Interaction of Drought Stress and Gibberellin Metabolism On

INTERACTION OF DROUGHT STRESS AND GIBBERELLIN METABOLISM ON

STEM ELONGATION IN TOMATO

ALEXANDER G. LITVIN

(Under the Direction of MARC VAN IERSEL & ANISH MALLADI)

ABSTRACT

Drought reduces plant and cell elongation. Our objective was to quantify the effects of drought on elongation and gibberellin homeostasis. We exposed tomatoes to drought to observe the effect on elongation and gibberellin metabolism-related gene expression.

Plants were maintained at substrate moistures to provide well-watered or drought conditions. To further investigate the effect of gibberellins on elongation, paclobutrazol

(PAC) was applied, reducing gibberellin production. Drought reduced height (P =

0.0012) under drought, and had an interactive effect with PAC on internode length (P =

0.0004), and cell size (P = 0.0067). We analyzed the transcription of SlGA20ox1, -2, -3, and -4, SlGA3ox2, and SlGA2ox2, -4, and -5, corresponding to gibberellin biosynthesis.

Transcription of LeEXP1, and -2, encoding for expansin enzymes, was also analyzed.

Down regulation of transcription due to stress was observed for SlGA20ox4, SlGA2ox5, and LeEXP1. These findings emphasize the inhibiting effect drought has on elongation during vegetative growth.

INDEX WORDS: Drought stress, gibberellin metabolism, gene expression, expansin,

GA2ox2, GA2ox4, GA2ox5, GA3ox2, GA20ox3, GA20ox4,

LeEXP1, LeEXP2, internode elongation, RNA, cell elongation,

environmental stress physiology.

INTERACTION OF DROUGHT STRESS AND GIBBERELLIN METABOLISM ON

STEM ELONGATION IN TOMATO

ALEXANDER G. LITVIN

B.S. California Polytechnic State University, San Luis Obispo 2009

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

2015

Alexander G. Litvin

INTERACTION OF DROUGHT STRESS AND GIBBERELLIN METABOLISM ON

STEM ELONGATION IN TOMATO

ALEXANDER LITVIN

Major Professor: Marc van Iersel Anish Malladi Committee: John Ruter Ron Pegg

Electronic Version Approved:

Suzanne Barbour Dean of the Graduate School The University of Georgia August 2015

DEDICATION

This thesis is dedicated to my family. My strength, perseverance, and all that I am grows from the foundation built on their love. This, like all my other accomplishments, would not be possible without their support and shared strength.

ACKNOWLEDGEMENTS

I would like to acknowledge the profound help of my advisors, Drs. Marc van

Iersel and Anish Malladi. Thank you for all the times you showed me how to grow and improve. Who I have become over the years under your guidance is due to your continual help. Sue Dove the technician at the greenhouse labs has been a major support for running any experiment and was paramount to getting things to work.

Finally I would like to thank the department of horticulture. It is a team effort getting each of us through our respective research, and it can sometimes seem all too overwhelming without the help of one other.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... v

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

CHAPTER

1 INTRODUCTION ...... 1

References ...... 3

2 LITERATURE REVIEW ...... 5

References ...... 12

3 PRELIMINARY STUDIES ON DROUGHT STRESS SUBSTRATE

MOISTURE THRESHOLDS FOR ‘MONEYMAKER’ TOMATO (Solanum

lycopersicum) ...... 16

Introduction ...... 16

Materials and Methods ...... 18

Results and Discussion ...... 23

Conclusion ...... 27

References ...... 29

4 DROUGHT STRESS DOWN REGULATES GIBBERELLIN

BIOSYNTHESIS AND REDUCES STEM ELONGATION IN TOMATOES

(Solanum lycopersicum) DURING VEGATATIVE GROWTH ...... 42

Abstract ...... 43

Introduction ...... 44

Materials and Methods ...... 47

Results and Discussion ...... 53

Conclusion ...... 58

References ...... 59

SUMMARY AND CONCLUSIONS ...... 74

vii

LIST OF TABLES

Page

Table 4.1: Multiple regression data for actual and predicted values for cell size modeled

by GA and expansin gene expression, paclobutrazol application, and substrate

moisture levels ...... 66

viii

LIST OF FIGURES

Page

Figure 3.1: Environmental conditions during summer preliminary trial ...... 33

Figure 3.2: Environmental conditions during fall preliminary trial ...... 34

Figure 3.3: Substrate volumetric water content (m3·m-3) during summer trial ...... 35

Figure 3.4: Effect of 0.2-0.4 m3·m-3 on plant height and internode length during summer

preliminary trial ...... 36

Figure 3.5: Leaf sizes during summer trial ...... 37

Figure 3.6: Substrate volumetric water content (m3·m-3) during fall trial ...... 38

Figure 3.7: Effect of 0.10-0.35 m3·m-3 on plant height and internode length during fall ..39

Figure 3.8: Accumulated shoot dry mass by substrate volumetric water content (m3·m-3)

during fall ...... 40

Figure 3.9: Water, osmotic, and turgor potentials from fall trial ...... 41

Figure 4.1: Substrate volumetric water content during (m3·m-3) GA trial ...... 67

Figure 4.2: Effect of drought stress and paclobutrazol drenches on plant height and

internode length ...... 68

Figure 4.3: Microscopy slides of drought and paclobutrazol effects on cell size ...... 69

Figure 4.4: Graphs of correlations between internode length and cell size, and between

plant height and internode length ...... 70

Figure 4.5: Relative genes expressions related to GA homeostasis and cell expansion ....71

Figure 4.6: Graph of correlation between expression levels of GA20ox3 and GA3ox2 ....72

Figure 4.7: Actual and predicted values for cell size modeled by GA and expansin gene

expression, paclobutrazol application, and substrate moisture levels ...... 73

CHAPTER 1: INTRODUCTION

Water availability influences plant growth and development throughout its life (Nuruddin et al., 2003). For agriculture, water availability is expected to decrease as a result of increased drought events due to climate change (Cook, et al. 2015). As water shortages become more common, drought stress will severely impact growth and yield by limiting transport of nutrients, hormonal activity, and metabolism (Soroushi et al., 2011), and as a result, reduce agriculture yields globally (Greenwood et al., 2010).

Weather events that do not allow reservoirs, ground water, or other sources of irrigation to replenish lead to drought (NOAA, 2013). Drought stress reduces yield, quality, and increases costs. Loss of yield from insufficient access to water has resulted in financial losses for farmers that exceeded $800 billion from 1980 to 2011 (NOAA, 2013).

Water relations of plants determine the current health and hydration of the plant and future potential growth and maturation of that plant (Nuruddin et al., 2003). Water availability drives the hormonal relationships that affect growth and survival (Nuruddin et al., 2003). Because of drought, water is becoming a limited factor in the production of horticultural crops. Increasing problems with water availability have produced a need to better understand the relationship of crops and their growth under drought stress conditions. Plants under drought stress limit metabolic functions and reduce overall yield by limiting cell division, expansion, transport of nutrients, hormonal activity, and metabolism (Soroushi, 2011).

Physiologically, plant water availability drives the capacity of any plant to swell, expand, maintain structure, transpire, and conduct any necessary metabolic functions critical from the start of its life cycle at germination, until death. Drought negatively affects growth by reducing cell division and elongation, crop load and maturation, and total yield (Vettakkorumakankav, 1999; Zhao, 2011).

Plants under drought stress conditions during critical growth periods can be substantially reduced in size due to reduced cellular elongation and division needed for growth and biomass accumulation (Liu, 2013). Hormone interactions during periods of drought stress play an important role in controlling growth during limited water availability (Liu, 2013). Gibberellins are an essential phytohormone group related strongly to cell division and elongation, as well as other roles involved in flowering, fruit set, and development (Weller, 1994). Plants under stress, or otherwise limited growing conditions, have shown stunted growth and poor fruit set (Xiao, 2010).

Tomatoes serve as a good model for studying the relationship between drought and cell elongation due to the extensive research published. Different levels of drought stress can show clear differences of reduced cell division and elongation in plants.

Changing environments can trigger stress signaling that can alter gene expression of enzymes, hormones, and other metabolic functions in order to acclimate (Olimpieri,

2011).

References

Cook, B.I., T.R. Ault, and J. E. Smerdon. 2015. Unprecedented 21st century drought

risk in the American southwest and central plains. Sci. Adv. 1:1-7.

Greenwood, D. J., K. Zhang, H.W. Hilton, and A.J. Thompson. 2010. Opportunities

for improving irrigation efficiency with quantitative models, soil water sensors and

wireless technology. J. Agr. Sci. 148:1-16.

Liu, T., S. Zhu, L. Fu, Y. Yu, Q. Tang, and S. Tang. 2013. Morphological and

physiological changes of ramie (Boehmeria nivea L. Gaud) in response to drought

stress and GA3 treatment. Rus. J. Plant Physiol. 60:749-55.

NOAA. 2013. National weather service drought factsheet. NOAA

rought.pdf>

Nuruddin, M., C.A. Madramootoo, and G.T. Dodds. 2003. Effects of water stress on

tomato at different growth stages. HortScience 38:1389-1393.

Olimpieri, I., R. Caccia, M.E. Picarella, A. Pucci, E. Santangelo, G.P. Soressi, and A.

Mazzucato. 2011. Constitutive co-suppression of the GA 20-oxidase1 gene in tomato

leads to severe defects in vegetative and reproductive development. Plant

Sci. 180:496-503.

Soroushi, H., T. Saki Nejad, A. Shoukofar, and M. Soltani. 2011. The interaction of

drought stress and gibberellic acid on corn (Zea mays L.). World Acad. Sci., Eng. and

Technol. 60:142-143.

Vettakkorumakankav, N.N., D. Falk, P. Saxena, and R.A. Fletcher. 1999. A crucial

role for gibberellins in stress protection of plants.” Plant Cell Physiol. 40:542-548.

