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This is to certify that the

dissertation entitled
Studies on Low Temperature Induced Gene Regulation and

Freezing Stress Tolerance in Arabidopsis
presented by

Daniel George Zarka

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Plant Biology

   

Major professor

Date July 30, 2001

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6/01 cJCIRCIDateDuepSS-QJS

STUDIES ON LOW TEMPERATURE INDUCED GENE REGULATION AND
FREEZING STRESS TOLERANCE IN ARABIDOPSIS

By

Daniel George Zarka

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Plant Biology

2001

ABSTRACT

STUDIES ON LOW TEMPERATURE INDUCED GENE REGULATION AND
FREEZING STRESS TOLERANCE IN ARABIDOPSIS

By

Daniel George Zarka

Low temperature is a significant environmental stress factor limiting
growth and productivity of agronomically important plants in many parts of the
world. Plant responses to low temperature are complex, involving coordinated
regulation of multiple biochemical pathways leading to expression of a number of
genes encoding proteins that contribute to cold acclimation. Genes whose
expression is increased during low temperature stress include those associated
with freezing stress as well as the cellular protective enzymes, proteins and other
compounds involved in osmotic adaptation to and tolerance of the cellular
dehydration caused by freezing stress. Other up regulated genes include the
several signaling proteins such as protein kinases and transcription factors that
play roles in adapting plants to tolerate freezing temperature. Some of the
regulatory DNA sequences that confer responsiveness to low temperature and
associated stresses have been identified and transcription factors that interact
with such cis-elements have been partially characterized. Work to identify
additional sequences and factors involved in the sensation of low temperature
and the transduction of the earliest gene response signals is continuing through

the use of reverse genetics and mutagenesis studies. In particular, the following

describes efforts to characterize some of the functions of the CBF genes as
transcriptional activators in the low temperature signal transduction pathway and
also to identify sequences responsible for regulation of their own activation.
Through the use of promoter deletion analyses, a 155bp fragment of the CBF2
promoter was found to be responsible for low temperature inducible expression
of the gene as well as responsiveness to mechanical agitation, ABA and the
translation inhibitor cycloheximide. Some transcript accumulation at warm
temperatures in the presence of cycloheximide also led to the idea that an
inhibitor of CBF as well as an inducer of CBF expression (ICE) may be working
to precisely regulate CBF expression.

Work to identify additional genes associated with the low temperature
signal transduction pathway upstream of the COR genes using a COR gene
promoter and marker gene fusion system and chemical mutagenesis was
performed. Markers included the BAR gene, codA, and the R gene. Several
mutants affected in their expression of both the R gene marker and one of the
COR genes were isolated but the change in expression was likely due to gene
silencing and not interruption of the cold signaling pathway. Plants exhibiting
loss of the endogenous COR78 expression were examined with an ion leakage
assay to assess the significance of the loss of this one COR gene on freezing
tolerance. No significant change was seen in these plants, however, a wealth of
information was obtained concerning the use of different reporter genes and

possible methods to improve the search for genes.

ACKNOWLEDGMENTS

I first and foremost want to acknowledge my wife Kelly for her endless
support throughout my graduate career. Her positive attitude, quick wit and
helpful encouragements inspire me.

I thank also my advisor Dr. Michael Thomashow for his patience and
support as well as the rest of my committee, Dr. Sears, Dr. Triezenberg and Dr.
Keegstra. They let me do a lot of things my own way.

I benefited from knowing all the members of the Thomashow lab
especially, Eric Stockinger, Sarah Gilmour, Keenan Amundsen, HuanYing Qin,
Ann Gustafson, Daniel Cook and Lenny Bloksberg and so I thank you.

And finally, to my parents for bringing me up right with love and support, I

will always be indebted.

TABLE OF CONTENTS

CHAPTER 1: Plant cold acclimation and freezing stress tolerance

Introduction .......................................................................................................... 1
Freezing, dehydration and damage ..................................................................... 4
Cold upregulated genes ..................................................................................... 11
Additional low temperature induced genes ........................................................ 17
Conclusion ......................................................................................................... 23
Literature cited ................................................................................................... 24

CHAPTER 2: Initial characterization of CBF gene function and identification of a
promoter region sufficient for low temperature regulated expression of CBF2

Summary ........................................................................................................... 35
Introduction ........................................................................................................ 36
Results ............................................................................................................... 40
Discussion ......................................................................................................... 62
Methods and materials ....................................................................................... 69
Literature cited ................................................................................................... 73

CHAPTER 3: Screen for mutations in the signal transduction pathway leading to
induction of COR genes

Summary ........................................................................................................... 77
Introduction ........................................................................................................ 78
Results ............................................................................................................... 81
Discussion ......................................................................................................... 91
Methods and materials ....................................................................................... 97

Literature cited ................................................................................................. 101

Chapter 4: Perspectives and Future Directions

Introduction ....................................................................................................... 1 04
Future directions ............................................................................................... 105
Conclusions ...................................................................................................... 107
Literature cited .................................................................................................. 109

vi

LIST OF FIGURES

Figure 1.1. Expression of CBF1 and COR genes in Arabidopsis plants. .......... 20

Figure 2.1. Expression pattern of CBF and COR genes after exposure to
low temperature ................................................................................................. 42

Figure 2.2. Pattern of gene transcript accumulation in response to
dehydration, ABA and mechanical agitation ...................................................... 44

Figure 2.3. Expression pattern of CBF and COR15a genes after cold
shock (CS) and a slow temperature downshift ................................................... 46

Figure 2.4. Expression of CBF genes and their targets after an extended
period of cold ..................................................................................................... 46

Figure 2.5. Diagram depicting the three CBF promoters and CBF2
promoter fragments used in promoter analysis experiments ............................. 48

Figure 2.6. Sequence from CBF2 promoter deletions and mutations from

-419 to +152 ........................................................................................................ 49
Figure 2.7. Staining of transgenic plants containing whole CBF

promoter/GUS fusions ........................................................................................ 50
Figure 2.8. Whole promoter CBF/GUS fusions demonstrate low

temperature induced promoter activity ............................................................... 51
Figure 2.9. Seedling staining indicating CBF promoter/GUS gene activity. ....... 51
Figure 2.10. Expression pattern of deleted CBF promoter/GUS fusions ............ 54
Figure 2.11. Monomer and dimer promoter fragment expression pattern .......... 56
Figure 2.12. Effects of ABA and drought treatment on CBF expression ............ 57
Figure 2.13. Turnover rate of CBF gene transcript ............................................ 58
Figure 2.14. Relative promoter activity after cold, ABA and drought

treatments ........................................................................................................... 58
Figure 2.15. Response of CBF promoter to hypo-osmotic shock. ...................... 59
Figure 2.16. Responsiveness of CBF promoters to mechanical agitation ......... 61

vii

Figure 2.17. Effects of cycloheximide treatment ................................................ 62

Figure 2.18. Effect of site directed mutagenesis on ABA and low

temperature responsive expression ................................................................... 63
Figure 3.1. Expression pattern of COR15a promoter/GUS fusions. .................. 82
Figure 3.2. Determination of transgene copy number in transformed

plants .................................................................................................................. 86
Figure 3.3. Gene expression pattern in transgenic plants ................................. 87
Figure 3.4. Gene expression in mutagenized plants ......................................... 89

Figure 3.5. lon leakage assay to determine cellular fitness or loss of
fitness after a three day cold acclimation period ................................................ 90

viii

Chapter 1

PLANT COLD ACCLIMATION AND FREEZING STRESS TOLERANCE

Introduction

Anchored in the environment in which they germinate, plants are often
subjected to adverse growth conditions including extremes in temperature,
drought and salinity. To survive, plants have developed a complex signaling
system that allows them to sense and respond to an ever-changing environment.
Tolerance of freezing is considered a primary limiting factor for growth,
productivity and distribution for many important crop plants and wild species in
the northern latitudes throughout North America, Europe, Asia and the high
altitudes of South America. Considerable time and energy has been directed
toward efforts to enhance freezing tolerance in agriculturally important plants.
However, limited advancement has been achieved in the progress toward
increasing freezing stress tolerance in most plants due in part to the complexity
of the problem that is being addressed. Multiple stress factors, both biotic and
abiotic are often occurring at the same time and affect plants that reside in
habitats that experience freezing temperatures. Understanding these influences
individually is difficult enough but the problem is exacerbated by unknown roles
of the potentially hundreds of genes involved in the acquisition of freezing
tolerance and the need to battle the multiple stresses associated with a low

temperature environment.

