20

40

60

AM

AS

S

(nm

ol

g-1

s-1

)

300

600

900

AR

EA

(cm

2)

75

150

225

ND

VI

0.3

0.6

0.9

1

3C

(‰

)

-30

-20

-10

%N

2

4

6

TO

UG

H

(N)

0.2

0.4

0.6

SL

A

(cm

-2 g

-1)

75

150

225

ns ns

nsA

BB

C

B

C

AAB

ns

nsA

BB

B

B

A

C

BC

AAA

B

Appendix Figure 2.3

40

Appendix Figure 2.3 Comparison of Helianthus annuus mature accession means for traits measured.

Traits measured include, A (photosynthesis on a area basis), gs (stomatal conductance), iWUE

(instantaneous water use efficiency), AMASS (photosynthesis on a mass basis), AREA (area of most

recent fully expanded leaf), NDVI (the normalized difference vegetative index), 13

C (integrated water

use efficiency), N (percent leaf nitrogen), TOUGH (leaf toughness) and SLA (specific leaf area). P-values

for significance are indicated by *, where * P = 0.05, ** P = 0.01 and *** P > 0.001. From left to right

means represent GRIN accession identifiers PI-642777 (HA-412-HO), PI-560141 (RHA-373), PI-578872

(HA-383) and PI-607506 (RHA 415).

41

R6

80

0.03

0.06

0.09

ANN TEPH

R800

0.3

0.6

0.9

**

Appendix Figure 2.4 Species means for normalized difference vegetative index (NDVI) components at

the seedling stage. NDVI components consist of reflectance at red 680 nm (R680) and near infrared 800

nm (R800). Species are abbreviated as Helianthus annuus (ANN) and H. niveus ssp. tephrodes (TEPH). P-

values for significance are indicated by *, where * P = 0.05, ** P = 0.01 and *** P > 0.001.

42

R6

80

0.05

0.10

0.15

ANN TEPH

R800

0.3

0.6

0.9

***

Appendix Figure 2.5 Species means for normalized difference vegetative index (NDVI) components at

the mature stage. NDVI components consist of reflectance at red 680 nm (R680) and near infrared 800 nm

(R800). Species are abbreviated as Helianthus annuus (ANN) and H. niveus ssp. tephrodes (TEPH). P-

values for significance are indicated by *, where * P = 0.05, ** P = 0.01 and *** P > 0.001.

43

A

( m

ol

m-2

s-1

)20

40

60

gs

(mm

ol

m-2

s-1)

0.4

0.8

1.2

iWU

E

(mm

ol

/ m

ol)

20

40

60

AM

AS

S

(nm

ol

g-1

s-1

)

250

500

750

AR

EA

(cm

2)

0.3

0.6

0.9

ND

VI

0.3

0.6

0.9

1

3C

(‰

)

-30

-20

-10

%N

2

4

6

TO

UG

H

(N)

0.25

0.50

0.75

SL

A

(cm

-2 g

-1)

100

200

300

ns ns

ns ns

ns ns

ns ns

A

B

AA

AA

A

A

AA

C BCBCB

BCBC

BCBC

Appendix Figure 2.6

44

Appendix Figure 2.6 Comparison of Helianthus niveus ssp. tephrodes mature accession means for traits

measured. Traits measured include, A (photosynthesis on an area basis), gs (stomatal conductance), iWUE

(instantaneous water use efficiency), AMASS (photosynthesis on a mass basis), AREA (area of most

recent fully expanded leaf), NDVI (the normalized difference vegetative index), 13

C (integrated water

use efficiency), N (percent leaf nitrogen), TOUGH (leaf toughness) and SLA (specific leaf area). P-values

for significance are indicated by *, where * P = 0.05, ** P = 0.01 and *** P > 0.001. From left to right

means represent GRIN accession identifiers PI-642777 (HA-412-HO), PI-560141 (RHA-373), PI-578872

(HA-383) and PI-607506 (RHA-415). For TEPH the accession identifiers are PI-664653 (AMES-27850),

PI-613758 (AMES-6852), PI-650018 (AMES-27422), PI-664643 (AMES-27421), PI-650017(AMES-

27420), PI-650019 (AMES-27830), PI-650020 (AMES-27831), PI-650021 (AMES-27832), PI-664651

(AMES-27847) and PI-664654 (AMES-27851).

45

Appendix Table 2.1 Distribution of means for normalized difference vegetative index (NDVI)

components R680 and R800 with associated standard error between two species of Helianthus (sunflowers).

Means represent the pooled values across accessions for each species.

H. annuus H. niveus ssp.

tephrodes

Ontogenetic

Stage

Reflectance

band

Reflectance (%) F df P

Seedling R680 0.056 ± 0.002 0.069 ± 0.004 9.61 1, 31 0.0054

R800 0.476 ± 0.007 0.492 ± 0.010 1.72 1, 31 0.2038

Mature R680 0.062 ± 0.006 0.127 ± 0.008 33.49 1, 37 <0.0001

R800 0.593 ± 0.045 0.708 ± 0.039 2.59 1, 37 0.1210

46

CHAPTER 3

CULTIVATED HELIANTHUS ANNUUS AND WILD RELATIVES (H. ARGOPHYLLUS AND H.

NIVEUS SSP. TEPHRODES) DIFFER IN GERMINATION RESPONSE TO SIMULATED DROUGHT

STRESS1

__________________________ 1Milton, E.F., E.W. Goolsby and L. A. Donovan, to be submitted to Helia

47

Abstract

Wild sunflowers may be a source of desirable traits for improvement of cultivated sunflower

Helianthus annuus. We tested the expectation that two wild species of sunflower (Helianthus), one native

to the Algodones Dunes region of the Sonoran Desert (H. niveus ssp. tephrodes) and one native to

western coastal Texas (H. argophyllus), would have higher percent germination and quicker germination

under simulated drought stress conditions as compared to the cultivated common sunflower (H. annuus).