Weller, J.L., J.J. Ross, J.B. Reid. 1994. Gibberellins and phytochrome regulation of stem elongation in pea. Planta 192:489-496.

Xiao, Y., D. Li, M. Yin, X. Li, M. Zhang, Y. Wang, J. Dong, J. Zhao, M. Luo, X.

Luo, L. Hou, L. Hu, and Y. Pei. 2010. Gibberellin20-oxidase promotes initiation and elongation of cotton fibers by regulating gibberellin synthesis. J. Plant

Physiol. 167:829-37.

Zhao, M., F. Li, Y. Fang, Q. Gao, and W. Wang. 2011. Expansin-regulated cell elongation is involved in the drought tolerance in wheat. Protoplasma 248:313-23.

CHAPTER 2: LITERATURE REVIEW

Drought and Tomatoes

Tomato plants are symptomatically sensitive to drought stress, making them early indicators of drought conditions (Gong, 2010). Drought stress causes a decline in the overall height and internodal growth. This is caused by a reduction in cell division and elongation, negatively affecting yield and quality (Khan, 2006). Plants that have undergone stress are usually shorter and exhibit some degree of stunted growth

(Nuruddin, 2003). Under drought conditions, leaves may also be smaller, wrinkled, and darker green (Koornneef, 1990). This reduction in growth may not just harm the plant’s vegetative growth, but can negatively affect size and quality of fruit (Nuruddin, 2003).

Fruit yield of drought stressed plants drops in comparison to non-stressed plants

(Nuruddin, 2003). Depending on the timing of the onset of stress, insufficient flowering can ensue, leading to a reduction in total fruit yield, or fruit may drop as a survival mechanism in order to carry the remaining fruit to maturity (Serrani, 2007). Lack of sufficient water reduces cell division and expansion, severely limiting plant size

(Nuruddin, 2003). Additionally, drought stress will reduce turgor pressure within the cells, causing overall firmness of the fruit to decline, and eventually resulting in dropped fruit (Nuruddin, 2003).

Diminishing availability of water during key growth stages, or overall during the life cycle, may cause extensive damage not only limiting the overall size of plants, but also the commercial grading of the fruit and subsequent revenue for growers (Nuruddin,

2003). Understanding the physiological and metabolic changes in plants under drought is required to determine possible options for mitigating damage caused by drought.

Limited plant water uptake under drought conditions reduces available water for gas exchange at the stomates. Stomatal closure not only reduces transpiration, but also slows CO2 diffusion into the leaves. This drop in CO2 exchange slows photosynthetic rates (Kakumanu, 2012). This decreases the rate of other metabolic functions throughout the plant and can affect expression of transcription factors (Gong, 2010). Transcription of genes that promote cell division and expansion become downregulated. Upon initiation of drought stress, turgor pressure decreases as a failure to maintain water in vacuoles, and the plant exhibits the onset of wilting. Vacuoles in cells then require additional solutes to pull water in and maintain cell turgor. The G1 rest phase of the cell cycle is upregulated to maintain cells in a state of arrested development, reducing cell division (Kakumanu,

2012). During a gradual onset of drought stress, expression of certain genes become upregulated, with other genes being downregulated accordingly in order to reduce oxidative damage and excess energy as starch degradation declines. Prolonged stress can reduce flowering, fruit and seed set, and induce fruit drop (Kakumanu, 2012).

Gibberellins Phytohormone Group

The phytohormone group of gibberellins regulates many of the same plant growth processes as drought stress (Fleet, 2005). Gibberellins are bio-synthesized from mevalonic acid through the isoprenoid pathway to create the precursors of gibberellins

(terpenes) (Nataraj, 1999). They affect many aspects of growth regulation and are strongly associated with cell division, cell elongation, germination, flowering, and fruit size (Serrani, 2007). As cell growth and stages of the life cycle in a plant are affected by

drought stress, gibberellins are of interest because of their association with stress signals and drought stress tolerance (Serrani, 2007).

In the synthesis of the gibberellins, there are two pathways to create the isoprenoid precursor for biosynthesis; either through the mevalonic acid pathway which processes acetyl-CoA into mevalonic acid and then into isopentenyl diphosphate (IPP), or through the methylerythritol phosphate (MEP) pathway which converts glyceraldehyde

3-phosphate and two carbons into 1-deoxy-D-xylulose 5-phosphate, then into MEP, and finally into IPP (Sponsel, 2010). IPP can react with dimethylalyl diphosphate (DMAPP) to create geranyl diphosphate (GPP). IPP is added constitutively in steps until forming farnesyl diphosphate (FPP) and lastly, geranyl geranyl diphosphate (GGPP) is formed for use in direct synthesis of gibberellins (Sponsel, 2010).

Within the plastids of the cell, GGPP is converted into ent-kaurene by the enzymes ent-copalyl-diphosphate synthase (CPS) and ent-kaurene synthase (KS). Ent- kaurene then moves to the endoplasmic reticulum where it is converted into GA12, the first actual gibberellic component (Sponsel, 2010). GA12 can go through either a 13- hydroxylation pathway or a non 13-hydroxylation pathway in the cytosol. The dominance of one pathway over another is most commonly dependent on plant species. Each pathway leads to the conversion of hormone precursors into the bioactive GA forms of either GA4 for 13-hydroxylation, or GA1 for non 13-hydroxylation pathways (Garcia-

Hurtado, 2012). In tomatoes, the 13-hydroxylation pathway is preferred with ent-7a- hydroxykaurenoic acid converting to GA12 aldehyde as opposed to the non 13- hydroxylation pathway (Karssen, 1990). In cases of overexpression of genes encoding

GA20 oxidase, the normal pathway may shift to a non-13-hydroxylation pathway

(Garcia-Hurtado, 2012).

Interruptions along GA pathways produce phenotypes exhibiting deficiency in bioactive GA levels (Karssen, 1990). Down regulation of expression of genes encoding enzymes along this pathway ultimately reduce bioactive gibberellins (Sponsel, 2010).

Such a down regulation in the biosynthesis pathway of gibberellins give rise to deficient phenotypes that are characterized by stunted growth, small dark wrinkled leaves, and reproductive issues such as sterility (Koorneef, 1990). These phenotypes can occur naturally from genotypic gibberellin deficient mutants, or by the use of gibberellin inhibitors like that of paclobutrazol (PAC) (Ranwala, 1998).

Many enzymes are involved in the production of bioactive gibberellins, while

GA2 oxidase enzymes degrade the bioactive forms and convert them into non-active GAs

(Xiao, 2010). Expression of SlGA20ox, encoding the enzyme GA20 oxidase, is positively correlated with bioactive levels of gibberellins, especially GA4 (Xiao, 2010).

Overexpression of this enzyme increases gibberellin content, increasing growth of the plant (Marti, 2010). First observed in Arabidopsis, transcription of the GA20ox genes regulate the production of the GA 20 oxidase (Hedden, 2000). Further interactions with other hormones such as auxins may affect expression and overall GA levels (Marti,

2010). Increases in auxin levels, or the inhibition of auxin transport can also upregulate gibberellin biosynthesis. Gibberellin biosynthesis is also negatively regulated by its own activity (Bethke, 1998).

Many commercial versions of gibberellins are currently used today for altering the production of many crops. Among them, GA3 and GA4+7 are widely used in

commercial applications (Ranwala, 1998). Both gibberellin applications have been effective in increasing overall heights and petiole/leaf size through increasing cell division and expansion (Ranwala, 1998). When applied and compared to a control, both types showed significant increases over the control (Ranwala, 1998). Similarly, when a growth retardant like PAC and prohexadione-Ca was applied, internode lengths decreased substantially with much smaller and darker leaves (Ranwala, 1998). The application of gibberellins to these cultivars overcame the effects of the growth retardants, increasing the growth of the plant to a normal phenotype. While the reduction caused by such growth inhibitors is substantial, the application of GA1 and/or GA20 effectively overcame the symptoms of arrested development successfully (Zeevaart,

1993). Adding gibberellic acid to tomatoes has shown that higher gibberellin levels increased vegetative growth, but also phosphorous concentration in the leaves, the number and size of fruit, lycopene content, and overall biomass of the shoots, leaves, and fruit (Khan, 2006). Although not all gibberellins are effective in overcoming growth retardants, some clear successes have been identified. The inhibition of gibberellic action under stress conditions results from an interruption in the bio-synthesis of gibberellins

(Zeevaart, 1993). Under conditions of stress, normal biosynthesis of gibberellins can be reduced resulting in dwarfed plants. It is suggested that this interruption and decline in the metabolic pathways of gibberellins would be best measured by looking at the concentration of gibberellins that are at the end of the metabolic pathway. GA8 is a final product of the pathway, whose concentrations in shoot tips, basal stems, leaves, and roots are reduced under drought stress (Rood, 2010).

Paclobutrazol Inhibited Gibberellin Biosynthesis

Inhibitors of GA production limit growth by decreasing cell division and expansion

(Ranwala, 1998). The site of action for inhibition of GA biosynthesis occurs early in the pathway by inhibiting normal activity of enzymes involved in the production of GA precursors (Vettakkorumakankav et al, 1999). PAC is one such chemical, belonging to the family of triazoles. It acts on the monooxygenase ent-kaurene oxidase, reducing its ability to convert ent-kaurene into ent-kaurenoic acid, an important early step in the GA biosynthesis pathway (Cowling et al, 1998; Vettakkorumakankav et al., 1999). This inhibits GA synthesis, reducing cell division and expansion (Hedden and Kamiya, 1997).

Stem tissue, leaf development, and reproductive organs experience reduced growth rates from lower GA levels. This would indicate a strong relationship between gibberellin biosynthesis and plant growth. PAC is used to study the impact of GAs by reducing gibberellin production.