Freezing temperature is one part of a set of stresses that must be
tolerated in order to survive in an environment that frequently produces sub-
optimal growth temperatures. For many plants growing in temperate zones,
environmental factors such as wind and ice formation can increase the tendency
for dehydration. Water logging and ice encasement cause anaerobic stresses.
High light combined with low temperature can lead to photoinhibition. In addition
plants that over winter may be subject to several diseases (Gaudet, 1994;
Tronsmo et al., 1993; Wise 1995).

Tolerance of these low temperature stresses can vary considerably
between species of plants. If one considers just the effect of cold temperatures,
plants can be classified into different groups based on their ability to cope. Most
tropical plants and many important crop plants such as rice and corn show injury
or/and loss of viability at temperatures between 0°C and 15°C and are
considered chilling sensitive (Anderson et al., 1994; Taylor et al., 1974). Plants
such as cultivated potato (Solanum tuberosum) can tolerate low nonfreezing
temperatures but are injured or killed when ice begins to form in their tissues
(Sukumaran and Weiser, 1972). Other plants can survive ice formation in their
tissues but are killed by high sub-zero temperatures. For example, many types
of citrus plants are injured or killed at temperatures of -3°C to -6°C (Yelenosky
and Guy, 1989). When acclimated, many of the cereals and temperate
herbaceous species of plants can survive freezing to temperatures ranging from

-10°C to -30°C (Fowler and Gusta, 1979; Scorza et al., 1983). Some of the most

hardy woody perennials can survive -30°C to -50°C in nature or even immersion
in liquid nitrogen (-196°C) when fully acclimated (Sakai, 1960).

In plants with the same genetic potential to acclimate to freezing
temperature, variations can be seen in the maximum degree to which a single
individual or type of plant can acclimate depending on the immediate
surroundings of the plant or other stresses affecting its growth. For example,
depending on growth conditions from year to year, some types of winter wheat

may have a maximal tolerance that varies from -16°C to -24°C (Fowler 9! al.,

1983). Lengthening the time over which the plant acclimates can often increase
the degree of tolerance. Although time is only one of many factors contributing to
tolerance, the longer the acclimation period the more likely a plant will be able to
reach its maximum tolerance level.

Because the ability to gain freezing tolerance requires time, even the most
freezing tolerant species may be sensitive to low temperatures in a
nonacclimated state such as in the spring after growth has resumed or until
growth stops in late summer or fall. At the end of a growing season however,
plants respond to cues such as decreasing temperature and changes in day
length in such a way as to increase their freezing tolerance, which allows them to
survive the prolonged periods of low temperatures. This adaptive change
leading to an increase in tolerance to freezing has been termed cold acclimation.
Tolerance to freezing that is gained by cold acclimation is transient though and is
rapidly lost during environmental conditions in which temperatures rise above

freezing. Therefore cold acclimation can be considered a dynamic physiological

process that increases the level of a plant’s tolerance to low temperature to
different extents depending on the level of conditioning prior to the freezing
stress. The level of acclimation is determined by factors such as the rate of
cooling, the minimum temperature reached and duration at that temperature, the
rate of thawing and interaction with other stresses in the environment. The
genetic potential and physiological strategies used to survive these stresses vary
greatly between plants. For some plants, preventing freezing by allowing water
within the tissues to supercool without forming ice crystals increases the chance
of survival. Others form ice but prevent its spread with barriers or prevent
movement of water from within cells out to the ice. Mechanisms to survive
dehydration are important because once extracellular ice is formed, the crystal
grows by drawing water from the cells. Because the water potential of ice is
lower than that of liquid water within the cell, water moves down a chemical
potential gradient from inside the cell to extracellular spaces which leads to the
cellular dehydration stress. Therefore, factors that influence a plant’s ability to
survive freezing temperature include whether supercooling or ice formation
occurs, the location of ice formed, whether mechanisms exist to prevent the
spread of ice or the movement of water to ice and the ability to tolerate
dehydration stresses.

Freezing, dehydration and damage

When the air or soil temperatures drop below the freezing point of water,
ice crystals can begin to form in plant tissues. lce normally forms in vessels of

the xylem in leaves and stems and intercellular spaces first due to relative

negative osmotic potential of the sap. The temperature required for formation of
ice, even here, may be several degrees below 0°C because the water molecules
need a nucleation site to first start forming the ice crystals. Location of
nucleation sites in plant tissues can be controlled by the composition and
structure of cell walls. Once ice has formed, subsequent nucleation occurs on
the surface of the ice crystal itself. Ice crystals then spread throughout the
vessels and extracellular spaces. As the ice crystal grows and accumulates
water, it excludes the solutes and these accumulate in the remaining liquid. lce
formation continues until the chemical potential of the unfrozen water is in
equilibrium with the ice (Mazur, 1970). Because solutes are excluded from ice,
the extracellular unfrozen solution becomes more concentrated. This creates a
gradient in the water potential between intracellular and extracellular solutions,
which causes water to move out of the cytoplasm until water potentials
equilibrate again. This results in cytoplasmic dehydration.

In a nonacclimated cell after ice nucleation has occurred, large amounts of
water cross the plasma membrane. As ice forms, large crystals cause tissues to
swell and the cytoplasm experiences dehydration, which may be extreme.
Damage resulting from freezing most often will result in death for the cell.
Acclimation reduces damage through several adaptive changes that take place in
the cell such as increasing the solute concentration within the cytoplasm to
decrease water movement out of the cell, changing the composition of the
plasma membrane to tolerate freeze thaw processes and avoidance of freezing

in the first place by supercooling. Supercooling is achieved in plants by

minimizing nucleation sites. It avoids the damage caused by cytoplasmic
dehydration, however, the protection cannot exceed the critical threshold for ice

nucleation, which is about -38°C. The degree of success minimizing nucleation

sites determines the temperature to which water in a particular plant or tissue can
supercool.

While many types of damage to cells can affect viability, the immediate
target of freezing induced damage appears to be the membrane systems in plant
cells (Pearce and Willison, 1985; Steponkus, 1984). Many forms of membrane
damage have been recognized as a consequence of freezing induced cellular
dehydration and therefore a key function of the cold acclimation process is to
stabilize membranes. Damage can result from expansion-induced lysis or local
changes in phase from lamellar to non-lamellar (Pearce, 1985; Pearce and
Willison, 1985; Pearce, 1988; Steponkus and Webb, 1992; Steponkus et al.,
1993; Uemura et al., 1997). In the case of expansion-induced lysis, as water
diffuses out of the cell, extracellular ice forms. The plasma membrane loses
tension and through a process of endocytotic vesiculation loses parts of the
membrane. When the cell thaws, the influx of additional water causes expansion
and eventual lysis of the cell (Steponkus, 1984).

