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CHIGAN S
EligT LANSING, MICH 48824-1048

This is to certify that the
dissertation entitled

Genetic engineering for dehydration-stress tolerance in
cucumber (Cucumis sativus L.) by expressing the Arabidopsis
thaliana-transcriptional regulators CBF1 and CBF3 and the
mannose-6—phosphate reductase gene M6PR from celery

(Apium graveolens L.)

presented by

Mohamed Saleh Tawfik

has been accepted towards fulfillment
of the requirements for the

Ph.D. ’ Horticulture

 

 

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2/05 c:/Cli_=i-C/DateDuo.indd-p.15

 

Genetic engineering for dehydration-stress tolerance in
cucumber (Cucumis sativus L.) by expressing the Arabidopsis
thaliana-transcriptional regulators CBF1 and CBF3 and the

mannose-6-phosphate reductase gene M6PR from celery
(Apium graveolens L.)

By

Mohamed Saleh Tawfik

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Horticulture Department

2005

ABSTRACT

Genetic engineering for dehydration-stress tolerance in cucumber (Cucumis
sativus L.) by expressing the Arabidopsis thaliana-transcriptional regulators CBF1
and CBF 3 and the mannose-6-phosphate reductase gene M6PR from celery

(Apium graveolens L.)

By

Mohamed Saleh Tawfik

Salinity and drought conditions are major factors affecting plant productivity and
distribution worldwide. To engineer resistance to dehydration stress in cucumber
(Cucumis sativus L.), transgenic cucumber were generated with genes associated with
enhanced abiotic stress tolerance: the mannose-6-phosphate reductase (M6PR) gene from
celery for mannitol production, and the CBF1/BREE] b and CBF 3/DREBI a, abiotic
stress-associated transcriptional regulators from Arabidopsis thaliana. To transgenic
M6PR cucumbers produced detectable mannitol, indicating functionality of the M6PR
gene in cucumber. However, mannitol accumulation in the T1 progeny was highly
variable making this trait-difficult to work with. Eleven lines of cucumber were produced
with the CBF genes, integration and expression was verified in the T0, T1 and T2
generation. Under greenhouse conditions, T1 and T2 CBF—cucumber plants accumulated
elevated levels of proline and soluble sugars, a signature for CBF expression in
Arabidopsis, indicating ability of the CBF gene to induce stress related responses in

cucumber. Proline and soluble sugars accumulation were highly correlated, suggesting

coordinated regulation in the transgenic plants. In the absence of salt or drought stress,
the CBF cucumbers showed equivalent growth compared to the nontransgenic controls.
In the presence of salt and drought stress, transgenic plants had less reduction in growth.
Plant performance and fruit production was evaluated under field conditions. Prior to
salinity-stress, transgenic and nontransgenic cucumber lines grew equivalently. CBF-
cucumber plants accumulated significantly higher levels of compatible solutes in leaves
(proline and soluble sugars) and roots (proline) compared to the nontransgenic controls.
Transgenic plants also had elevated levels of K+ and CaH ions and a decreased NaVK+
ratio in root tissues, suggesting a wider range of adaptive responses in the transgenic
plants than has been reported previously. In the absence of salinity, CBF lines had less
fresh weight than the nontransgenic controls; however, dry weight and fruit yield were
equivalent to the nontransgenics. In the presence of salinity stress, CBF-transgenic plants
showed significantly less reduction in fresh weight, dry weight, fruit number and fruit
weight. These results suggest that expression of the CBF/DREB in cucumber, a species
known for sensitivity to salinity and drought conditions, may offer an effective approach

to enhance salinity and drought tolerance.

Acknowledgments

I would like to thank my committee members for their endless support and
encouragement during the last five years, Dr. Mike F. Thomashow, Dr. Wayen H.
Loescher, and Dr. Mathieu N gouajio. I particularly would like to thank my major
professor Dr. Rebecca Grumet, for her guidance; you were the best supervisor any
student could ever ask for; your support carried me over all the difficulties I had.

I also would like to thank the previous and the current members of the ABSP project
for their unconditional support for the last decade. I particularly would like to thank Dr.
John Dodds, Dr. Catherine Ives, Dr. Johan Brink, and Dr. Karim Maredia, your help and
support is beyond my capability to express it in words. Also I would like to thank Dr.
Colm Lawler, for his trust and friendship.

I also‘would like to thank the long list of friends and colleagues in the plant science
community at MSU, being part of this elite group is an honor, you enriched my life and
made my last five years something to cherish and hold on to for the rest of my life.
Thanks to all the current and previous members of the Grumet’s lab, your presence made
the lab a fun place to be there every day.

Finally, I would like to finish by thanking my wonderful family, my Dad, Mom and

my wonderful sisters, for their everlasting love and support.

iv

Table of content

List of Tables
List of Figures
Chapter I
Literature review
Innoducfion
Causes and effects of plant water stress
A- Drought effects on plants
B- Salinity effects on plants
Improvement of dehydration stress tolerance in plants
A- Conventional breeding for drought and salt tolerance
B- Genetic engineering for dehydration stress
1- Genes involved in Na” ion exclusion from the cyt0plasm
2- Genes that control osmolyte and compatible solute
3- Genes that encode stress induced proteins
4- Genes involved in transcriptional regulation of stress
response mechanism in plants
a- The CBF system
b- Regulation of CBF
c- Effect of overexpression of CBF/DREB on the plant transcriptome
d- Effect of CBF/DREB-overexpression on plant metabolome
e- Conservation of the CBF/DREB system among plant species
f- Engineering dehydration-stress tolerance in plants using
the CBF/DREB system
The objectives of this dissertation
Literature cited

page
viii

ix

\lQ-bwi—H

10
16
21

23
23
25
28
31
32

33
37
39

Chapter H
Expression of the Arabidopsis thaliana transcriptional regulators, CBF1
and CBF 3, confers dehydration stress tolerance in cucumber

(Cucumis sativus L.) plants.

Introduction 55
Materials and methods 59
Plant constructs and transformation 59
DNA and RNA isolation, PCR, and Northern blot analysis 60
Salinity experiments 61
Drought experiments 62
Sugar and proline analyses 63
Chlorophyll fluorescence 63
Results 64
Discussion 80
Literature cited 86
Chapter III

Cucumber (Cucumis sativus L.) plants expressing the Arabidopsis thaliana-
transcriptional regulators, CBF] and CBF 3, are more tolerant to salinity stress

under field conditions

Introduction 91
Materials and methods 96
Plant materials 96
Field salinity Experiment 96
Soluble sugars, proline and mineral analysis 98
Results 99
Discussion 108

Literature cited 114

vi

page
Chapter IV
Introduction of the celery mannose-6-phosphate reductase (M6PR) gene for

mannitol production into cucumber (Cucumis sativus L).

Introduction 118
Materials and methods 123
Plant constructs, transformation and seed production 123
DNA isolation and PCR analysis 123
ELISA analysis 123
Mannitol analysis 124
Results and discussion 125
Literature cited 131
Conclusions and future work 134

Appendix 139

vii

List of Table

Table l-lA. Genetic engineering for dehydration stress tolerance in plants:
A. Ion exclusion/sequestration/balance

Table l-lB. Genetic engineering for dehydration stress tolerance in plants:
B. Osmotic adjustment/dehydration stress tolerance

Table l-lC. Genetic engineering for dehydration stress tolerance in plants:
C. Stressed induced proteins

Table l-lD. Genetic engineering for dehydration stress tolerance in plants:
D. Transcription factors and other proteins involved in gene regulation

Table 2-1. Segregation analysis of T1 transgenic cucumber lines expressing
the Arabidopsis CBF1 and CBF3 genes. Presence or absence of the gene was
determined by PCR analysis

Table 2-2. Levels of soluble sugar, free proline, above ground fresh weight
and dry weight, and plant height in CBF-transgenic and control lines 15 days
post initiation of N aCl treatment

Table 3-1. Number of nodes, branches, male and female flowers of T2
355:CBF and non-transgenic cucumber lines prior to initiation of salinity
treatment in the field, 3 weeks post transplanting.

Table 3-2. Fresh weight, dry weight, fruit number and total fruit yield of CBF—
expressing transgenic families and the nontransgenic control lines 24 days post
initiation of salinity treatment.

Table 4-1. Segregation analysis of T1 transgenic cucumber lines expressing
the celery-M6PR gene. Presence or absence of the gene was determined by
PCR analysis

Table 4-2. Mannitol accumulation in segregating Tl-M6PR plants.

viii

11

12

13

14

65

70

100

107

125

130

List of Figures

Figure 2-1. Expression of A. thaliana-transcriptional regulators CBF1 and
CBF3 genes in T2 transgenic cucumber lines.

Figure 2-2. Free proline content (A-C) and total soluble sugars (D-F) of two
transgenic lines (A4, B3), non-transformed control (C) and nontransgenic

segregants (AZ).

Figure 2-3. Total soluble sugar, proline content, above ground fresh and dry
weight of ten transgenic lines (CBF1 and CBF 3), non-transformed control (C)

and azygous segregants (AZ) 15 days post initiation of 100 mM NaCl treatment.

Figure 2-4. A- Correlation between soluble sugar and proline content in
cucumber plants 15 days post initiation of 100 mM NaCl treatment.

Figure 2-5. Expression of A. thaliana-transcriptional regulators CBF1 and
CBF 3 genes in four T2 cucumber expressing lines and non-transgenic lines
(azygous) growing the presence or absence of 100 mM NaCl for 2 weeks.

Figure 2-6. Total soluble sugar and free proline content of CBF] and
CBF3-expressing cucumber lines and nontransgenic controls under well-
irrigated and drought conditions.

Figure 2-7. Chlorophyll fluorescence (F v/F m) in CBF-expressing cucumber
plants and azygous non-transgenic plants, under well-irrigated and drought-
stressed conditions.

Figure 2-8. Effect of drought conditions on above ground fresh weight (A),
dry weight (B) weight, and plant height (C) of CBF], and CBF3-transgenic
lines and non-transformed controls C, and non-transgenic segregants AZ.

Figure 3-1. Accumulation of proline ug/gdw (A, B) and soluble sugars
(C, D) in cucumber plants growing in the field in the absence (A and C)
or presence (B and D) of 100 mM NaCl.

Figure 3-2 Accumulation of proline mg/gdw (A) and soluble sugars
mg/gdw (B) in root tissues of cucumber plants growing in the field in
the absence or presence of 100 mM salinity stress.

Figure 3-3. Potassium (A), sodium (B), Na/K ratio (C) and calcium

content (D) in roots of cucumber plants growing in the field for 24 days,
in the presence or absence of salinity treatment.

ix

66

67

69

72

73

75

77

78

101

103

104

Figure 3-4. Above ground fresh (A) and dry weight (B) accumulation, average
fruit number (C) and total fruit yield (D) per plot in cucumber plants growing
in the field in the presence or absence of salinity treatment for 24 days. 106

Figure 4-1. PCR analysis of the presence of the M6PR gene in To cucumber
plants. 126

Figure 4-2. Gas chromatography for mannitol analysis in one of the cucumber
To plants (M3) (top) and nontransgenic wild-type cucumber plants (bottom) 127

Figure 4-3. Gas chromatography for mannitol analysis in wild type (top) and
T1 plant from family M3 (bottom). 129

Chapter I

Literature Review
Introduction

Environmental factors that impose water-deficits stress place a major limitation on
plant productivity (Bray, 1994; Bohnert and Jensen, 1996). Water deficit is intrinsic to
most abiotic forms of stress, such as drought, salinity and freezing temperatures (Bohnert
and Jensen, 1996). Deleterious effects can be manifested as a reduction in transpiration
and photosynthesis, a reduction in growth rate due to reduced cell enlargement, and
reduction in the synthesis of metabolites and structural compounds (Zhang et al., 1999).
Cellular water deficit also disrupts membrane integrity, which causes loss of cellular
water potential and denaturation of cellular proteins (Bray, 1997). To overcome these
limitations and to improve production efficiency of plants, development of dehydration
stress-tolerant crops is essential (Khush, 1999). In this literature review, I will summarize
causes and effects of dehydration stresses in plants and efforts to develop dehydration

stress tolerance in plants via conventional breeding and genetic engineering approaches.

1- Causes and effects of plant water stress

. Water is the driving force of living organisms; it works as a medium for the
biochemical activities in all living cells (Xiong and Zhu, 2002) and is involved in
biosynthesis and assembly of molecules into organized structures (Tanford, 1978). In
plant cells, water potential (WW), is responsible for generating the needed turgor pressure
for cell expansion. The water potential of a given cell is composed of pressure potential.

(Y’p, which reflects the physical pressure generated by cell wall) and osmotic potential

('I’s which is generated by the solute concentration inside the cell). Plant water deficit
results fi'om inability of plants to acquire their water needs, resulting in loss of turgor
and/or osmotic stress. This could be due to the unavailability of water under drought and
freezing temperatures, or the presence of highly negative osmotic pressure, due to high
salt concentrations in the growing environment. Cellular water deficit, if prolonged, could
be lethal to plant cells, interfering with basic metabolic pathways and changes in
membrane shape and integrity (Bray, 1997).

The ability of plants to respond to and survive water deficit is a complex
phenomenon, which requires adjustment at the molecular, cellular and whole plant level
(Greenway and Munns, 1980; Ingram and Bartels, 1996; Zhu et al., 1997). At the
molecular level, osmotic stress will trigger cascades of signals involving CaH and
reactive oxygen molecules as primarily signals to activate pathways critical for plant
survival under the stress conditions (Knight et al., 1997; Knight and'Knight, 2001). At
the cellular level, responses include metabolic adjustment to produce compatible solutes
(Cherry, 1989), activation of transporters at the plasma and vacuolar membranes for ion
sequestration or exclusion (Blumwald and Poole, 1985; Shi et al., 2000), and activation
of enzymes involved in detoxification of free radicals (Mittova et al., 2002; Bor et al.,
2003; Mittova et al., 2004; Badavvi etal., 2004). At the whole plant-level, responses
include closure of plant stomatal apparatus coupled with an inhibition of vegetative

growth and increase in root growth (Maggio et al., 2003).

I-A- Drought effects on plants

Drought is a serious environmental factor that affects plant production worldwide.
For example, it is estimated that about 25% of the total cultivated areas with rice in the
world is under rain-fed; the shortage of water from one season to another is a serious
threat to yield stability (Babu et al., 2001 ). Drought stress occurs when the rate of water
uptake from the soil is less than plant transpiration rate (Bonhert and Jensen, 1996). One
of the first responses of plants to dehydration stress is triggered by the increase of ABA
concentration, which generates a cascade of signals that leads to a decline in stomatal
conductance to minimize transpiration and to keep it in balance with water absorption
from soil (Zeevaart and Creelrnan, 1988; Chandler and Robertson, 1994). At this stage
the plant can still maintain turgor, and partial stomatal closure can occur several times on
daily basis, especially during mid-day. If unfavorable conditions continue for a long time,
then the stomatal apparatus loses the ability to compensate for the lack of water and
stomatal conductance declines sharply (Quarrie, 1989). High abscisic acid (ABA)
concentrations in plant tissues under drought conditions contribute to reduction of leaf
area and plant height, and pollen abortion (Quarrie, 1989). Persistence of drought stress
eventually causes a dramatic reduction in all processes contributing to plant yield and
reduction in plant growth in general (Cherry, 1989). Persistence of drought conditions
eventually forces the plant to concentrate on survival and water conservation mechanisms
(Cherry, 1989).

ABA application or an endogenous transient increase in ABA due to drought causes
cytosolic pH changes and membrane depolarization; this increases the concentration of

free cytosolic Ca2+ ions in guard cells in response to transient drought perception (Leung

and Giraudat, 1998). Free cytosolic Ca” activates cyclic adenosine 5’-diphosphate ribose
(cADPR), which plays a major role in ABA response (Allen and Schroeder, 1998).
Another major player that is activated due to dehydration stress is phospholipase C
(PLC), reSponsible for releasing inositol 1,4,5-triphosphate (1P3), which in turn mediates
the release of Ca2+ ions into the cytosol (Hirayama et al., 1995). Recent reports
conducted on Arabidopsis cell suspensions suggests that this transient increase in 1P3 is

independent of ABA but still requires Ca2+ binding (Takahashi et al., 2001).

I-B Salinity effects on plants

Stress caused by high salinity in soil or in the irrigation water is a serious factor
limiting the productivity of major agricultural crops as the majority of the agriculturally
important plants species are sensitive to high salt concentrations (McWilliam, 1986;

.. Zhang and Blumwald, 2001). Soil salinity affects about 5% of all cultivated land,
approximately 77 million ha (Jain and Selvaraj, 1997; Tester and Davenport, 2003).
Areas that are affected with salinity are increasing; for exertiple, 1/3 of the irrigated land
worldwide is currently affected by salinity (Tester and Davenport, 2003). Salinization of
soil is expected to reach up to 30% in the next 25 years and up to 50% of the arable land
by the year 2050 (Wang et al., 2003).

Plant response to salt-induced water deficit depends on several factors including
genotype, length and severity of water loss, stage of deveIOpment, and environmental
factors such as temperature and humidity (Bray, 1994). High salinity causes both
hyperosrnotic and hyperionic stress effects, which if sufficiently severe, could result in

plant death (Bohnert et al., 1999; Hasegawa et al., 2000). The plant cell membrane serves

as an impermeable barrier to macromolecules and also most molecules of low molecular
mass. Thus, high salt conditions can lead to increased extracellular solute concentration,
which causes a flux of water out of the cells, resulting in a decrease in turgor pressure and
an increase in concentrations of intracellular solutes (Lichtentaler, 1995).

In addition to the lack of water, exposure to high salinity leads to “toxic sodium
effect” where by excess toxic Na+ in the cytoplasm causes a deficiency of essential ions
such as K+ and Ca+ (Bohnert and Jensen, 1996; Hasegawa et al., 2000). High
concentration of Na+ ions in the cytosol causes metabolic toxicity; this is in part due to
the ability of sodium ions to compete with K+ ions for binding sites for several enzymes
(Tester and Davenport, 2003). High Na+ and Cl' concentrations also disrupt enzyme
function, protein synthesis, structure and solubility and membrane structure and fimction
(Blum, 1988). It also has been suggested that accumulation of salts in older leaves
reduces supply of hormones to the growing tissues, which contribute to poor growth in
salt stressed conditions (Munns, 1993).

To survive such conditions, some plants have developed mechanisms to deal with
excess sodium ions either by compartrnentation of the toxic ions into the vacuole (Apes
at al., 1999; Blumwald et al., 2000; Zhang and Blumwald, 2001; Zhang et al., 2001) or
by exclusion from the cell (Shi et al., 2000; Zhu, 2002; Shi et al., 2003). Interestingly,
halophyte (plants that normally grow in saline areas and can tolerate up to 0.5 M NaCl
concentration before suffering injuries) are able to use ions fiom the surrounding
environment for osmotic adjustment by internally distributing them in a way to keep the
Na+ ions away from the cytosol (Zhu, 2000). Alternatively, some plants tend to

accumulate compatible solutes in order to overcome high salinity problem (Tarczynski et

al. 1993; Bohnert and Jansen, 1996; Sakamoto and Murata, 2000), while other species
have the ability to activate proteins that are involved in damage repair in plant cells
(Ingram and Bartels, 1996; Campbell and Close, 1997).