In plants with reduced GA production, as seen in PAC treated plants, growth becomes stunted from reduced cell division and elongation. Leaves remain small, wrinkled, and darkly colored. If fruit set occurs, yield will be low, with many fruits dropping early (Koorneef, 1990). Further investigation into the relationship between gibberellin activity and environmental stresses are needed in understanding of the effect of drought on production.

Drought Stress and Gibberellins

Symptoms arising from drought stress and GA deficiency can appear phenotypically similar. During prolonged drought, plants are reduced in height, leaf development, and

flowering/fruit development (Olimpieri, 2011). Similarly, a reduction in gibberellin content produces dwarfed plants with reduced stem elongation, leaf development, and problems with flowering and fruit set (Vettakkorumakankav, 1999). In some cultivars of barley, dry weight of the plant may be reduced by 30% due to drought stress. Under severe stress, development and expansion of cells slows down (Vettakkorumakankav,

1999). Though gibberellins are capable of inducing stem elongation and plant development to the point of offsetting drought symptoms, the application of gibberellins does not remedy the negative effects of a prolonged drought. Drought stress results in down regulation in the expression of genes involved in gibberellin biosynthesis. This has been previously reported in the reduced production of GA20 oxidase enzymes (Zeevaart,

1993). This suggests that during drought stress, plants down regulate gibberellin biosynthesis in order to prevent aggressive growth. Gibberellins can be reduced under stress conditions, leading to a decline in elongation as water availability decreases (Liu,

2013). Plants that have reduced GA production have shown advantages for plantings.

Dwarfed plants are able to tolerate and survive in stressed conditions. These dwarf plants are reported as more suitable for environments where drought/heat stress occurs more frequently (Vettakkorumakankav, 1999). Additionally, their compact size allows them to be grown more densely, allowing for better efficiency of resources during drought (Peng,

1999).

References

Bethke, P. and R.L. Jones. 1998. Gibberellin signaling. Current Opinion in Plant

Bio. 1:440-446.

Fleet, C. and T. Sun. 2005. A DELLAcate Balance: The role of gibberellin in

plant morphogenesis. Current Opinion in Plant Bio. 8:77-85.

Garcia-Hurtado, N., E. Carrera, O. Ruiz-Rivero, M.P. Lopez-Gresa, P. Hedden, F.

Gong, and J.L. Garcia-Martinez. 2012. The characterization of transgenic tomato

overexpressing gibberellin 20-oxidase reveals induction of parthenocarpic fruit

growth, higher yield, and alteration of the gibberellin biosynthetic pathway. J.

Expt. Bot. 63:5803-5813.

Gong, P., J. Zhang, H. Li, C. Yang, C. Zhang, X. Zhang, Z. Khurram, Y. Zhang,

T. Wang, Z. Fei, and Z. Ye. 2010. Transcriptional profiles of drought-responsive

genes in modulating transcription signal transduction, and biochemical pathways

in tomato. J. Expt. Bot. 61:3563-3575.

Hedden, P. and A.L. Phillips. 2000. Gibberellin metabolism: new insights

revealed by the genes. Trends in Plant Sci. 5:523-530.

Kakumanu, A., M.M.R. Ambavaram, C. Klumas, A. Krishnan, U. Batlang, E.

Myers, R. Grene, and A. Pereira. 2012. Effects of drought on gene expression in

maize reproductive and leaf meristem tissue revealed by RNA-seq. Plant Physiol.

160:846-867.

Karssen, C.M. 1995. Hormonal regulation of seed development, dormancy, and

seed germination studied by genetic control. In Seed Dev. and Germination, J.

Khan, M.M.A., C. Guatam, F. Mohammad, M.H. Siddiqui, M. Naeem, and N.

Khan. 2006. Effect of gibberellic acid spray on performance of tomato. Turk. J.

Bio. 30:11-16.

Koornneef, M., T.D.G. Bosma, C.J. Hanhart, J.H. Van Der Veen, and J.A.D.

Zeevaart. 1990. The isolation and characterization of gibberellin-deficient mutants in tomato. Theoretical and Applied Genetics 80:852-857.

Liu, T., S. Zhu, L. Fu, Y. Yu, Q. Tang, and S. Tang. 2013. Morphological and physiological changes of Ramie (Boehmeria Nivea L. Gaud) in response to drought stress and GA3 treatment. Rus. J. Plant Physiol. 60:749-755.

Martí, E., E. Carrera, O. Ruiz-Rivero, and J.L. García-Martínez. 2010. Hormonal regulation of tomato gibberellin 20-oxidase1 expressed in arabidopsis. J. Plant

Physiol. 167:1188-1196.

NOAA. 2013. National weather service drought factsheet. NOAA www.drought.gov

North Carolina State University. 2013. General facts about agriculture. College

Relations College of Agr. Life Sci. North Carolina State U.

Nuruddin, M., C.A. Madramootoo, and G.T. Dodds. 2003. Effects of water stress on tomato at different growth stages. HortScience 38:1389-1393.

Olimpieri, I., R. Caccia, M.E. Picarella, A. Pucci, E. Santangelo, G.P. Soressi, and

A. Mazzucato. 2011. Constitutive co-suppression of the GA 20-oxidase1 gene in

tomato leads to severe defects in vegetative and reproductive development. Plant

Sci. 180:496-503.

Peng, J., D.E. Richard, N.M Hartley, K.M. Devos, J.E. Flintham, J. Beales, L.J.

Fish, A.J. Worland, F. Pelica, D. Sudhakar, P. Christou, J.W. Snape, M.D. Gale, and N.P. Harberd. 1999. ‘Green revolution’ genes encode mutant gibberellin response modulators. Nature 400:256-261.

Ranwala, N.K.D. and D.R. Decoteaur. 1998. Involvement of gibberellins in phytochrome-regulated stem and petiole elongation in watermelon plants.

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Rood, S.B., K. Zanewich, C. Stefura, and J.M. Mahoney. 2000. Influence of water table decline on growth allocation and endogenous gibberellins in black cottonwood. Tree Physiol. 20:831-36.

Schuppler, U., P. He, P.C.L. John, and R. Munns. 1998. Effect of water stress on cell division and cell-division- cycle 2-like cell-cycle kinase activity in wheat leaves. Plant Physiol. 117:667-678.

Serrani, J.C., R. Sanjuan, O. Ruiz-Rivero, M. Fos, and J.L. Garcia-Martinez.

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145:246-57.

Soroushi, H., T.S. Nejad, A. Shoukofar, and M. Soltani. 2011. The Interaction of drought stress and gibberellic acid on corn (Zea Mays L.). World Acad. Sci., Eng.

Technol. 60:142-143.

Sponsel, V. 2010. Gibberellins: regulators of plant height and seed germination.

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Sunderland, MA, pp.545-619

Vettakkorumakankav, N.N., D. Falk, P. Saxena, and R.A. Fletcher. 1999. A crucial role for gibberellins in stress protection of plants. Plant Cell Physiol.

40:542-548.

Weller, J.L., J.J. Ross, J.B. Reid. 1994. Gibberellins and phytochrome regulation of stem elongation in pea. Planta 192:489-496.

Xiao, Y., D. Li, M. Yin, X. Li, M. Zhang, Y. Wang, J. Dong, J. Zhao, M. Luo, X.

Luo, L. Hou, L. Hu, and Y. Pei. 2010. Gibberellin20-oxidase promotes initiation and elongation of cotton fibers by regulating gibberellin synthesis. J. Plant

Physiol. 167:829-37.

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323.

CHAPTER 3: DROUGHT STRESS SUBSTRATE MOISTURE THRESHOLDS FOR ‘MONEYMAKER’ TOMATO (Solanum lycopersicum)

1 Introduction

Water shortages reduce agriculture yields globally (Greenwood et al., 2010).Water availability for agriculture is expected to decrease with increasing drought periods as a result of climate change (Cook, et al. 2015), and the influence of water availability on plant physiology affects growth and maturation throughout a plant’s life cycle (Nuruddin et al., 2003). Drought can reduce cell division and elongation as well as crop growth and yield (Nuruddin et al., 2003; Zhao et al., 2011; Vettakkorumakankav et al., 1999). This reduction in elongation from drought ultimately results in shorter and smaller plants

(Mahajan and Tuteja, 2005; Alem et al., 2015). Water availability influences the hormonal relationships that affect the growth and survival of the plant (Nuruddin et al.,

2003). As water shortages become more common, drought stress may severely impact crop growth and yield by limiting transport of nutrients and affecting hormonal activity and metabolism (Soroushi et al., 2011).

Temperature and drought can affect cell division and elongation in meristem and leaf development (Ehleringer, 1982; Hasanuzzaman et al., 2013), which can result in smaller leaves with reduced CO2 exchange and subsequent metabolic functions

(Chartzoulakis et al., 2002). The severity of drought stress determines the resulting reduction in growth (Galmes et al., 2007). During a mild drought, plants may acclimate in

order to maintain metabolic functions. Reduction in cell elongation and photosynthetic rates are plant responses that may result from acclimation, and the decrease can become more severe with increasing stress. Additionally, acclimation to environmental stress can result in morphological changes, affecting cell size, organs, and whole plants (Xu et al.,

1997). A severe stress may limit a plant’s ability to acclimate. As reduction in photosynthesis becomes more severe, stomates close as a response to the stress and dramatically limit CO2 exchange (Watkinson et al., 2003; Kakumanu et al., 2012). This decreases the rate of other metabolic functions throughout the plant and increases expression of stress-related genes encoding key regulatory enzymes (Gong et al., 2010).

Much of the surface area of mature tissues results from the expansion that occurred in the cells of that tissue (Van Volkenburgh, 1999). Elongation, division, and water content of cells can be reduced by drought stress and are hypothesized to be indicative of stress severity (Hsiao, 1973; Massacci, 1996; Farooq et al. 2009). In fact, cell elongation is very sensitive to drought stress, and its symptoms are easily observed across a range of severity (Massacci, 1996; Galmes et al., 2006; Zhao et al., 2011).