Non-lamellar, or hexagonal ll phase transitions result from the
destabilization of membrane protein lipid interactions (Williams, 1990). The
appearance of fracture-jump lesions, identified through the use of a freeze
fracture electron microscopy technique, is an indication of changes in lipid

interactions in the membrane bilayer (Webb and Steponkus, 1993). Membrane

stabilization can be aided by changes in lipid composition (Steponkus et al.,
1993; Uemura et al., 1997). Other soluble molecules including carbohydrates
that accumulate during growth at low temperature can also stabilize membranes
(Strauss and Hauser, 1986; Crowe et al., 1992; Anchordoguy et al., 1987) and
thus help cells survive.

Other potential effects resulting from freezing include inhibition of catalytic
activity of multimeric enzymes due to dissociation of its subunits and loss of
conformational stability at temperatures below the freezing point of water leading
to protein denaturation (Li et al., 1999). Certain molecular chaperones may play
roles in maintaining conformation and assembly of proteins during freezing stress
and thus allow tolerance of the stress. Members of the family of HSP70 protein
molecular chaperones are influenced by low temperature. Increases in
expression could be due to a decrease in temperature or due to the loss of
cellular homeostasis caused by processes affected by low temperature, such as
impairment of protein biogenesis or cold denaturation of cold labile proteins.
Increases in expression are most pronounced at chilling temperature but several
of the stress 70 and other molecular chaperones are upregulated several hours
after a freeze/thaw stress (Li et al., 1999; Anderson et al., 1994; Parswell and
Lindquist, 1993).

Oxidative stress tolerance is also an important component of freezing
tolerance. Reactive oxygen species can be deleterious to membranes and
photosystems (Tanino and McKersie, 1985; Wise, 1995). Enhancement of

antioxidative mechanisms may minimize damage and are normally expressed at

higher levels in low temperature stressed plants. McKersie et al. (1993) have
shown that transgenic alfalfa that over expressed Mn-superoxide dismutase
(SOD) cDNA had enhanced freezing tolerance and higher growth rate after a
sub-lethal freezing stress. Less benefit was seen after more severe freezing
temperature and higher levels of active oxygen indicating the SOD system may
have been overwhelmed. The levels of other potential oxidative stress protective
enzymes including ascorbate peroxidase, glutathione reductase and catalase
(Bridger etal., 1994; Hull et al., 1997; Prasad, 1997; Prasad et al., 1994; Jahnke
et al., 1991; Walker and McKersie, 1993) increase after exposure of cells to low
temperature. Working in combination these may help reduce the damage
caused to photosynthetic systems by reactive oxygen molecules (Krivosheeva et
aL,1996)

As previously stated, cold acclimation in plants is an adaptive response to
low non-freezing temperatures that leads to an increase in tolerance to below
freezing temperatures. In addition to low temperature, dehydration stress can
lead to an increase in freezing tolerance (Guy et al., 19923). This is not
surprising since during periods of freezing, extracellular ice formation causes
cellular dehydration. Analysis of the major water stress induced genes and the
low temperature induced genes reveals similarities in the families of genes that
are induced. One of the major families is the late embryogenesis abundant or
LEA. Many of the Arabidopsis COR genes fall into this family and will be
discussed later. Dehydration stress also results in the endogenous synthesis

and accumulation of abscisic acid (ABA) (Write and Hiron, 1969) and may trigger

the acclimation response. ABA levels are known to increase in a variety of plants
during cold acclimation. This led to the hypothesis of Chen and Gusta (1983)
that cold acclimation is activated through an ABA mediated pathway. This idea
was further strengthened with the observation that exogenous applications of
ABA to plants kept at warm temperatures enhanced their freezing tolerance
(Chen et al., 1983; lshikawa et al., 1990; Orr et al., 1986). And with the
commonality of molecular responses and genes associated with drought and
cold acclimation, ABA appeared to be a common agent that mediated responses
to both types of stresses (Close et al., 1989; Gilmour et al., 1992; Horvath et al.,
1993; Nordin etal., 1993; Yamaguchi-Shinozaki and Shinozaki, 1993a). In
addition, Arabidopsis plants carrying mutations in ABA synthesis (aba1) or in the
plant’s ability to respond to ABA (abi 1) are less freezing tolerant than wild type
(Gilmour and Thomashow, 1991; Heino et al., 1990; Mantyla et al., 1995).
However, ABA does not seem to be required for activation of several cold
responsive genes since their expression is still induced by low temperature in
both the aba1 and abi1 mutants (Gilmour and Thomashow, 1991; Nordin et al.,
1991 ). The discovery of separate cis-acting DNA regulatory elements in the
promoters of cold responsive genes also supports the idea of independent cold
induced and ABA regulated pathways (Baker et al., 1994; Yamaguchi-Shinozaki
and Shinozaki, 1994). lshitani et al. (1997), isolated mutants that increase or
decrease expression of cold regulated genes in response to both cold and ABA.
These results suggest that the cold and ABA regulatory pathways are not

completely independent pathways but are rather part of a network that shares

some components. Identification of more factors in the signal transduction
pathways should help clarify the relationship between cold and ABA.
Regardless of the mechanism by which the low temperature signal is
transmitted, certain facts about the cold acclimation process are known. For
instance, during the acclimation process, a plant’s metabolism changes
considerably. Rates of respiration, photosynthesis and synthesis of proteins and
other compounds are all up-regulated (Guy, 1990). Solutes accumulate during
low temperature growth that can change osmotic potential, which influences the
freezing or ice nucleation point in cells. The major changes in osmotic potential
are due to changes in sugars (Levitt, 1980; Sakai and Larcher, 1987). Some
studies on plants that demonstrate this include the observation that an increase
in soluble sugar content of cabbage leaves, led to acquisition of freezing
tolerance. The slow increase of sucrose, glucose and fructose correlates with
the degree of freezing tolerance. The loss of freezing tolerance after one day of
deacclimation is associated with large reductions in sugar content (Sasaki et al.,
1996). In alfalfa, the accumulation of sucrose, stachyose and raffinose correlates
with development of freezing tolerance. In addition, differences in freezing
tolerance between a cold tolerant and a cold sensitive cultivar are closely
associated with accumulation of raffinose and stachyose (Castonguay et al.,
1995). In winter cereals, fructans are storage carbohydrates that act to regulate
sucrose levels and provide osmoregulation. In response to freezing they are
hydrolyzed into soluble sugars, which provided some protection against freezing

(Olien and Clark, 1995; Livingston and Henson, 1998).

10

Membrane lipid composition also plays an important role in cold
acclimation. Early work showed that as plants acclimated to low temperature,
lipids became more unsaturated (Lynch and Steponkus, 1987; Steponkus, 1984).
More recently, the composition of lipids that make up the membranes has been
shown to change as a result of acclimation to cold. ln Arabidopsis thaliana, the
proportion of phospholipids increase in percent over total lipids while the
proportions of cerebrosides and free sterols decrease (Uemura et al., 1995). In
cold acclimated winter rye and oats, the proportions of phospholipids increase
significantly as a result of increases in proportions of phosphotidylcholine and
phosphotidylethanolamine. The relative proportion of diunsaturated species also
increases and there is a decrease in the proportion of cerebrosides in the plasma
membrane (Lynch and Steponkus, 1987; Uemura and Steponkus, 1994). These
changes all contribute to the increase in freezing tolerance that occurs during
acclimation (Steponkus et al., 1988).

Cold up-regulated genes

In 1970, Weiser suggested that cold acclimation required transcriptional
activation of a set of genes that are not normally expressed under non-cold
acclimating conditions. Guy et al. (1985) were the first to demonstrate the
accuracy of this supposition when they showed that exposure of spinach leaves
to low temperature (5°C) led to induction of newly translatable mRNAs. A
number of plant genes have since been isolated from both monocotyledonous
and dicotyledonous species that are up-regulated in their mRNA levels by low

temperature treatments. Many of these genes encode proteins with known

11

enzymatic activities that may contribute to freezing tolerance. They include some
relating to primary metabolism or stress metabolism such as the housekeeping
genes B-tubulin (Chu et al., 1993) and EF-1or, which is involved in general up-
regulation of protein synthetic capacity (Berberich et al., 1995; Dunn et al., 1993;
Hong et al., 1997). Alcohol dehydrogenase is up regulated and may aid in
anaerobic metabolism during periods of ice encasement (Jarillo et al., 1993).

