interaction of drought stress and gibberellin metabolism
TRANSCRIPT
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.
INTERACTION OF DROUGHT STRESS AND GIBBERELLIN METABOLISM ON
STEM ELONGATION IN TOMATO
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
INTERACTION OF DROUGHT STRESS AND GIBBERELLIN METABOLISM ON
STEM ELONGATION IN TOMATO
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
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Fleet, C. and T. Sun. 2005. A DELLAcate Balance: The role of gibberellin in
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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.
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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
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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.
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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
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NOAA. 2013. National weather service drought factsheet. NOAA
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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,
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Ranwala, N.K.D. and D.R. Decoteaur. 1998. Involvement of gibberellins in
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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.
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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.
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-
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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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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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
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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).
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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).
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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.
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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.