Both wild species have been hypothesized to be drought resistant based on their native habitat, with H.

niveus spp. tephrodes receiving limited rainfall and H. argophyllus growing in the sandy saline

environment of coastal Texas. In a growth chamber study, we compared three representative accessions of

each species for germination under four osmotic treatments imposed with polyethylene glycol 6000 (PEG

6000) to simulate varying levels of drought stress: 0, -0.4, -0.8 and -1.2 MPa. As expected, all three

species responded to increasing simulated drought stress with decreased percent germination and delayed

germination. However, the expectation that both wild species would fare substantially better than

cultivated H. annuus in response to increasing stress was not supported. Helianthus argophyllus did

respond to increasing stress with less of a delay and more uniformity for germination than H. annuus, but

it had only a trend for greater percent germination in the -1.2 MPa treatment. In contrast, H. niveus ssp.

tephrodes responded to increasing stress with lower percent germination, more of a delay and less

uniformity than H. annuus. Our results provide little evidence that either wild species is likely to be a

potential donor for desirable genetic variation as pertaining to improved drought resistance in cultivated

sunflower.

INDEX WORDS: Sunflower, Drought, Germination Polyethylene Glycol, Algodones dunes

48

Introduction

Characterizing traits that allow species to live in biologically taxing habitats is often the first step

to discovering potential novel germplasm to be utilized for increased production in agricultural settings.

With the onset of global warming, precipitation patterns are expected to change, causing agricultural

production to rely on fewer and less predictable rainfall events. Given these changes in precipitation

patterns and the increase in mean global temperature, both frequency and severity of drought are expected

to increase significantly (IPCC 2007). Drought is widely accepted as one of the most limiting and

complex abiotic stresses in agriculture (Boyer 1982; Blum 1996; Passioura 1996; Verslues, Agarwal et al.

2006). It is currently estimated that one fifth of the world’s agricultural areas are irrigated while

producing roughly two fifths of the food supply (Döll 2002). Because water is a limited resource,

developing more drought resistant cultivars is imperative for maintenance of yield under marginal

agricultural conditions in order to meet the increasing food supply demands in our growing global

population. A potential avenue for increased drought resistance is through the introduction of drought

adapted wild germplasm into cultivated stock. Since cultivated plants have often lost genetic variation and

stress resistance during the domestication and selection for fast growth and yield, wild congeners are

receiving renewed attention as source of allelic variation for enhanced abiotic stress resistance (Richards

1996; Jackson and Koch 1997; Tanksley and McCouch 1997; Koziol, Rieseberg et al. 2012).

The timing and onset of drought can affect final yield in differing degrees of severity depending

on the ontogenetic stage in which the crop is being impacted (Passioura 1996). Passioura (Passioura

1996) argues that phenology, or the developmental timing of a crop as it relates to water availability, is

the most important consideration for determining drought resistance. In many crop species it has been

shown that the two most susceptible times for crop failure due to drought are the germination to seedling

stage and the flowering stage (Ahmad, Ahmad et al. 2009). In terms of crop establishment, undoubtedly

the most critical developmental stage is germination (Ashraf and Mehmood 1990). Drought can impact

germination by reducing percent germination or deferring it. A loss of uniform germination, emergence

and stand establishment can be a significant barrier to production and yield (Harris, Joshi et al. 1999;

49

Iqbal and Ashraf 2006). One method to overcome this barrier has been the development of pre-sowing

seed treatments. Proposed treatments such as hydropriming (water soaking), halopriming (inorganic salt

solution soaking) and osmopriming (organic osmotica solution soaking) have been shown to enhance

germination in the face of water stress imposed by both drought and soil salinity (Hegarty 1978; Ashraf

and Foolad 2005; Kaya, Okçu et al. 2006). However, pre-sowing treatments can be both economically

costly and time consuming and have varying effects depending on crop and soil moisture at sowing

(Taylor and Harman 1990). Wild congeners of crops, particularly from water stressed environments may

be a more effective source of drought resistance for improvement of germination characteristics under

drought stress.

Sunflower (Helianthus) is a genus of 49 species of both perennial and annual species and contains

one of the few food crops native to North America (Heiser and Smith 1969; Schilling and Heiser 1981).

The genus as a whole displays a wide geographic distribution occupies an array of diverse habitats and

possess an abundance of variability for agronomically important traits from yield to abiotic and biotic

stress resistance (Heiser and Smith 1969). Two examples of the variability within this genus are silver

leaf sunflower, H. argophyllus and the dunes sunflower, H. niveus ssp. tephrodes. Both of these species

are hypothesized to be drought resistant based on their native arid habitats and the presence of leaf

characteristics associated with species adapted to water limited environments, such as dense white leaf

pubescence (Baldini and Vannozzi 1998; Murillo‐Amador, López‐Aguilar et al. 2002; Seiler, Gulya et al.

2006; Seiler, Gulya et al. 2006).

Compared to cultivated H. annuus, H. argophyllus and H. niveus ssp. tephrodes grow in more

water limited environments. However these environments are very different in terms of water limitation.

Helianthus argophyllus grows along the coastal Texas plain, from inland fields to saline dunes along the

coast while H. niveus ssp. tephrodes in the extremely hot and dry Algodones dunes region of the Sonoran

Desert. Previous field studies using inbred lines derived from H. argophyllus × H. annuus have provided

evidence of its usefulness as a source of variation for performance under drought conditions (Baldini and

Vannozzi 1998; Baldini and Vannozzi 1999). The results of these studies indicate that one H. argophyllus

50

× H. annuus line had a higher water use efficiency, and harvest index in drought conditions than

cultivated sunflower, but the hybrid line performed more poorly under well watered conditions (Baldini

and Vannozzi 1998; Baldini and Vannozzi 1999). Another study evaluated lines of H. argophyllus × H.

annuus hybrid seedlings for short term root and shoot growth under water stress conditions induced by

polyethylene glycol (PEG) and found that H. argophyllus derived lines performed similarly to cultivated

H. annuus and wild H. debilis (El Midaoui, Serieys et al. 2003).Conversely, very little is known about the

drought physiology of H. niveus ssp. tephrodes.