The levels of cold up-regulated heat shock proteins also increase. These may be
used for protein folding and assembly (Parswell and Lindquist, 1993). The heat
shock proteins may play roles in protecting proteins from denaturation at low
temperature or they may be up regulated to deal with increased demand for
protein folding and assembly caused by the up regulation of general protein
synthetic machinery during cold acclimation.

Other up regulated genes or proteins probably play protective roles such
as sucrose phosphate synthase and A“’pyrroline-5-carboxylate synthase, which
are involved in sucrose synthesis and proline synthesis, respectively, and
contribute to solute accumulation (Guy et al., 1992b; Reimholz et al., 1997;
lgarashi et al., 1997).

A third group of up-regulated genes or proteins include those that are
regulatory such as RNA-binding proteins (Carpenter et al., 1994; Dunn et al.,
1996; Molina et al., 1997), protein kinases and 14-3-3 proteins (Holoppa and
Walker-Simmons, 1995; Hong et al., 1997; Monroy and Dhindsa, 1995; Jarillo et
al., 1994), and the CBF genes which activate expression of the four cold

regulated COR genes from Arabidopsis and presumably many other genes

12

associated with increased freezing tolerance (Gilmour et al., 1998; Jaglo-Ottosen
etaL,1998)

In addition to the genes whose functions or partial functions are known,
many more genes have been identified that encode polypeptides whose
functions are not known. Some recent reviews have listed collections of genes
with known functions, as well as those with speculative functions and unknown
functions (Thomashow, 1999; Pearce, 1999). Many of the polypeptides whose
function is not known are either novel or share similarities to LEA proteins. LEA
proteins are late embryogenesis abundant and have been found to accumulate
during development just prior to the time when seeds undergo dehydration and
enter a stress resistant dormant state. Several classes of LEA proteins have
been identified and separated into groups based on differences in the types of
motifs found in their sequence.

These LEA genes have been isolated from plants of all types and a major
group belongs to the dehydrin family (Close et al., 1989). Dehydrins (also called
the LEA D11 or group 2 family of developmentally induced seed proteins) (Baker
et al., 1988), can reach levels as high as 1% of total soluble protein during water
stress or low temperature stress (Close et al., 1989; Bray, 1993). Some common
features among many of these polypeptides are that they are hydrophilic and
remain soluble upon boiling. LEA proteins have relatively simple amino acid
composition with repeated sequence motifs and regions capable of forming

amphipathic a-helices. The dehydrins are recognized by a highly conserved

IYsine (K) rich sequence, EKKGIMDKIKEKLPG, called the K segment (Close,

13

1996). The numbers of K segments found in dehydrins vary from one to eleven

or more and are predicted to form an amphipathic a-helix which may function to

stabilize subcellular structures and membranes (Close, 1996; Dure, 1993). A
possible mechanism for protection may be through retention of the interaction
between the aqueous cytosolic material, proteins and membranes, and
prevention of membrane-membrane contact during periods of low water activity
such as that caused by freezing induced dehydration (Close, 1997; Pearce,
1999), which could lead to the types of damage described earlier. A second
sequence often found in these proteins is (V/T)DEYGNP known as the Y-
segment. Outside of the conserved sequences there is little conservation of
sequence between different member proteins in the family. However they
frequently contain high content of glycine and other polar amino acids.

In wheat, cold induced members of one family of dehydrins are localized
in the cytoplasm and nucleus (Houde et al., 1995). The polypeptide sequence
from a member of the family, WCS120, has 6 K-segments and 11 Y-segments
and is rich in glycine and threonine. The WSC120 gene has been shown to be
up-regulated by cold within 24 hours and remains at elevated levels while the
plant is in the cold, however mRNA levels decrease rapidly during deacclimation
(Houde, 1992).

The cold regulated gene wcor410 is also associated with acclimation to
freezing stress in several gramineae species (Danyluk et al., 1998).

Immunolocalization studies have shown the protein to be closely associated with

14

the plasma membrane. It has been proposed that WCOR410 helps to prevent
membrane destabilization during freeze induced dehydration.

It has not yet been possible to directly test the role of the dehydrins or
other LEA proteins in mutant plants with knock-outs in part due to the
redundancy in families of LEA genes. However, a heterologous expression
system has been used to test activity of several isolated LEA proteins. Yeast
expressing Em, a group 1 wheat LEA (Swire-Clark and Marcotte, 1998), LE25, a
tomato group 4 LEA (lmai et al., 1995), LE4, a tomato group 2 and HVA1, a
barley group 3 LEA (Zhang et al., 2000), all show improved survival after freezing
stress. Generalized roles ascribed to these proteins, based in part on structure
and function in the yeast, include ion scavenging and stabilizing membranes and
proteins. Continued success with expression of plant genes in yeast could help
further dissect the mechanisms of stress tolerance and protection by the different
types of stress proteins.

Several other cold-responsive proteins, some of which are classified as
LEA or LEA-like and others as novel proteins, are also highly hydrophilic and
boiling stable, including the COR (cold regulated) gene family of proteins in
Arabidopsis. At least four groups of COR genes have been isolated that show
elevated levels of mRNA transcripts within four hours of transfer to low
temperature (Thomashow et al., 1990; Gilmour and Thomashow, 1991; Gilmour
et al., 1992). The mRNA transcript levels remain high for the duration of low
temperature treatments and rapidly decrease upon transfer to warm temperature.

Key representatives from each group include COR6. 6, COR15a, COR47, and

15

COR78. The COR genes have also been termed LTI (low temperature induced),
KIN (cold inducible), RD (responsive to desiccation) and ERD (early dehydration-
inducible) (Nordin et al., 1993; Wang et al., 1994; Welin et al., 1994; Welin et al.,
1995; Wilhelm and Thomashow, 1993; Yamaguchi-Shinozaki and Shinozaki,
1993b). Each of the groups has been found to exist as tandem gene pairs
physically linked in the genome and with members of each pair being
differentially regulated. At least one member of each pair is induced in response
to low temperature. Many of these genes also respond to other treatments
associated with water deficit including dehydration, high salt concentrations and
ABA.

One of the more thoroughly studied COR genes is COR15a, which has
been shown to play a direct role in freezing tolerance. COR15a encodes a
15kDa polypeptide that is targeted to the chloroplast where it is processed to a
9.4kDa polypeptide. Constitutive expression of COR15a in transgenic
Arabidopsis was shown to increase the freezing tolerance of both chloroplasts
and isolated leaf protoplasts by 1°C to 2°C. Currently, COR153 is believed to
enhance freezing tolerance by helping to stabilize membranes which prevents
the formation of or lowers the temperature at which lamellar to hexagonal ll
phase membrane transition occurs (Steponkus et al., 1998). The improvement in
freezing tolerance in the plants overexpressing COR15a is subtle since no
obvious enhancement is observed at the whole plant level (Artus et al., 1996).
Such has also been the case for some other cold-induced genes that have been

overexpressed in transgenic plants (Zhu et al., 1996; Kaye et al., 1998).