High salinity conditions are also responsible for generating reactive oxygen radicals
which, if not dealt with, could lead to unbalance in cellular 0; processing (Rental and
Knight, 2004). Strong correlation was found between the ability of plants to adapt to high
salinity levels and the increased activity of antioxidant enzymes such as superoxide
dismutase (SOD), peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX) and
glutathione reductase (GR) (Mittler and Zilinskas, 1994; Liu et al., 1999; Mittova et al.,
2002; Bor et al., 2003; Mittova et al., 2004; Badawi et al., 2004).

In general, the process of adaptation to salinity is coupled with activation of different
signaling pathways in plants that lead to changes in gene expression (Hasegawa et al.,
2000; Xiong et al., 2002; Zhu, 2002; Shinozaki et al, 2003; Seki et al., 2003). For
example, imposing dehydration stress conditions on A. thaliana resulted in the activation
of transcriptional factors that are involved in plant adaptation to salt-induced-
dehydration-stress (Stockinger et al., 1997; J aglo-Ottosen et al., 1998; Liu et al., 1998;
Kasuga et al., 1999; Haake et al., 2002; Chinnusamy et al., 2003). Microarray data from
Arabidopsis plants grown under drought and salinity revealed the presence of 277
upregulated transcripts (5 fold) under drought conditions and 194 cDNAs that are
induced under salinity conditions (Seki et al., 2002); the upregulated transcripts, 128 are
strictly induced under drought conditions, 119 transcript are also induced under salinity
conditions (Seki et al., 2002). Not surprisingly, it was found that about 51% of the

drought induced transcripts are also induced under high-salinity conditions; similarly,

about 72% of the high-salt induced transcripts are also induced under drought conditions
(Seki et al., 2002).

These results strongly indicate the correlation between drought and salinity signaling
mechanisms in plants, which also could explain why many genes encoding for late
embryonic abundant proteins (LEA), heat-shock proteins (HSp), osmoprotectant
biosynthesis, carbohydrate metabolic enzymes, detoxification enzymes, transporters, ion
channels and membrane modification enzymes are activated under both hi gh-salinity and
drought stresses (Thomashow, 1999; Knight and Knight, 2001; Shinozaki and
Yamaguchi-Shinozaki, 2003; Seki et al., 2002; Shinozaki et al., 2003). The same thing is
also true for many genes coding for transcriptional regulators, mitogen activated protein
kinases (MAPKs), and phosphatases that are involved in regulating plant response to high
salinity and dehydration (Thomashow, 1999; Knight and Knight, 2001; Shinozaki et al.,

2003)

II- Improvement of dehydration stress tolerance in plants

II-A Conventional breeding for drought and salt tolerance

Drought and salt response, and apparent tolerance of a species, vary according to the
type, concentration and distribution of salts in the root growing zone, duration of stress,
and developmental stage of the plant (Jain and Selvaraj, 1997). These are also influenced
by other environmental factors such as temperature, humidity, reduced oxygen in poorly
drained or structured soil, and elevated C02 in the surrounding environment (Pasternak,
1987). Traditional breeding strategies to develop drought and salt tolerant plants has had

only limited success, probably due to a combination of difficulties in establishing

selection conditions and the complexity of the resistance mechanisms (Flowers and Yeo,
1995). For example, when evaluating yield performance of a crop under saline
conditions, one should consider the variation in salinity levels within a field, the
possibility of interaction between salinity level and other environmental factors such as
soil fertility, drainage quality and water loss due to transpiration (Flowers, 2004). Thus,
using yield components, as main criteria for selection requires a long period and multiple
locations for testing, and evaluation (Blum, 1989).

Quesada et al., (2002) attributed the lack of success in breeding for salt tolerance to
the quantitative nature of most of the processes involved in salt tolerance. In maize,
Ribaut et al. (1997) measured several yield components (grain yield, car number, kernel
number and kernel weight) in plants growing in three different water-stress regimes.
They found that the correlation between the grain yield under well-watered and severe
stress conditions was very low (0.31). Therefore, selection based on yield component
values would not be effective. They also reported that no major quantitative trait loci
(QTL) expressing more than 13% of the phenotypic variance were detected for any of the
studied traits. Ribaut et al. (1997) concluded that there were inconsistencies in the QTL
genomic positions across the three different water regimes. Working with interspecific
crosses between salt-sensitive tomato plants (Lycopersicon esculentum) and salt-tolerant
L. pimpinellifolium plants, Foolad (1999) reported the presence of a weak correlation
(0.22) between seed germination rate and the percentage of plant survival under salt
stress. The overall results indicated that salt tolerance during seed germination was

independent of salt tolerance during vegetative growth (F oolad, 1999). In general, QTL

that are linked to salt and drought tolerance at one developmental stage are not
necessarily linked to tolerance at other stages (Cushrnan and Bohnert, 2000).

In spite of these complexities, a number of salt-tolerant varieties of crops such as
wheat and rice have been developed (Shannon and Noble, 1990; Forster, 2001). For
example, crossing durum wheat and the wild relative Aegilops tauschii, increased K+/Na+
discrimination in the synthetic hexaploid hybrid wheat (Pritchard et al., 2002). This
increase in K/Na discrimination was significantly correlated with fresh weight
accumulation in wheat plants under salt-stressed conditions. In rice, Senadhira et al.,
(2002), reported the production of dihaploid lines of rice from crossing of two lndica
breeding lines, one of which is superior in yield, the other is superior in salinity tolerance.
Field trials conducted for 5 years revealed that some of the dihaploid lines performed
better than other cultivars grown in saline-prone lands. Some lines showed several
desirable traits such as high yield, salinity tolerance, and early maturation.

Interestingly, the use of in vitro tissue culture and somaclonal variation techniques
has resulted in development of salt-tolerant plants (Safarnejad et al., 1996; Boscherini et
al., 1999). In alfalfa, Safarnejad et al. (1996) isolated somatic clones of alfalfa, which
showed increased salt tolerance, greater accumulation of proline, and a greater increase in
glutathione reductase compared to the parental line. In tomato, Boscherini et al. (1999)
identified a somatic clone that showed enhanced tolerance to salinity compared to wild
type when tested at different NaCl concentrations (0, 75, 150, 300 mM NaCl). Leaf and
flower necrosis was observed only in wild type plants. Plants coming from the somatic

clone retained higher leaf turgor compared to the wild type when tested at 150 mM NaCl.

II-B Genetic engineering for dehydration stress

Differences in gene expression profiles among dehydration stress sensitive and
tolerant plants indicate that the ability to withstand these unfavorable conditions is
conferred by genetically encoded mechanisms (Bray, 1994). A variety of approaches
have been used in order to engineer enhanced salinity and drought tolerance in different

plant species (Table 1-1).

II-B-l Genes involved in Na+ ion exclusion from the cytoplasm

One way for plants to avoid Na+ ion toxicity is to exclude Na+ fiom the cytosol. Ion
transport across membranes in plant cells (plasma membrane and tonoplast) is driven by
proton gradients generated by proton pumps located at the different membranes (Sze et
al., 1999). The main pump in the cell plasma membrane is the plasma membrane H+-
ATPase (PM H+-ATPase), which is responsible for generating the gradient between the
cytosol and the extracellular environment, making the cytosol more basic and the outer
environment more acidic (Palrngren, 1998). At the vacuole, there are two major pumps
that are responsible for generating an acidic pH inside the vacuole; those pumps are the
vacuolar H+-ATPase (V-ATPase) and the vacuolar H+ pumping pyrophosphatase (H+-
PPase) (Sze et al., 1999). Plant cells use the electrochemical gradient that is generated by
the different pumps to load/unload different materials into or out of the cytosol.

In the case of sodium compartrnentation, Na+ ions are loaded from the cytosol

directlyth the vacuole before it reaches a critical toxic concentration. The presence of

10

Table 1. Genetic Engineering for dehydration stress tolerance in plants:

A. Ion
F Resistance

content

Canola
Tomato
Rice
Wheat
rates; grain yield

no; fresh and dry weight;

Conditions of stress treatment

and with 200 mM NaCl for 4D

Salinity: Hydrdponic-culture mM NaCl)
Salinity: Germination supplemented with 0300 mM NaCl
Salinity: Field trail (in soil with EC. of 1.2. 10.6 and 13.7 dSm-i) soild were irrigated 4

were

C G F References

 

 

Table 1. B. Osmotic
Resistance

PSCR Tobacco
(Pyrroline-SCarboxylateReducase) Rice

PDHase (proline dehydrogenase)

MtlD (mannitol-1phosphate Tobacco
dehydrogenase)
Wheat

Eggplant

CodA (Coline oxidase)

BetB. Betaine aldehyde dehydrogenase

BADH Betaine aldehyde dehydrogenase

(Betaine aldehyde dehydrogenase)

fluorescence; roots biomass;

TPS1 (trehalose—1-phospho trasferase)

T6PSIT6PP

S-adenosylmethionine decarboxylase
SAM-DC

Sperrnidine biosynthesis

mg..-

12

 

tolerance

Conditions of stress treatment C G F References

or
Irrigation with 50 150 ml (non-stress) H20 every 4D

grain yield Drought In assay with 10% (w/v) PEG
Drought 21 ‘D water withheld at 10 leaf stage to 15% relative water content

assay on
for 2D

In vitm on medium with 75mM NaCl for 35D

 

 

 
     
  
      

  
  

 

Table 1. C. Stress induced

   

| C G F References

 
   

Resistance Conditions of stress treatment

   

 
 
  

 
 

Rice recovery;

  

on or
and root weight Plants trays with 200mM NaCl 10D then with SOmM NaCl for 30D

         

OT

 
     
 

were

 

Wheat every

  
 
 

Tomato

    
   
 
   
  
    

 
 

medium with 125 or mM NaCI for 28D

assay on

Oil

for 100

 

tolerance

 

 

 

 

Table 1. D- T

kinase)

factors and other involved in

Resistance Conditions of stress treatment

were grown same

were
seedling assay on agar plates supplemented with 500mM sorbitol for

011

were paper
soaked with either 250 mM for 4D or mannitol (300 and 400 mM)

C G F References

X

 

 

large, acidic membrane-bound vacuoles in plant cells allows cells to efficiently
compartmentalize excess Na+ ions into the vacuole by the vacuolar Na+/H+ antiporter
(Blumwald and Poole, 1985). The difference in the H+ is initially established by the H+-
ATPase pump (Blumwald et al., 2000). In salt tolerant species, an increase in transcript
level of Na+/H+ antiporters was observed upon exposing plants to high salt levels (Tester
and Davenport, 2003). Apse et al. (1999) overexpressed the A. thaliana AtNHXI gene
(coding for a vacuolar Na+/H+ antiporter) in Arabidopsis and showed that transgenic
plants were able to tolerate up to 200 mM NaCl treatment (Table l-lA). Tomato plants
overexpressing the AtNHXI gene accumulated 20-28 fold more sodium in their
vegetative tissues compared to wild type plants (Zhang and Blumwald, 2001) and
AtNHXI-overexpressing canola plants accumulated up to 6% of their dry weight as
sodium compared to almost 0.01% in non-transgenic plants (Zhang et al., 2001).

Salt exclusion can be facilitated by the use of the SOS (Salt Overly Sensitive) genes,
which encode a plasma membrane Na+/11+ antiporter(SOS1), a serine/threonine protein
kinase ($052) and a myristoylated Ca2+-binding protein (5053). Identification of those
genes has also furthered our understanding of Ca2+ signaling in plant response pathways
to salinity. Sudden change in Na+ ion concentration in the cytosol is immediately
coupled with a transient change in the Ca2+ concentration in the cytosol; this transient
change in cytosolic Ca2+ is known as the Ca signature (Knight et al., 1997). The calcium
ions then bind to the myristoylated Ca2+-binding protein, encoded by SOS3, which then
mediates downstream responses (Liu and Zhu, 1998; Ishitani et al., 2000; Zhu, 2002).
S053 interacts with and activates the $052, which is a serine/threonine protein kinase

(Halfter et al., 2000; Liu et al., 2000; Zhu, 2002). Both SOS3/SOSZ proteins are

15

responsible for regulating the plasma membrane Na+/H" antiporter, which is encoded by
the SOS] gene in Arabidopsis (Shi et al., 2000).

Overexpression of the plasma membrane Na+/IV antiporter gene SOS] in a mutated
strain of yeast lacking the Na+/H+ antiporters had a slight but significant increase on yeast
survival on medium supplemented with 100 mM NaCl. Co-expression of SOS3/SOS2 as
well as SOS] genes in the same mutated strain of yeast had a dramatic effect on yeast
survival on medium supplemented with NaCl (Zhu, 2002). Moreover, overexpression of
SOS] gene in Arabidopsis significantly improved plant salt tolerance (Shi et al., 2003).
Shi et al., (2003) reported that transgenic plants overexpressing SOS] gene accumulated
less Na+ in their tissues than their non-transgenic counterparts (Table l-lA).

Recently, there have reports indicating that the 8082 protein kinase may have
multiple effects on other genes (Cheng etal., 2004; Qiu et al., 2004). Qiu et al., (2004)
demonstrated that the tonoplast Na+/H+ transporter in Arabidopsis is also one of the
targets of the SOS regulatory pathway, which means that there might be more branches to
the SOS pathway. Interestingly, Cheng et a]. (2004) showed that 8082 protein kinase
also regulates the vacuolar I-I‘L/Ca2+ antiporter CAXl. Co-expression of SOS2 specifically
activated CAX] gene in yeast. Using the yeast two-hybrid assay, $082 was found to

interact with the N terminus of CAXI.

II-B—2 Genes that control osmolyte and compatible solute content in plants
An important adaptation to osmotic stress is the ability to accumulate compatible
solutes (e. g., proline, glycine-betaine, alcohol sugars, fructans and trehalose) in the

cytoplasm under dehydration-stress conditions (Bohnert and Jansen, 1996). Compatible

16

solutes, which are sometimes called osmoprotectants, are non-toxic organic metabolites
of low molecular weight that act to raise the osmotic potential of the cell, or to stabilize
membranes or macromolecular structure (Bohnert and Jensen, 1996). Engineering
strategies for developing salt stress tolerance has been performed with genes encoding
production of proline, glycine betaine, mannitol, fructans, and trehalose (Holmberg and
Bulow, 1998; Taiz and Zeiger, 1998).

Several groups have demonstrated that proline accumulating in response to water or
salt stress can act as an osmoprotectant in plant cells under salt stress (Kishor et al., 1995;
Taiz and Zeiger, 1998; Zhu et al., 1998; Hong et al., 2000; Ain-Lhout et al., 2001). Mali
and Mehta (1977) were among the first to report on the rapid accumulation of proline in
rice varieties upon exposure to drought stress. Water stress-tolerant rice plants showed a
5.4 fold increase in free proline compared to 1.2 fold increases in sensitive varieties.
Increased proline accumulation in response to dehydration stresses has been observed in
numerous species, and has been reviewed extensively in the literature (Cherry, 1989).
One of the first attempts to engineer plants to overproduce proline came from Kishor et
al. (1995), which who overexpressed in tobacco the moth bean Al-pyrroline-S-
carboxylate synthetase (PSCS), a bifunctional enzyme that catalyzes the conversion of
glutamate to proline. The transgenic plants accumulated 10 to 18 fold more proline than
the non-transgenics. Proline accumulation was accompanied by an increase in salinity
tolerance measured as increased germination percentage and seedling fresh weight when
grown in 200 mM NaCl (Table l-lB). Zhu et al. (1998) also reported similar results in
rice (Oryza sativa L) when overexpressing the same enzyme under a stress inducible

promoter. Moreover, Ronde et al. (2000) overexpressed the Al-pyrroline-S-carboxylate

l7

reductase, (in antisense orientation) in soybean plants. Antisense-soybeans failed to
survive a 6-day drought stress at 37 C° in contrast to wild type plants that survived the
treatment. Similarly, removal of the feedback inhibition of PSCS enzyme resulted in an
increased accumulation of proline in plant tissue that correlated with an increased
tolerance to osmotic stresses (Hong et al., 2000).

Glycinebetaine is a common compatible solute in many different organisms including
certain plant species (Sakarnoto and Murata, 2000). Betaine in vitro acts as an
osmoprotectant by stabilizing the structure of proteins and the highly ordered structure of
membranes against the adverse effects of water deficit conditions such as high salinity
and extreme temperature (Gorham, 1996). Many plant species grown in saline and arid
areas accumulate glycinebetaine in response to drought and salinity (Grumet and Hanson,
1986; Saneoka et al., 1995; Sakarnoto and Murata, 2000). Hayashi et al. (1997) achieved
enhanced salt-tolerance in lirabidopsis by overexpressing the soil bacterium Arthrobacten
globiformis codA gene (choline oxidase, a key enzyme in glycine-betaine production).
Overexpression of the E. coli betA gene (which encodes choline dehydrogenase) in
tobacco confers salt-tolerance (Holmstrom et al., 2000). Growth of control plants was
totally inhibited by watering with 200 mM NaCl solution, whereas transgenics were not
affected (Table l—lB). J ia et al., (2002) transformed tomato plants with the Atriplex
hortensis-BADH gene, which encodes betaine aldehyde dehydrogenase, to convert
betaine aldehyde into glycine-betaine. Transgenic tomato plants grew normally under
120mM NaCl and exhibited enhanced root development compared to non-transgenic
plants (Table 1B). More recently, cabbage plants overexpressing the bacterial betA gene,

exhibited higher tolerance to NaCl stress compared to nontransformed plants

18

(Bhattacharya etal., 2004). Transgenic cabbage plants showed better growth response
and greater stability in maintaining plant water relations at high levels of salinity.

Sugar alcohols also work as osmolytes in plant cells exposed to salt stress (Cherry,
1989; T arczynski et al., 1993; Bohnert and Jensen, 1996; Bohnert et al., 1999) and can
also serve as scavengers for reactive oxygen species (Halliwell et al., 1988). Tarczynski
et al. (1993) first reported stress protection of transgenic tobacco plants by
overexpressing the osmolyte sugar alcohol mannitol (Table 1-1 B). Since then several
reports have been published indicating that overexpression of genes involved in
production of sugar alcohols in plants can confer stress tolerance (Karakas et al., 1997;
Liu et al., 1999; Zhifang and Loescher, 2003). Karakas et al. (1997) transformed tobacco
plants with a gene encoding mannitol-l-phosphate dehydrogenase (MtlD). Transgenic
plants were not affected under salt stressed conditions that caused a dry weight reduction
of 44% in the nontransgenic controls. More recently, Tilahun et a1. (2003) showed that
overexpression of the bacterial gene mtlD in wheat resulted in plants that were more
tolerant to high salt stress. When subjected to 150 mM NaCl, transgenic T2 wheat plants
showed 50% reduction in fresh weight and 30% reduction in dry weight compared to
77% and 73% reduction in fresh and dry weight respectively in non-transgenic wheat
(Table 1B). In contrast to using the bacterial gene mtlD, Everard et a]. (1997) isolated a
gene encoding mannose-6-phosphate reductase (M6PR), key enzyme for mannitol
production from celery. Zhifang and Loescher (2003) introduced the M6PR into
Arabidopsis plants and showed that transgenic Arabidopsis plants were able to grow
normally and complete normal development and seed production in the presence of 300

mM NaCl. Similarly, expression of the E. coli GutD gene (encoding for gluctiol-6-

19

phosphate dehydrogenase) a key enzyme for biosynthesis of the sugar alcohol sorbitol,
caused increased accumulation of sorbitol in transgenic maize, and enhanced salt-
tolerance compared to non-transgenics (Liu et al., 1999).