Experimentally, PEG can be used to create osmotic stress treatments that simulate drought stress

for germination, although it is less desirable for studies of seedling growth (Michel 1973; Iqbal and

Ashraf 2006). For crops including sunflower, simulated drought stress has been shown to decrease

germination at osmotic potentials more negative than approximately -0.6 MPa, although the threshold for

sensitivity varies among species and cultivars (Somers, Ullrich et al. 1983; El Midaoui, Talouizte et al.

2001; Kaya, Okçu et al. 2006; Ahmad, Ahmad et al. 2009). Germination percentages in PEG solutions

have been correlated to seedling emergence in soils under osmotic stress, suggesting that germination

studies utilizing PEG may be a promising method for screening for drought resistance at early stages of

crop stand development (Somers, Ullrich et al. 1983). To our knowledge, the germination characteristics

of wild non-H. annuus sunflowers species under PEG induced simulated drought stress have not been

previously examined.

The broad objective of this study is to compare two putatively drought resistant wild sunflower

species H. argophyllus and H. niveus ssp. tephrodes to cultivated H. annuus for germination

characteristics in response to simulated drought. We quantify percent germination and mean germination

time (MGT) for all three species under non-water limiting conditions (0 MPa), mild water stress (-0.4

MPa), moderate water stress (-0.8 MPa) and severe water stress (-1.2 MPa) in order to determine if wild

species have the potential to serve as candidates for breeding for improved germination percentage and

uniformity in drought prone agricultural lands. We hypothesize that all three species will have reduced

percent germination and delayed MGT as simulated drought increases, but that the wild sunflower species

51

will have a greater percent germination and less of a delay in mean germination time than H. annuus in

the more stressed treatments.

Materials and Methods

Germination response to simulated drought stress was assessed in a growth chamber study at the

University of Georgia greenhouse facility, Athens, GA, in January of 2012, using seeds germinated in

petri dishes. The growth chamber conditions were dark and 25ºC. The study was a randomized complete

block design with three species, three accessions per species, four simulated drought stress treatments,

three spatial blocks with the growth chamber, and two petri dishes per species/line/block (petri dishes

within block were averaged to a single data point) for a total of n =108 experimental units for statistical

analysis.

The three study species were cultivated H. annuus, H. argophyllus and H. niveus ssp. tephrodes.

All achenes (here after called seed) were obtained from the USDA National Plant Germplasm System and

accession names for each of the following species represent USDA National Genetic Resources Program

(GRIN, http://www.ars-grin.gov/). For H. annuus the accession identifiers are PI-642777 (HA-412-HO),

PI-560141 (RHA-373) and PI-578872 (HA-383). For H. argophyllus the accessions identifiers are PI-

468651 (ARG-1575), PI-435623 (ARG-400) and PI-649862 (No. 81). For H. niveus spp. tephrodes the

accessions identifiers are PI-664653 (AMES-27850 and NIV-2442, PI-613758 (NIV-1243), and PI-

650018 (AMES-27422). Seeds were scarified by excising the blunt end with a razor blade prior to

treatments to enhance imbibition and synchronize germination in the control treatment. Ten seeds per

accession per species were placed in a 100×15 mm Petri plate containing two sheets of Whatman # 1 filter

paper and 6 mL of treatment solution. Seeds were moved to new plates and filter papers every other day

to maintain consistency in the simulated drought stress treatments.

The simulated drought stress treatments were created with polyethylene glycol, molecular weight

6000 (PEG-6000). PEG-6000 was used to simulate drought at four water stress levels: 0 (control), -0.4, -

0.8 and -1.2 MPa (Michel and Kaufmann 1973). For osmotic treatments, PEG -6000 was dissolved into

de-ionized water solutions totaling 1 L, utilizing 165.72 g (-0.4 MPa), 222.84 g (-0.8 MPa) and 265.56 (-

52

1.2 MPa). PEG-6000 treatment solutions were assessed for osmotic potential by measuring the osmolality

in a Wescor Vapro 5520 vapor pressure osmometer (Westcor Inc. Logan, Utah) and then converting

osmolality to solute potential ( ) using the van’t Hoff relationship at 25º C (Nobel 1991).

Germination was assessed daily for a total of seven days. Seeds were scored for germination based upon

radicle length. To account for species differences in seed weight (Kaya, Okçu et al. 2006), successful

germination was as assessed as reaching a radicle length of 2, 1.5, and 1.5 mm for H. annuus, H.

argophyllus, and H. niveus ssp. tephrodes, respectively. For each petri plate, the percent germination for

the entire 7 day interval was calculated at the total number of seeds that germinated per plate divided by

the total number of viable seed per plate (i.e. seed that had not been lost to mold). Although a few seeds

were lost to mold, there was no bias by species or treatment. For each petri plate, the mean germination

time (MGT) was calculated using the following formula (Battle and Whittington 1969):

MGT = ∑(G × T)

F

where T is the day in which germination was recorded, G is the number of seeds germinated on day T and

F is the total number of seeds which germinated. Statistical analyses of percent germination and MGT

were carried out using a general linear model (PROC GLM) in the statistical software package SAS®

(PROC GLM; SAS Institute, Cary, NC, USA). Main effects comparisons for among species means were

analyzed using the analysis of variance technique (ANOVA) using a significance of P<0.05. For each

species, differences among accessions were additionally assessed with ANOVA using the SLICE

command to partition water stress treatment comparisons to within species. Significant differences among

accessions within species for a given treatment were assessed by comparing LSMeans, a significant

difference between accessions for a given treatment was determined if P < 0.05.

While MGT provides a useful response variable, it is possible to provide additional information

by further analyzing and assessing the progression of germination as median germination time and

uniformity of germination. To do this, germination times for each species and treatment combination were

modeled with logistic regressions in SAS® (PROC GLM; SAS Institute, Cary, NC, USA) according to

53

the following formula:

( ) = (1)

Logit(y) is the log of the probability of germination; m and b are the regression slope and intercept,

respectively; and x represents day. The cumulative probability of germination is given by the formula:

=

( )

(2)

The probability density function is given by applying the first derivative to the cumulative probability

function:

=

[( × )( )] [( )( × )]

( )

(3)

Median germination time for each species and treatment combination was calculated by dividing

regression intercepts (b) by regression slopes (m). To statistically compare species’ responses to specific

water stress treatments, a standard unit approach was applied to remove the effect of interspecific

variation, in which all control germination times were standardized by setting control germination times

for each species equal to one another, and all treatments germination times were scaled accordingly.