16

Teml

new

stat

l
col:

I1
30*

P.
m,»
I v

Additional Low Temperature Induced Genes and Roles in Low
Temperature Signal Transduction
The mechanisms by which plants perceive a low temperature signal and
how they trigger responses are poorly understood. What many of the
components are that make up the earliest steps of the signal transduction
pathway are unknown. Sensation of cold may be relayed through changes in
membrane physical properties (Nishida and Murata, 1996; Vigh et al., 1993) but
direct evidence in higher plants is lacking. Recognition of changes in the redox
status of photo-system II has also been suggested as a sensing mechanism for
cold (Gray et al., 1997). The role of either in plants is yet to be determined.
Calcium and protein phosphorylation play an important part in the
acquisition of freezing tolerance. Calcium may play a role as a second
messenger relaying the information of a temperature change. Phosphorylation
may then affect the regulation of low temperature signal transduction (Knight et
al., 1996; Monroy and Dhindsa, 1995; Monroy et al., 1993; Tahtiharju et al.,
1997; Polisensky and Braam, 1996). In Arabidopsis, a cold shock elicits an
immediate rise in cytosolic free calcium concentrations (Knight et al., 1996;
Polisensky and Braam, 1996). This may result from the cold-induced opening of
membrane calcium channels or influx from the vacuole (Burk et al., 1976; Monroy
and Dhindsa, 1995; Monroy etal., 1993; Knight etal., 1996). Chemical
treatments to block calcium channels or inhibit protein kinases severely affect the
ca'pacity of plants to develop cold-induced freezing tolerance and limit expression

of some cold regulated genes (Knight et al., 1996; Monroy and Dhindsa, 1995;

17

Monroy et al., 1993; Tahtiharju et al., 1997). However, Monroy and Dhindsa
(1995) have shown in alfalfa the addition of a calcium ionophore to cells caused
an influx of calcium and induced the expression of two cas (cold acclimation
specific) genes at 25°C.

Following the increase in calcium flux, transcript levels for several protein
kinases have been shown to increase (Jonak et al., 1996; Mizoguchi et al., 1996;
Monroy and Dhindsa, 1995; Tahtiharju et al., 1997). In Arabidopsis a MAP
kinase and MAP kinase kinase kinase are induced in response to low
temperature (Mizoguchi et al., 1996). Calcium dependent protein kinases in
alfalfa (Monroy and Dhindsa, 1995) and Arabidopsis (Tahtiharju et al., 1997)
were also activated by low temperature. Application of protein kinase inhibitors
blocks the induction of cold acclimation and cold regulated gene expression while
addition of a phosphatase inhibitor stimulated the induction of 03315. Total
phosphatase activity and phosphatase 2A is inhibited by low temperature
treatment and 2A is also decreased by calcium ionophore treatment (Monroy et
al., 1998), demonstrating a potential link between activation of a calcium
cascade, protein phosphorylation and induction of freezing tolerance.

Low temperature stress responsive promoter elements involved in the
transcriptional control of a subset of cold upregulated genes are known. Within
the promoter of the Arabidopsis COR genes is a DNA sequence element, the
CRT (C-repeat), which has a core sequence CCGAC that is sufficient for low
temperature responsiveness of these genes. It is also found in the wheat

wcs120 and Brassica napus BN115 low temperature regulated genes and

18

|
a!“
UIIUI

bl t

C.)

loc

undoubtedly many others. This core sequence interacts with the C-repeat
binding factor (CBF) under different stress conditions and makes up part of a low
temperature signal transduction pathway.

CBF is part of a small gene family (CBF1, CBF3 and CBF2; also termed
DREB for dehydration responsive element binding protein (Liu et al., 1998))
located in direct repeats in the genome. The CBFs contain an acidic activation
domain, a DNA binding domain motif found in APETALA2 and other plant
transcription factors and a possible nuclear localization sequence. CBF1 was
first shown to be a functional transcription factor in yeast with its ability to activate
reporter genes containing the CRT in the promoter region (Stockinger et al.,
1997)

Overexpression of CBF1 in transgenic Arabidopsis upregulates the
expression of a family of COR genes that have the CRT in their promoter without
a low temperature stimulus (Figure 1.1). When freezing tolerance was assayed
by an ion leakage test, transgenic plants overexpressing CBF1 showed a
dramatic increase in freezing tolerance over non-transgenic plants that had not
been cold acclimated and transgenic plants overexpressing just a single COR
gene, COR15a. At a whole plant level, an increase in freezing tolerance is also
observable (Jaglo-Ottosen et al., 1998). These experiments demonstrate that
CBF-mediated cold induced genes play significant roles in freezing and stress
tolerance. The isolation of CBF was a major advancement that helped identify
an important early component of a low temperature signal transduction pathway

and was discovered using a reverse genetic approach. Using repeats of the

19

elFI

P011

Nonacclimited Cold-acclimated
B1 6 T8 RLD A6 B1 6 T8

A6
COR6.6 *
COR15a , ..

COR47 - ‘ g
In
Q

RLD

 

CBF1

      
   

 

COR78

  

elF4A

 

Figure 1.1. Expression of CBF1 and COR genes in Arabidopsis plants. Total
RNA was prepared from leaves of nonacclimated and 3-day cold acclimated
plants and examined for CBF1 and COR gene transcript accumulation by RNA
blot hybridization analysis. Amounts of COR gene transcripts are detectable in
nonacclimated CBF1 overexpressing plants (A6, B16) at a higher level than
nontransgenic plants (RLD). ln nonacclimated A6 plants, COR transcripts
accumulate to levels approximating that seen in cold acclimated plants.
Overexpression of CBF1 did not affect transcript concentrations of elF4A
(eukaryotic initiation factor 4A) (Metz et al., 1992), a constitutively expressed
gene that is not responsive to low temperature and was used here to compare
RNA loading. Transgenic plants overexpressing COR15a (T8) only increased
transcript accumulation for COR15a.

20

N

”I

8C2

Elpi

CRT in the promoter of a reporter gene in a yeast one-hybrid screen, Stockinger
et al., (1997) was able to identify CBF1. Additional links backward in the COR
gene signaling pathway will help better define the significance of this particular
pathway. A more complete understanding of how a low temperature signaling
network involving multiple signaling pathways may work will not be possible
however until additional components associated with cold acclimation are
defined.

Recently, three mutational screens have been used in Arabidopsis to help
dissect freezing tolerance pathways. The first approach involved isolating
mutants that were defective in their ability to cold acclimate. Warren et al.,
(1996) isolated seven mutants that were unable to develop freezing tolerance
even after extended periods of cold acclimation. These mutants were named sfr
for sensitive to freezing. The reason for sensitivity in some of the sfr mutants
appeared to be due to an inability to accumulate soluble sugar during cold
acclimation (McKown et al., 1996). One of the mutants, sfr6, was shown to be
deficient in CBF-mediated induction of COR genes which confirms the
importance of the CBF pathway in cold acclimation (Knight et al., 1999). The
additional sfr mutants did not affect COR gene expression. Most of the sfr
m utants still retain some capacity to cold acclimate suggesting the mutation
blocks one signaling pathway but also indicating involvement of multiple
pathways.

A second mutational approach was used to generate plants with aberrant

expression of a reporter gene driven by the COR78 promoter (lshitani et al.,

21

elemer‘.
Dahlia

II ncr .
lal int:
osmoti
genes

i031 E

U‘iEI-E

wild I.

trig-,7:

1997). Since the COR78 promoter has cold, drought and ABA responsive
elements, this approach allows identification of signal components in all three
pathways, at least as they are transmitted through to the COR genes. Several
hundred mutants with altered reporter gene activity were identified. The mutants
fall into three general classes. The cos mutants show constitutive expression of
osmotically responsive genes. The los mutants show loss of expression of these
genes, and the has mutants show hyper-expression. Two of the has mutants,
hos1 and hosZ, are less freezing tolerant after cold acclimation even though they
over-accumulate COR gene transcripts when exposed to cold (lshitani et al.,
1997, Lee et al., 1999). The H081 locus appears to be a negative regulator of
cold signal transduction but may be a positive regulator for other stress signal
pathways. H082 also appears to be a negative regulator of cold acclimation
through the COR pathway but another low temperature requiring process,
vernalization, is unaffected. H082 may play a positive role in regulation of
factors subsequent to COR gene expression such as changes in sugar
metabolism or membrane composition. Analysis of the different types of mutants
isolated by this approach suggest a network of multiple signaling pathways that
share components in different pathways to activate the COR genes in response
to several different stress factors.