Fructans are soluble polymers of fructose that are produced by approximately 15% of
flowering plant species. Accumulation of fi'uctans in the cell vacuole helps maintain
water potential gradients of cells by raising the osmotic potential of the cell (Pilon-Smits
et al., 1995). Pilon-Smits et a1. (1999) overexpressed a gene from Bacillus subtilis (SacB
gene) to produce bacterial fi'uctans in sugar beet. The growth of transgenics was
significantly enhanced under drought conditions compared to the nontransgenics, as
measured by higher dry leaf and root weights.

The disaccharide trehalose (a-D-glucopyranosyl-l , or -D-glucopyranoside) is present
in a large variety of microorganisms and plants where it can serve as a reserve
carbohydrate and as an osmoprotectant (Vogel et al., 2001). The occurrence of trehalose
has also been documented in several desiccation-tolerant plants (Muller et al., 1995). It
protects membranes and proteins in cells exposed to salt-stress induced dehydration
(Penna, 2003). Pilon—Smits et al. (1998) showed that transgenic tobacco plants
overexpressing the Escherichia coli OtsA and OtsB genes (encoding trehalose-6-
phosphate-synthase and trehalose-6-phosphatase respectively) responded better to
dehydration stresses compared to non—transgenics. Under drought conditions the
transgenics yielded 30-39% more dry weight compared to non-transgenics. They also
reported that detached leaves from transgenic tobacco plants had a higher capacity to

retain water than the wild type. Using the OtsA and OtsB genes under salt inducible

20

promoter, Garg et al. (2002) reported successful production of transgenic rice plants with

an elevated tolerance to 100 mM NaCl (Table l-lB).

II-B-3 Genes that encode stress induced proteins such as the LEA (late

embryogenesis abundant) and COR (cold regulated) proteins

Another important group of genes that play a role in plant adaptation and resistance to
dehydration-stress induced conditions are known as LEA (late embryo genesis abundant)
and COR (cold regulated) genes. LEA proteins were first identified and characterized in
cotton as a set of proteins that accumulate in embryos at the late stage of seed
development when seeds are undergoing the dehydration process necessary for long term
survival in a dormant state (Dure et al., 1981). Transcription of genes encoding LEA
proteins is also activated in other tissues such as leaves subjected to osmotic stresses
(Zhang et al., 2000). LEA proteins are divided into 5 major groups according to sequence
homology (Swire-Clack and Marcotte, 1999). Group 1 proteins that might play a role in
binding or replacement of water, group 2 and 4 proteins that may play a role in
maintaining protein and membrane structure under severe dehydration, and finally group
3 and 5 that are thought to have a role in ion sequestration in plant cells (Swire-Clack and
Marcotte, 1999). The class 2 LEA proteins (also known as the lea D11 family) are
dehydrins that accumulate in plant cells in response to dehydration stresses and low
temperatures. The dehydrins, which range from 82 to 575 amino acids in length, share
several conserved domains (Close, 1997). The first, named the K domain, is an a-helix
domain composed of 15 amino acids (EKKCIMDKIKEKLPG) which exists in single or

multiple COpies. The second domain is the S-segment, consisting of a stretch of 6-10 Ser

21

residues. The third is the Y-segment (T/VDEYGNP), located at the N-terminus (Close,
1996).

It has been hypothesized that LEA proteins play a role in desiccation tolerance during
seed development and in response to dehydration and salinity stress (Hoekstra et al.,
2001 and Close, 1997). This role is probably achieved through maintenance of protein or
membrane structure, sequestration of ions, binding of water, and function as molecular
chaperones (Bray, 1997). Two classes of LEA proteins have been shown to have a direct
functional role in salt and dehydration tolerance in plants. Rice plants transformed with
the HV A1 gene from barley (a group 3 LEA protein) showed an increased tolerance to
dehydration and salinity (Table 1-1C), compared to the non-transgenics (Xu et al., 1996).
Improved salinity tolerance was also reported in yeast cells expressing the tomato LEA
protein LE25, a group4 LEA protein (Irnai et al., 1996). Another group of LEA proteins
that has a role in water binding or replacement is the LEAI group; yeast cells
overexpressing the LEA] protein, Em, had enhanced growth when subjected to medium
with high osmolarity (Swire-Clark and Marcotte, 1999).

Plants exposed to low non-freezing temperature undergo a phenomenon known as
cold acclimation, a process that is necessary for many plant species to survive freezing
temperatures. Thomashow and co-workers identified a group of genes, designated as
COld Responsive (COR), that are induced upon cold acclimation (Gilmour et al., 1992;
Lin and Thomashow, 1992; Hovarth et al., 1993). The COR genes were also identified by
other groups and are also known as LT 1 (low temperature-induced), KIN (cold-
inducible), RD (responsive to desiccation) and ERD (early dehydration-inducible). COR

genes comprise four families, each of which is composed of two genes that are physically

22

linked in the genome in tandem array (Thomashow, 1999). COR15, 78, 6.6 and 47
encode hydrophilic polypeptides. The COR47 hydrophilic polypeptide belongs to group 2
LEA proteins (Thomashow, 1999).

COR genes may help plant cells to tolerate potentially damaging effects of
dehydration associated with freezing-induced drought injury (Steponkus et al., 1998). As
temperatures drop below 0 °C, ice formation initiates in the intercellular spaces of plant
tissues, resulting in a drop of water potential outside the cell. The water potential gradient
will facilitate the movement of unfrozen water. At —1 0 °C, more than 90% of the
osmotically active water moves out of the cell causing dehydration injuries (Thomashow,
1999). Over expression of the COR15a gene in Arabidopsis thaliana protoplasts resulted
in a small, but significant increase in protoplast survival upon freezing over the range of —
4.5 to —7.0 °C (Artus et al., 1996). COR15a, which encodes a chloroplast-targeted
polypeptide, enhances the freezing tolerance of chloroplasts by protecting membranes

from freeze-induced dehydration.

II-B-4 Genes involved in transcriptional regulation of stress response
mechanism in plants

II-B-4a The CBF system

In general, plant responses to abiotic stresses and water deficits are multigenic, where
cascades of biochemical and cellular changes are necessary for plants to adapt to
environmental changes (Bohnert and Jensen, 1996). Thus induction of cascades of
responses may be more effective in increasing stress tolerance than single gene changes.

In some cases, individual changes had only a modest effect on stress tolerance. For

23

 

 

 

example, introduction of glycinebetaine into Arabidopsis, Brasica napus and tobacco
(Huang et al., 2000) caused only a small increase in stress tolerance. Although transgenic
plants accumulated 8-18 fold more glycinebetaine than the non-transgenic controls, there
was only a moderate improvement in salt tolerance in tobacco plants as measured by
fresh weight of shoots. In addition, only B. napus showed a slightly better photosynthetic
rate in response to salinity (Huang et al., 2000). Overexpression of the cold induced
CORI5a gene in Arabidopsis thaliana did not improve the freezing tolerance at the
whole plant level (Artus et al., 1996).

An alternative approach to the induction of a single gene is to induce an array of
adaptive plant responses through the use of key transcription factors (Thomashow, 1999).
The COR genes are characterized by the presence of a common cis-acting element within
their promoter region that confers stress-induction. Baker et al. (1994) reported that the
5’ region of the Arabidopsis thaliana c0r15a gene includes a cis-acting element that
confers cold-, drought-and ABA-regulated gene expression. Yarnaguchi-Shinozaki and
Shinozaki (1994) identified a 9 bp cis-acting element (TACCGACAT) at the promoter
region of the COR 78/RD29A gene (COR 78 and RDZ9A are alternative designations of the
same gene); and named it drought responsive element (DRE). The DRE stimulated gene
expression in response to low temperature, dehydration, and high salinity in Arabidopsis
(Y amaguchi-Shinozaki and Shinozaki, 1994). Stockinger et al. (1997) identified a 5 bp
DNA regulatory element (CCGAC) at the promoter of the COR gene family and
designated it as the C-repeat (CRT). The CRT element, which also occurs in the DRE
element, was found to be essential for COR gene transcription in response to low

temperature (Stockinger et al., 1997).

24

 

Using the yeast one-hybrid system, Stockinger et al. (1997) isolated an A. thaliana
cDNA clone encoding the transcription factor CBF] [CRT (C-repeat)/DRE (Drought
Response Element) Binding Factor]. CBF1 is a 24 kDa protein, with a nuclear
localization domain and an activation domain; it also has an AP2 domain that has a
DNA-binding site. Transcripts of the CBF gene family increase dramatically within 15
minutes after transferring Arabidopsis plants to low non-freezing temperature. This
increase is followed by an increase in COR gene transcripts (Gilmour et al., 1998),
indicating that CBF gene expression is an early step in the COR gene transcriptional
cascade. Similarly, the Shinozaki group cloned two cDNAs encoding for DREB [DRE
(Drought Response Element) Binding] proteins DREB] a and DREBZb and showed that
expression of DREB] a was activated by low temperature, while DREBZb transcript was
activated by dehydration (Liu et al., 1998). CBF], CBFZ and CBF 3 are alternative

designation of DREB] b, DREB] c and DREB] a.

II-B-4b Regulation of CBF

Gilmour et al. (1998) proposed that due to the rapid induction of CBF genes upon
cold treatment (about 15 minutes) and the lack of a CRT/DRE element in the promoter of
CBF/DREB] genes, another protein that regulates expression must be present in warm
conditions. This protein would bind to the CBF promoter and induce CBF expression
upon cold treatment. This hypothetical protein was designated as Inducer of CBF
Expression, ICE (Gilmour et al., 1998; Thomashow, 2001; Thomashow et al., 2001).

Upon exposure of plants to low non-freezing temperatures, a modification in ICE or in

25

 

another associated protein would occur, this would allow ICE to bind to CBF promoters
and upregulate CBF expression.

A breakthrough in understanding cold and freezing tolerance mechanisms was
achieved when Chinnusamy et al. (2003) isolated and identified ICE], an upstream
transcription activator that positively upregulates transcription of the CBF gene family.
ICE] gene encodes a 53.5 kD nuclear localized MY C-likc basic-Helix-Loop-Helix
(bHLH domain) transcription factor. ICEl has an acidic domain near the N-terminus with
a typical bHLH DNA binding domain and a dirnerization domain near the C-terminus.
ICEI binds to a cis-element (CANNTG) about 1 kb upstream of the CBF 3 promoter.
Zarka et al., (2003) found that there are at least two regulatory elements in the promoter
region of CBF2, Inducer of CBF Expression region 1 and 2 (ICErl and ICEr2
respectively), including the core sequence (CANNTG). Results from Chinnusamy et al.
(2003) indicate that there are 5 MYC recognition sites at the promoter of CBF 3 .
Knockout and overexpression experiments revealed the role of the ICE] gene in chilling
and freezing tolerance in Arabidopsis plants. Mutated Arabidopsis plants (iceI) were
impaired in their response to chilling and freezing temperatures, while ICE] -
overexpressing plants were significantly more chilling and freezing tolerant than wild
type (Chinnusamy et al., 2003).

Interestingly, although I CE 1 -overexpressing plants were chilling and freezing
tolerant, they did not show an elevation in CBF 3 gene expression level in warm
temperature; an increase in CBF 3 expression was only observed under cold temperature
(Chinnusamy et al., 2003). In ice] -mutants, the expression levels of CBF] and CBF 2

were similar to wild type plants, although there was some delay in expression at l and 3

26

 

hours of cold treatment (Chinnusamy et al., 2003). Results from Chinnusamy et al.
(2003) indicate that expression of members the CBF/DREB] gene family might be
regulated differently due to the observation that members of the CBF/DREB] family are
differentially induced under different conditions (Haake et al., 2002) and that members of
CBF/DREB] family are differentially expressed under the same stress condition (N ovillo
et al., 2004). For example, screening of the Arabidopsis genome revealed the presence of
another CBF homolog, CBF 4, which has a 63% amino acid similarity to the CBF gene
farme (Haake et al., 2002). The expression of CBF4/DREBId is not induced under low
temperatures, but is rapidly induced in response to drought and ABA treatment.
Recently, Novillo et al. (2004) investigated the contribution of each CBF/DREB]
member in cold adaptation. Using knockout mutants of CBF2, Novillo et al. (2004) found
the surprising results that cbf2 plants had an elevated level of CBF] and CBF 3 transcript
even under warm temperature. This resulted in induction of target genes and increased
freezing, dehydration, and salinity tolerance (N ovillo etal., 2004). Northern blot analysis
revealed a delay in expression between the different members of the CBF gene family,
with CBF] and CBF 3 expression preceding the expression of CBF 2. These results are
consistent with the fact that the transcript level of CBF 2 is almost 5 times higher than the
transcript levels of CBF] and CBF 3 under warm condition (Fowler and Thomashow,
2002). Novillo et al., (2004) concluded that CBF2 might act as a negative regulator of
both CBF] and CBF 3 . Under warm condition, the steady state of CBF2 transcript would
negatively regulate the expression of CBF] and CBF 3, to make sure that their expression

is tightly controlled. Upon cold exposure, ICE and other proteins would be activated to

27

rapidly increase the expression of CBF] and CBF 3, resulting in increased environmental-

induced dehydration tolerance (Novillo et al., 2004).

II-B-4c Effect of overexpression of CBF/DREB on the plant transcriptome

Examination of gene expression changes allows for comparison between the effects
of cold treatment and CBF overexpression. Transcriptome-profiling experiments in
Arabidopsis, using Affymetrix-Gene—Chip (8297 genes) revealed changes in transcript
level of 306 genes (about 4% of the total genes tested) in response to cold (Fowler and
Thomashow, 2002). Of these 306 genes, 218 genes showed a 3-fold or greater transcript
increase and 88 genes showed a two-fold transcript decrease in Arabidopsis plants treated
at 4 C° (Fowler and Thomashow, 2002). Of the upregulated genes, more than 70%
increased transiently, with expression reaching the highest level during the first 24 hours
of cold treatment, followed by a dramatic decline in transcript level (Fowler and
Thomashow, 2002). Transiently-upregulated genes were classified into several groups,
transcription factors (CBF1 and 3, other AP2 domain containing proteins, zinc finger
proteins, MYB proteins, MADS box containing proteins, and other ethylene responsive
element binding factors); cell metabolic regulation (e.g., carbohydrate and osmolyte
biosynthesis along with other genes involved in starch catabolism); transporters (water
channels, ion and sugar transporters); cellular communication (protein kinases and
proteases); cellular defense and detoxification mechanisms (LEA proteins and other
enzymes involved in radicals detoxification); and finally a class of proteins with
unknown function (45 genes). The remaining genes were categorized as long-term

upregulated genes. Long-term upregulated genes continue to accumulate several fold

28

(compared to plants growing in warm temperature) even when tested after 7 days (Fowler
and Thomashow, 2002). This group of genes also included transcription factors, genes
involved in metabolic pathways, transporters, cell signaling, cell maintenance and
detoxification as well as 20 genes of unknown function. Genes that were down regulated
by cold included genes involved in photosynthesis and metabolism, signal transduction,
heat shock and transcriptional regulation (Fowler and Thomashow, 2002).

Seki et al. (2002) also reported that more than 40% of transcripts induced under cold
temperature (53 transcript were induced under cold treatment in total) are also induced
under both drought and high salinity, among those are different members of the COR
genes (COR15a, COR4 7 and COR 78). Sequencing of the promoter region of the 22 genes
that are induced under drought, cold and high salinity conditions indicated the presence
of the CRT/DRE element in 16 genes and 8 of which also had the ABRE element in their
promoter region (Seki et al., 2002).

Comparison of transcriptome profiling between warm-grown Arabidopsis plants
constitutively expressing CBF], CBF 2 or CBF 3 and control nontransgenic plants
revealed that not all genes upregulated under cold temperature are also upregulated when
overexpressing CBF. Thus, the CBF regulon represents only a part of the genes that are
cold upregulated (Fowler and Thomashow, 2002). It was found that 60 genes (more than
80% of those genes are transiently upregulated) upregulated under cold were not
upregulated in plants overexpressing any of the CBF genes; those were suggested to be
CBF-independent. Of the 41 genes upregulated in CBF overexpressing plants, 30 were
also upregulated by cold, which makes them the members of the CBF regulon (Fowler

and Thomashow, 2002). Genes that were upregulated include genes were known to have

29

CDT /DRE elements, such as COR6. 6, COR15b, COR4 7, COR 78/RD29. Interestingly,
genes that may be involved in osmolyte accumulation such as galactinol synthase for
galactinol and raffinose production and P5 CS for proline production were also increased
in response to CBF expression (J aglo-Ottosen et al., 1998; Kasuga et al., 1999; Gilmour
et al., 2000; Seki et al., 2001; Seki et al., 2002).

It is known that many of the genes that are induced by the CBF genes in response to
dehydration induced stress conditions are also induced by the application of abscisic acid
ABA (Y amaguchi-Shinozaki and Shinozaki, 1994; Ishitani et al., 1997; Liu et al., 1998;
Thomashow, 1999; Shinozaki and Yamaguchi-Shinozaki, 2000). Thomashow (1999)
suggested that ABA concentration increases transiently in response to cold non-freezing
temperature. This non-accumulative response helped establish the argument that ABA
doesn’t play a role in cold acclimation, and that cold acclimation occurs via two separate,
ABA-dependent and ABA-independent pathways. The ABA-independent pathway
includes the CBF/DREB] genes (Liu et al., 1998; Thomashow, 1999; Shinozaki and
Yamaguchi-Shinozaki, 2000; Shinozaki et al., 2003). More recently, Knight et al. (2004)
showed that the activation of the CBF pathway could also occur via the ABA pathway;
100 uM ABA treatment for 1 hour was sufficient to increase the transcript and the protein
level of the CBF], CBF 2 and CBF 3 genes. Knight et al. (2004) suggested that the CBF1-
3 genes, which are activated by cold and ABA treatment, might give them a distinctive
role in plant adaptation under both fieezing and drought conditions, in contrast to CBF 4
which is strictly activated under drought and ABA treatment (Haake et al., 2002). This
possibly adds another layer of complexity to signaling in plants under abiotic stress

conditions.