Results

For all three species, percent germination decreased with increasing simulated drought stress (i.e.

a more negative osmotic potential) as expected (Figure 3.1, Table 3.1). For both H. argophyllus and H.

annuus, the decline was only evident in the -1.2 MPa treatments indicating that the threshold for response

is between -0.8 and -1.2. In the maximum stress treatment, H. argophyllus tended to have a higher percent

germination than H. annuus (P = 0.06). Helianthus niveus ssp. tephrodes however, showed a decline in

germination in the -0.8 MPa treatment, indicating a more sensitive threshold for response to simulated

drought stress as compared to H. annuus and H. argophyllus. Additionally, H. niveus ssp. tephrodes had

lower percent germination in the -1.2 MPa treatment as compared to either H. niveus ssp. tephrodes in -

0.8 MPa, or H. argophyllus and H. annuus in the -1.2 MPa treatment. A comparison of accessions within

54

species for percent germination found differences for H. annuus and H. niveus ssp. tephrodes (See

Appendix Figure 3.1 and 3.2).

For all three Helianthus species, MGT was also delayed (i.e. longer) with increasing simulated

drought stress, as expected (Figure 3.2, Table 3.1). For all three species, germination in the -0.8 and -1.2

MPa treatments was delayed as compared to lower stress treatments, indicating a threshold between -0.4

and -0.8 MPa. Helianthus argophyllus germinated faster than H. annuus in the maximum stress treatment.

For H. niveus ssp. tephrodes the delay in germination the -0.8 MPa treatment was greater than that for H.

annuus, but that difference disappeared in the -1.2 MPa treatment. A comparison of accessions within

species for mean germination time found accession differences for H. annuus and H. niveus spp.

tephrodes (Appendix Figure 3.2).

A logistic regression analysis of time to germination provides further information by

simultaneously addressing median time to germination as well as uniformity (height of peak) of

germination (Figure 3.3). Consistent with the mean germination time ANOVA results (Figure 3.2, Table

3.1), the plot of the probability of germination on a particular day indicates that median germination time

was delayed with increasing drought stress for all three species (Figure 3.3, Table 3.2). Likelihood ratio

contrasts of standardized species germination time regressions (standardized to 0 MPa) detected species

differences for proportion to germinate on a given day under water stress,(Figure 3.3, Table 3.2).

Comparisons between H. annuus and H. argophyllus indicates that H. annuus has a greater uniformity of

germination (higher peak) under moderate water stress (-0.8 MPa) with no difference observed under

mild (-0.4 MPa) and severe (-1.2 MPa) water stress (Figure 3.3, Table 3.2). In contrast, H. niveus ssp.

tephrodes has poorer uniformity (lower peak) and delayed germination as compared to than both H.

annuus and H. argophyllus in the -0.4, -0.8 and -1.2 MPa treatments (Figure 3.3).

Discussion

We expected the cultivated H. annuus and the two wild Helianthus species to have decreased

percent germination and longer germination times in response to increasing simulated drought stress, but

that the effects would be more pronounced for the cultivated species. As expected, all three species

55

demonstrated decreased percent germination and longer germination times with increasing simulated

drought stress and the species differed in the responses. However, the expectation that both wild species

would be more resistant to simulated drought stress, and thus have less of a reduction in percent

germination and reduced lengthening of germination time as compared to H. annuus, was not supported.

Overall H. annuus and H. argophyllus faired similarly for germination under simulated drought stress,

while H. niveus ssp. tephrodes germination was the least resistant to simulated drought stress.

For cultivated H. annuus, our germination responses are generally consistent with reports from

other experiments that used PEG to create osmotic stress to simulate drought stress. Although the

molecular weight of PEG used in other studies varied (6000 to 20000), all of the molecular weights were

sufficiently large to create an osmotic stress external to the seed and not impose toxicity (Lawlor 1970;

Somers, Ullrich et al. 1983; El Midaoui, Talouizte et al. 2001; Iqbal and Ashraf 2006; Kaya, Okçu et al.

2006; Ahmad, Ahmad et al. 2009). Most studies found no decline in cultivated H. annuus germination in

the -0.4 to -0.6 MPa treatments, suggesting a threshold for decline below that range (Somers, Ullrich et al.

1983, El Midaoui, Talouizte et al. 2001; Kaya, Okçu et al. 2006; Ahmad, Ahmad et al. 2009), although

one study did find a decrease in germination at -0.6 MPa (Iqbal and Ashraf 2006). For cultivated H.

annuus in our study, the threshold for a decline in percent germination in response to simulated drought

stress was below -0.8 MPa. In previously published studies the percent germination was only from 0-

37% % in treatments with approximately -1.2 MPa stress level, while cultivated H. annuus in our study

was 76 % (El Midaoui, Talouizte et al. 2001; Iqbal and Ashraf 2006; Kaya, Okçu et al. 2006). Our study,

which did find accession differences in percent germination under the greatest water stress (-1.2 MPa), is

congruent with accession differences reported for other studies of cultivated H. annuus (Somers, Ullrich

et al. 1983; El Midaoui, Talouizte et al. 2001).

We observed in all species of Helianthus, that as water stress was increased, MGT also

lengthened, with mild water stress (-0.4 MPa) having the least effect on MGT and severe drought having

the greatest effect (Figure 3.2). As with percent germination, several other studies report a similar trend in

MGT for cultivated H. annuus (El Midaoui, Talouizte et al. 2001; Iqbal and Ashraf 2006; Kaya, Okçu et

56

al. 2006; Ahmad, Ahmad et al. 2009). Studies found that MGT ranged from 2 to 4 days for the no water

stress control (0 MPa) with our study being at the faster end of the range, 2.3 days (El Midaoui, Talouizte

et al. 2001; Iqbal and Ashraf 2006; Kaya, Okçu et al. 2006). At mild water stress (-0.3 to -0.4 MPa) mean

germination time was either significantly different (El Midaoui, Talouizte et al. 2001) increasing from 2.6

to 3.6 day or exhibited no deviation from control which is most consistent with our observations (Kaya,

Okçu et al. 2006). Under moderate water stress (-0.6 to -1.0 MPa), cultivated H. annuus mean

germination time has a reported range of 3 to 6 days (El Midaoui, Talouizte et al. 2001; Kaya, Okçu et al.