A third mutagenesis approach involved the isolation of constitutively
freezing tolerant (cft) mutants. These mutants are more freezing tolerant than
wild type in the absence of cold acclimation. Xin and Browse (1998) isolated

twenty-six mutants with increased freezing tolerance. One of the mutants,

22

eskimof, tolerates freezing to —10.6°C without cold acclimation due to its high
levels of proline and soluble sugars. It does not express the COR genes in the
absence of cold acclimation. Characterization of additional mutants should
provide insights into other aspects of freezing tolerance signaling pathways (Xin
and Browse, 2000).

Conclusion

Over the last several years, advances in technology including methods in
molecular biology, and the completion of sequencing of the Arabidopsis genome
and partial sequencing of genomes from several other plants types and the ability
to study genome wide expression of genes through micro-arrays has allowed
leaps in our understanding of the signaling pathways. A great deal of information
however is still lacking and it will be up to those continuing and advancing these
technologies to dissect in finer detail the specific mechanisms and interactions
involved in signal transduction. Already the picture is complex with the possibility
of several different pathways working at the same time to provide freezing
tolerance to plants. The reason for the redundancy is not clear but is sure to

provide fodder for additional research in the future.

23

the 5
gene
Plan

Ara:

and

Char
Plar

CIOp

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34

Chapter 2

INITIAL CHARACTERIZATION OF CBF GENE FUNCTION AND
IDENTIFICATION OF A PROMOTER REGION SUFFICIENT FOR LOW
TEMPERATURE REGULATED EXPRESSION OF CBF2

Summary
Arabidopsis COR (cold regulated) genes are induced in response to many
different environmental stresses. Promoter analysis of these genes has led to the
identification of cis-acting elements including a cold and dehydration responsive
DNA regulatory element termed the CRT (C-repeat)/DRE (dehydration-
responsive element) (Baker et al., 1994; Yamaguchi-Shinozaki and Shinozaki,
1994). Subsequently, a transcription factor that binds to the CRT/DRE was
identified and termed CBF (C-repeat binding factor)/DREB (dehydration
responsive element binding protein) (Stockinger et al., 1997; Liu et al., 1998). A
small family of these transcriptional activators (CBF 1, CBF2, and CBF3) was
later identified, all of which bind to the CRT/DRE and activate expression of
reporter genes in yeast that contain the CRT/DRE as an upstream activator
sequence. In addition, overexpression of CBF1 and CBF3 in Arabidopsis plants
grown at non-acclimating temperatures induces COR gene expression and
increases freezing tolerance.
In this study, the CBF genes and their promoters were further examined to
characterize their role in the low temperature signaling cascade and to identify
the means for their own regulation. Transcript levels for all three CBF genes

increased within 15 min of transferring plants to low temperature followed by the

35

accumulation of COR gene transcripts at two to four hours. The CBF transcripts
also accumulated in response to mechanical agitation, drought, ABA, and
cycloheximide. Promoter deletion analysis identified a 155 base pair region that
is sufficient for responsiveness to low temperature and the other stresses. The
CBF genes do not appear to be autoregulated and likely require additional
transcriptional regulators, possibly both positive and negative, to mediate their
expression.

Introduction

During their life cycle, plants frequently experience many different
environmental stresses that can significantly impact their productivity and ability
to survive. Because of their immobility, they must make metabolic and structural
changes to cope with these environmental conditions. An inducible genetic
system responsive to stress stimuli enables many plants to survive. A number of
genes that respond at a transcriptional level to stresses such as low temperature,
drought and high salinity have been described. The function of a few is known.

In Arabidopsis, a group of four sets of gene pairs, each pair represented
by a tandem repeat, have been described which are abundantly expressed in
response to low temperature. These are termed the COR (cold regulated)
genes. Transcripts for the COR genes begin to accumulate within two to four
hours after experiencing cold temperatures. The COR153 protein, the best
characterized of the COR gene products, has been localized to the stromal
compartment of the chloroplast and may play a role in minimizing damage

between chloroplast and plasma membranes during freeze thaw cycles

36

(Steponkus et al., 1998). Functions of the other COR genes products are not
known. Part of the stress responsive induction of the Arabidopsis COR genes is
mediated at a transcriptional level (Baker et al., 1994). In fact, the promoters
from many stress inducible genes have been analyzed for responsiveness to low
temperature, drought and high salinity and different mechanisms for regulation
have been proposed (lshitani ef al., 1997). ABA is often associated with stress
responses in plants. ABA levels do increase in many plants in response to low
temperature (Chen and Gusta, 1983). The COR genes, for example, are
responsive to applications of ABA (Thomashow, 1994). The cold inducible
Arabidopsis RAB18 gene is also dependent on ABA for activation and the gene
is not induced by low temperature in the ABA mutants aba1 or abi1 (Lang and
Palva, 1992). RAB18 has an ABA-responsive cis-acting DNA sequence element
in its promoter that may be responding to the binding of a bZlP type of
transcription factor (Guiltinan et al., 1990, Meshi and lwabuchi 1995, Shen et al.,
1996). However, using the same ABA mutants, but this time looking at COR
gene response, a second pathway was discovered that was not directly
dependent upon ABA.

With certain COR genes, ABA induced expression is impaired in the ABA
mutants, however, cold induced expression is not affected (Gilmour and
Thomashow, 1991, Nordin et al., 1991). Subsequent deletion analysis of the
Promoter from COR15a led to the discovery of the cis-acting DNA sequence
element required for the cold induced expression. This element was termed the

CRT (C-repeat) (Baker et al., 1994) and contains the core sequence CCGAC.

37

Colt

This core sequence was also present in a cis-acting sequence motif discovered
by Yamaguchi-Shinozaki and Shinozaki (1994). Their element was termed the
DRE (dehydration responsive element) because it could elicit dehydration-
induced expression from genes whose promoters contained this element. The
identification of a transcription factor, CBF 1, which interacts with the CRT/DRE
(Stockinger et al., 1997), has advanced our understanding of an important part of
the low temperature signal transduction pathway. Liu et al., (1998) subsequently
identified the same factor and named it DREB1b (Dehydration Responsive
Element Binding protein 1b). Insights gained from the low temperature stress
pathways can aid our understanding of other stress responses and pathways.
Ultimately, this may facilitate the development of applications to enhance plant
responsiveness to stress. For example, the activation of a cold acclimation
response can be achieved by expression of a single transcription factor such as
CBF1. This gene could have great agronomic potential in crops that are subject
to unexpected frost during their growing season. Understanding the basic
science behind freezing tolerance and regulation of genes such as CBF is also
important because the induction of a freezing tolerance state can be costly to a
plant. If it were not, evolution might have produced constitutively hardy plants.
Such plants would be expected to have a considerable selective advantage in
regions or seasons that experience sudden frosts.

In fact, constitutive expression of CBF, while it improves freezing tolerance
of non-acclimated plants considerably and even improves freezing tolerance of

cold acclimated plants (Jaglo-Ottosen et al., 1998), has often resulted in plants

38

IES
PIC
ier
CB
def
SIII

WII.‘

liar
IO v.

dele

with a retarded growth phenotype (Liu et al., 1998; Gilmour et al., 2000). This
has been experimentally overcome in part with the use of an inducible promoter
that expresses CBF at higher than endogenous levels only after cold treatment.
In this example, the CBF gene was controlled by the COR78 promoter, which
resulted in hyper-responsiveness to cold and osmotic stress and less growth
retardation (Kasuga et al., 1999). Identification of additional factors responsible
for regulation may afford a fine-tuning of the expression levels for some of these
transcription factors to prevent the negative results that are sometimes seen.