30

II-B-4d Effect of CBF/DREB-overexpression on plant metabolome

The CBF/DREB gene families are transcriptional activators that directly or indirectly
work as master switches in regulating transcript in plants in response to dehydration-
inducing conditions (Thomashow, 2001). Interests in exploring large scale changes in
plant metabolome, as a direct result of CBF/DREB expression, has been recently
investigated in Arabidopsis (Cook et al., 2004).

Cook‘et al. (2004) included three ecotypes of Arabidopsis thaliana, differing in
tolerance to freezing temperatures [Cape Verde Islands-1 (Cv-l), Wassilewskija-Z (W s-
1), and Columbia (CM]; the first is less freezing tolerant than the latter two. Using a GC—
time—of-flight MS method to assess large scale changes in metabolic profile, at least 325
low molecular weight compounds (carbohydrates, amines, organic acids, and other polar
molecules) increased 2-fold or more in Ws-plants in response to 14 days of cold
treatment. Of those 325 compounds, 114 increased at least 5-fold higher compared to the
control non-cold acclimated plants. On the other hand, in the Cv-l ecotype, only 269
compounds increased in response to cold acclimation, of which 244 were common with
the Ws-l ecotype. The finding that only 53 out of the 269 compounds had at least 5-fold
increase in Cv-l cold acclimated plants compared to 114 in the freezing tolerant Ws-l
ecotype may explain why these ecotypes differ in their ability to withstand freezing
temperatures.

Cook et al. (2004) went on to compare the metabolome profile of CBF 3/DREBI a
overexpressing Arabidopsis plants and cold acclimated-wild-type Col plants. These
experiments revealed that of the 325 metabolites that increased in response to low

temperature, 256 also significantly increased in CBF 3/DREBI a overexpressing plants. Of

31

those compounds, 102 increased at least 5-fold in the CBF3/DREBla overexpressing
plants. These results clearly demonstrate the similarity between the metabolome of
CBF3-expressing plants and the metabolome of cold-acclirnated wild-type plants. Cook
et al. (2004) suggested that the dramatic increase in proline and low-molecular-weight

soluble carbohydrates is a signature of the CBF regulon.

II-B-4e Conservation of the CBF/DREB system among plant species

CBF1/DREB] b homologs have been identified in Arabidopsis thaliana (Stockinger et
al., 1997; Haake et al., 2002), canola (J aglo et al. 2001), barley (Choi et al., 2002),
tomato (J aglo et al. 2001), rice (Choi et al., 2002; Dubouzet et al., 2003), strawberry and
scur cherry (Owens et al., 2002), suggesting that the CBF/DREB system is highly
conserved throughout the plant kingdom, including both dicots and monocots.

J aglo et al. (2001) reported the presence of other CBF/DREB] homologs in
Arabidopsis (DREBIe and DREBIf), and other plants. Canola (Brassica napus), which
like Arabidopsis a member of the Brassica family, encodes two different CBF -1ike
proteins that share approximately 76% homology to the Arabidopsis CBF] gene;
similarly, BnCBF] and BnCBF 2, accumulated in canola plants within 30 minutes after
transferring plants into cold nonfreezing temperature (J aglo et al., 2001). The expression
of the BnCBF] and BnCBF 2 was followed by accumulation of the B. napus-CORI5a
ortholog, B22115.

J aglo et al. (2001) also reported the presence of CBF homologs in the more distantly
related, chilling sensitive species tomato (Lycopersicon esculentum L). Similar to the

Arabidopsis CBF], CBF 2 and CBF 3 genes, Zhang et al. (2004) found that LeCBF],

32

LeCBF2 and LeCBF 3, are organized in tandem and all lack introns. Interestingly,
LeCBFI was induced only in response to cold non-fieezing conditions after about 30
minutes, reaching maximum induction after 2 hours, while LeCBF 2 and LeCBF 3 were
not induced under cold. LeCBF] , LeCBF 2 and LeCBF 3 responded very weakly to
mechanical agitation, drought, salinity and ABA treatment (Zhang et al., 2004). Owens et
al. (2002), reported that CBF1-orthologs also exist in sour cherry (Prunus cerasus L.) and
strawberry (F ragaria X ananassa Duchesne). The putative orthologs of sour cherry
PcCBF] and strawberry F aCBF ] shared about 48% similarity at the amino acid level to
the Arabidopsis CBF1 protein (Owens et al., 2002).

Distantly related plants such the monocots rye (Secale cereale L.), wheat (T riticum
aestivum L.), barley (Hordeum vulgare L.) and rice (Oryza sativa) also have CBF-like
homologues (J aglo et al., 2001; Choi et al., 2002). The rye and wheat polypeptides had
30% and 34% sequence homology, respectively, to the Arabidopsis CBF1 polypeptide.
Choi et al. (2002) identified a sequence, OsCBF 3, from the rice genome database similar
to the CBF 3 gene fi'om Arabidopsis. This in turn was used to screen a barley BAC
library, leading to identification of a barley CBF 3 ortholog. Expression of the barley CBF

gene, HvCBF 3, was found to be cold induced (Choi et al., 2002).

II-B-4f Engineering dehydration-stress tolerance in plants using the
CBF/DREB system

Jaglo-Ottosen et al. (1998) achieved a breakthrough by demonstrating that
overexpression of a transcription factor in plants would result in activation a cascade of

genes directly/indirectly involved in abiotic stresses tolerance. Transgenic, non-

33

acclimated Arabidopsis plants overexpressing the CBF] gene were more freezing tolerant
than their non-acclimated control counterpart as determined by electrolyte leakage (a test
used to determine membrane damage) and whole plant fieezing test assays. ELso values
(the fieezing temperature that results in release of 50% of tissue electrolytes) indicated
that CBF] overexpressing Arabidopsis plants were significantly more tolerant to freezing
than wild type (3.3 °C difference). Similarly, Kasuga et al. (1999) overexpressed
DREBIA in Arabidopsis and showed that DREBIA enhanced tolerance to drought, salt
and fieezing in transgenic plants: 69.2% of the transgenics survived a 14 day dehydration
treatment, whereas no wild type plants survived (Table 1-1 D). When exposed to —6 C°
for 2 days, followed by 5 days exposure to 22 C°, less than 10% of the nonacclimated
control plants survived while more than 75% of the transgenics survived. Moreover,
78.6% of the transgenics were able to survive a treatment of dipping in 600 mM NaCl
solution for 2 h, before transplanting into pots, while only 17.9% of the wild-type plants
survived this treatment (Kasuga et al., 1999).

Arabidopsis plants overexpressing CBF 4 gene also were tolerant to freezing and
drought stresses (Haake et al., 2002). Freezing tests of nonacclimated plants revealed that
while only 1% of wild type plants survived freezing at —10°C for 20 hours, the range of
survival was 52-100% in the transgenic plants, depending on the expression level of
CBF 4. The same trends were achieved when plants were subjected to 9 days of
dehydration; only 2% of the wild type plants survived compared to 87% of transgenic
plants (Table 1-1D).

Transgenic Arabidopsis plants overexpressing CBF] (J aglo-Ottosen et al., 1998)

exhibited numerous physiological changes associated with CBF expression and increased

34

 

dehydration stress resistance including increased membrane stability, increased
compatible solute accumulation and the ability to scavenge for reactive oxygen species
(J aglo-Ottosen et al., 1998; Kasuga et al., 1999; Gihnour et al., 2000; Hsieh et al. 2002a).
Non-acclirnated transgenic Arabidopsis plants had lower EL50 values in response to
freezing temperatures than their nontransgenic counterparts indicating that CBF/DREB-
overexpressing plants suffered less membrane damage.

CBF/DREB-overexpressing plants also accumulated higher levels of osmolytes e. g.,
(proline and soluble sugars) compared to the nontransgenic controls (J aglo-Ottosen et al.,
1998; Kasuga et al., 1999; Gilmour et al., 2000; Hsieh et al. 2002b). CBF3-
overexpressing Arabidopsis lines had approximately 3-fold higher level of total soluble
sugars and 5-fold higher proline levels compared to the nontransgenics (Gihnour et al.,
2000). This increase in proline was accompanied with an increase in the transcript level
of A-pyrroline-S-carboxylate synthetase (P5 CS), a key enzyme in proline synthesis.
Haake et al. (2002) also reported similar results when overexpressing the CBF 4 gene in
Arabidopsis, a gene that is only activated under drought and ABA treatment.

In canola, non-cold acclimated plants overexpressing the Arabidopsis-CBF1, CBF2
or CBF 3 genes had EL50 value of —6 C° while their nontransgenic counterparts had a
value of -3 C° (J aglo et al., 2001). The EL50 values were —l2.7 C° and -8.1 C°, for the
transgenic and the nontransgenic controls, when plants were cold acclimated before
performing the electro-leakage test (J aglo et al., 2001). Similarly, constitutive expression
of the Arabidopsis-CBF genes in canola plants resulted in activation oan115 and B2128
(orthologs to the Arabidopsis COR] 5a and COR6.6 genes) without cold acclimation.

Thus, canola appears to have a cold-response pathway that is very close to Arabidopsis

35

Arabidopsis CBF genes also can confer increased dehydration stress resistance to
other less closely related species (Hseih et at., 2002a; Hseih et al., 2002b; Owens et al.,
2002; Kasuga et al., 2004). Kasuga et al. (2004) used the stress-inducible rd29A
promoter to drive the expression of DREBIA/CBF 3 gene in tobacco plants. Tobacco
plants expressing DREBIA/CBF 3 under the dehydration inducible promoter rd29A were
more drought tolerant compared to wild type plants and had higher photosynthetic
activity under drought and cold-nonfieezing temperature. Similarly, tomato plants
overexpressing the Arabidopsis CBF] gene were more dehydration stress tolerant than
wild type plants; 83.3% of the transgenic plants survived a 4 week drought treatment
while less than 6% of the nontransgenics survived this treatment (Hseih et al., 2002b).
The transgenic plants had 3-4 fold more proline under nonstressed conditions compared
to the nontransgenic controls. Proline levels increased under stress conditions in both
transgenic and nontransgenic plants but were 30-60% higher in plants overexpressing the
Arabidopsis-CBF] gene (Hseih et al., 2002b). Interestingly, whereas overexpression
experiments of LeCBF] in Arabidopsis resulted in accumulation of COR gene transcripts
and increased the EL50 values by —2.5 °C in the overexpressing lines, no differences in
the electro-leakage were observed in transgenic tomato lines overexpressing AtCBF] ,
AtCBF 3 or LeCBF] (Zhang et al., 2004).

Owens et al. (2002), overexpressed the A. thaliana-CBF] gene in strawberry and
tested the plants for their ability to withstand freezing temperature compared to
nontransgenic plants. The EL50 values indicated that CBF] expressing strawberry plants

were significantly more tolerant to freezing temperatures than wild type (-10.3 °C in the

transgenic compared to -6.4 °C in wild type).

36

III- The objectives of this dissertation

The above examples clearly indicate that various types of genes can be used to
engineer dehydration stress tolerance in plants. The objective of this work was to
investigate the possibility to engineer enhanced dehydration stress tolerance in cucumber
plants following two approaches. Cucumber plants are known to be sensitive to salinity
and drought (Mass and Hoffinan, 1977). Salinity and drought has been reported to have
strong negative effects on cucumber plants, especially on seed germination and seedling
emergence, leaf expansion rate, photosynthesis, fruit set, as well as fruit growth rate and
fruit quality (Chartzoulakis, 1994; Navazio and Staub, 1994; Ho and Adams, 1994;
Tazuki, 1997; Serce et al., 1999; Drozdova et al., 2004). At the time I started this work,
there was no published data on the evaluation of genetically engineered plants for
enhanced salt or drought tolerance under field conditions. To our knowledge, there are
only two published reports, both of which came in late 2004 that evaluated genetically
engineered plants for enhanced dehydration stress tolerance under field conditions (Table
1-1). Quan et al. (2004) reported enhanced grain yield production by transgenic maize
plants expressing a gene for betaine aldehyde dehydrogenase following drought stress
period of 21 days, and Xue et al. (2004) tested wheat plants expressing the Arabidopsis
tonoplast H‘L/Na+ antiporter gene for their ability to grow in saline soil and reported
higher grain production in the transgenic plants compared to the nontransgenic controls.
The aim of this thesis is to:
l-Investi gate and test the possibility to improve dehydration-stress tolerance in cucumber

(Cucumis sativus L.), plants by following two strategies

37

(a) Induction of mannitol production in cucumber plants by using the celery M6PR
gene, or

(b) Induction of an array of adaptive responsive mechanisms that are associated with
dehydration-stress tolerance in plants by expressing the A. thaliana-transcriptional
regulators CBF/DREB] genes in cucumber plants.
2- Evaluate and validate the performance and mu yield of transgenic cucumber plants

for their ability to withstand dehydration stress under greenhouse and field conditions.

38

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54

Chapter II

Expression of the Arabidopsis thaliana transcriptional regulators, CBF]

and CBF3, confers dehydration stress tolerance in cucumber (Cucumis

! .

sativus L.) plants.

Introduction

Environmental factors that impose dehydration stress affect species distribution and
plant productivity (Bray, 1994; Bohnert and Jensen, 1996). As the driving force of living
organisms, water serves as a medium for the biochemical activities in living cells, is
involved in biosynthesis and assembly of molecules into organized structures, and is
responsible for generating the needed turgor pressure for plant cell expansion (Tanford,
1978; Xiong and Zhu, 2002). When plants fail to acquire sufficient water, the resultant
water deficit causes loss of turgor and/or osmotic stress. Dehydration stress can be caused
by several abiotic stresses including drought, freezing temperatures and salinity.

Soil salinity is estimated to affect about 77 million ha (Jain and Selvaraj, 1997; Tester
and Davenport, 2003) and may reach up to 50% of the total arable land by the year 2050
(Wang et al., 2003), making it a priority to find ways to alleviate this problem. Similarly,
due to climate changes worldwide, a long-term trend of higher temperatures with a
decrease in rainfall is expected to negatively impact agricultural production, especially in
arid and semiarid regions (Hillel and Rosenzweig, 2002). These trends, along with the
need to increase food production for a continuously growing world population will

increase drought- and salinity-affected areas.

55

In general, plant responses to changing environmental conditions are mediated by
alterations in gene expression (Guy et al., 1985; Greenway and Munns, 1980; Bohnert
and Jensen, 1996; Ingram and Bartels, 1996; Zhu et al., 1997). The first group of genes
whose expression is altered in response to dehydration-inducing conditions include those
encoding transcriptional factors (TF), mitogen activated protein kinases (MAPKs),
dephosphorylation enzymes, and chromatin remodeling proteins (Thomashow, 1999;
Knight and Knight, 2001; Shinozaki et al., 2003). Regulation of the first group of genes
generates signals that lead to adaptive responses including the induction of genes
involved in biosynthesis of osmolytes and compatible solutes, late embryogenesis
abundant (LEA) proteins, transporters, and detoxification enzymes (Shinozaki and
Yamaguchi-Shinozaki, 1997; Seki et al., 2001; Fowler and Thomashow, 2002; Seki et
al., 2002). Thus, induction of response-cascades has been suggested as an effective
approach to enhance dehydration stress tolerance (Thomashow, 1999).

Study of processes that lead to freezing and drought tolerance in Arabidopsis thaliana
revealed critical information about plant response to dehydration stress conditions.
Exposure of Arabidopsis plants to low, non-fieezing temperatures or to drought stress
conditions leads to increased expression of a group of genes, the COld Responsive (COR)
and Responsive to Drought (RD) genes that are induced under both cold acclimation and
drought conditions (Gihnour et al., 1992; Hovarth et al., 1993; Lin and Thomashow,
1992; Thomashow, 1999). The COR and RD genes are characterized by the presence of a
cis-acting element (CCGAC) within their promoter regions known as the CRT/DRE
element [CRT (C-repeat)/DRE (Drought Response Element)], which confers

responsiveness to low temperature, dehydration and salinity (Baker et al., 1994;

56

Yarnaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997). The CRT/DRE
element is bound by the CBF/DREB (CRT Binding Factor/DRE binding) transcription
factors which are characterized by a putative nuclear localization domain, an activation
domain and an AP2 DNA binding domain (Stockinger et al., 1997; Liu et al., 1998).

Transgenic Arabidopsis plants overexpressing CBF] showed elevated expression of
target COR transcripts and were more tolerant to freezing stress than their non-transgenic
counterparts as determined by electrolyte leakage and whole plant assays (J aglo-Ottosen
et al., 1998). Similarly, transgenic Arabidopsis plants overexpressing DREB] a had
enhanced drought and salinity resistance (Kasuga et al., 1999). Transgenic plants also
exhibited several physiological changes associated with increased dehydration stress
resistance including an increase in proline and total soluble sugars accumulation
compared to their nontransgenic counterparts (J aglo-Ottosen et al., 1998; Kasuga et al.,
1999; Gilmour et al., 2000; Jaglo et al., 2001; Seki et al., 2001; Seki et al., 2002; Haake
et al. 2002; Kasuga et a]. 2004). More recently, elevated expression of CBF was shown
to cause large-scale-metabolome changes in Arabidopsis including marked increase in
soluble sugars and amino acids leading to the suggestion that increased accumulation of
some soluble carbohydrates (galactinol, glucose, raffinose and fructose) and proline is a
signature of the CBF regulon (Cook et al., 2004).

Induction of CBF genes also confers increased dehydration stress resistance to other
less closely related species. Tobacco plants expressing CBF 3/DREB1A had higher
photosynthetic activity under drought conditions compared to wild type plants (Kasuga et
al., 2004); tomato plants overexpressing the Arabidopsis-CBF I/DREB] B gene showed

elevated tolerance to drought (Hseih et al., 2002a, b) and salinity (Lee et al., 2003); and

57

strawberry plants expressing the Arabidopsis CBF1/DREBIB gene had less membrane
damage in response to freezing temperatures than their nontransgenic counterparts
(Owens et al., 2002).

In the present work, we sought to investigate and test the possibility to improve
dehydration—stress tolerance in cucumber (Cucumis sativus L.) by expressing the A.
thaliana transcriptional activators CBF] and CBF 3. Cucumber is known to be sensitive

to salinity and drought especially in arid and semi-arid areas (Mass and Hoffman, 1977).