Hormones have a large role in stress signaling, and can alter the expression of genes related to cell division and elongation, and affecting the metabolism of the cells. Cell elongation depends on a variety of cellular processes and enzymatic activities. Among these processes is the loosening of the cell wall to increase plasticity of the cell wall and adequate turgor pressure to drive expansion (Cosgrove et al., 2002). The expression of genes encoding for expansin enzymes (Huang et al., 2008) and expansin activity, an important regulator in the expansion of cells, can decrease due to drought stress (Tuteja,

2007; Huang et al., 2008).

The objective of these studies was to determine substrate VWC thresholds that clearly demonstrate either well-watered or drought stress conditions, using plant height and internode length as indicators of drought severity. These VWC thresholds can then be used in future trials to determine the effects of drought on gene expression and physiological responses. This study focused on stem elongation under varying levels of drought stress using tomato plants as a model. Tomatoes have been extensively studied, resulting in a genetically well-characterized model species with a large genomic database

(Matsukura et al., 2008). Tomato plants exhibit an easily observable response to drought, thus making them early indicators of stress conditions (Gong, 2010). Additionally, their commercial importance and production in arid regions, such as California and the

Mediterranean, makes tomato an economically important crop to study (Pena, 2005).

Understanding the impact of drought on tomatoes may help future growers better mitigate the detrimental effects from prolonged drought periods.

2 Materials and Methods

2.1 Cultivation

Tomato ‘Moneymaker’ was seeded into 15-cm pots, and grown in a glass greenhouse in

Athens, Georgia. Pots were filled with soilless substrate (70% peat, 30% perlite; Fafard

1P; Fafard, Agawam, MA) with a controlled release fertilizer (Harrell’s 16-6-11;

Harrell’s, Lakeland, FL) incorporated at a rate of 5.93 kg·m-3. A few seeds were planted in each pot to ensure at least one seedling per pot. After 2 weeks, seedlings were thinned to 1 plant per pot. Heating was provided by two propane heaters hung from the ceiling

and air was circulated by horizontal air flow fans. Cooling of the greenhouse was done by an evaporative cooling pad spanning one side of the greenhouse, and air was pulled through by exhaust fans on the opposing side. The positioning of the pad at one end of the bench likely created a temperature gradient as air flowed through the greenhouse, with cooler and more humid air (lower vapor pressure deficit) close to the cooling pad.

2.2 Substrate water content and datalogging

Irrigation was managed using a data logger (CR1000; Campbell Scientific, Logan, UT).

VWC of the substrates was measured with capacitance sensors (GS-3; Decagon Devices

Inc., Pullman, WA). Sixteen sensors were connected to the data logger, and readings from each sensor were used to determine substrate VWC. Each GS-3 sensor was inserted into the pot from the side, placed such that the three prongs of the sensor were vertically aligned down the side of the pot and inserted parallel to substrate level. A relay driver

(SDM-CD16AC; Campbell Scientific) operated by the data logger administered valve control. Each valve controlled one experimental unit, with five sub repetitions (plants) per valve. Each pot was irrigated with a dribble ring connected to a 2 L·h-1 pressure compensating emitter. Volumetric water content thresholds for each plot were programmed into the data logger. Each time the data logger program was executed, the data logger compared the measured VWC to the irrigation threshold. When the measured

VWC for a given experimental unit dropped below its respective threshold, the corresponding irrigation valve was opened for 10 s, providing 5.5 mL of water/plant per irrigation. The data logger program ran every 10 minutes, providing irrigation to individual plants on a need basis, up to 144 times per day.

2.2.1 Treatments and Data Gathering

Drought stress treatments were evaluated during a summer and fall trial. The summer study set out to determine general thresholds for possible VWC thresholds representative of well-watered and drought stressed conditions. The first trial was conducted from June

20 until July 18, 2013 (29 d). Thresholds for VWC were 0.40, 0.35, 0.30, 0.25, and 0.20 m3·m-3 (Fig. 3.3), with each treatment replicated three times, was set on June 25 (day 1).

After reaching respective VWC target thresholds 3-4 d later, plants were given 9 d to acclimate to their respective VWC treatments. Plant height, internode length, node count, and leaf area were measured in conjunction with visual observations of wilting twice weekly from day 14 until day 25, and at these times visual assessments of wilting also was done. Total height of the plants was measured from the substrate level to the apical meristem. The increase in plant height over the course of the summer study was calculated as final height, measured on day 25, minus initial height as measured on day

18.

For the second trial, tomatoes were seeded on September 11 and the trial ran until

October 24, 2013 (44 d). The number of treatments was reduced to four, which were decided based the results from the summer trial. Treatment VWC thresholds (0.35, 0.25,

0.15, and 0.10 m3·m-3), selected based on the results from the summer study, were initiated 7 d after germination (day 1) to allow time for all seedlings to establish (Fig.

3.6).

Initial measurements of plant height were taken on day 27, once most VWC thresholds were achieved, with final measurements taken on day 44. All treatment groups

dried down to their respective VWC threshold with the exception of the 0.10 m3·m-3 threshold. To determine the ability of the plants to recover from drought stress treatments, some plants in each experimental unit were left after the study and irrigated to a VWC of 0.35 m3·m-3.

At the end of each study, plants were harvested for destructive measurements.

Leaf size was measured in summer by selecting 3 leaves adjacent to measured internodes using a leaf area meter (LI-3100, LICOR, Lincoln, NE). In the fall, samples were taken for leaf water potential and biomass measurements. Leaf discs were cut using a 5 mm diameter biopsy punch (Miltex, Inc., York, PA) from fully expanded leaves at approximately 3 PM, after the conclusion of all non-destructive measurements. The leaf discs were then inserted into a thermocouple psychrometer (Model 76, J.R.D. Merrill

Specialty Equipment, Logan, UT) for leaf water potential (Ψleaf) measurements. Samples were equilibrated in a water bath (Neslab RTE-221, Thermo Fisher Scientific, Waltham,

MA) at 25.0 °C for 4 h before measurement of Ψleaf. Psychrometer output was measured with a data logger (CR7X, Campbell Scientific, Logan, UT). The psychrometer thermocouples were then placed in a freezer overnight to disrupt the membranes in the leaf tissue and osmotic potential was measured the following day after re-equilibration in the water bath (25 °C) for 4 h. Turgor pressure within the leaves was then calculated as the water minus osmotic potential. Fresh weight and dry weight of the entire shoots were also measured. Shoots were dried for 3 d in a drying oven at 80 °C before dry weight measurements.

2.3 Environmental conditions

Environmental conditions were measured with a quantum sensor (QSO-sun; Apogee

Instruments, Logan, UT) and a temperature and humidity sensor (HMP60, Vaisala Inc.,

Woburn, MA) connected to the data logger. The temperature and humidity data were used to calculate the vapor pressure deficit (VPD). The quantum sensor monitored the photosynthetic photon flux (PPF) throughout the study. The daily maximum PPF and cumulative daily PPF (daily light integral, DLI) were determined by the data logger.

The data logger recorded measurements at 20 minute and daily intervals.

Substrate volumetric water content (VWC) readings from each sensor, cumulative irrigation volume per plant, and environmental data were recorded. Minimum, maximum, and average temperature and VPD, as well as DLI and maximum PPF were recorded at midnight.

During the summer trial, mean temperatures in the greenhouse were 24.5 ± 0.9 °C

(mean ± sd), with humidity at 83.9 ± 3.6%. Mean VPD was 0.48 ± 0.13 kPa. DLI averaged 11.3 ± 5.9 mol·m-2·d-1, ranging from 4.6 to 21.3 mol·m-2·d-1. The low VPD was due in largely to high humidity (Fig. 3.1). Temperatures in the greenhouse during the second study were lower than in summer with a mean of 21.2 ± 0.6 °C. VPD ranged from

0.19 to 3.60 kPa (mean 0.76 ± 0.24 kPa) with higher daily maximum values being observed near the end of the study. These high VPD values correspond with low daily minimum relative humidity during the latter part of the study. The DLI during the fall study ranged from 3.6 to 17.1 mol·m-2·d-1 and averaged 10.6 ± 4.0 mol·m-2·d-1 (Fig. 3.2).

2.4 Experimental design and data analysis

Due to an error in the setup of the summer trial, there was a failure to impose a randomized design. An error in programmed VWC thresholds caused treatment groups to be sorted together in descending order from 0.40 m3·m-3 to 0.20 m3·m-3, across the length of the bench. All plots with the 0.40 m3·m-3 threshold were closest to the cooling pad, while the 0.20 m3·m-3 plots were closest to the exhaust fan. Because of this, and a presumed temperature and vapor pressure deficit gradient across the bench, treatment effects cannot be statistically separated from a possible location effect, so data were not analyzed statistically.

The fall study was designed using a randomized complete block with four blocks and four treatments for a total of 16 experimental units, with five plants per experimental unit. Treatment effects were evaluated by one-way ANOVA (proc anova, SAS 9.4, SAS

Systems, Cary, NC). In the event of missing data, data was analyzed using a general linear model (GLM) in SAS (proc glm). Mean separation was done using Tukey tests.

3 Results and Discussion

In the summer, the substrate dried down gradually until VWC thresholds were reached, within 3-4 d (Fig. 3.3). The increase in plant height was lowest with the highest VWC threshold (0.40 m3·m-3) and comparable to that with the lowest VWC threshold of 0.20 m3·m-3 (Fig. 3.4). Plants grown at 0.35 m3·m-3 VWC threshold had the greatest increase in plant height (44.65 ± 17.8 cm).