Subsequent to the initial identification of CBF 1, members of the CBF
family (CBF 1, CBF2, and CBF3, also termed DREB1b, DREB1c and DREB1a,
respectively, (Liu et al., 1998)) were shown to be transcriptionally regulated.
Promoter/reporter fusions and transgenic plants were used to demonstrate low
temperature induction. In addition, Gilmour et al., (1998) demonstrated that the
CBF genes were responsive to mechanical agitation but were unable to
determine responsiveness to drought and ABA. However Liu et al., 1998, and
Shinwari et al., 1998 suggested they were not induced by drought nor treatments
with ABA.

In this study, analysis of expression patterns for the CBF genes was
performed in order to determine the role of CBF in the low temperature signal
transduction cascade and to examine the responses of the CBF gene promoters
to various stimuli. Activity of the CBF2 promoter was further examined with

deletion and nucleotide base substitution experiments in order to determine DNA

39

sequences that may be important for its responsiveness as well as that of the
other CBF genes.

Results

Mapping of CBF and COR genes. Analysis of DNA blot hybridization
and subsequent sequencing of a clone from a genomic library screen indicated
three CBF genes were physically linked in direct repeats in the Arabidopsis
genome (Gilmour et al., 1998). To determine the map position of the CBF gene
cluster and that of four CRT-containing COR genes, COR6. 6, COR15a, COR47,
and COR78, cosegregation analysis of molecular markers from the Lister and
Dean recombinant inbred lines (Lister and Dean, 1993) was conducted. The
results indicated that the CBF genes were located on chromosome 4 at 75.6cM.
The four COR genes were distributed throughout the genome unlinked to any
other COR gene or the CBF genes. COR6.6 was located on chromosome 5 at
32.1cM, COR 15a was on chromosome 2 at 76.9cM, COR47 was on
chromosome 1 at 27.4cM and COR78 was on chromosome 5 at 106.5cM.

Expression of CBF genes. The CBF genes have been shown to activate
expression of a reporter gene carrying a CRT element in its promoter in yeast
(Stockinger et al., 1997, Gilmour et at, 1998). Whether the CBF genes
themselves were constitutively expressed or induced in response to low
temperature was another question. A constitutively expressed gene would
suggest that the CBF protein was activated in response to low temperature while
low temperature induced accumulation of gene transcripts would suggest either a

transcriptionally regulated component or an mRNA stability factor. To partially

40

answer the question, Arabidopsis plants were grown at nonacclimating conditions
and then transferred to low temperature for various periods of time. RNA was
then extracted and blot hybridization was performed using a cDNA for the entire
CBF1 coding sequence, which hybridizes to all three CBF genes. The results
indicated the CBF transcripts increased rapidly within 10—15 minutes after
transfer to low temperature. The transcripts continued to increase until about 2
hours of low temperature treatment, then decreased to a new lower level over the
24-hour period of the experiment (Figure 2.1A). Between 2-4 hours after the
induction of CBF, transcripts for the COR genes began to accumulate.

To test whether elevation of transcript levels was due to dehydration
stress, we used cultured Arabidopsis cells that were suspended in an aqueous
medium and thus were constantly hydrated. Transcript levels for the CBF and
COR genes were examined and also found to be elevated in this liquid cell
suspension when treated with low temperature (Figure 2.1 B).

The CRT/DRE DNA regulatory element found in the promoter of COR
genes stimulates gene expression in response to both low temperature and
dehydration stresses but not to ABA (Yamaguchi-Shinozaki and Shinozaki,
1994). To determine if the CBF promoters might also be responsive to these
treatments, RNA blot hybridization analysis of treated samples were examined.
When using the entire CBF1 cDNA as a hybridization probe, the apparent CBF
transcript levels increased within 15 minutes of placing detached leaves on dry

filter paper or after spraying plants with a solution of ABA (100pM). The levels of

transcript accumulated to a peak at about 30 minutes and then decreased to a

41

6X

0f,I

we

‘I f
E

A Time (h) at 4°C
0.25.512 4 624

"v”

 

 

   

CBF1

  

 

 

COR15a

rRNA

 

B Time (h) at 4°C

 

 

 

 

o 30
CBF1
COR15a .
COR78 —
COR66
rRNA

Figure 2.1. Expression pattern of CBF and COR genes after exposure to low
temperature. In A, plants were exposed to low temperature for between Oh and
24h. After treatment, transcript accumulation for the indicated genes was
detected by RNA blot hybridization. In B, suspension cultured cells were treated
for 30h at low temperature and transcript accumulation was detected for the
indicated genes. The rRNA panel shows an ethidium bromide stained gel picture
of ribosomal RNA representative of the loading for the blots.

level similar to untreated plants by 24h for dehydration treatments or 4h for ABA
treatments (Figure 2.2). These results initially appeared to indicate that CBF
might be responsive to both ABA and dehydration. However, control
experiments in which leaves were detached under water to prevent dehydration
or/and in the air and then placed onto wet filter paper or plants sprayed only with

water had a similar pattern of expression as the experimental treatments. This

suggested that much of the induction seen in the dehydration and ABA test

42

treatments might actually have been due to another stress such as the
mechanical agitation associated with the treatment.

The effects of mechanical stress were tested by growing seedlings in
covered Petri dishes, to prevent changes in environmental water status, and then
tapping the plate on its side for five minutes to produce a mechanical agitation.
RNA blot hybridization was then performed and CBF transcripts were observed
to increase within 15 minutes and then subside to near untreated levels within an
hour after the tapping treatment (Figure 2.2). COR15a gene expression also
rose in all of the experiments within 2-4h. The increase in COR15a expression is
presumably due to the large increase in CBF expression that resulted from the
mechanical agitation stress applied to the plants, however, additional regulatory
factors may also be involved. In the dehydration and ABA treatments, COR153
transcript remained higher than controls at 24h possibly due to an increase in
endogenously produced ABA, which induced COR15a expression through the
ABREs present in the promoter of the gene (Baker et al., 1994).

Further studies on CBF gene expression. Many previous experiments
used to examine the expression of the CBF genes were carried out only after
directly shifting plants from a warm growth chamber (~20°C) to cold (4°C)
(Gilmour et al., 1998). Therefore, the question arose whether there was a need
for this large temperature change to activate the CBF low temperature response
pathway. This, in fact, does not appear to be the case (Figure 2.3). Plants that

were slowly cooled at a rate of 2°C per hour over an 8h period and then held at

4°C for an additional 16h began to accumulate CBF gene transcripts at about 3h

43

A Deh dration
Test Control
(hr) 0.25 .5 1 2 4 6 24.25 .5 1 2 4 6 24

CBF1 * "II '

COR15a

 

 

 

nlflw

 

 

   

rRNA

 

B ABA

Test Md
(hr)0.25.512 4 62425512 4 624

 

 

 

 

 

 

 

CBF1

COR15a

rRNA

C Mechanical Agitation

(hr)0.25 .5 1 2 4 6
CBF1 '

COR15a v.5 .

rRNA I 4 1.....- I

Figure 2.2. Pattern of gene transcript accumulation in response to dehydration,
ABA, and mechanical agitation. Arabidopsis seedlings were grown on agar
medium and then treated as described in the text. Levels of CBF1 and COR15a
transcripts were determined by RNA blot analysis. All zero time samples are of
control plant material collected prior to treatment. (A) RNA isolated from
detached leaves incubated on dry filter paper (Test) or wet filter paper (Control)
for the indicated times. (B) RNA isolated from seedlings sprayed with a solution
containing ABA (Test) or without ABA (Control). The samples are from times
after spray treatment. (C) RNA isolated from seedlings mechanically agitated for
5 min and then incubated for the indicated times with no agitation. The rRNA
panel shows an ethidium bromide stained gel picture of ribosomal RNA
representative of the loading for the blots.