58

Materials and methods

Plant constructs and transformation

The Agrobacterium plant transformation constructs containing CBF] or CBF 3, under
control of the CaMV 35S promoter were kindly provided by M. F. Thomashow (J aglo-
Ottosen et al., 1998). Cucumber transformation was performed using the following
procedure derived from the methods of Tabei et al. (1998). Cucumber seeds, cv. Straight
8 (Hollar Seeds, Rocky Ford, Co.) were decoated and surface sterilized for 15 min with
15 % Clorox solution (1% sodium hypochlorite solution) with 1-2 drops of Tween 20
(Sigma-Aldrich, St. Louis, MO). Seeds were rinsed several times with sterilized distilled
water and placed overnight in the dark on honnone-free MS basal salt mixture
(Murashige and Skoog, 1962) supplemented with 30g/l sucrose and 2.5 g/l Phytagel
(Sigma-Aldrich, St. Louis, MO). Cotyledon explants were prepared by separating from
the embryo, removing the outer edges, and cutting into 4-6 explants. Explants were co-
cultivated with Agrobacterium tumefaciens strain C58 (Deblaere et al., 1985) , by
dipping in a 1/20 diluted overnight culture for 10 min. Explants were blotted on sterilized
filter paper then cultured onto C1 shoot induction medium [MS basal salt mixture
medium (Sigma-Aldrich, St. Louis, MO) supplemented with 2 mg/l benzyl amino purine
(BAP) (Sigma-Aldrich, St. Louis, MO), 1 mg/l abscisic acid (ABA) (Sigma-Aldrich, St.
Louis, MO), 30 g/l sucrose and 2.5 g/l Phytagel]. Plates were wrapped in aluminum foil
and incubated in the dark on culture shelves at 25 C°. Three days later, explants were

rinsed several times with sterilized H20, blotted on filter paper and cultured onto C2

medium [Cl medium supplemented with 400 mg/l Timentin® (GlaxoSmithKline,

Research Triangle Park, NC)]. A week later, explants were transferred on to a selection

59

medium C3 [C2 medium supplemented with 75 mg/l kanamycin (Sigma-Aldrich, St.
Louis, MO)] and were monitored for 3-4 weeks for shoot growth and elongation.
Emerging shoots were then placed onto selection medium C4 [C3 medium supplemented
with 8 g/l agar (Sigma-Aldrich, St. Louis, MO)]. Shoots were continuously grown on
media supplemented with 75 mg/l kanamycin (Sigma-Aldrich, St. Louis, M0) for
selection. Shoots were rooted in 250 ml Magenta boxes (Magenta Corp, Chicago, IL)
containing 50 ml of MS medium with 30 g/l sucrose and 7 g/l agar.

PCR-verified transgenic plants were transferred into 10 cm pots filled with Baccto
soil mix (Michigan Peat Co., Houston TX) and kept in the growth chamber for 2-3 weeks
(16/8 h light, temperature was maintained at 22 C°, Relative humidity was maintained
around 50%). Acclimated shoots were transferred to the greenhouse for 2-3 weeks before
transferring into 3.6 1 pots filled with Baccto soil mix for seed production. Transgenic
CBF1 or CBF3-cucumber plants were self pollinated to produce fruits in the greenhouse;
6-8 weeks old fruits were harvested and seeds were extracted, dried and stored in a dry

area.

DNA and RNA isolation, PCR, and Northern blot analysis

DNA was extracted fiom 200 mg young leaf samples using the Wizard DNA
purification kit (Promega, Cat # A7951, Madison, WI). For RNA isolation, young leaf
tissues were collected and immediately frozen and ground in liquid nitrogen and
extracted using Concert Plant RNA Reagent (Invitrogen, Carlsbad, CA). RNA quantity
and quality was determined by measuring absorbance at 260 nm and by gel

electrophoresis (Sarnbrook and Russell, 2001). Quantitation of RNA was performed with

60

a Molecular Imager FX Pro multi-imager system (Bio-Rad® Laboratories, Hercules, CA).
PCR was carried out using CBF]- and CBF3-specific primers (Stockinger et al., 1997).
Northern hybridization analysis was conducted using 32P labeled CBF] and CBF 3 cDNA

firll length probes following the procedure of Sarnbrook and Russell (2001).

Salinig experiments

T1 segregating progeny of transgenic To plants were planted in the greenhouse and
screened by PCR for the presence of the introduced CBF genes at the 2-3 -leaf stage.
Transgenic seedlings, non-transgenic T1 segregant progeny, (Azygous) and wild type
‘Straight 8’ (WT) plants were transplanted into 15cm clay pots filled with vermiculite
(Therm-o-Rock East Inc, Grade no. 3A, Washington, PA). To minimize evaporation, the
soil surface was covered with plastic disks that fitted at the top of the clay pots. In the
first greenhouse experiment, two families (A4 and B3) were tested with three levels of
NaCl (0 mM, 100 mM and 200 mM) in a randomized complete block design with three
replications. Salt treatment was initiated seven days after seedling transplant with a
stepwise increase of 50mM NaCl at two day intervals to reach 200mM NaCl. Plants were
fertilized with 300 ppm 20:20:20 fertilizer once a week throughout the experiment.
Young leaves (3-4 cm diameter) were collected every four days for sugar and proline
analysis as described below.

In the second and third greenhouse experiments, 10 and 9 CBF-families were tested
(Table, 1), at three NaCl levels (0, 50 and 100 mM), in a randomized complete block
design with six replications. Salt treatment was initiated 15 days after transplanting, with

a stepwise increase of 50 mM NaCl at three day intervals to reach 100mM NaCl. Total

61

soluble sugars and proline were measured at time 0 (immediately before the salt
treatment) and 15 days post initiation of salt treatment (dps). Growth parameters (total
above ground fresh and dry weight, plant height and number of leaves) were measured at

15 dps. Statistical analysis was performed using Microsoft Windows® EXCEL and SAS®

9.1.2 programs.

Drought experiments

T2 segregating progeny of transgenic families were planted in the greenhouse and
screened by PCR for the presence of the CBF gene at the 2-3-leaf stage. Transgenic
seedlings, non-transgenic segregants, and wild type ‘Straight 8’ plants were transplanted
into 3.6 1 pots filled with Baccto soil mix (Michigan Peat Co., Houston TX). A split plot
design with water as the main effect was used with three replications in experiment 1 and
four replications in experiments 2 and 3. Genotypes were assigned randomly within each
main plot. Drought treatment was carried out by withholding water until the first sign of
wilting (about nine days in experiments 1 and 2 and 12 days in experiment 3) followed by
one day of irrigation to full soil saturation. In the first experiment, this cycle was repeated
three times, in the second experiment this cycle was repeated twice and in the third
experiment this cycle was performed one time. Plant heights were determined after each
cycle of drought; total above-ground flesh and dry weight was determined at the end of
the experiment. Leaf tissue samples were collected immediately before the beginning of

the treatment, as well as after each drought cycle for proline and sugar analysis.

62

Sugar and proline analyses

One young leaf (3-4 cm diameter) was collected from each plant at each sample date,
freeze-dried for 48 hr, ground, and split into two aliquots, one for proline and one for
sugar analysis. Proline analysis was carried out using the procedure described by Troll
and Lindsey, (1955) (Appendix A). Total soluble sugar analysis was performed by the

phenol/sulfuric acid method of Dubois et al., (1956) (Appendix A).

Chlorophyll fluorescence

Components of chlorophyll fluorescence were quantified using a portable Plant
Efficiency Analyzer fluorometer (Hansatech, Norfolk, UK). Measurements were
performed in the greenhouse, using attached leaves. Three leaves, 7-9 cm in diameter,
and well-exposed to sun light, were chosen per plant for sequential measurements. After
30-40 min dark adaptation period, minimal fluorescence (F0), maximal fluorescence
(Fm), variable fluorescence (Fv) and fluorescence efficiency (Fv/Fm) were measured
immediately before the beginning of the drought treatment, and after 2, 6 and 12 days of

drought.

63

Results

Introduction and expression of the Arabidopsis CBF] and CBF3 transgenes in

cucumber plants

Thirteen transgenic cucumber plants were produced, six with CBF] and seven with
CBF 3 . Presence and expression of CBF genes was verified in the To plants by PCR and
northern blot analysis, respectively (data not shown). Successful transfer of the CBF] and
CBF 3 genes into T1 and T2 progeny was verified using PCR analysis; x2 analysis of
segregating progeny was consistent with single gene insertion in each case (Table 2-1).
Analysis of CBF expression in transgenic T2 plants showed varying transcription levels

among the different lines (Figure 2-1).

Transgenic cucumber plants expressing CBF genes have elevated levels of proline

and total soluble sugars in leaves compared to the non-transgenic controls

Greenhouse experiments were performed to evaluate the response of CBF] and
CBF3-transgenic cucumber plants to salt stress. Seeds were only available for two lines,
A3 and B4. In the absence of salt stress, CBF-expressing cucmnber plants accumulated
significantly higher levels of both fi'ee proline (up to 5 fold higher) and total soluble
sugars (2 fold higher), compared to the nontransgenic controls (Figure 2-2A, D;
ANOVA, P< 0.01 and P<0.05 for proline and soluble sugars, respectively). Under salt
stress conditions, CBF-cucumber plants accumulated higher levels of proline and soluble

sugars compared to the non-stressed conditions (ANOVA, P<0.01). The azygous (non-

64

Table 2-1. Segregation analysis of T1 transgenic cucumber lines expressing the
Arabidopsis CBF] and CBF 3 genes. Presence or absence of the gene was determined by
PCR analysis

 

 

Line Gene construct T1 segregation 12 (3:1)
(trans: non) -
A1 35S.‘.'CBFI 46 I 11 0.71 ns
A3 35S.'.'CBFI 49 Z 13 0.30 ns
A4 35S.’.'CBFI 51 2 16 0.09 ns
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Bl 35S.’.‘CBF3 44 I 12 0.21 ns
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BS 35S.‘.'CBF3 49 I 10 1.63 ns
B6 35S.’.'CBF3 47 I 9 0.69 ns
B7 35S.'.'CBF3 40 I 12 0.02 ns

 

 

 

 

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transgenic segregant) and wild-type plants had equivalent levels of proline and soluble
sugars accumulation, indicating the observed differences from CBF-plants were due to
the presence of CBF genes. Exposure of cucumber plants to 200 mM NaCl did not cause
an increase in either proline or sugar concentration (Figure 2-2C, F), compared to the 100
mM NaCl level (Figure 2-2 B, E). In addition, plants irrigated with 200 mM NaCl
exhibited severe leaf discoloration and necrosis; therefore, 100 mM NaCl was the

maximum concentration used in subsequent experiments.

Additional CBF families (10 and 9 families) were tested in the second and third
experiments, equivalent results were obtained in both experiments. Significant
differences were observed between transgenic and nontransgenic families for proline and
soluble sugar accumulation prior to the initiation of salt treatment; on average, CBF-
expressing cucumber plants had 24.5 a 3.1 mg/gdw soluble sugar and 8.9 a 0.4 ug/gdw
proline, compared to an average of 15.8 a 1.2 mg/gdw soluble sugars and 2.3 :t 0.3
pg/gdw proline in the nontransgenic controls (ANOVA, P<0.01 and P<0.05 for proline
and soluble sugar, respectively). Fifteen days post initiation of salinity treatment, proline
and total soluble sugar accumulation was significantly greater in the transgenic families
compared to the nontransgenic controls at all salt levels (Figure 2-3A and B; Table 2-2).
There were no significant differences between the CBF] and CBF 3 lines (Table 2-2).

The transgenic cucumber plants showed a progressive increase in proline and soluble
sugar accumulation at both 50 and 100 mM NaCl levels, while proline and sugars levels

in the nontransgenic controls did not increase above 50 mM NaCl (Figure 2-3). The

68

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amount of accumulation of the two types of compounds within a given genotype was
highly correlated (r2=0.89; Figure 2—4A), indicating that they are part of a coordinated
response. Salinity treatment caused a marked increase in soluble sugar and proline
accumulation in CBF-plants beyond what would be expected from an additive effect of
the two separate components, CBF in the absence of stress, and salt stress in the absence
of CBF (Figure 48, C arrows). Thus, the effect of the CBF gene on solute accumulation
increased in response to salt stress.

Given the increased accumulation of compatible solutes in 3SS:CBF plants in
response to salinity stress, CBF transcript levels were compared in the presence or
absence of salinity stress (Figure 2-5). The lack of differences is consistent with expected
results for expression driven by the constitutive CaMV3SS promoter, and this also
indicates that there were not differences in CBF mRNA stability associated with salt

StI'CSS.

CBF-expressing cucumber plants showed less reduction in fresh and gg weight

under salt stress conditions

Growth data were collected at the end of experiments 2 and 3. At 0 mM NaCl, there were
no significant differences between CBF— transgenic families and the nontransgenic
controls for height, and above ground flesh and dry weight (Figure 2—3 C, D; Table 2-2).
At 50 mM NaCl, neither the CBF nor the nontransgenic lines showed significant
differences in growth. At 100 mM NaCl, nontransgenic controls exhibited a significant
reduCtion in fresh weight (48%), dry weight (56%), and height (16%) relative to non-salt

stressed plants. While 100 mM NaCl also affected growth of the transgenic cucumber

71

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lines (a reduction of 30%, 16%, and 22% in fresh weight, dry weight, and height .
respectively), growth inhibition was significantly less severe relative to the nontransgenic
controls for fresh weight (ANOVA, P< 0.05) and dry wei ght (AN OVA, P< 0.01). Plants
expressing CBF1 or CBF 3 genes had almost twice the dry weight as the nontransgenic
controls. CBF] and CBF 3 lines performed equivalently (Table 2—2). Grovvth in the
presence of 100 mM NaCl appears to be correlated with levels of soluble sugars and

proline levels (r2 = 0.79 and 0.78, respectively).

Transgenic CBF-cucumber plants have elevated level of compatible solutes and less

reduction in photosvnthetic capacity in response to drouth stress

CBF1 and CBF3-expressing cucumber plants were also tested under drought stress
conditions. The same trends were observed in all three experiments. Under well~irri gated
conditions, significant differences in proline levels between the CBF~expressing families
and the nontransgenic controls were observed while significant differences in soluble
sugar were observed later in the experiment (Figure 2-6A, C; AN OVA, P<0.01). After
the first cycle of drought (until the first symptoms of wilting of lower leaves), CBF-
expressing cucumber plants accumulated approximately twice the amount of soluble
sugar and approximately 5-fold higher levels of proline than their nontransgenic
counterparts (Figure 2-68, D; ANOVA, P<0.01). Although afier a second cycle of
drought proline and soluble sugar levels remained higher in the CBF-plants than the non-
transgenics, soluble sugar level in the transgenic plants did not increase compared to the
first cycle and levels of proline declined (Figure 6D). Moreover, the wilted lower leaves

did not recover after the second re-irrigation. No differences in proline or soluble sugar

74

 

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75

levels were observed between the azygous and the wild-type plants. Chlorophyll
fluorescence was used to determine the maximum photochemical efficiency in cucumber
plants under drought stress conditions. Measurements were taken immediately before the
beginning of drought treatment and after 2, 6 and 12 days of drought in experiment 3.
There were no significant differences between the CBF-transgenic cucumber plants
and the nontransgenic controls under well irrigated conditions. Significant differences
were detected after 12 days of drought treatment (Figure 2-7), at which time stressed
plants began to show the first obvious signs of wilting in the lower leaves for both the
transgenic and the nontransgenic controls. The photosynthetic activity of the upper, non-
wilted leaves of CBF-expressing plants had 50% higher fluorescence value than the
nontransgenic controls, reflecting greater stability of photosystem II (PSII) under drought

stress conditions (Figure 2-7).

CBF-expressing cucumber plants showed less ggowth reduction under drought

stress conditions

Growth parameters (plant height, above ground flesh and dry weight) were collected
at the termination of the drought experiments. Under well-irrigated conditions, no
significant differences were observed between CBF-transgenic families and the
nontransgenic controls for height, above ground flesh weight and dry weight (Figure 2-
8). In response to a cycle of drought, transgenic-CBF lines showed significantly less
reduction in plant height and dry weight than did the nontransgenic controls (average
reduction of 45% and 46% for height and dry weight vs. 25% and 15%, for CBF lines;

ANOVA, P< 0.05). The CBF-transgenic lines did not show significantly less reduction in

76

 

 

Chlorophyll fluorescence under well-irrigated conditions

 

 

 

 

 

 

 

 

 

 

 

800 1
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i l
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o l
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8
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0 2 4 6 8 10 12
Days post initiation of drought
Chlorophyll fluorescence under drought-stressed conditions

i
ii
5‘
5
29
:2:
0
9.3
8
0
O
3
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0 2 4 , 6 8 10 12
Days post initiation of drought

 

 

 

Figure 2-7. Chlorophyll fluorescence (Fv/Fm) in CBF-expressing cucumber
plants and azygous non-transgenic plants, under well-irrigated and drought-
stressed conditions. Each data point represents the mean of four replicates
with three plants/replicateltreatment1- SE. '

77

 

 

250 w —

 

   
  

     

Emmi-emu

E, 200 l :“flwh‘smifl
3..
.g 150 1
J: 100
m
E 50 -‘

o l

 

Controls

Dry weight (g)

 

Controls

Height (cm)

 

 

 

Transgenic lines Controls

 

 

Figure 2-8. Effect of drought conditions on above ground fresh weight (A),
dry weight (B) weight, and plant height (C) of CBF1, and CBF3-transgenic
lines, non-transformed controls (CC), and non-transgenic segregants (AZ).
Measurements were recorded after one cycle of drought stress. Light bars:
well-irrigated-plants; dark bars: drought stressed-plants. Values are the
mean 1 SE of three replicates/treatment with 3 plants/replicate.

78

flesh weight (39%) than the nontransgenic controls (50%) (ANOVA, P< 0.12).
Equivalent trends were observed after two cycles of droughts; with significantly less

reduction in height (48% vs. 29%) and dry weight (45% vs. 24%) for the CBF lines

compared to the nontransgenic controls.

79

Discussion

Several lines of transgenic cucumber plants expressing CBF1/DREBb and
CBF3/DREBa genes under the control of the constitutive CaMV3SS promoter were
produced and tested for physiological changes and response to dehydration stresses.
Previous studies with the CBF gene family demonstrated that overexpression of CBF in
Arabidopsis caused significant increase in accumulation of compatible solutes, especially
soluble sugars and proline (J aglo-Ottosen et al., 1998; Kasuga et al., 1999; Gihnour et
al., 2000; Seki et al., 2001; Seki et al., 2002; Haake et al. 2002; Cook et al., 2004).

Under nonstressed conditions, cucumber plants expressing CBF1 and CBF 3 genes
had higher soluble sugar and proline levels than the nontransgenic controls; these levels
continued to increase throughout the experiment, indicating that the heterologous-CBF1
and CBF 3 genes induce pathways in cucumber similar to those in A. thaliana. The levels
of increase in soluble sugar accumulation (2—3 fold) and proline (5-fold) were comparable
to that observed in CBF-overexpressing Arabidopsis plants (Gilmour et al., 2000;
Gilmour et al., 2004). Similar findings were also observed when overexpressing
CBF/DREB genes in the heterologous species, tomato and tobacco (Hsieh et al., 2002a;
Hsieh et al., 2002b; Lee et al., 2003; Kasuga et al., 2004).