Previous research showed that elongation of plant height can be controlled by water availability (Burnett et al., 2005). As plants are exposed to reduced substrate VWC,

stem elongation is reduced, resulting in shorter plants (Alem et al., 2015). The results of the current study revealed no significant reduction in elongation with decreasing VWC, which may be due in part to a temperature gradient across treatments (Kaspar & Bland,

1992; Reddy et al., 1992). Plants grown at the 0.40 m3·m-3 threshold on average had 1.5 fewer nodes (17.1 nodes/plant) than those in other treatments (18.7 nodes/plant) which were grown under warmer temperatures. Previous reports of temperature effects on node development and plant height have shown to limit the rate of development and elongation. Resulting in increased node count and elongation as temperature rises, and that this development and elongation slows as temperatures decrease (Reddy et al., 1992;

Wu et al., 2015). This may help further explain the unexpectedly small increase in height at the 0.40 m3·m-3 VWC threshold, as it is unusual for substrate moisture content to effect growth in well watered plants compared to drought stressed plants as seen in the summer trial.

Although the change in plant height decreased with increasing VWC, final internode length increased with increasing VWC. Those plants maintained at a 0.40 m3·m-3 VWC threshold had the longest internodes (4.55 ± 0.68 cm), while the lowest

VWC thresholds resulted in internode lengths of 4.05 ± 0.50 cm and 4.1 ± 0.21 cm for the 0.25 and 0.20 m3·m-3 thresholds, respectively. Longer internodes with increasing

VWC thresholds agree with previous reports on the effect of substrate water content on elongation (Hsiao, 1973; Mahajan and Tuteja, 2005; Alem et al., 2015), and suggest that plant elongation at higher VWC thresholds would have been expected to be greater given proper randomization of treatments.

Wilting of leaves is a common symptom of drought stress and can be seen in tomato plants under mild drought stress (Nuruddin et al., 2003). The wilting is caused by reduced turgor pressure within cells and this pressure is needed to maintain the plant rigid and upright (Hsiao, 1973; Mahajan and Tuteja, 2005). Wilting was observed to varying degrees throughout the study and was more severe during midday hours. Recovery of mild wilt occurred for most plants beginning in the early evening. Plants at the 0.20 m3·m-3 threshold showed more wilting as compared to plants with higher VWC thresholds. With the exception of some sub replicates at the 0.20 m3·m-3 threshold, most plants were able to recover in the evening.

Leaves adjacent to the measured internodes were larger in the plants grown at

0.40 and 0.35 m3·m-3 VWC thresholds compared to those at lower thresholds (Fig. 3.5).

Leaf development and expansion are affected by the health and size of the plant (Chutia and Borah, 2012; Van Volkenburgh, 1999), and drought stress can reduce cell division and elongation, limiting leaf area development (Farooq et al., 2009). The increased size of the leaves at the 0.40 and 0.35 m3·m-3 thresholds may have also been due to increased water content caused by the closure of stomates from the cool air coming from the nearby cooling pad. Wind and temperature can lead to plants closing stomates, reducing transpiration, and result in extra turgor pressure to drive cell expansion (van

Volkenburgh, 1999).

From the results of the second study, drought stress reduced elongation of plant height (p < 0.0001). Plants in the 0.25 m3·m-3 threshold were significantly taller compared to those in the 0.15 and 0.10 m3·m-3 thresholds. There was no significant difference in height between the 0.35 and 0.25 m3·m-3, and between the 0.35 and 0.15

m3·m-3 thresholds. The three highest VWC thresholds resulted in significantly taller plants than the 0.10 m3·m-3 threshold. Internode lengths showed no significant differences among the 0.35, 0.25, and 0.15 m3·m-3 thresholds. Internodes of plants grown at 0.10 m3·m-3 were shorter than in any other treatment (p < 0.0001), which was in line with results with plant height measurements. Continued dry down of substrates of plants at the 0.10 m3·m-3 threshold showed a decreasing rate of water uptake as drought stress progressed (Fig. 3.6). The VWC never reached the 0.10 m3·m-3 threshold, suggesting that the plants were not able to dry down the substrate to this level. With decreasing water content in drier substrates, hydraulic conductivity decreases, limiting water flow through the substrate to the roots. This decrease in hydraulic conductivity has been suggested to be the main reason for limiting plant water uptake in dry substrates (O’Meara et al.

2014).

The 0.10 m3·m-3 threshold resulted in the lowest shoot dry mass of all substrate water content treatments (p < 0.0001) (Fig. 3.8). Shoot dry mass of the three other thresholds (0.35, 0.25, and 0.15 m3·m-3) followed the same trend as plant height (Fig.

3.7), with the exception that there were no significant differences in shoot dry mass among these three treatments. By comparison, plants at the 0.10 m3·m-3 VWC threshold had 95% less shoot dry mass than the average of the three highest treatments, again indicating that the drought stress with a 0.10 m3·m-3 VWC threshold was excessive.

Based on the results of the second study, drought stress was seen in some degree among the three highest VWC thresholds, but most noted among those plants at the 0.10 m3·m-3 threshold.

Osmotic potential was significantly lower in the 0.10 m3·m-3 threshold (Fig. 3.9) compared to that of the 0.15 m3·m-3 (p = 0.0216). Water and osmotic potentials tend to be lower as drought stress increases (O’Neil, 1983; Naor et al., 1995), and although the most stressed treatment (0.10 m3·m-3 VWC threshold) generally did show lower water, osmotic, and turgor potentials compared to other treatments, there no significant differences among the three treatments with higher VWC thresholds. The resulting turgor pressure calculated as the difference between water and osmotic potential, did show a tendency for those plants at the 0.10 m3·m-3 VWC threshold to have lower turgor than the higher thresholds, but this effect was not significant. Lower turgor pressure under drought was previously reported when osmotic adjustment was not enough to mitigate the effects of drought stress on water potential (Naor et al., 1995). Normally in drought conditions, a positive correlation would be seen between height and water potential (Burnett et al.,

2005). The current study does not support previous findings that suggest higher water potentials for higher VWC thresholds.

4. Conclusion

Our results suggest that both 0.35 and 0.25 m3·m-3 can be considered well-watered treatments due to longer elongation measurements and mass accumulation in relation to other treatments. Plants grown at 0.35 m3·m-3 in the summer and fall studies had longer internodes in comparison to thresholds with lower substrate VWC, and this further confirmed this VWC threshold as being adequate for a well water-watered treatment.

The most severe drought stress treatment (0.10 m3·m-3) during the fall study greatly inhibited growth, and this stress level was decided to be too excessive for further

use. All plants except for those in the 0.10 m3·m-3 threshold were able to show partial or full recovery. Due to this evidence, it was decided that the next threshold level up (0.15 m3·m-3) would have to suffice as a drought stress treatment, as it would serve little purpose to conduct a full scale study on severely stressed, non-growing plants.

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Fig. 3.1. Daily mean, minimum, and maximum temperature, and vapor pressure deficit during the summer study. Daily light integral measurements were recorded at the end of each day.

Fig. 3.2. Daily mean, minimum, and maximum temperature, and vapor pressure deficit during the fall study. Daily light integral measurements were recorded at the end of each day.

Fig. 3.3. Mean substrate moisture content averaged across replicates every 24 h for each treatment level during the summer. Error bars indicate standard error. Non-visible error bars are within the limits of the symbol. Treatments were imposed on Day 1, and subsequent substrate drying occurred until treatment VWC thresholds were achieved approximately 3-4 d.

Fig. 3.4. Increase in plant height over time and final internode length in response to substrate volumetric water content measured from day 14 until day 25 during the summer trial.

Fig. 3.5. Mean leaf size of leaves attached to 6th node as affected by substrate volumetric water content.

Fig. 3.6. Substrate volumetric water content (VWC) for each threshold during the fall.

VWC thresholds for treatments were set on Day 1, resulting in a substrate dry down to target thresholds.

Fig. 3.7. Total plant height and internode length of tomato (Solanum Lycopersicum) in response to substrate volumetric water content thresholds at the end of the fall study.

Means with the same letter are not significantly different (P = 0.05).

Fig. 3.8. Shoot dry mass of tomato (Solanum Lycopersicum) plants grown with different substrate volumetric water content thresholds. Only the most severe drought stress treatment (0.10 m3·m-3) differed significantly from the other treatments (P < 0.0001).

Fig. 3.9. Water, osmotic, and turgor potentials of tomato (Solanum Lycopersicum) plants in the fall study. Means with the same letter are not significantly different (P = 0.05).

There were no significant treatment effects on water and turgor potential.

CHAPTER 4

DROUGHT STRESS DOWNREGULATES GIBBERELLIN BIOSYNTHESIS AND

REDUCES STEM ELONGATION IN TOMATOES (Solanum lycopersicum) DURING

VEGETATIVE GROWTH1

1Litvin, A.G., M.W. van Iersel, and A. Malladi. To be submitted to Journal of ASHS

Abstract. Drought stress reduces leaf and cell expansion. Since gibberellins (GA) play an important role in controlling cell elongation, the objective was to quantify the effects of drought stress on elongation and regulation of GA metabolism. We exposed

‘Moneymaker’ tomatoes to drought stress to observe the effect on internode elongation and GA metabolism-related gene expression. Plants were grown from seed in 15-cm pots filled with a peat-perlite substrate in a greenhouse for 25 d. Irrigation was automated using a data logger, which maintained volumetric water contents (VWC) of 0.35 and 0.15

(m3·m-3) for well-watered and drought stressed conditions, respectively. To further investigate the effect of GAs on elongation, paclobutrazol (PAC), a GA biosynthesis inhibitor was applied to reduce GA production. The transcript levels of SlGA20ox1, -2, -

3, and -4, SlGA3ox2, and SlGA2ox2, -4, and -5, corresponding to enzymes in the later steps of GA biosynthesis and. LeEXP1, and -2, encoding for expansin enzymes related to the loosening of cell wall necessary for cell expansion, were analyzed. Drought stress reduced plant height (p = 0.0012), internode length (p < 0.0001), and cell size (p = 0.002) compared to well-watered conditions. Down regulation of transcript levels due to drought stress was observed for SlGA20ox4, SlGA2ox5, and LeEXP1, but not for any other genes.