2006). Once again our observations were on the lower end at 3 days and were significantly different from

the control and mild water stress. Germination under severe water stress (-1.2 to -1.6 MPa) exhibited the

most variation among studies with mean germination times ranging from 4.5 to 10 days (El Midaoui,

Talouizte et al. 2001) and complete absence of germination (Kaya, Okçu et al. 2006) with our results most

closely resembling those of El Midaoui, Talouizte et al. (2001) at 4.8 days.

These results are in agreement with the detrimental effects of simulated drought stress as seen in

percent germination for cultivated H. annuus. Thus, drought can cause significant delay of germination or

prevent it entirely. Inconsistent germination can cause a lack of uniformity in stand establishment which

is a significant barrier to production and yield (Harris, Joshi et al. 1999; Iqbal and Ashraf 2006). One

potential resource for increasing germination consistency is by identifying wild congeners of crops from

arid and semi-arid regions that may impart reduced MGT and greater percent germination under

simulated drought stress.

To our knowledge wild H. argophyllus and H. niveus ssp. tephrodes haven not been explored as a

source of desirable alleles for enhancing drought resistance during the germination phase. The Texas

coastal sand dune species, H. argophyllus, could be a resource for drought resistance and salt tolerance

because of its native arid dunes habitat and close proximity to the Gulf of Mexico. It is thought that

greater salt tolerance may also confer greater drought resistance at the seedling stage, as it has been

shown for two accessions of cultivated H. annuus (Ashraf and O'Leary 1996). The salt-tolerant accession

was able to produce greater above and below ground biomass as well as maintain a greater stomatal

57

conductance, rate of transpiration and instantaneous photosynthetic rate under PEG induced water stress

(Ashraf and O'Leary 1996). A greater ability to maintain germination under increasing PEG simulated

drought stress has also been show in contrasts of salt tolerant and salt sensitive species. Studies

comparing percent germination in species growing along a salt gradient have found that more salt tolerant

species maintained a higher percent germination with increasing simulated drought stress (Dodd and

Donovan 1999; Tobe, Li et al. 2000). In our study, H. argophyllus and cultivated H. annuus germination

responded similarly to all levels of simulated drought stress. However, it should be noted that H.

argophyllus did show a trend (P = 0.06) for higher percent germination under severe simulated drought

stress. Thus, evaluating wild germplasm thought to be salt tolerant, such as H. argophyllus, may be a

logical first step in identifying species with potential drought resistance.

The desert dune endemic H. niveus spp. tephrodes has been hypothesized to be drought resistant

based on its native arid habitat (Murillo‐Amador, López‐Aguilar et al. 2002; Seiler, Gulya et al. 2006;

Seiler, Gulya et al. 2006). However, we report a significantly lower percent germination in H. niveus spp.

tephrodes than H. annuus at -0.8 and -1.2 MPa water stress levels. These results suggest that H. niveus

spp. tephrodes may not be a desirable target for improvement of germination under water stress in

cultivated sunflower. The germination patterns do suggest, however, that H. niveus spp. tephrodes

germination may respond to environmental cues in a manner that provides an adaptive advantage in its

native desert dune habitat. For example, a study of xerophytic species from arid and semi-arid regions in

China found that species from most arid regions experienced the greatest decline in percent germination

in response to increasing simulated drought stress with PEG (Zeng, Wang et al. 2010). Semi-arid species

were capable of at least some germination at water stress of -1.5 to -2.4, while species from more arid

environments were only capable of germination at less stressful levels of -1.2 to -1.8MPa. These results

support our findings, and suggest that H. niveus spp. tephrodes may be triggered by germination cues

more conducive to favorable growth conditions such as greater water availability to avoid seedling

mortality.

58

We found mean germination time to increase in wild H. argophyllus and H. niveus spp. tephrodes

with increasing water stress. At mild water stress we observed no differences in mean germination time

between wild species and cultivated H. annuus, but under severe water stress H. argophyllus had a longer

mean germination time than H. annuus. The logistic regressions, which simultaneously assess median

germination time and uniformity, highlights that both H. argophyllus and H. niveus ssp. tephrodes did

more poorly than H. annuus under moderate water stress, and H. niveus ssp. tephrodes did more poorly

than H. annuus under severe water stress.

Conclusion

The aim of this study was to evaluate drought resistance at the germination stage for two wild

species of sunflower hypothesized to be drought resistant based on their native distribution in the arid

climates of coastal Texas and the Sonoran Desert. Results from this study provide little evidence that

either wild species is likely to be a potential donor for desirable genetic variation as pertaining to

improved drought resistance in cultivated sunflower.

59

References

Ahmad, S., R. Ahmad, et al. (2009). "Sunflower (Helianthus annuus L.) response to drought stress at

germination and seedling growth stages." Pakistan Journal of Botany 41(2): 647-654.

Ashraf, M. and M. Foolad (2005). "Pre‐sowing seed treatment—a shotgun approach to improve

germination, plant growth, and crop yield under saline and non‐saline conditions." Advances in

Agronomy 88: 223-271.

Ashraf, M. and S. Mehmood (1990). "Response of four Brassica species to drought stress."

Environmental and Experimental Botany 30(1): 93-100.

Ashraf, M. and J. W. O'Leary (1996). "Effect of drought stress on growth, water relations, and gas

exchange of two lines of sunflower differing in degree of salt tolerance." International Journal of

Plant Sciences 157(6): 729-732.

Baldini, M. and G. Vannozzi (1998). "Agronomic and physiological assessment of genotypic variation for

drought tolerance in sunflower genotypes obtained from a cross between H. annuus and H.

argophyllus." Agricoltura Mediterranea 128(3): 232-240.