Accumulation of soluble sugar was highly correlated with proline accumulation,
indicating that syntheses of these compounds are coordinately regulated in the CBF- ‘
expressing plants. In Arabidopsis, the promoters of genes for key enzymes involved in
proline and sugar biosynthesis (e.g., P5 CS and galactinol synthase) have binding sites for
CBF and have been shown to be upregulated in response to CBF expression (Seki et al.,

2001; Seki et al., 2002; Fowler and Thomashow, 2002; Gilmour et al., 2000; Vogel et al.,

80

2005). While orthologous genes in cucumber may also include CBF-binding sites, direct
transcriptional regulation by CBF would not fully explain the observed increase in
proline and sugars in CBF-cucumbers in response to salt stress. Exposure to salinity
increased the levels of soluble sugar and proline in CBF-plants beyond a simple additive
effect of the individual contributions of salinity and CBF. The increase in proline and
sugar accumulation in response to salt stress was not a direct result of salt-induced
increase in CBF-transcript levels, indicating that the enhanced accumulation, is at least in
part downstream of CBF per se. It is possible that post- transcriptional or translational
regulation of key enzymes that are critical for production and accumulation of sugars and
proline is affected. Alternatively, the initial induction of dehydration stress-related
responses by CBF may result in cascades of signals that indirectly, lead to changes in
transcription rate of key proline and sugar biosynthetic genes.

Transcriptional profiling in CBF-overexpressing Arabidopsis plants indicated
activation of several classes of genes including genes encoding transcriptional regulators
(e.g., AP2 containing proteins, zinc-finger containing proteins, MYB-family
transcriptional activators), genes involved in stress-signaling (MAP-kinases, calcineurin
and calcineurin-like proteins), and genes involved in metabolism and catabolism (Fowler
and Thomashow, 2002, Sike et al., 2001; Maruyama et al., 2004; Vogel et al., 2005). The
responding genes including transcriptional factors could be clustered into groups whose
expression increased or decreased at different time periods following transfer to the cold,
suggesting sequential induction (Fowler and Thomashow, 2002, Sike et al., 2001;
Maruyama et al., 2004; Vogel et al., 2005). These observations indicate involvement of

multiple regulatory systems which can be initially triggered by CBF. Indeed, not all of

81

the upregulated genes include the CDT/DRE element in their promoters, suggesting that
these genes may be secondarily regulated by one of the CBF-induced transcription factors
(Fowler and Thomashow, 2002; Vogel et al., 2005). The up—regulation of these genes
could eventually affect other pathways in plants that could also induce production of
compatible solutes. Thus, CBF-induced pathways may influence subsequent responses,
perhaps priming the CBF-cucumbers to allow for enhanced response to stresses.

There appears to be a limitation, however, to the increase in proline and sugar in
response to stress, even in the CBF -p1ants. In our conditions, the transgenic cucumber
plants did not accumulate higher levels of soluble sugars and proline when treated with
200 mM NaCl or when imposing a second drought cycle. This may indicate a limitation
in the adaptive responses that can be induced by the CBF/DREB genes, or limitation of
the ability of a species to respond to the unfavorable stress conditions. In non-transgenic
plants, accumulation of soluble sugar and proline did not increase beyond levels obtained
with the 50 mM salt treatment, or with imposition of drought, while levels of soluble
sugars and proline continued to increase in the CBF-expressing cucumber plants (at 100
mM NaCl and after 2 cycles of drought), suggesting that the CBF increased the range of
response in cucumber plants to a higher limit.

These differences in range of response were also reflected in plant growth. In the
presence of 50 mM NaCl, no significant differences in above ground flesh and dry
weight and plant height were observed among CBF-transgenic and nontransgenic lines,
indicating that at 50 mM NaCl, wild-type cucumber plants were able to adapt and adjust
to this salinity level. In the presence of 100 mM NaCl, the cucumber plants expressing

the Arabidopsis-CBF1 and CBF 3 genes showed significantly less reduction in above-

82

ground flesh and dry weight, compared to the nontransgenic controls. These results
coupled with the drought stress results, indicate that CBF allowed for increased salt and
drought stress resistance in cucumber.

Moreover, at the time of first wilt of lower leaves, CBF—expressing cucumber plants
also exhibited less reduction in F v/Fm (a measure of stability of photosystem II) in
response to drought stress, than did the nontransgenic controls, indicating an additional
physiological effect of the CBF transgene. Similar effects were also reported in tomato
and tobacco plants expressing CBF1/DREBb (Hseih et al., 2002; Lee et al., 2003; Kasuga
et al., 2004). Increased photosynthetic stability may result flom altered expression of
CBF-target genes, or an indirect effect of increased osmoprotectant. The presence of
higher levels of proline has been reported to correlate with higher protection of
photosystem II (De-Ronde eta1., 2004). Similarly, greater stability of PS II was also
reported with the production of the compatible solute glycine betaine (Hayashi et al.,
1997; Sakamoto et al., 1998; Kishitany et al., 2000; Holrnstom et al., 2000; Quan et al.,
2004), trehalose (Garg et al., 2002; J ang et al., 2003), and mannitol (Loescher et al.,
personal communication), or by overexpressing the yeast invertase gene in tobacco
plants, which results in accumulation of glucose and fluctose up to 8-fold (Fukushima et
aL,2001)

Several studies with CBF-overexpressing Arabidopsis plants have reported negative
impacts on plant growth (Lui et al., 1998; Kasuga et al., 1999; Gilmour et al., 2000).
Severity of growth retardation was positively correlated with CBF expression levels (Liu
et al., 1998; Gilmour et al., 2000), and was minimized the by use of a stress-inducible

promoter (Lee et al., 2003, Kasuga et al., 2004; Pellegrineschi et al., 2004). Additional

83

phenotypic differences in plants constitutively overexpressing CBF 3 , included darker
leaves, shorter petioles, and delayed bolting in Arabidopsis (Gilmour et al., 2000), and
shorter intemodes and less fluit and seed production in CBF-overexpressing tomatoes
(Hsieh et al., 2002). Expression of the CBF gene in cucumber did not have visible retard
growth under the conditions tested. The lack of negative effects in this study could be due
to differences in expression levels of CBF] and CBF 3 genes in 3SS:CBF-cucumber
plants compared to the levels 35S:CBF Arabidopsis, or to differences in the nature of the
downstream responses in cucumber in response to CBF expression.

Despite enhanced stress resistance in tomato, efforts to clone COR homologs flom
tomato were not successful, even under low stringency conditions, indicating the absence
of COR genes as a CBF target in that species (Hsieh et al., 2002). Furthermore,
microarray analysis of Arabidopsis and tomato plants expressing CBF genes, revealed the
presence of marked differences in induced gene expression between these species,
including the failure to observe predicted orthologs to Arabidopsis CBF-regulon genes.
These results further suggests that responses may differ in heterologous systems, and may
have differential impacts on growth and development.

In conclusion, the present study demonstrates that expression of CBF in cucumber, a
species known for sensitivity to salinity and drought conditions, may offer an effective
approach to enhance salinity and drought tolerance. Our results shows that 35$:CBF—
expressing cucumber plants were able to adapt to a higher range of dehydration-induced
stresses than did their nontransgenic counterparts without apparent costs on plant growth.
This increase in stress resistance was also accompanied by coordinated physiological

responses including accumulation of compatible solutes and maintenance of photosystem

84

 

II stability. Further studies to evaluate plant performance as well as fluit production under

field conditions are needed to begin to assess agricultural potentials.

85

 

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90

 

Chapter III

Cucumber (Cucumis sativus L.) plants expressing the Arabidopsis
thaliana-transcriptional regulators, CBF] and CBF3, are more tolerant

to salinity stress under field conditions.

Introduction

Stress caused by high concentrations of NaCl in soil or irrigation water negatively
influences productivity of major agricultural crops (McWilliam, 1986; Zang and
Blumwald, 2001). Statistical assessments of naturally salt-affected areas worldwide vary,
but in general it is estimated that close to 1 billion hectares (approximately 7% of the
world’s land area) have naturally saline soil (Ghassemi et al., 1995). In addition to the
naturally affected areas,it is estimated that 77 million hectares have become salt affected
due to extensive agricultural practices, mainly in irrigated areas worldwide. It has been
estimated that these numbers will increase to affect up to 50% of the total arable land by
the year 2050 (Wang et al., 2003). Moreover, the demand to increase food production for
the continuously grong world population will result in the need for more land for
agricultural production, leading to an increase in salinity affected areas (Ghasserrri et al.,
1995). Economic damage due to soil salinization has been difficult to assess, but
Ghassemi et al. (1995) estimated that economic damage due to soil salinization at the
Colorado River Basin is about 750 million US dollars/year and was about 330 and 208

million US dollars/year for the Punjab area and the Murray—Darling Basin in Australia.

91

Excess NaCl in soil and irrigation water causes hyperosmotic and hyperionic stress
effects, which if sufficiently severe can result in plant death (Bohnert et al., 1999;
Hasegawa et al., 2000). The hyperosmotic effect results from concentration of
extracellular solutes, which causes a flux of water out of the cell, a decrease in the
osmotic potential within the cell, and in the cellular turgor pressure (Lichtentaler, 1995).
The hyperionic effect resulting from exposure to high salinity leads to “toxic sodium
effect”, whereby excess Na+ in the cytoplasm causes an unbalance of other essential ions
such as K+ and Ca+ (Bohnert and Jensen, 1996; Hasegawa et al., 2000). High
concentration of Na+ ion in the cytosol causes metabolic toxicity, in part due to the
competition between K+ and Na+ for binding sites for several enzymes (Tester and
Davenport, 2003). High Na+ and Cl' concentrations interfere with enzyme function,
protein synthesis, structure and solubility, and membrane fluidity and function (Blum,
1988)

Traditional breeding programs to develop salinity tolerance in plants have had modest
success due to difficulties in establishing selection criteria, limited availability of sources
of genetic resistance, quantitative nature of resistance, and the variety of mechanisms
involved in salinity tolerance (Flowers and Yeo, 1995; Quesada et al., 2002). For
example, when evaluating yield performance of a crop under saline conditions, many
factors can influence performance, including variation in salinity levels within a field, or
possibility of interaction between salinity level and other environmental factors such as
soil fertility, drainage quality, and water loss due to transpiration (Flowers, 2004). Thus,
using yield components as main criteria for selection requires a long period and multiple

locations for testing, and evaluation (Blum, 1989). Furthermore, results from several

92

groups indicate that QTL linked to salinity tolerance at a given developmental stage may
differ fiom those linked to tolerance at another developmental stage (Greenway and
Munns, 1980; Foolad, 1999; Cushman and Bohnert, 2000; Quesada et al., 2002; FoOlad,
2004).

Physiological and molecular studies aimed toward understanding plant response to
salinity stress have indicated the complexity of this phenomenon, where an entire cascade
of biochemical and cellular changes is necessary to adapt to high salinity stress (Bohnert
and Jensen, 1996). Gene expression analysis, in the model plant A. thaliana under
different dehydration inducing conditions (drought, salinity and fi'eezing temperatures),
revealed changes in expression patterns of several groups of genes. One of the first
groups that shows immediate changes are those that encode transcription factors (TFs),
mitogen activated protein kinases (MAPKs), dephosphorylation enzymes, and chromatin
remodeling proteins (Thomashow, 1999; Knight and Knight, 2001; Hasegawa et al.,
2000; Xiong et al., 2002; Zhu, 2002; Shinozaki et al, 2003; Seki et al., 2003; Vogel et al.,
2005). This primary response is followed by activation of multiple mechanisms that are
essential for plant adaptation to dehydration stresses (Bohnert and Jensen, 1996; Ingram
and Bartels 1996; Campbell and Close, 1997).

The multigenic nature of plant response to salinity suggests that induction of multiple
adaptive mechanisms at the same time might be a good strategy to engineer salinity
tolerance in plant species. The CBF/DREB [CRT (C-repeat) Binding Factor /DRE
(Drought Response Element Binding)] gene family encodes transcriptional activators that
work as master switches in regulating plant response to dehydration-inducing conditions

(Thomashow, 1999; Shinozaki et al., 2003). Expression of the CBF gene family is

93

activated in response to low temperatures, drought or high salinity (Stockinger et al.,
1997; J aglo-Ottosen et al., 1998; Liu et al., 1998; Kasuga et al., 1999; Haake et al. 2002).

Transgenic Arabidopsis plants overexpressing CBF/DREB genes showed elevated
levels of resistance to dehydration stresses relative to their nontransgenic counterpart, as
determined by electrolyte leakage and whole plant test assays (J aglo-Ottosen et al., 1998;
Kasuga et al., 1999). Similarly, in growth chamber trials, transgenic Arabidopsis plants
overexpressing CBF3/DREBIa had enhanced resistance to drought and salinity stresses
(Kasuga et al., 1999; Kasuga et al., 2004); expression of CBF1/DREB] b gene in tomato
increased the resistance levels to salinity and drought (Hsieh et al., 2002; Lee et al.,
2003); and expression of either CBF1/DREB] b or CBF 3/DREBI a genes in cucumber
plants reduced their susceptibility to salinity and drought stress compared to their
nontransgenic control counterparts (Tawfik and Grumet, 2001 and 2003). In general, the
increase in dehydration stress resistance is accompanied by increased membrane stability
and/or accumulation of compatible solutes, especially proline and soluble carbohydrates
(J aglo-Ottosen et al., 1998; Kasuga et al., 1999; Gilmour et al., 2000; J aglo et al., 2001;
Seki et al., 2001; Seki et al., 2002; Haake et al. 2002; Hseih et al., 2002b; Tawfik and
Grumet, 2003; Kasuga et al. 2004). This elevation in compatible solute accumulation in
CBF/DREB expressing transgenic plants, especially in soluble sugars and proline, has
been described as a signature for CBF/DREB expression (Cook et al., 2004).

Despite these successful examples which clearly demonstrate the potential value of
the CBF/DREB system in increasing dehydration stress tolerance in plants, enhanced
tolerance has not yet been demonstrated in field conditions. Indeed, Flowers (2003) stated

that “after years of research using transgenic plants to alter salt tolerance, the value of this

94

approach has yet to be established in the field”. To our knowledge, there are only two
published reports that evaluated genetically engineered plants for enhanced dehydration
stress tolerance under field conditions. Quan et al. (2004) reported enhanced grain yield
production by transgenic maize plants expressing a gene for betaine aldehyde
dehydrogenase following drought stress period of 21 days. Xue et- al. (2004) tested wheat
plants expressing the Arabidopsis tonoplast H’L/Na+ antiporter gene for their ability to
grow in saline soil and reported higher grain production in the transgenic plants
compared to the nontransgenic controls.

In this work we sought to evaluate the performance of CBF-expressing cucumber
plants under salinity stress in field conditions (Tawfik and Grumet, 2001; Tawfik and
Grumet, 2003). Cucumber is known to be sensitive to salt (Mass and Hoffman, 1977).
Salinity delays seed germination and seedling emergence, decreases leaf expansion rate
and water potential, and decreases plant photosynthesis and yield (Chartzoulakis, 1994;
Tazuki, 1997). Previously, we demonstrated that cucumber plants expressing CBF] and
CBF 3 genes had elevated levels of resistance to salinity and drought conditions in
greenhouse conditions. In the current study, CBF] and CBF3-expressing cucumber plants
were tested for their ability to withstand continuous irrigation with 100 mM NaCl for 25
days under field conditions. Transgenic 35S:CBF cucumber plants had higher levels of
proline and soluble sugars in leaves and accumulated higher levels of K+ and Ca4+ ions
in roots relative to non-transgenic controls in the presence or absence of salinity stress. In
response to salinity, the CBF-cucumber lines exhibited significantly less reduction in
growth and fi'uit yield than did the non-transgenic controls, demonstrating the potential

effectiveness of CBF in conferring salt stress resistance in the field.

95

Materials and methods

Plant materials

The transgenic 35S-CBFI and 35S-CBF 3 cucumber lines were produced as described
in chapter 11. Of the 13 CBF-expressing lines, four lines were selected for the field trial;
two lines expressing CBF1, A1 (high level of CBF1 expression) and A5 (low level of
CBF1 expression); and two lines expressing CBF 3 gene, Bl (low level of CBF 3
expression) and, B5 (high level of CBF 3 expression) (Chapter 11). Two types of controls
were included: wild-type parental cultivar “Straight 8” (Hollar Seeds, Rocky Ford, Co.)
and azygous sibling progeny of the transgenic CBF-cucumber lines. T2 segregating
azygous progeny and commercial “Straight 8” seeds were planted in trays (51 x 40 x 6.5
cm, 32 cells/tray, Hummert "‘ International, Earth City, Mo) in the greenhouse and
screened by PCR for the presence of the CBF genes at the 2-3-leaf stage. DNA was
extracted from 200 mg young leaf samples of seedlings using the Wizard DNA
purification kit (Promega, Cat # A7951, Madison, WI). PCR was carried out using CBF1-
and CBF3-specific primers (Stockinger et al., 1997). Non-transgenic segregants from the

different CBF-lines were pooled for the azygous control plots.

Field salinig experiment

The field experiment was arranged in a split plot design, with four replications with
salt treatment as the main plot and genotype as the sub-plot. Six genotypes were tested
[four T2 CBF-families, parental Straight 8 (wild-type WT) and azygous segregant T2
progeny (Az)], with 6 plants per subplot. To allow for regulation of salinity levels,

seedlings were transplanted into 50 x 30 x 15 cm plastic bags filled with 22.5 kg

96

playground sand (Sandastic Co., IL) and perforated with drainage holes along the edges.
Plants were spaced 60 cm apart along rows of 1.5 m wide black plastic mulch; between
row spacing was 2.1 m. Two control “Straight 8” plants separated each plot. The
experimental plots were surrounded by two bOrder rows of control “Straight 8” plants on
all sides. Border plants were directly transplanted into the soil.

Irrigation was applied manually every other day using a 750 liter water tank
connected to a tractor. The plants were allowed to acclimate for three weeks before
starting salt stress treatment. Two salinity levels were used (0 mM and 100 mM NaCl),
and stepwise salt application was carried out with an increase of 25 mM every other day
until reaching the 100 mM NaCl level. Once the desired salinity level was reached,
irrigation was applied daily, until run through, (approximately 2.0 l/day). Plants were
fertilized with 150 ppm 20:20:20 (N: P: K) fertilizer twice a week throughout the
experiment.

All measurements and leaf sampling were conducted using the middle four plants of
each plot. Sampling for sugar and proline content was done four times during the
experiment, at one-week intervals starting just before the initiation of salt application.
Two 3-4 cm diameter leaves were collected from the main stem of each plant; sampled
leaves of a given plot were combined for proline and sugar analysis. Samples were taken
early in the morning and placed immediately in liquid nitrogen. Growth measurements
(number of nodes on the main stem, number of branches, and number of male and female
flowers) were recorded just before initiation of the salinity treatment. Fruit were
harvested three times, at 12, 18, and 24 days post initiation of salinity treatment. Twenty

four days post initiation of salt treatment, vines were harvested and above ground fresh

97

and dry weight was measured. After removing the above ground parts, the sand
surrounding the roots was washed away with water; roots were removed and then rinsed
several additional times with water before being placed in plastic bags on ice. Once in the
lab, roots were washed several times with deionized water, blotted on paper towels, and

then stored at -80 C° until firrther analysis.

