INTERACTION OF DROUGHT STRESS AND GIBBERELLIN METABOLISM

INTERACTION OF DROUGHT STRESS AND GIBBERELLIN METABOLISM ON

STEM ELONGATION IN TOMATO

by

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.



by

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

© 2015

Alexander G. Litvin

All Rights Reserved



by

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

iv

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.

v

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.

vi

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

vii

Abstract ......................................................................................................43

Introduction ................................................................................................44

Materials and Methods ...............................................................................47

Results and Discussion ..............................................................................53

Conclusion .................................................................................................58

References ..................................................................................................59

SUMMARY AND CONCLUSIONS ................................................................................74

viii

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

ix

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

x

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

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

1

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).

2

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).

3

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

<http://www.nws.noaa.gov/om/csd/graphics/content/outreach/brochures/FactSheet_D

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.

4

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.

5

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,

6

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

7

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

8

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

9

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).

10

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

11

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).

12

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.

13

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.


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

<http://www.nws.noaa.gov/om/csd/graphics/content/outreach/brochures/FactShee

t_Drought.pdf

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

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

<http://www.cals.ncsu.edu/CollegeRelations/AGRICU.htm>.

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

14

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.

HortScience 33:493-494.

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.

2007. Gibberellin regulation of fruit set and growth in tomato. Plant Physiol.

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.

15

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

In Plant Physiol., L. Taiz and E. Zeiger, (eds.) Sinauer Associates, Inc,

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.

Zeevaart, J.A., D.A. Gage, and M. Talon. 1993. Gibberellin A1 is required for

stem elongation in spinach. Proc. Nat. Acad. Sci. 90:7401-7405.


elongation is involved in the drought tolerance in wheat. Protoplasma 248:313-

323.

16

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

17

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).

18

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

19

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.

20

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

21

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.

22

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).

23

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,

24

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.

25

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

26

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.

27

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

28

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.

29

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33

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.

34

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.

35

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.

36

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.

37

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

water content.

38

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.

39

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).

40

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).

41

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.

42

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

43

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.

44

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

45

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,

46

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

47

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

48

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

49

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).

50

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.

51

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).

52

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

53

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

54

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).

55

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

56

= 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).

57

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.

58

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.

59

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66

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

67

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.

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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).

69

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).

70

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).

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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.

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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.

73

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.

74

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.

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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.

Documents

INTERACTION OF DROUGHT STRESS AND GIBBERELLIN METABOLISM