Paclobutrazol increased expression of SlGA20ox1 and -3, and SlGA3ox2. Application of

PAC reduced elongation and it is presumed that the up regulation of genes involved in

GA metabolism is a response by the plants attempting to compensate for lower GA production due to PAC-induced inhibition of GA biosynthesis. These findings suggests that drought stress effects on elongation are at least partly due to effects on GA production.

Water availability for agriculture is expected to decrease while drought becomes more common as a result of climate change (Cook, et al. 2015). Water availability affects growth and development throughout a plant’s life cycle (Nuruddin et al., 2003), and a decrease in availability results in reduced agricultural yields globally (Greenwood et al.,

2010). Drought stress severely impacts growth and yield and can limit cell division, expansion, transport of nutrients, hormonal activity, and general metabolism in the plant

(Soroushi et al., 2011).

The severity of drought stress determines the physiological responses of the plant including reduction in growth (Galmes, et al., 2007; Kim et al., 2012). Under mild drought stress, plants may acclimate to maintain metabolic functions. Reductions in stem elongation and photosynthetic rates can result from acclimation, and can intensify with increasing stress (Xu et al., 1997). More intense drought stress may limit a plant’s ability to acclimate, resulting in more severe plant responses, such as stomatal closure and large reductions in photosynthesis (Watkinson et al., 2003; Kakumanu, 2012). This decreases the rate of other metabolic processes throughout the plant and alters the expression of genes related to stress signaling (Gong, 2010).

Cell expansion and division can be reduced by drought stress and are indicative of stress severity (Hsiao, 1973; Massacci et al., 1996; Farooq et al. 2009). Cell expansion, an increase in cell volume, is very sensitive to drought, and reduced cell size is easily observed across a range of drought severities (Massacci et al., 1996; Galmes et al., 2006;

Zhao et al., 2011). Loosening of the cell wall to increase plasticity and the presence of

adequate turgor pressure are key factors that facilitate cell expansion (Cosgrove et al.,

2002).

Cell expansion is affected by a cell’s ability to loosen its cell wall. This cell wall loosening is driven by expansin enzymes, which increase plasticity of the cell by degrading the connections of microfibrils and hemicelluloses in the cell wall, allowing cell turgor pressure to expand the cell. Multiple hormones within the plant system, including GAs, stimulate this expansin activity (Cosgrove, 2000; Keller and Cosgrove

1995). The activity of expansins and expression of genes encoding them can decrease due to drought stress, limiting cell expansion (Zhao et al., 2011).

Stem elongation, dependent on cell division and elongation, was reduced in studies of sweet sorghum (Sorghum bicolor L.) under drought stress (Massacci et al.,

1996). Cell division and elongation in response to increasing drought stress severity has been reported in American sweetgum (Campbell, 1974). In tomatoes, internode length becomes reduced under drought stress (Morales et al., 2015). These studies highlight the effect of drought has on elongation in relation to the magnitude of the stress (Hsiao,

1973).

Gibberellins are plant hormones that promote cell expansion and division. The main regulating enzymes of the final steps of GA metabolism are GA 20-oxidases, GA 3- oxidases, and GA 2-oxidases (Hedden and Kamiya, 1997; Hedden and Phillips, 2000;

Yamaguchi and Kamiya, 2000). Both GA 20-oxidases and GA 3-oxidases act in succession on GA precursors to form bioactive GAs, while GA 2-oxidases are responsible for the catabolism of the bioactive GAs. The three gene families (GA20ox,

GA3ox, and GA2ox) encoding these enzymes help control the main regulatory steps in

GA metabolism (Hedden and Phillips, 2000). Growth related responses of synthesized

GAs can be affected by signaling mechanisms. DELLA, a key component in GA signaling, acts as repressor of most GA-related processes (Daviere and Achard, 2013).

Bioactive GAs bind to their receptor GID1, which in turn binds to DELLA and targets it for degradation. This relieves the repression of DELLA on GA responses (Hedden and

Thomas, 2012; Thomas, 2005).

Symptoms of GA deficiency can appear phenotypically similar to that of drought stress. During prolonged drought, plants display reduced height, leaf development, and flowering/fruit development (Olimpieri, 2011). Similarly, a reduction in endogenous GA content results in dwarfed plants with reduced stem elongation, leaf development, and aberrant flowering and fruit set (Vettakkorumakankav, 1999). Drought stress results in down regulation in the expression of genes involved in GA biosynthesis. This has been previously reported in the reduced production of GAs (Zeevaart, 1993). Gibberellins can be reduced under stress conditions, leading to a decline in elongation as the extent of stress increases (Liu, 2013). This offers an adaptive advantage as the plants displaying reduced growth are better able to tolerate stress and survive in these conditions. These plants with reduced growth are reported as more suitable for environments where drought/heat stress occurs more frequently (Vettakkorumakankav, 1999).

Gibberellin biosynthesis can also be inhibited through the application of chemicals that inhibit the activity of enzymes involved in GA biosynthesis

(Vettakkorumakankav et al., 1999). Paclobutrazol (PAC), a triazole, inhibits ent-kaurene

oxidase, reducing its ability to convert ent-kaurene to ent-kaurenoic acid, an important early step in the biosynthesis of GA precursors (Cowling et al., 1998;

Vettakkorumakankav et al., 1999). As a result, PAC reduces endogenous production of bioactive GAs, reducing cell division and expansion (Hedden and Kamiya, 1997). Such a down regulation in the biosynthesis pathway of gibberellins give rise to phenotypes in tomatoes that are characterized by stunted growth, small dark wrinkled leaves, and reproductive issues such as sterility (Koorneef, 1990). Stem tissue, leaf expansion, and reproductive organs have shown reduced growth rates from lower GA levels in tomatoes

(Hafeez-ur-Rahman et al, 1989; de Moraes et al, 2005). This indicates a strong relationship between gibberellin biosynthesis and plant development.

Stem elongation is a simple and quantitative proxy for a variety of drought stress responses (Alem et al., 2015; Hsiao, 1973; Nuruddin, 2003). The objective of this study was to determine the effects of drought stress on stem elongation and GA metabolism- related genes in tomato plants in order to better understand the morphological and transcriptional effects of drought stress. To further elucidate the role of GAs in cell expansion and elongation, the effect of paclobutrazol on gene expression and stem elongation was studied as well.

Materials and Methods

Plant material and growth conditions. The study was conducted from June 23 until July

17, 2014 (25 d). Tomato ‘Moneymaker’ was seeded into 15 cm round pots, grown in a

glass greenhouse in Athens, Georgia. Pots were filled with soilless substrate (70% peat,

30% perlite; Fafard 1P; Fafard, Agawam, MA) with a 16N-2.6P-9.1K controlled release fertilizer (Harrell’s 16-6-11; Harrell’s, Lakeland, FL) incorporated at a rate of 5.93 kg/m3.

Initially several seeds were planted in each pot, but were thinned to one seedling per pot after one week. Temperature control in the greenhouse was provided by evaporative cooling, or when necessary, two ceiling-mounted propane heaters and horizontal air flow fans.

Photosynthetic photon flux density (PPFD) inside the greenhouse was measured with a quantum sensor (QSO-sun; Apogee Instruments, Logan, UT) connected to a data logger (CR1000; Campbell Scientific, Logan, UT). Temperature and humidity were measured by a HOBO data logger (HOBO U12 Temp/RH, Onset, Bourne, MA) inside a radiation shield. Temperature and humidity data were used to calculate vapor pressure deficit (VPD). Daily maximum PPF and the cumulative daily PPF (daily light integral,

DLI) were determined by the data logger. Temperatures averaged 24.5 ± 2.4 °C (mean ± s.d.), ranging from 19.6 to 32.9 °C. The mean VPD during the study was 0.55 ± 0.15 kPa. DLI ranged from 6.9 to 36.8 mol·m2·d-1, averaging 20.3 ± 7.2 mol·m2·d-1.

Substrate volumetric water content. Substrate volumetric water content (VWC) was measured using capacitance sensors (GS-3; Decagon Devices Inc., Pullman, WA).

Twenty sensors were connected to a data logger (CR1000; Campbell Scientific), and readings from each sensor were used to compute VWC. Each GS-3 sensor was inserted into the pot from the side, placed such that the three prongs of the sensor were vertically aligned down the side of the pot and inserted parallel to the substrate level. Irrigation

control was managed using the data logger. A relay driver (SDM-CD16AC controller;

Campbell Scientific) operated by the CR1000 data logger administered valve control.

Each valve irrigated one experimental unit, with four pots as sub repetitions per valve.

Each pot was irrigated with a dribble ring (Dramm, Manitowoc, WI) connected to a 2

L·h-1 pressure-compensating emitter (Netafim USA, Fresno, CA). VWC thresholds for irrigation, corresponding to either well-watered or drought-stressed conditions, were programmed into the data logger to automate irrigation. When VWC for a given experimental unit dropped below its respective threshold, the corresponding irrigation valve was opened for 20 s, providing 11.1 mL/plant per irrigation cycle. The data logger program ran every 10 min, providing irrigation to individual experimental units on a need basis, up to 144 times per day.