Soluble sugars, proline and mineral analysis

Leaf samples collected from the field were placed in 13x100 mm glass tubes and
freeze-dried for 48 hr. Samples from a given plot were pooled, ground, and split into two
aliquots for proline and sugar analyses. Root samples were freeze dried for 48 hr.
Samples of a given plot were pooled, ground with a morter and pestel in liquid nitrogen,
and split into aliquots for proline, sugar and mineral analysis. Proline and soluble sugars
analyses were carried out following the procedure described by Troll and Lindsley (1955)
and Dubois et al., (1956), respectively. Mineral analyses were performed on ashed root
samples prepared by placing 2.0 g of freeze dried roots into ceramic crucibles and
incinerating at 500 °C for 16 hrs to insure complete ashing. Afier cooling, ash weight was
determined. Weighed samples of approximately 100 mg ash were added to 25 ml of 3N
HNO3 digestion solution. The samples were incubated for 1 hour at room temperature
and the solution filtered through 90mm x 100, No.2, Whatrnan® filter paper (Whatrnan®
International Ltd, Maidstone, England) into labeled vials. The concentration of Na+, K+
and Ca” ions were determined by the MSU soil analysis laboratory using a DC. Plasma

Emission atomic analyzer (Pye Unicam SP9).

98

Results

CBF-expressing cucumber plants and the nontransgenic controls had guivalent in

growth under field conditions before salinig treatment

Six genotypes were tested [two T2 CBF1 -fami1ies (A1 and A5), two CBF3-families
(B1 and B5), parental “Straight 8” (wild-type WT) and azygous segregant T2 progeny
(Az)]. Prior to initiation of salinity treatment, several grth parameters were measured
(Table 3-1). All lines performed equivalently; no significant differences were observed in
vine length, number of nodes, nrnnber of branches, or number of male and female flowers

between transgenic cucumber plants and the nontransgenic controls (Table 3-1).

Transgenic cucumber plants expressing CBF genes have elevated level of proline

and total soluble sugars compare to the non-transgenic controls

In the absence of salinity treatment, transgenic CBF- cucrnnber plants accumulated
significantly higher levels of proline (5-10 fold) compared to the nontransgenic controls,
and then levels increased throughout the growing season (Figure 3-1). As the season
progressed levels of soluble sugars also were significantly higher in the non-salt stressed
CBF-cucumber plants compared to their nontransgenic counterparts. When irrigated with
100 mM NaCl CBF-cucumber plants accumulated significantly higher levels of proline
and total soluble sugars compared to both non salt stressed conditions. CBF lines
accumulated higher levels of proline and soluble sugars than salt stressed nontransgenic
controls (ANOVA, P< 0.01). Twenty one days following initiation of salinity, the CBF1
and CBF3-transgenic cucumber families, on average, had more than 15 fold higher levels

of proline and 4 times higher levels of total soluble sugars compared to the nontransgenic

99

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101

counterparts. Salinity did not cause a significant increase in levels of compatible solute
accumulation in the azygous or the parental “Straight 8” wild-type controls. No
significant differences were found between CBF1 vs. CBF3-cucumber lines (ANOVA,
P< 0.76). Azygous plants did not differ from parental “Straight 8” plants.

Accumulation of compatible solutes was also tested in root samples at the termination
of the experiments, 24 days post initiation of salinity stress treatment. In the absence and
presence of salinity stress, CBF-cucumber roots accumulated higher levels of proline than
their nontransgenic counterparts (ANOVA, P<0.05; Figure 3-2 A). No significant
differences in accumulation of soluble sugar were detected between CBF-transgenic and

the nontransgenic cucumber plants (Figure 3-2 B).

Transgenic cucumber plants expressing CBF genes have elevated level of potassium

ions in their roots compared to the non-transgenic controls

Analysis of ashed root samples revealed differences in ion composition between the
transgenic and the nontransgenic controls. CBF -1ines exhibited 10-15 fold higher levels
of K+ ions than the nontransgenic controls in the presence and absence of salinity stress
(Figure 3-3A). On the other hand, Na+ levels did not differ significantly between the
transgenic and nontransgenic plants and did not increase significantly in response to
salinity treatment (Figure 3-3B). Transgenic cucumber plants had markedly lower Na+/K+
ratio in roots under both non—salt stress, and salt stress conditions compared to the
nontransgenic controls. The average Na+/K+ ratio did not change in the transgenic lines in
response to salinity stress (Figure 3-3C). Calcium levels also differed in the CBF

cucumber transgenic lines with approximately 2-fold higher levels than in the

102

 

 

 

   

        
   
   

        
     
  

 

   
  
 
 
  

 
      

 

    

         

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103

 

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104

nontransgenic lines under both salt stressed, and non-salt stressed conditions (Figure 3-

3D).

CBF-cucumber plants had less reduction in fresh and dry weight under salt stress
conditions

Under non-salt stressed conditions, CBF-expressing cucumber families had lower
fresh weight than the nontransgenic controls at the time of harvest; dry weight was
equivalent for the CBF and non-transgenic controls (Figure 3—4; Table 3-2). Salinity
treated CBF-cucumber plants on average did not show a significant reduction in fresh
weight (690 g vs. 646 g in the absence or presence of salinity), while nontransgenic
cucumber plants had an average reduction of 38% in fresh weight in response to salinity
treatment. Similarly, on average, salinity treated CBF-expressing plants did not show a
significant reduction in dry weight, while azygous and wild type Straight 8 plants had an

average reduction of more than 50%.

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the nontransgenic controls

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fruits or fi'uit weight between the CBF-expressing cucumber plants and the nontransgenic
controls (Figure 3-4; Table 3-2). Salinity treated CBF-expressing lines did not show
significant reduction in fruit number or weight, compared to a 35% reduction in fruit
number and 50% reduction in fruit weight for the nontransgenic controls. Salt stressed

CBF1 lines (Al and A5) had higher yields than the CBF3 lines (Bland B5).

105

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107

Discussion

The CBF gene has been demonstrated to confer dehydration stress resistance in
several species in growth chamber and greenhouse studies (Kasuga et al., 1999; Hsieh et
al., 2002; Lee et al., 2003; Pellegrineschi et al., 2004; Kasuga et al., 2004). This study
tested the performance of the 35$:CBF-cucumber plants under field conditions.
Consistent with greenhouse experiments (Chapter II), field grown transgenic 35S:CBF-
cucumber plants had elevated levels of proline and soluble sugars relative to the
nontransgenic controls. CBF-cucumber plants progressively accumulated higher levels of
the compatible solutes throughout the growing season, even in the absence of salinity
stress.

Irrigation with 100 mM NaCl caused significant elevation in soluble sugars and
proline levels in the leaves of CBF-cucumber plants while levels of sugar and proline did
not change in the nontransgenic controls, indicating enhanced ability of the CBF-lines to
respond to salinity stress. The higher levels of proline in roots of the transgenic CBF-
lines indicate that CBF caused osmotic adjustment throughout the plant.

In addition to the increases in compatible solutes, marked changes in ion composition
in root tissues also were observed. Levels of K+ ion in the transgenic lines were about 10-
15 fold higher than the nontransgenic controls, in the presence or absence of salt stress;
levels of Ca++ were approximately two-fold higher. To our knowledge this is the first
report that shows the influence of CBF expression on ion composition in plants,
suggesting an effect on ion transport properties in roots. K+ and CaH are major nutrients
and are the two most abundantly distributed cations in plant tissue (Devlin and Witham,

1983). In addition to serving as a primary contributor to cell turgor, K” is also involved in

108

many physiological processes in plants (Zhu et al., 1997). Ca++ is critical for stabilizing
and maintaining cell walls and is involved in various signal transduction pathways
(Knight and Knight, 2001).

Exposure of plants to high salinity leads to a “toxic sodium effect”, whereby excess
Na+ in the cytoplasm causes a deficiency of essential ions such as K)“ and Ca2+, and
competes with K+ ions for binding sites for several enzymes (Bohnert and Jensen, 1996;
Hasegawa et al., 2000; Tester and Davenport, 2003). Living cells tend to accumulate K+
and exclude Na+, to maintain sufficient levels of K+ to perform essential functions that
sodium either cannot fulfill or actively inhibits (Epstien, 1998). The increase in K+
content in CBF-cucumber roots was comparable in the presence or absence of salinity
stress, suggesting that excess Na+ ions did not prevent the elevated K+ accumulation by
the CBF-cucumber roots. Accumulation of Na+ did not differ between transgenic and
nontransgenic plants, and was not significantly increased in the presence of salinity
stress, suggesting selectivity in K+ ion uptake.

A possible explanation for the enhanced in K+ and Cal+ accumulation by the CBF-
cucumber roots may be due to differential expression of different transporters and
channels responsible for ion uptake from the soil or subsequent movement through the
plant. Recent transcriptional profiling ofArabidopsis plants overexpressing CBF/DREB
genes (Seki et al., 2001; Fowler and Thomashow, 2002; Vogel et al., 2005) provided an
Opportunity to examine global gene expression profile changes due to CBF/DREB.
Among the upregulated genes are putative and known transporter proteins and
membrane channel proteins that might play a role in preferential selectivity to K“ and

Ca++ ions over the toxic Na+ ions (Fowler and Thomashow, 2002; Vogel et al., 2005).

109

One of the genes that was upregulated in CBF2 overexpressing Arabidopsis plants
encodes a CaH/ATPase transporter, which contains a CRT element in its promoter region
(V ogel et al., 2005). Calcium has been shown to maintain or enhance the selective
absorption of potassium by plants at high concentration of of sodium (Epstien, 1998),
thus such a transporter might facilitate enhanced Ca“ and K+ uptake. Whether a similar
CBF-inducible transporter is present in cucumber roots, or whether other transporters
may be affected, remains to be determined. In general, the samples used for the
Arabidopsis transcriptional analyses performed to date have been primarily composed of
shoot tissue (V ogel et al., 2005). More comprehensive analysis of root tissue may lead to
identification of additional relevant genes as possible targets for CBF/DREB.

Another possible explanation for K+ accumulation may be related to the elevated
levels of proline accumulation in CBF-cucumber roots. It has been observed that in
response to several abiotic and biotic stimuli in plants, that there is a correlation between
compatible solute accumulation and KJr ion content (Garcia et al., 1993; Hare and Cress,
1997; Backor et al., 2004). Garcia et al. (1993) found that lower Na/K ratios were
obtained upon treating rice roots with several osmoprotectant, including proline. Backor
et al. (2004) recently found that in heavy-metal resistant strains of lichen photobionts
(T rebouxia erici) levels of proline correlated with ability to block K+ ion efflux. Garg et
al. (2002) observed that transgenic rice engineered for trehalose accumulation were able
to maintain a higher level of selectivity for K+ over Na+ uptake in the roots. Thus
expression of CBF genes, either directly (through altered gene expression) or indirectly
(by changing levels of compatible solutes), might affect processes involved in ion

homeostasis in plants.

110

Prior to initiation of salinity treatment, no significant differences were observed
between the transgenic and the nontransgenic plants as measured by vine length, number
of nodes, number of branches, female and male flowers. At the end of the experiment, in
the absence of salinity, CBF lines had less fi'esh weight than the nontransgenic controls;
however, dry weight and fruit number and fi'uit weight were equivalent to the
nontransgenics. These results suggest the possibility to obtain positive effects of CBF for
stress resistance without a negative impact on fruit yield, although this would need to be
verified in more extensive testing situations.

In the presence of salinity stress, CBF-transgenic plants showed enhanced tolerance
to stress conditions compared to the nontransgenic lines as measured by fresh weight, dry
weight, fiuit number, and fiuit weight. CBF1-cucumber lines did not show reduction in
yield, compared to 34% reduction in fi'uit number and more than 50% reduction in fruit
weight in the nontransgenic lines. Thus the CBF genes conferred increased salt stress
tolerance to cucumber plants.

The growing demand to increase food production worldwide, requires a multi-
disciplinary approach that will include adding new land to the agricultural production
area, the use of low quality saline water and the reuse of drainage waters, as well as
developing new salinity tolerant plants capable of adapting to a wider range of
dehydration-inducing stresses. Introduction of the CBF gene into cucumber activated a
variety of salt adaptive responses including increased in compatible solute accumulation
and maintenance of higher I(+/Na+ balance. The CBF-transgenic cucumbers showed

enhanced resistance to salinity stress, with minimal or no reduction in growth and fruit

11]

yield in the absence of salt stress, making this approach very promising to engineer

dehydration resistance in crops.

112

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117

Chapter IV

Introduction of the celery mannose—6-phosphate reductase (M6PR) gene

for mannitol production into cucumber (Cucumis sativus L).

Introduction

Plant response to unfavorable conditions requires adjustment at the molecular,
cellular and whole plant level (Greenway and Munns, 1980; Ingram and Bartels, 1996
and Zhu et al., 1997). One of the many mechanisms that plants have developed to
overcome the low osmotic potential associated with salinity and drought conditions, is
the ability-to accumulate compatible solutes in the cytoplasm (Tarczynski et al., 1993;
Bohnert and Jansen, 1996; Shen et al., 1997; Sakamoto and Murata, 2000). Compatible
solutes (e.g., proline, sugar alcohols, fi'uctans, trehalose, quaternary ammonia
compounds, and tertiary sulfonic compounds), are non-toxic organic metabolites of low
molecular weight that can decrease the osmotic potential of cells without interfering with
cellular metabolism. The compounds can also serve as osmoprotectants to help stabilize
membranes and macromolecular structures (Bohnert and Jensen, 1996, Stoop et al., 1996,
Zhang et al., 1999). Thus, attempts to engineer enhanced salinity tolerance in plants have
included the use of genes encoding key enzymes for biosynthesis of compatible solutes
such as marmitol (Tarczynski et al. 1993; Karakas et al., 1997; Abebe et al., 2003;
Zhifang and Loescher, 2003), proline (Kishor et al., 1995; Zhu et al., 1998; Ronde et al.,
2000), and glycine-betaine (Holmstrom et al., 1994; Hayashi et al., 199 7; Sakamoto et

al., 1998; Holmstrom et al., 2000; Kishitani et al., 2000; Jia et al., 2002)

118

Sugar alcohols such as mannitol, galactitol and sorbitol represent the chemically
reduced form of aldoses or ketose sugars (Loescher and Everard, 1996, Loescher and
Everard, 2000). it also has been suggested that sugar alcohols may play an important role
in scavenging active oxygen species and preventing peroxidation of lipids, which can
lead to membrane damage (Halliwell et al., 1988; Smirnoff and Cumbes, 1989;
Tarczynski et al., 1993; Bohnert and Jensen, 1996; Stoop et al., 1996; Bohnert et al.,
1999). Synthesis of mannitol also was suggested to work as a supplemental mechanism to
dissipate reducing power (N ADPH) accumulated during the light reactions of
photosynthesis (Loescher, 1987).

Mannitol is the most common form of sugar alcohol in nature, and it has been
reported in numerous plant species, including many crops such as carrot, parsley, celery,
green beans, cabbage, pumpkins, coffee and olive trees (Loescher et al., 1992; Stoop et
al., 1996). Plant species that produce mannitol as one of their primary photosynthetic
products tend to have a substantial dehydration stress tolerance as in celery, coffee and
olive trees (Loescher et al., 1992; Stoop et al., 1996).

A role for mannitol in adaptation to dehydration stress is supported by changes in
mannitol production in response to salinity and drought stress. Exposure of celery plants
to 300 mM NaCl resulted in a shift in the pool size of sucrose, mannitol and starch
towards nearly exclusive accumulation of mannitol (Everard et al., 1994). Similar results
were observed when testing celery plants grown in saline hydroponic culture equivalent
to 30% sea water (Stoop and Pharr, 1994). No differences in dry weight were observed
between salt stressed and non stressed plants, suggesting that the total carbohydrate

assimilation was not affected by salinity. Furthermore, salt stress induces expression of

119

key mannitol biosynthetic enzymes and down regulates mannitol eatabolic enzymes (
Zamski et al., 1996; Loescher and Everard, 2000; Zamski et al., 2001; Zhifang and
Loescher, 2003).

Several studies reported enhanced stress protection of transgenic plants by
introducing bacterial genes for sugar alcohol production. Expression of the Echerichz'a
coli mannitol-l-phosphate dehydrogenase gene (mtID) in tobacco resulted in the
accumulation of mannitol in leaves and roots of transgenic tobacco plants as detected by
NMR and mass spectroscopy (Tarczynski et al. 1992; Tarczynski et al., 1993; Karakas et
al., 1997) The mannitol—accumulating tobacco plants had an elevated level of tolerance to
100 mM NaCl, as indicated by flesh weight, plant height and root biomass. In addition to
salt stress resistance, mtID-expressing tobacco plants had higher relative water content in
their leaf tissues, in response to drought stress (Karakas etal., 1997). Eggplant (Solanum
melongena L.) seedlings expressing the mt] D gene exhibited increased tolerance to
salinity stresses as measured by increased germination rate and higher fresh and dry
weight compared to the nontransgenic controls at 200 mM NaCl (Prabhavathi etal.,
2002). Transgenic T2 mtID-wheat plants subjected to 150 mM NaCl showed less
reduction in fresh and dry weight than did the non-transgenic wheat (Abebe et al., 2003).

Similarly, expression of the E. coli GutD gene encoding glucitol-6-phosphate
dehydrogenase, a key enzyme for biosynthesis of the sugar alcohol sorbitol in maize
plants, also increased sorbitol accumulation and enhanced salt tolerance compared to the
nontransgenic controls (Liu et al., 1999). Rice plants (Oryza sativa L.) expressing the E.

coli GutD and the mtID genes, were able to accumulate both sorbitol and mannitol in

120

their vegetative tissue and were more salt tolerant than their nontransgenic counterparts
(T ilahon et al, 2003).

In addition to the use of the bacterial genes, mannitol biosynthetic genes from celery
also have been used to engineer mannitol accumulation (Zhifang and Loescher, 2003). In

celery, mannitol is synthesized from fi'uctose-6-phosphate in three steps:-

Fructose-G-P <—> mannose-S-P __, mannitol-1-P —> mannitol
PMI M6PR Pase

Fructose-6-P (fructose-6-phosphate), mannose—6-P (mannose-6-phosphate), mannitol-l -P (mannitol-l-phosphate), PMI

(phosphomannose isomerase), M6PR (mannose-6-phosphate reductase), Pase (phosphatase)

In celery, fi'uctose-6-phosphate and mannose-6-phosphate are in an equilibrium state;
fructose-6-phosphate is converted into mannose-6-phosphate by phosphomannose
isomerase (PMI). Conversion of mannose-6-phosphate into mannitol-l-phosphate which
is performed by mannose-6-phosphate reductase (M6PR) is the first committed step in
the pathway and appears to be the primary site of regulation of mannitol biosynthesis
(Everard and Loescher, 1997; Zhifang and Loescher, 2003). Mannitol-l-phosphate is
then converted into mannitol by phosphatase enzyme (Pase).

The M6PR gene was fist cloned by Everard et al. (1997) fi'om celery plants (Apium
graveolens L.). Expression of the M6PR gene, in Arabidopsis thaliana, a non-mannitol
accumulating species, enabled plants to grow and complete their normal life cycle
(including seed production) in the presence of 300 mM NaCl (Zhifang and Loescher,

2003).