Baldini, M. and G. P. Vannozzi (1999). "Yield relationships under drought in sunflower genotypes

obtained from a wild population and cultivated sunflowers in rain-out shelter in large pots and

field experiments." Helia 22(30): 81-96.

Battle, J. P. and W. J. Whittington (1969). "The relation between inhibitory substances and variability in

time to germination of sugar beet clusters." The Journal of Agricultural Science 73(03): 337-346.

Blum, A. (1996). "Crop responses to drought and the interpretation of adaptation." Plant Growth

Regulation 20(2): 135-148.

Boyer, J. S. (1982). "Plant productivity and environment." Science 218(4571): 443-448.

Dodd, G. L. and L. A. Donovan (1999). "Water potential and ionic effects on germination and seedling

growth of two cold desert shrubs." American Journal of Botany 86(8): 1146-1153.

Döll, P. (2002). "Impact of climate change and variability on irrigation requirements: a global

perspective." Climatic Change 54(3): 269-293.

60

El Midaoui, M., A. Talouizte, et al. (2001). "Effect of osmotic pressure on germination of sunflower seeds

(Helianthus annuus L.)." Helia 24(35): 129-134.

El Midaoui, M., H. Serieys, et al. (2003). "Effects of osmotic and water stresses on root and shoot

morphology and seed yield in sunflower (Helianthus annuus L) genotypes bred for Morocco or

issued from introgression with H. argophyllus T. & G. and H. debilis Nutt." Helia 26(38): 1-15.

Harris, D., A. Joshi, et al. (1999). "On-farm seed priming in semi-arid agriculture: development and

evaluation in maize, rice and chickpea in India using participatory methods." Experimental

Agriculture 35(01): 15-29.

Hegarty, T. (1978). "The physiology of seed hydration and dehydration, and the relation between water

stress and the control of germination: a review." Plant, Cell & Environment 1(2): 101-119.

Heiser, C. B. and D. M. Smith (1969). The North American sunflowers (Helianthus), Seeman Printery.

IPCC (2007). Climate change 2007: The physical science basis. Contribution of working group I to the

fourth assessment report on the intergovernmental panel on climate change. Solomon, S., Qin, D.,

Manning, M. R., Marquis, M., Averyt, K. B. Tignor, M., Miller, H. and Chen, Z.

Iqbal, N. and M. Y. Ashraf (2006). "Does seed treatment with glycinebetaine improve germination rate

and seedling growth of sunflower (Helianthus annuus L.) under osmotic stress." Pakistan Journal

of Botany 38(5): 1641-1648.

Jackson, L. E. and G. W. Koch (1997). The ecophysiology of crops and their wild relatives. Ecology in

Agriculture. L. E. Jackson. San Diego Academic Press: 3-37.

Kaya, M. D., G. Okçu, et al. (2006). "Seed treatments to overcome salt and drought stress during

germination in sunflower (Helianthus annuus L.)." European Journal of Agronomy 24(4): 291-

295.

Khodarahmpour, Z. (2011). "Effect of drought stress induced by polyethylene glycol (PEG) on

germination indices in corn (Zea mays L.) hybrids." African Journal of Biotechnology 10(79):

18222-18227.

61

Koziol, L., L. H. Rieseberg, et al. (2012). "Reduced drought tolerance during domestication and the

evolution of weediness results from tolerance-growth trade-offs." Evolution 66(12): 3803-3814.

Lawlor, D. W. (1970). "Absorption of polyethylene glycols by plants and their effects on plant growth."

New Phytologist 69(2): 501-513.

Michel, B. E. and M. R. Kaufmann (1973). "The osmotic potential of polyethylene glycol 6000." Plant

Physiology 51(5): 914-916.

Murillo‐Amador, B., R. López‐Aguilar, et al. (2002). "Comparative effects of NaCl and polyethylene

glycol on germination, emergence and seedling growth of cowpea." Journal of Agronomy and

Crop Science 188(4): 235-247.

Nobel, P. S. (1991). Physicochemical and environmental plant physiology, Academic Press, Inc.

Passioura, J. (1996). "Drought and drought tolerance." Plant Growth Regulation 20(2): 79-83.

Richards, R. (1996). "Defining selection criteria to improve yield under drought." Plant Growth

Regulation 20(2): 157-166.

SAS Institute Inc. 2011. Base SAS® 9.3 Procedures Guide. Cary, NC: SAS Institute

Inc.

Schilling, E. E. and C. B. Heiser (1981). "Infrageneric classification of Helianthus (Compositae)." Taxon

30(2): 393-403.

Seiler, G., T. Gulya, et al. (2006). "Exploration for wild Helianthus species from the desert Southwestern

USA for potential drought tolerance." Helia 29(45): 1-10.

Seiler, G. J., T. Gulya, et al. (2006). "Plant exploration to collect wild Helianthus niveus subspecies for

sunflower improvement". Proceedings Sunflower Research Workshop.

Somers, D., S. Ullrich, et al. (1983). "Sunflower germination under simulated drought stress." Agronomy

Journal 75(3): 570-572.

Tanksley, S. D. and S. R. McCouch (1997). "Seed banks and molecular maps: Unlocking genetic

potential from the wild." Science 277(5329): 1063-1066.

62

Taylor, A. and G. Harman (1990). "Concepts and technologies of selected seed treatments." Annual

Review of Phytopathology 28(1): 321-339.

Tobe, K., X. Li, et al. (2000). "Effects of sodium chloride on seed germination and growth of two Chinese

desert shrubs, Haloxylon ammodendron and H. persicum (Chenopodiaceae)." Australian Journal

of Botany 48(4): 455-460.

Verslues, P. E., M. Agarwal, et al. (2006). "Methods and concepts in quantifying resistance to drought,

salt and freezing, abiotic stresses that affect plant water status." The Plant Journal 45(4): 523-539.

Zeng, Y. J., Y. R. Wang, et al. (2010). "Is reduced seed germination due to water limitation a special

survival strategy used by xerophytes in arid dunes?" Journal of Arid Environments 74(4): 508-

511.