Volumetric water content and PAC treatments. A randomized complete block design with four treatments (2 VWC levels, with and without PAC application) and five blocks was used. Each experimental unit had four subsamples. Volumetric water content threshold treatments were initiated on July 3 (day 1) and designated as either well-watered (0.35 m3·m-3) or drought-stressed (0.15 m3·m-3) based on preliminary studies. Within each

VWC treatment, half the experimental units received PAC, which was applied as a drench at 4 mg of active ingredient per plant diluted in 2 mL water on day 1. All plants were then lightly irrigated following PAC application. The rate of PAC application was determined from previous reports on stem elongation of tomatoes (de Moraes et al., 2005;

Hafeez-ur-Rahman et al., 1989; Serrani et al., 2007).

Growth Measurements. Total height of the plants from the substrate level to the apical meristem as well as the internode length between the 4th and 5th nodes from the base of the plant was measured daily. Initial height measurements were taken on day 5. Internode lengths were measured from day 8 until day 15, starting after substrate dry down.

For water potential measurements, leaf discs were cut using a 5 mm diameter biopsy punch (Miltex, Inc., York, PA) from fully expanded leaves at approximately 12

PM. The leaf discs were quickly inserted into thermocouple psychrometers (Model 76,

J.R.D. Merrill Specialty Equipment, Logan, UT), which were then sealed. Samples were temperature equilibrated in a water bath (25 °C) for 4 h (Neslab RTE-221, Thermo Fisher

Scientific, Waltham, MA) before measurement of the psychrometer output with a datalogger (CR7X, Campbell Scientific). Samples were then placed in a freezer overnight to disrupt the cellular membranes. Osmotic potential was measured on these samples the following day, after re-equilibration in the water bath (25 °C) for 4 h. Each psychrometer was individually calibrated to convert their output to water and osmotic potential. Turgor pressure within the leaves was then calculated as the difference between water and osmotic potential. Dry weight of the entire shoots was recorded at the end of the study, after drying for 3 d in a drying oven at 80 °C.

Microscopy. At the conclusion of the study, internodes were harvested for cell size analysis. Harvested internodes were immediately fixed in FAA solution

(formaldehyde:acetic acid:ethyl alcohol:water, 10:5:50:35) (Berlyn and Miksche, 1976).

Slides were prepared by slicing internode tissue longitudinally (40-50μm thick) using a vibratome (Micro-Cut H1200, BIO-RAD, Hercules, CA) and staining with toluidine blue.

Immediately after preparation, slides were viewed under a digital microscope (BX51;

Olympus Corporation, Waltham, MA) and images were obtained for measurement of cell size. Images were analyzed using ImageJ (U.S. National Institutes of Health, Bethesda,

MD) to determine cell area. Individual cells of parenchyma tissue in each image were measured by outlining the cell in ImageJ and converting the pixel count to an area.

Gene Expression

Tissue Collection and Storage. Actively growing internodes located directly above those used in elongation measurements were harvested on day 17 at approximately 10 AM to determine differences in gene expression among treatments. These internodes were carefully removed using pruning shears and edges cleaned with a razor blade. Samples were immediately placed into 15 mL centrifuge tubes and snap frozen in liquid nitrogen.

Samples were then placed in a -80 °C freezer and held until further analysis.

Identification of Gibberellin Metabolism & Cell Expansion-Related Genes. Genes associated with the regulation of later stages of GA metabolism (GA20ox, GA3ox, and

GA2ox), GA signaling (DELLA), and those encoding expansins (LeEXP) were identified from NCBI Genbank (National Center for Biotechnology Information, Bethesda, MD).

PCR primers were designed for selected genes using the NCBI Nucleotide Primer Blast tool (Blast, NCBI).

RNA Extraction, cDNA Synthesis, and qRT-PCR. Tissue samples were ground in liquid nitrogen, and total RNA was extracted using the E.Z.N.A. Plant RNA Mini Kit following manufacturer’s protocol (Omega Bio-Tek, Norcross, GA). Total RNA was eluted in 8 µL of DEPC (diethylpyrocarbonate) treated water and quantified using a NanoDrop 8000

(Thermo Fisher Scientific, Waltham, MA). The samples were stored at -80 °C until cDNA synthesis.

Total RNA (1 µg) from each sample was used for cDNA synthesis following the protocol by Malladi and Hirst (2010). Genomic DNA contamination was removed with

DNAse (Promega, Madison, WI). The cDNA synthesis was performed using oligo dT primers and ImProm II reverse transcriptase (Promega, Madison, WI). The cDNA was diluted 6-fold, and stored at -20 °C.

Gene expression was analyzed by qRT-PCR using a Stratagene Mx3005P PCR

(Agilent Technologies, Santa Clara, CA). Diluted primers specific to the genes were used along with the SYBR green master mix (Applied Biosystems, Foster City, CA).

Amplification and normalization of data followed previous protocols (Dash et al., 2012).

Gene expression was normalized to that of ACTIN and TUBULIN genes.

Experimental Design and Statistical Analysis. When data was collected from all subsamples for height, internode, and shoot dry mass, those data were averaged before analysis. Data was analyzed using one-way ANOVA (α = 0.05) in SAS (PROC ANOVA,

SAS 9.4, SAS Systems, Cary, NC). In the event of missing data, data was analyzed using a general linear model in SAS (PROC GLM). Tukey’s HSD was subsequently used for mean separation. Linear regression analysis was conducted to analyze correlations among

measured variables. Multiple regression analysis with backward selection (p < 0.05,

PROC REG; SAS) was then used to evaluate the effects of gene expression, VWC, PAC and their interactions on cell size.

Results and Discussion

Morphological and Physiological Effects. Volumetric water content treatments were initiated on July 3 (day 1) and plants under drought stress reached their VWC thresholds by day 10 (Fig. 4.1). Over the course of the study, irrigation volume averaged 1431 ± 174 mL for well-watered, and 111 ± 66 mL/plant for drought stressed plants. Treatment with

PAC resulted in no significant differences in water use.

Total plant height was reduced by both drought stress (p = 0.0012) and PAC (p <

0.0001), consistent with previous findings that elongation is reduced by decreasing water availability and PAC (Burnett et al., 2005; Alem et al., 2015; de Moraes et al, 2005).

Drought stress and PAC reduced plant height by 2.4 and 7.1 cm, respectively. There was an interaction effect of substrate VWC and PAC on internode length (p = 0.0004).

Drought stress reduced internode length of the non-paclobutrazol treated (NO-PAC) plants by 8.2 mm, and in PAC-treated plants by 1.5 mm (Fig. 4.2). Under both well- watered and drought stress conditions, PAC reduced plant height by ~45%, and internode elongation by ~65%, consistent with previous reports (Hafeez-ur-Rahman et al, 1989; de

Moraes et al, 2005).

Drought and PAC displayed an interactive effect on cell size of growing internodes (p = 0.0067). Drought stress reduced cell size of plants not treated with PAC by 62%, but had no significant effect on cell size of plants treated with PAC (Figs. 4.2

and 4.3). We hypothesize that the effect PAC was already so pronounced in limiting cell size, drought stress may have had little to no additional effect. Cell size, internode length and plant height were strongly and positively correlated (Fig. 4.4), demonstrating inter- relationships among these parameters (Schouten et al., 2002). Cell size is a key component of stem elongation (Huber et al., 2014; Pearson et al., 1995). The elongation of individual cells results in a phenotypic response on the whole plant level. Phenotypic parameters of plant height and internode lengths are then determined at the micro level by the responses of individual cells to metabolic and environmental conditions which affect their elongation (Huber 2014; Nuruddin et al., 2003).

Water (average of –0.42 MPa), osmotic (-0.88 MPa) and turgor potential (0.46

MPa) were not significantly affected by drought or PAC. The lack of an effect of drought stress was unexpected, since water, osmotic, and turgor potential were previously reported to be decreased under drought (O’Neil, 1983; Naor et al., 1995; Chartzoulakis et al., 2002). High coefficients variability of at least 19.8% in the data may have contributed to a lack of significance.

Well-watered plants without PAC had significantly higher shoot dry mass than other plants. As with other responses, PAC application and VWC treatments had an interactive effect on dry mass (p = 0.024); drought stressed, PAC-treated plants had the lowest shoot dry mass. No significant difference was observed between the drought- stressed NO-PAC and well-watered PAC treatments. As previously reported, plant size and mass can be reduced by the effect of limited water availability inhibiting cell division and elongation (Farooq et al., 2009; Hsiao, 1973), and PAC further exaggerates this effect by acting on GA metabolism to reduce cell size (de Moraes et al., 2005).

GA biosynthesis. Expression of genes in several gene families (GA20ox, GA3ox, and

GA2ox) encoding key GA metabolism-related enzymes displayed varied responses to the treatments (Fig. 4.5). GA20-oxidases generally catalyze the synthesis of bio-active GA precursors (Hedden and Phillips, 2000; Li et al., 2012). SlGA20ox1 and 2 showed no significant differences across treatments, but drought stress down-regulated the expression of SlGA20ox4 by 48% (p = 0.0185, Fig. 4.5). Lower transcript levels of

SlGA20ox4 in response to drought stress suggest a role of this gene in regulating plant responses under drought. SlGA20ox3, another gene within the same family, did not significantly change in expression under drought, but did display a 5.5 fold up-regulation in response to PAC (p < 0.0001). GA3-oxidase enzymes produce bio-active GAs by acting upon the products of reactions catalyzed by the GA20-oxidases. SlGA3ox2, a member of a gene family encoding GA3-oxidases, displayed a 4 fold up-regulation in response to PAC (p = 0.0031). The strong, positive correlation between SlGA20ox3 and

SlGA3ox2 (Fig. 4.6) may be due in part to co-regulation of these genes (Hedden and

Kamiya, 1997; Hedden & Phillips, 2000; Hedden & Thomas, 2012), in response to low

GA endogenous levels. The upregulation of SlGA20ox3 and SlGA3ox2 following PAC application may be an attempt by the plant to upregulate GA biosynthesis in response to low endogenous GAs caused by PAC. This suggests that these genes may be involved in maintaining GA homeostasis.