121

In this work, I investigated the possible use of the M6PR gene to engineer enhanced
dehydration stress tolerance in cucumber plants. Transgenic M6PR-cucumber plants were
produced in our laboratory, analyzed for the presence of M6PR gene by PCR and tested

for accumulation of mannitol in leaf tissue.

122

Materials and methods

Plant constructs, transformation and seed production

The Agrobacterium plant transformation construct containing the M6PR gene, under
the control of the CaMV 35S promoter (Zhifang and Leoscher, 2003) was kindly provided
by W. H. Leoscher (Michigan State University). Cucumber transformation was

performed based as described in Chapter II.

DNA isolation and PCR analysis

DNA was isolated from young leaf samples; 200 mg leaf tissue was ground in liquid
nitrogen and extracted using the Wizard DNA purification kit (Promega, Cat # A7951,
Madison, WI). DNA quality was determined by gel electrophoresis (Sambrook and
Russell, 2001). PCR was carried out according to the procedure of Sambrook and Russell
(2001), using M6PR specific primers (RG278, forward, CACAGCACACACACCAC and

RG279, reverse CACACATTCCCCTCCACA).

ELISA analysis

ELISA test was used to test for the presence of the NPT 11 protein in the To transgenic
plants using NPTII-ELISA® kit (Agdia Inc., Elkhart, IN). One leaf disc of each plant was
collected and placed in 96 well ELISA plate and stored in -80 C° until further analysis.
Just before starting the ELISA procedure, leaf discs were left at room temperature

followed by re-fi'eezing for 2-3 times to ensure cell wall leakage of tissues.

123

 

 

Mannitol analysis

One leaf (6-8 cm diameter; approximate fresh weight 1.5 g) was collected from To
and T1 plants, freeze-dried for 48 hr, ground and placed in 13x100 mm disposable glass
tubes (Cat No. 47729-572, VWR international, West Chester, PA). Total soluble
carbohydrates were extracted according to the procedure of Loescher et al. (1997)
(Appendix A). Standards were previously prepared using 0.1 g of fructose, glucose,
sucrose, rafflnose, mannitol, myo-inositol, all mixed together and dissolved in 100 ml
H20 (this should give 1000 ppm) to establish elution peak time as discussed in more

details at appendix A.

124

Results and discussion

Introduction and expression of the celery (Apium gaveolens}M6PR gene in

cucumber plants

Six transgenic cucumber plants were produced, and the presence of the M6PR gene
was verified in the To plants by PCR (Figure 4-1). Successful transfer of the M6PR gene
into T1 progeny was verified using PCR analysis (Table 4-1). )8 analysis of segregating

progenies was consistent with a single gene insertion for the six families.

Table 4-1. Segregation analysis of T1 transgenic cucumber lines expressing the celery-
M6PR gene. Presence or absence of the gene was determined by PCR analysis

 

 

Line T1 segregation (trans: non) 12 @1)
M1 29 : 8 0.22 ns
M2 31 :18 5.30 ns
M3 42:9 4.6 ns
M4 38:12 0.03 ns
M5 34:8 3.86 ns
M6 22:11 2.75 ns

 

 

 

125

H20

4.
12M1M2M3M4 7 8 9M511§E

415 bp

 

Figure 4-1. PCR analysis of the presence of the M6PR gene in To cucumber plants. Lane
1-11: putative transgenic cucumber plants. Lane 12-14: PCR mix, plasmid control and
H20. The arrow indicates the expected 415 bp product size.

126

 

 

 

mm A. (61mm MOM)

   
 

Mannitol 8.495

21.258

 

 

 

 

-" 25.080

M6PR M3

 

3.
8

r; J 1} 29.227

 

 

 

 

35000

30000‘

25000

150001

100001

FlDt A. (mammal)

 

%om

11.145

12.455

21.259

 

Wild-type

 

7‘
or

.5
O

...
.N
G

...
U

31 7 25.164

27.5 m

 

 

127

Figure 4-2. Gas
chromatography for
mannitol analysis in
one of the cucumber
To Plants (M3) GOP)
and nontransgenic
wild-type cucumber
plant (bottom). The
arrows indicate the
8.5 elution time which
represents the
expected mannitol
peak based on elution
time of the mannitol
standard. The peak at
8.5 is absent in the
wild type cucumber
plant.

Transgenic cucumber plants expressing the M6PR gene have elevated level of
mannitol in leaves compared to the non-transgenic controls

Mannitol accumulation in the To progeny was tested using gas chromatography
(Figure 4-2). All six putative PCR-verified transgenic cucumber plant showed the

expected mannitol peak (elution time 8.5 min) indicating marmitol presence.

Mannitol also was observed occasionally in the T1 M6PR plants (Figure 4-3; for
example, M3), the M6PR T1 plants did not consistently show mannitol accumulation
(Table 4-2). Mannitol accumulation in four families was further investigated. To test
whether the inconsistency in observed mannitol accumulation was due to catabolism of
mannitol or translocation into sink tissue, leaf and sink tissues were collected from
greenhouse growing cucumber plants at two different time points; early in the day (before
10:00 am) and later in the day (between 3-5:00 pm). Mannitol was not detectable in
majority of samples, and no obvious pattern could be determine relative to old vs. young
tissue, leaves vs. flowers or time of the day. Due to the inconsistence in mannitol

accumulation of the T1 progeny, this project was not pursued further.

128

 

 

 

 

 

 

 

 

 

  
      

 

 

 

 

 

counts 1 FlD1A.(o1oacm1eom.uoh1) g
‘ .-. Control
35000 1
1
“°°°° 1
4
25000 4
1
20000 '1
15000
§
10000 1'9
L
o I
'1“ T"'+T T 1'" I' '1 n 1‘ 1‘ '“1
75 10 12.5 15 17.5 20 22.5 25 27.5 ....
K
3
counts F101 A. mmsmm ..N; M6PR M3
35000 3
1
30000 1
1
25000 '1 ID
..
1 *0.
1 C
20000 1 g
1 E
15000 1
.1
10000 1 l
4 8 ._ g
‘ v- 0 h
- a ..- § 3 ... .. o 5
_ Q Q q
M—a—A—n—e—M—
o 1
1'0 13 23 23 m'

 

Figure 4-3. Gas chromatography for mannitol analysis in wild type (top) and T1 plant
fi'om family M3 (bottom). The arrows indicate the 8.5 elution time which represent the
expected mannitol peak based on elution time of the mannitol standard. The first peak
which represent mannitol is absent in the wild type plants and indicated by the black
arrow.

129

Table 4-2. Mannitol accumulation in segregating Tl-M6PR plants. Different tissues were
analyzed (young leaves, fully expanded leaves and female flowers) were collected at two

time points (before 10:00 am and after 3:00 pm) from M6PR plants growing in the
greenhouse.

 

Number of plants showing mannitol accumulation

 

 

PCR
$16121 positive Young leaves fully expanded leaves Flowers
. - Early Late Early Late Early Late
Ml 12:4 2:14 1:14 0:14 1:14 3:10 1:13
M3 9:5 0:14 0:14 4:10 2:12 7:4 2:12
M4 14:3 4:13 2:15 6:11 2:15 0:17 -—---
M5 10:8 2:16 1:17 3:15 6:12 1:16 3:15

 

130

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Conclusions and future work

In the current work, I produced several lines of transgenic cucumber plants
expressing the Arabidopsis thaliana transcriptional regulators CBF1/DREBb and
CBF3/DREBa genes under the control of the constitutive CaMV35S promoter (Chapter
2). Greenhouse experiments indicated that CBF1 and CBF 3 cucumber plants showed
array of adaptive responses to the imposed salinity and drought stress, including the
accumulation of compatible solutes (e. g., proline and soluble sugar), less reduction in
photochemical efficiency as measured by chlorophyll fluorescence, less growth
reduction, and accumulation of higher above ground dry matter compared to non-
transgenic controls. These observations are similar to other reports that introduced CBF
genes into tomato, tobacco and rice; however, CBF homologues have been identified in
all of those species. This raises the question of whether cucumber has CBF homologues. I
tried to look for CBF homologues in cucumber by following more than one approach; for
example, trying southern hybridization using CBF] and CBF 3 as probes with low
stringency washing conditions, designing degenerate primers for conserved common
motifs among the known CBF genes from different species, and finally using the
cucumber genomic library to screen for CBF homologues. The fact that I could not clone
any homologues does not role out the possibility that cucumber may have CBF
homologues. One thing to pursue in the future would be expand efforts to identify CBF-
homologs from cucumber and ask if they are true functional homologues of the
Arabidopsis-CBF gene family, by testing for expression, phenotypic and physiological

changes in Arabidopsis plants overexpressing these homologues. It would also be

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beneficial to understand how these CBF-homologues are regulated in response to
different stresses.

Despite the enhanced stress resistance in tomato, expression of predicted COR
homologs did not increase in response to overexpression of CBF. Given the fact that
there are marked differences between Arabidopsis and tomato, in gene expression in
response to CBF-overexpression, and the current failure to observe CBF-regulation of
predicted tomato orthologs to Arabidopsis CBF-regulon genes, further suggests that
responses may differ in heterologous systems, which in turn may make finding of CBF-
target genes in cucumber more challenging. One way to answer this would be by using
microarray analysis to compare gene transcription profiles of CBF-expressing and
nontransgenic control cucumber plants. The one drawback in this approach is that the
microarray chip would be from a different species (Arabidopsis). Another alternative
could be the generation of subtractive libraries from CBF-transgenic and nontransgenic

cucumber plants. I

Salinity treatment (50 and 100 mM NaCl) caused a marked increase in soluble sugar
and proline accumulation in CBF-plants beyond what would be expected from an
additive effect of CBF and salt stress. The same trend was also observed in some of the
work on CBF-expressing plants, although it was less pronounced than in our study. The
lack of differences in CBF levels in the transgenic lines in the presence or absence of
salinity indicated the presence of other levels of regulation downstream of CBF in
cucumber. Recently, Cook et al. (2004) showed metabolic changes in Arabidopsis in
response to cold acclimation and overexpression of CBF genes. The idea that CBF genes,

which encode transcriptional regulators, could activate waves of responses makes it of

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interest to monitor metabolomic changes in cucumber plants under different stress
conditions. Thus questions here would be what are the metabolic changes in cucumber
plants in the presence or absence of salinity? What are the changes in the CBF-cucumber
line in the presence or absence of salinity? How would these differ at different stress
levels? Informations from these experiments might also help identify possible CBF target
genes. These types of studies could provide insights into the secondary regulation in

plants and how this is relate to gene expression profiling.

We also reported for the first time that expression of the CBF system in cucumber
plants caused an increase in ion composition of cucumber roots. Cucumber roots
accumulated higher levels of K+ and Ca++ ions in the presence or absence of salinity
stress, suggesting that excess Na+ ions did not prevent the accumulation of K" to higher
levels in the roots of CBF-cucumber plants and maybe an increased selectivity in K+
uptake or due to differential expression of different transporters and channels responsible
for ion uptake or movement through the plant. The fact that CaH/ATPase transporter was
one of the upregulated genes in CBF-overexpressing plants raises questions about ion

transport regulation in CBF-expressing plants.

Calcium has been shown to maintain or enhance the selective absorption of potassium
over sodium in plants under salt stress (Epstein, 1998). This may be through activation of
Na+/H+ antiporters at the plant plasma membrane or maybe by activating some high K
selective channels. Another question is whether changes in Na+/1C ratio is an indirect
side effect of accumulation of compatible solutes in plant tissue? If so then the

phenomenon would be similar to that in previous reports (Chapter III). One way to

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answer these questions would be by using tissue specific microarray analyses, or more
comprehensive analysis of root tissue may lead to identification of additional genes as
possible targets for CBF/DREB. Another possibility would be to generate subtractive
libraries from roots of CBF-cucumber plants and compare it with nontransgenic control

cucumber plants.

Finally, when we first started this work we had few questions to ask; can CBF confer
dehydration stress tolerance in a heterologous system? What are the limitation of the
transgenic cucumber plants in response to salinity and drought? What are the possible
effects of the CBF gene on growth and yield data of cucumber plants in the presence or
absence of salinity stress? Although my current work was an attempt to answer many of
these questions, our findings also raised new questions:

1- What are the metabolic changes in cucumber plants in the presence or absence of
salinity and drought? How does it differ in CBF cucumber lines in the presence or
absence of dehydration stresses?
2- How would these differ at different stress levels? Can these metabolic changes be used
to predict possible CBF-target genes in cucumber?
3- What cause the accumulation of higher K+ levels in roots of CBF-cucumber plants; it is
due to changes in K+ uptake, K+ selectivity, or in ion movement in plant tissue?
4- The changes in the Na+/K+ ratio an indirect side effect of accumulation of compatible
solutes in plant tissue?

In summary, results demonstrate the potential usefulness of the CBF/DREB system

in providing elevated levels of dehydration stress tolerance in salt sensitive species such

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as cucumber and also raise new questions about the function of CBF in regulating stress

tolerance related phenotypes.

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Appendix A: Proline, soluble sugar and mannitol protocols

Proline analysis

One leaf (3-4 cm diameters) was collected from each plant, and freeze-dried for 48
hrs and ground to assure uniformity. Each sample was split into two equal weights for
proline and sugar analysis. Proline analysis was carried out using the ninhydrin reagent
procedure described by Troll and Lindsley, (1955). Tissue samples (0.1-0.2 g dry weight)
were placed in 13x100 mm disposable glass tubes (Cat No. 47729-572, VWR
international, West Chester, PA) and proline extracted fiom dried tissue by overnight
soaking in 3.0 m1 of distilled H20 followed by heating at 80 C° for 30 min. The
supernatant was then transferred into a fresh 13x 100 glass tube and the heating step was
repeated with fresh 3 ml of water H20 for 30 min. Tissues and debris were removed from
the samples by centrifuging for 1 min at 2500 g. the supernatant was then transferred into
fresh glass tubes. 400 pl of the extract solution was transferred to microcentrifuge tubes
containing 400 pl of the ninhydrin reagent (prepared as described below) and 400 pl of
glacial acetic acid. One gram of Ninhydren reagent (Sigma-Aldrich, St. Louis, MO) was
prepared by dissolved in 16 m1 of concentrated phosphoric acid in the dark; then bringing
the volume to 40.0 ml with glacial acetic acid (MERCK KGaA, Darmstadt, Germany).
The sample tubes were closed and placed in a boiling water bath for an hour. The tubes
were removed from the water bath, and cooled to room temperature for 10-15 min. The
samples were transferred into a new 13x100 mm disposable glass tube, and 2.5 m1 of
toluene added to each tube. The tubes were periodically gently shaken by hand for a few
second to allow the red color to dissolve in the toluene phase. Absorbance of the toluene

phase was measured spectophotometrically at A515. The standard curve was prepared by

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dissolving 10 mg of LProline (Sigma-Aldrich, St. Louis, MO) into 10 ddeO m1
(lmg/ml, lOOOppm). l, 2, 4, 6, 8, and 10 pl of the standard was added into a fresh
microcentrifuge tube then brought up to 400 pl with water and processed in as described

above.

Total soluble sugar analysis

Total soluble sugar analysis was performed by the phenol/sulfuric acid method as
described by Dubois et al., (1956). Three ml of 80% ethanol was added to 0.1-0.2 g of the
dried samples in 13x100 mm glass tubes and incubated overnight at 4 C°. The next day,
total soluble sugars were extracted by boiling the samples for 30 min; the supernatant was
transferred into a fresh 13x 100 glass tube and the heating step was repeated with fresh 3
ml of 80% ethanol. Tissues and debris were removed from the samples by centrifuging
for 1 min at 2500 g. The supernatant was then transferred into fresh glass tubes followed
by adding 3 m1 of chloroform (MERCK KGaA, Darmstadt, Germany); samples were
mixed by inverting several times and centrifuged for 5 min at 2500 g. The clear upper
aqueous phase was transferred into a fresh 13x100 mm disposable glass tubes. 100 pl of
the extract solution was added to 1.9 ml HzO, followed by 50 pl 80% phenol (Sigma-
Aldrich, St. Louis, MO); 5 ml of concentrated sulfuric acid was quickly added. Samples
were lefi for 10 min before gently vortexing at low speed. Absorbance of the mix was

measured at A485.

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Mannitolggalvsis

One leaf (6-8 cm diameter; approximate fresh weight 1.5 g) was collected from To
and T1 plants, freeze-dried for 48 hr, ground and placed in 13x100 mm disposable glass
tubes (Cat No. 47729-5 72, VWR international, West Chester, PA). To total extract
soluble carbohydrates, samples were boiled in 3 ml 80% ethanol for 30 min and the
supernatant transferred into fresh 13x100 mm glass tubes. The pellet was re-extracted by
boiling again with 2 ml 80% ethanol for another 30 min. Afier the extraction, 5 ml water
was added to each sample, and samples were transferred into 15 ml disposable Corning
tubes (Corning Inc., Corning, NY). Five ml of chloroform (MERCK KGaA, Darmstadt,
Germany) was added to each sample; the samples were capped and shaken by hand and
then centrifuged for 5 min at 2500 g. The clear upper aqueous phase was transferred into
a fresh 13x100 mm disposable glass tubes and placed in heating blocks at 50 C° in the
fume-hood until the samples were completely dried (approximately 2-3 hrs.). One ml of
derivatization solution [pyridine (Sigma-Aldrich, St. Louis, MO) kept over NaOH pellets
(Sigma-Aldrich, St. Louis, MO)] was added to each tube. The tubes were capped tightly
and vortexed until all the dried sugar crystal were dissolved. The tubes were placed in a
heating block at 70-80 C° for an hour; samples were vortexed every 30 min, then
transferred to a tube rack and allowed to cool to room temperature for 10-20 min. One ml
of room temperature hexamethyldisilazane (HMDS) (Sigma-Aldrich, St. Louis, MO) was
added to each sample. Samples were allowed to stand for 20-30 seconds before adding
100 pl of trifluoroacetic acid (TFAA) (Sigma-Aldrich, St. Louis, MO), followed by brief
vortexing. Samples were incubated for an hour at room temperature, then 1.0 m1 of clear

upper aqueous phase was transferred into GC Vials (Alltech Corporation, Deerfield, IL),

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capped tightly and placed in the auto-sampler tray for GC-Series H analysis (Hewlett
Packard 5890-11 gas chromatography, Palo Alto, CA) which was fitted with a DB-17
capillary column (J &W Scientific, Folsom, Ca, USA). Standards were previously
prepared using 0.1 g of fructose, glucose, sucrose, raffinose, inositol, all mixed together
and dissolved in 100 ml HzO (this should give 1000 ppm). Different volumes of the
standard mix were transferred into 13x100 mm glass tubes (1.0, 0.5, 0.1, 0.05, 0.025 and
0.01 ml) and were placed in heating blocks at 50 C° in the fume-hood until the samples
were completely dried (approximately 2-3 hrs.) followed by the same derivatization steps

that was described earlier.

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