63

Species

ANN ARG TEPH

% G

erm

inati

on

0

20

40

60

80

100

0 Mpa

-0.4 Mpa

-0.8 Mpa

-1.2 Mpa

A A A

BC

A A A

B

A A

C

D

Figure 3.1 Percent germination after seven days for three species of Helianthus (sunflower) H. annuus

(ANN), H. argophyllus (ARG) and H. niveus ssp. tephrodes (TEPH) for polyethylene glycol – 6000

induced water stress treatments. Water stress treatments are 0 MPa (control), -0.4 MPa (mild water

stress), -0.8 MPa (moderate water stress) and -1.2 MPa (severe water stress). Dissimilar letters represent

significant differences based on least square means at P < 0.05 level, error bars represent SE.

64

Species

ANN ARG TEPH

Mea

n G

erm

inati

on

Tim

e (d

ay

s)

0

2

4

6

0 Mpa

-0.4 Mpa

-0.8 Mpa

-1.2 Mpa

A

A

B

D

A

A

B

C

A

A

C

D

Figure 3.2 Mean germination time after seven days for three species of Helianthus (sunflower) H. annuus

(ANN), H. argophyllus (ARG) and H. niveus ssp. tephrodes (TEPH) for polyethylene glycol – 6000

induced water stress treatments. Water stress treatments are 0 MPa (control), -0.4 MPa (mild water

stress), -0.8 MPa (moderate water stress) and -1.2 MPa (severe water stress). Dissimilar letters represent

significant differences based on least square means at P < 0.05 level, error bars represent SE.

65

TEPH

Day

0 1 2 3 4 5 6 7 8

0.0

0.2

0.4

0.6

ARG

Pro

po

rtio

n g

erm

ina

ted

0.0

0.2

0.4

0.6

ANN

0.0

0.2

0.4

0.6

0 MPa

-0.4 MPa

-0.8 MPa

-1.2 MPa

Figure 3.3 Probability density functions for observed germination frequency by day for three species of

Helianthus (sunflower) H. annuus (ANN), H. argophyllus (ARG) and H. niveus ssp. tephrodes (TEPH)

for polyethylene glycol – 6000 induced water stress treatments. Water stress treatments are 0 MPa

(control), -0.4 MPa (mild water stress), -0.8 MPa (moderate water stress) and -1.2 MPa (severe water

stress).

66

Table 3.1 Percent germination (% Germ) and mean germination time (MGT) (means + SE) for three

species of Helianthus (sunflowers), H. annuus (ANN), H. argophyllus (ARG) and H. niveus ssp.

tephrodes (TEPH) for polyethylene glycol – 6000 induced drought stress treatments. Water stress

treatments are 0 MPa (control), -0.4 MPa (mild water stress), -0.8 MPa (moderate water stress) and -1.2

MPa (severe water stress). Significant p-values are indicated in bold.

ANN ARG TEPH

Treatment Trait F df P 0 MPa % Germ 95.00 ± 4.41 99.44 ± 0.56 99.38 ± 0.62 0.67 2, 3 0.5164

MGT. 2.39 ± 0.11 2.49 ± 0.04 2.65 ± 0.10 1.17 2, 3 0.3160

-0.4 MPA % Germ 98.27 ± 1.20 100.00 ± 0.00 97.16 ± 1.23 0.21 2, 3 0.8111

MGT 2.71 ± 0.11 2.77 ± 0.09 2.97 ± 0.16 0.93 2, 3 0.3967 -0.8 MPa % Germ 97.42 ± 1.39 98.88 ± 0.73 75.34 ± 4.85 17.83 2, 3 <0.0001

MGT 3.41 ± 0.14 3.64 ± 0.09 4.29 ± 0.19 10.96 2, 3 <0.0001

-1.2 MPa % Germ 75.99 ± 4.81 84.44 ± 3.95 41.23 ± 5.18 53.74 2, 3 <0.0001

MGT 4.84 ± 0.20 4.45 ± 0.17 5.03 ± 0.14 4.72 2, 3 0.0111

67

Table 3.2 Likelihood ratio contrasts of standardized species Helianthus annuus (ANN), H. argophyllus

(ARG) and H. niveus ssp. tephrodes (TEPH) germination time regressions for polyethylene glycol – 6000

induced drought stress treatments. Water stress treatments are 0 MPa (control), -0.4 MPa (mild water

stress), -0.8 MPa (moderate water stress) and -1.2 MPa (severe water stress). Significant p-values are

indicated in bold, df = 1.

ANN vs ARG ARG vs TEPH ANN vs TEPH

Treatment χ2 p-value χ

2 p-value χ

2 p-value

0 Mpa 0 1 0 1 0 1

-0.4 MPa 0.6690 0.4134 27.301 <0.001 34.071 <0.001

-0.8 MPa 7.6891 0.0050 141.02 <0.001 87.957 <0.001

-1.2 MPa 3.0298 0.0817 26.176 <0.001 14.472 <0.001

68

ANN

40

80

120

ARG

Per

cen

t G

erm

ina

tio

n (

%)

40

80

120

TEPH

40

80

120

Accession 0 MPa

-0.4 MPa-0.8 MPa-1.2 MPa

A AAB

C

A A AAB BABAB

BC

A A AAB A A AB

A A A

B

A A A A A

B

C

C

C

DE

AB

Appendix Figure 3.1

69

Appendix Figure 3.1 Percent germination after seven days for accessions of three species of Helianthus

(sunflower) H. annuus (ANN), H. argophyllus (ARG) and H. niveus ssp. tephrodes (TEPH) for

polyethylene glycol – 6000 induced water stress treatments. Water stress treatments are 0 MPa (control), -

0.4 MPa (mild water stress), -0.8 MPa (moderate water stress) and -1.2 MPa (severe water stress).

Dissimilar letters represent significant differences based on least square means at P < 0.05 level for

accessions within species only, error bars represent SE. From left to right means represent GRIN

accession identifiers. For ANN the accession identifiers are PI-642777 (HA-412-HO), PI-560141 (RHA-

373) and PI-578872 (HA-383). For ARG the accessions identifiers are PI-468651 (ARG-1575), PI-

435623 (ARG-400) and PI-649862 (No. 81). For TEPH the accessions identifiers are PI-664653 (AMES-

27850 and NIV-2442, PI-613758 (NIV-1243), and PI-650018 (AMES-27422).