Catabolism of bioactive GAs is carried out by GA2-oxidases encoded by multiple members of the GA2ox gene family. Among these, SlGA2ox5 was downregulated by 51% under drought stress (p = 0.0247). Within PAC treated plants, drought stress downregulated transcript levels of SlGA2ox2 by 71% compared to well-watered plants (p

= 0.0147, Fig. 4.5). The physiological significance of downregulation of these genes under drought is not clear and warrants further study. Expression of DELLA, a key gene involved in GA signaling, was similar across all treatments.

Different responses to drought or PAC of genes within the same family, as seen in this study, may be due to different roles of these genes in response to specific metabolic or environmental cues. This may create redundancies which allow for appropriate regulation of metabolism, depending on tissue and specific conditions.

The transcript levels of genes encoding for GA 20-oxidases, GA 3-oxidases, and

GA 2-oxidases may each influence the expansion of cells of stem tissue. Models accounting for the effects of VWC, PAC application, gene expression, and relevant interactions among them accounted for 81 to 87% of the variability in cell size (Fig. 4.7).

Drought stress and/or the application of PAC (Table 4.1) generally explained most of the variability in cell size. The interaction of VWC and PAC along with the expression of

SlGA20ox4 genes accounted for 86% of the variation in cell size. Higher expression levels of SlGA20ox4 were correlated with larger cell size. The positive slope of the regression model as gene expression increases suggests a relationship between bioactive

GAs and reduced cell elongation under drought. This correlation is supported in part by the downregulation of SlGA20ox4 under drought stress in this study and the associated reduced cell size of internode tissue. In one study overexpression of SlDREB, a repressive transcriptional factor, was found to reduce internode elongation by downregulating

SlGA20ox4 resulting in suppression of GA-biosynthesis. This down regulation of

SlGA20ox4 limiting bioactive GAs was suspected to be part of a drought tolerance mechanism (Li et al., 2012).

Interestingly, high expression of SlGA20ox3 and SlGA3ox2 was associated with smaller cells in plants treated with PAC, but not in non-PAC-treated plants. This may be due to a feedback mechanism for these genes in response to low GAs (Cowling et. al.,

1998, Li et al., 2012). Further research is needed to describe the different roles of these genes within their respective regulatory families.

Expression of SlGA2ox5 was downregulated under drought stress conditions. The positive slope of the regression lines within these models indicate that cell size increases as expression of these genes increased. This correlation may be the result of lower GA levels under stress conditions, reducing the need for catabolism of bioactive GAs.

Expansin gene expression. Expansins facilitate stem elongation, and tissue and leaf expansion (Reinhardt et al, 1998; Caderas et al, 2000; van Volkenburgh, 1999), as well as the expansion or softening of cells in other organs such as fruit (Rose et al, 1997;

Cosgrove et al, 2002; Powell et al, 2003). Both expansin genes analyzed in this study,

LeEXP1 and LeEXP2, were expressed in actively growing shoots (Fig. 4.5), but only

LeEXP1 was down regulated (by 33%) in response to drought stress (p = 0.0036). Down- regulation of expansin gene expression in response to drought was also reported in wheat

(Zhao et al., 2011). Previously, LeEXP1 was reported in the ripening tissues of tomato fruit and was believed to be specific to fruit ripening (Rose et al., 1997). Expression and regulation of LeEXP1 in stem tissue of tomato suggests a newly discovered role for this gene. Our data suggest that LeEXP1 is involved in regulation of cell expansion in internodes in response to water availability.

A model containing substrate VWC, and the interaction among VWC, PAC and

LeEXP1 gene expression described 84% of the variation in cell size (Fig. 4.7; Table 4.1).

Within treatments, low transcription levels of LeEXP1 were correlated with low cell size, consistent with the role of expansins in cell wall loosening.

Conclusion

The current study confirmed that cell expansion and consequently, internode and stem elongation are reduced in response to drought stress. The effect of drought was generally greater in non-PAC treated plants than in PAC treated plants. These data highlight the importance of GA metabolism in drought responses, specifically in relation to internode elongation. GA20ox4 and GA2ox5 genes encoding GA20-oxidase and GA2-oxidase enzymes in the later steps of the GA pathway were down-regulated in response to drought stress, demonstrating the relationship between GA metabolism and drought.

Paclobutrazol, on the other hand, increased the expression of GA-metabolism related genes GA20ox3 and GA3ox2, possibly as a feedback response to low endogenous GA levels resulting from PAC. Plants also down-regulated expression of the expansin gene

LeEXP1 which may slow elongation. The changes in gene expression may help in further understanding the hormonal balance plants maintain in response to their environment.

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Table 4.1. Coefficients and partial R2 of significant variables within multiple regression models describing the effects of volumetric water content (VWC), paclobutrazol (PAC), normalized gene expression (NGE), and their interactions on the size of tomato (Solanum lycopersicum) stem parenchyma cells.

coefficient Variable partial R2 GA20ox1 GA20ox2 GA20ox3 GA20ox4 GA3ox2 GA2ox2 GA2ox4 GA2ox5 LeEXP1 LeEXP2 NGE x x x 793.01 x x x x x x 0.09 VWC x x x x x x 251.93 230.89 x 0.08 0.08 PAC x x -2231 x x x x x x x 0.64 VWC*PAC x x x x x x x x x x

VWC*NGE x x -16.15 -37.50 -32.16 x x x x x 0.20 0.02 0.41 PAC*NGE x x x x x x x x x x

VWC*PAC*NGE x x x 20.65 15.51 x x 13.24 23.30 x 0.77 0.47 0.78 0.78 Intercept x x 5717 8597 2852 x x 3488 3318 x x non-significant

Fig. 4.1. Substrate volumetric water content (VWC) levels for each treatment group shows gradual dry down of drought treatments. Initiation of VWC thresholds for the study was performed on Day 1.

Fig. 4.2. Plant height, internode length, cell size, and shoot dry mass of tomatoes

(Solanum lycopersicum) recorded under the two levels of treatment for well-watered

(WW) and drought stressed (DS), for the application of paclobutrazol (PAC) and without

(NO-PAC). Means with the same letter are not significantly different (P = 0.05).

Fig. 4.3. Microscopy slides showing cell sizes of parenchyma stem tissue collected from actively growing internodes of tomatoes (Solanum lycopersicum) recorded under the two levels of treatment for well-watered (WW) and drought stressed (DS), for the application of paclobutrazol (PAC) and without (NO-PAC).

Fig. 4.4. Correlation of morphological parameters in tomatoes (Solanum lycopersicum) stem tissue: internode length versus cell size (left) and plant height versus internode length (right). Plants were exposed to two levels of treatment: well-watered (WW) and drought stressed (DS), and for the application of paclobutrazol (PAC) and without (NO-

PAC).

Fig. 4.5. Expression of genes related to cell expansion and GA biosynthesis and regulatory functions of internode tissue in tomatoes (Solanum lycopersicum). Gene expression is illustrated as relative to well-watered (WW) or drought stressed (DS) and with paclobutrazol (PAC), and without (NO-PAC). Significance for volumetric water content (VWC) and PAC application is indicated by * above the specific gene (p <0.05). ns= not significant. GA3ox2 was significantly affected by PAC application.

Fig. 4.6. Correlations between the gene expression of GA20ox3 and GA3ox2 in internode tissue in tomatoes (Solanum lycopersicum) recorded under the two levels of treatment for well-watered (WW) and drought stressed (DS), for the application of paclobutrazol

(PAC) and without (NO-PAC). Gene expression is expressed as a log transformation.

Fig. 4.7. Actual cell sizes of internode tissue in tomatoes (Solanum lycopersicum) and predicted in relation to respective gene expressions in GA metabolism, and the predicted cell size as modeled by that gene along with the two levels of treatment for well-watered

(WW) and drought stressed (DS), and for the application of paclobutrazol (PAC) and without (NO-PAC).Gene expression is expressed as a log transformation.

CHAPTER 5: SUMMARY AND CONCLUSION

Decreases in water availability resulted in reduced stem elongation and shoot dry mass accumulation in tomato (Solanum lycopersicum). This study showed that the increasing threat of drought within agriculture can affect growth and possibly yield. In chapter 3 limits of water availability, inducing reduction in the elongation rate of stem tissue, was studied and conditions for drought stress imposition were described in terms of substrate volumetric water content (VWC). This was then subsequently used to further study the effect of drought stress on elongation and gibberellin-related gene expression.

Consequently, GA homeostasis was affected by drought stress, reducing expression of genes encoding for key enzymes in GA biosynthesis. Decreasing water availability characterized within these trials as a reduction in VWC reduced plant height, internode, and individual cell elongation. Subsequently, final shoot dry mass was reduced due to drought stress.

Normal GA metabolism allows for GA-promoted cell elongation, and the effect of reduced GA production was characterized by the chemical inhibition of GA biosynthesis by paclobutrazol. This chemical inhibition of GA biosynthesis resulted a strong reduction in cell size, internode length and plant height. Indeed, the inhibiting effect of paclobutrazol resulted in similar shoot dry mass for well-watered plants as untreated drought stressed plants. The interactive effect of paclobutrazol with drought stress demonstrated additional factors are responsible for reduced growth under stress.

Expression of SlGA20ox3 and GA3ox2 were up regulated under paclobutrazol application and may be an attempt by the plant to upregulate GA biosynthesis in response to low endogenous GAs caused by PAC. This suggests that these genes may be involved in maintaining GA homeostasis.

Expression of the expansin gene LeEXP1 dropped in response to drought stress and this may have reduced cells’ ability to increase plasticity and to expand. This was the first documented case reporting the expression and role of LeEXP1 in stem tissue under drought stress. Further research is needed to study the role of LeEXP1 and other expansins in relation to cell elongation and how they may play a part in drought tolerance.