70

2

4

6

Mea

n G

erm

inati

on

Tim

e (d

ays)

2

4

6

ANN

H. argophyllus

Accessions

2

4

6

ARG

TEPH

0 MPa-0.4 MPa-0.8 MPa-1.2 MPa

AB

C

D

E

AAB

CD

E

ABCCD

D

E

AA

B

C

A

AB

B

C

AA

B

C

AA

B

D

A

B

DD

ABAB

C

D

Appendix Figure 3.2

71

Appendix Figure 3.2 Mean germination time after seven days for accessions of three species of

Helianthus (sunflower) H. annuus (ANN), H. argophyllus (ARG) and H. niveus ssp. tephrodes (TEPH)

for polyethylene glycol – 6000 induced water stress treatments. Water stress treatments are 0 MPa

(control), -0.4 MPa (mild water stress), -0.8 MPa (moderate water stress) and -1.2 MPa (severe water

stress). Dissimilar letters represent significant differences based on least square means at P < 0.05 level

for accessions within species only, error bars represent SE. From left to right means represent GRIN

accession identifiers. For ANN the accession identifiers are PI-642777 (HA-412-HO), PI-560141 (RHA-

373) and PI-578872 (HA-383). For ARG the accessions identifiers are PI-468651 (ARG-1575), PI-

435623 (ARG-400) and PI-649862 (No. 81). For TEPH the accessions identifiers are PI-664653 (AMES-

27850 and NIV-2442, PI-613758 (NIV-1243), and PI-650018 (AMES-27422).

72

CHAPTER 4

CONCLUSION

The research discussed herein examined the potential use of wild species for improving abiotic

stress resistance, specifically drought, in a closely related crop. The two studies compiled here surveyed

putative morphological and physiological drought resistance traits at various ontogenetic stages in two

potentially drought resistant wild species of sunflower, H. argophyllus and H. niveus ssp. tephrodes.

Helianthus argophyllus and H. niveus ssp. tephrodes are hypothesized to be drought resistant based on

their respective native saline and arid environments leaf characteristics. Helianthus argophyllus and H.

niveus ssp. tephrodes were compared with the closely related cultivated sunflower, H. annuus to assess

whether traits present in wild species may be useful for improved drought resistance over those already

present in H. annuus. From this work several conclusions can be drawn.

Helianthus niveus ssp. tephrodes, the Algodones dunes sunflower was assessed at three separate

ontogenetic stages to test the hypothesis that it possesses trait characteristics more conducive to drought

resistance compared to its cultivated relative H. annuus. Comparisons under non-water limiting

conditions at the seedling stage for root traits related to drought resistance suggest that H. niveus ssp.

tephrodes confers no distinct advantage over variation currently present in accessions of H. annuus tested.

Despite speculation that H. niveus ssp. tephrodes would root to a specified depth more quickly than H.

annuus, the opposite was found with H. annuus taking nearly half the time. This result was surprising

given the shifting nature of the dune system in which H. niveus ssp. tephrodes is accustomed to growing

and the adaptive advantage that earlier root proliferation and quicker elongation would provide in an arid

water-stressed environment. However, this result was observed under non-water limiting conditions, and

may not hold under water-stress.

For both the seedling and adult stages, leaf and canopy traits of H. niveus ssp. tephrodes are

indicative of drought resistance in terms of dissipation of excess heat and incident radiation. Helianthus

73

niveus ssp. tephrodes exhibits small thick leaves covered in a dense white pubescence and was capable of

greater light reflectance. At the seedling stage, the total canopy area of Helianthus niveus ssp. tephrodes

is greatly reduced as compared to H. annuus. All of these traits are characteristic of plants adapted to high

light and high water-stress conditions, such as those present in the Algodones dunes region of the Sonoran

Desert and are suggestive of Helianthus niveus ssp. tephrodes being a potential donor of leaf and canopy

traits conferring drought resistance.

At the mature stage, H. niveus ssp. tephrodes was also characterized for instantaneous gas

exchange and leaf chemistry to assess photosynthetic capacity and water use efficiency measures.

Overall, H. niveus ssp. tephrodes exhibits greater instantaneous water use efficiency, a measurement

which is sensitive to short term changes in environmental conditions. When H. niveus ssp. tephrodes and

H. annuus were compared for integrated water use efficiency, a proxy for water use over the entire

lifespan on the leaf, no difference was observed. It has been suggested, that integrated water use

efficiency is a more valid approach for indicating drought resistance capacity. In terms of photosynthetic

capacity, no difference was observed when measured on an area basis; however on a mass basis H.

annuus had a greater rate per investment in leaf biomass. This result is congruent with most annual crops

which have been selected for short lifespans, high growth rates and yield in non-resource limiting

conditions. The lower rate of photosynthesis on a mass basis for H. niveus ssp. tephrodes is likely

explained by having denser leaves. Given the potential for ameliorating potential heat load and excess

irradiance problems associated with cropping systems subject to drought, H. niveus ssp. tephrodes should

not be discounted on the basis of marginally reduced photosynthetic rates.

Lastly, H. niveus spp. tephrodes and another wild relative of H. annuus, H. argophyllus were

assessed for germination characteristics under various levels of simulated drought stress induced by

polyethylene glycol ranging from mild to severe. As expected, it was found that increasing water stress

decreased both percent germination and increased mean germination time for all three species. However,

observed results do not support the original hypothesis that wild species would experience reduced

detrimental effects due to simulated drought stress as compared to cultivated H. annuus. Both wild

74

species did more poorly than H. annuus as water stress increased. The results do suggest that H. niveus

spp. tephrodes may be relying on moisture availability cues consistent with optimal germination time

given its native desert habitat.

In conclusion, the studies compiled within, support the investigation and use of wild germplasm

related to crops as a potential resource in breeding for improved abiotic stress resistance, specifically

drought. Because drought is a multifaceted stress that has varying detrimental effects on plants throughout

ontogeny it is likely that suites of traits as opposed to one or two will be necessary for adequate

improvements.