 

 

 

 

44

ele
aul
lo I

Iov.

after the start of the experiment. The ambient temperature surrounding the plant
leaves at the point CBF mRNA was first visible by RNA blot hybridization was
about 14°C. The amount of CBF transcripts continued to increase and reached a
maximum at 4°C, similar to the amount of transcript accumulation for plants that
experienced a cold shock. The amount of CBF transcripts after about 10 hours
(about 2-3 hours after reaching 4°C) began to decrease to a new steady state
level (Figure 2.3).

In addition, when the levels of CBF and COR genes mRNA were
measured over an extended period of cold treatment (21days), this new lower
level for CBF persisted while COR gene transcripts remained high. Plants
containing a transgene with two copies of the CRT fused to GUS also maintained
a high level of transcript throughout the 21-day low temperature treatment
(Figure 2.4). This suggested that the lower level of CBF transcript was sufficient
to maintain the high level of COR gene expression. Alternatively, increased
Stability of the CBF protein during the long period of cold may decrease the need
for a high level of CBF transcript in order to maintain elevated levels of COR
gene or GUS marker gene transcripts.

Examination of CBF promoters for low temperature responsive
elements. Gilmour et al., (1998) demonstrated that the CBF genes are not
autoregulated, and examination of the three CBF promoter sequences revealed
no regions containing the core pentamer sequence of the CRT/DRE (CCGAC).

However, RNA gel blot analysis using CBF 1, CBF2, and CBF3 gene specific

45

Cold

 

Shggk Gradual Temperature Decrease
Time (hr) 1.54 01 2 3 4 5 6 7 8 9101224
Temp (°C> 44 201816141210 86 4 4 4 4 4

 

 
 
 
 

 

CBF1

 

COR15a
rRNA AAAAAA

Figure 2.3. Expression pattern of CBF and COR15a genes after cold shock and
a slow temperature downshift. Plants grown at 20°C were transferred directly to
a 4°C chamber for cold shock or cooled slowly (05°C per 15 min) and sampled
at the indicated times. RNA blot hybridization was used to detect transcripts at
the sampled time points using a probe for COR15a and a full length CBF1 probe.
The rRNA panel shows an ethidium bromide stained gel picture of ribosomal
RNA representative of the loading for the blots.

Experiment #1 Experiment #2 Experiment #3
Time at 4°C

Oh153691d3714210h1.53691d3714210h1.53691d371421

COR15a '

2xCRT:
GUS

CBF1
CBF2

CBF3
r RNA

 

Figure 2.4. Expression of CBF genes and their targets after an extended period
Of cold. Three independent sets of plants containing two copies of the CRT in a
Chimeric promoter fused to GUS (2xCRTzGUS) were grown at 22°C before
transfer to 4°C for 21 days. RNA was isolated within hours (h) or days (d) after
transfer to 4°C. RNA blots were then probed to determine expression of the
endogenous COR15a, the GUS marker and the three CBF genes using gene
Specific probes. The rRNA panel shows an ethidium bromide stained gel picture
of ribosomal RNA representative of the loading for the blots.

46

CB

WIII

probes indicated that all three genes were rapidly induced by exposure of plants
to low temperatures (4°C) (Gilmour et al., 1998). To investigate the hypothesis
that a cis-acting element in the promoter was responsible for low temperature
responsive gene expression, a set of promoter/reporter gene plasmid chimeras
consisting of promoter fragments from the CBF genes were fused to the GUS
reporter gene (Jefferson et al., 1986). Whole promoter gene fusions, containing
approximately 1kb of promoter sequence upstream of the translational start site,
were created for all three CBF genes. Six gene fusions with the 5' end of the
CBF2 promoter deleted and three gene fusions with the 3' end of the CBF2
promoter deleted were also created (Figure 2.5). The sequence and relevant
information about the region of the CBF2 promoter used in the deletion studies is
shown in Figure 2.6. Arabidopsis thaliana plants were then transformed by an
Agrobacterium mediated transformation method and T2 and T3 populations of
plants were analyzed for low temperature responsive expression of the GUS
reporter gene.

At least ten independent transgenic Arabidopsis plants for each whole
CBF promoter GUS fusion were analyzed for B-glucuronidase activity which
When positive after an assay, results in blue staining in tissues (Jefferson, 1987).
After both warm and low temperature treatments, staining was observed in
tissues of the plants (Figure 2.7). This seemed to contradict earlier results from
IRNA blots indicating the endogenous CBF gene transcripts were not elevated at
Warm temperatures. Therefore, RNA blots were prepared for the transgenic

reporter lines and hybridized with the GUS gene as a probe. For all transgenic

47

CI

Into
were
358

GUS

-987 CBF1 promoter +176 _—
-1091 CBF3 promoter +139 _—
-982 CBF2 promoter +152 _—
CBF2 promoter 5’ deletions
.41 9 , +152 I ‘

"363919 _—

' _—

'189 __
'94 _—.

‘53 , ‘—

CBF2 promoter 3’ deletions
'724 '35 L46” __

-724 -1_45 Ii
'724 . *244 — _—
Monomer -189-35_ _—
Dimer -1 89335-1 89-35 ——

Mutations '319 "Q1 2 +152 _—
m

'189 x3 ‘—

'139 ",1 _—

‘189 , "£4 . _—

Figure 2.5. Diagram depicting the three CBF promoters and CBF2 promoter
fragments used in promoter analysis experiments. Each fragment was inserted
Into a binary vector upstream of the GUS gene. Fragments that had 3’ deletions
Were first inserted into a binary vector upstream of the cauliflower mosaic virus
358 minimal promoter (-46 minimal promoter) then fused to GUS.

48

-419
rb

GAATGGAGAA
-369

r’
CATTATGTCT
-319

r'
GTTATTCGTG
TACAGCCACA

GGCATCAACC
CATGTCAGAT

ATCGCTTAGC

-68
r'

GCGTTCGACC

AGCTCTCACT
AAGAAAGATA
ACAGAGATCT
GAAACAGAGT

AAGACAGAAA

AATTATTTTA

GAAAAATAAT
CATTCATACA

AGTGAAGGGT
TCTCAGTGAT

TGTTTCTTAT

CCACAAATAT

GAGCATTTGA
CCATAGAAAA

ATTTAATAAT
ATAAGAAGTT

-244

CAGAAACTTC
TGACAGCCTT
<J

-145

-94
m2
CA

CCAAATATCT

+1

CCAACATTTC @lTAACTTCA

GAGAGAGAAA
TTCTACTTAC
ACTCTTCTGA

TAAATATTTT
TCTACTCTCA
TCA ATG

AAATATCTCG
GCGACAATTA

GGTTGTCGTA
AAAAAAATTC

-139 m1

qCAAGATGGG

TTTCACGGAT

TAAACAATTT

CCCTATAAAC
ATACCCTAAA

TCAfghGGACA

 

GATAATTACA

m3
TTCPICAGAGA

TCCGGCCAAT

~35

AACACTTACC
ATCATACCAT
TAAACCTTAT

AAACCGTGGG

m4

choIAAAc'rch

ATAAACAGCA

TGAATTAGAA
ACAAAAAAAG
CCAGTTTCTT

Figure 2.6. Sequence from CBF2 promoter deletions and mutations from -419 to
+152. The +1 indicates the transcription start site. Arrows are used to designate
the points of the 5' and 3' deletions. Mutations (m1, m2, m3, and m4) are boxed
and were created by deleting the boxed bases and replacing them with a
restriction site (Xba I, TCTAGA). A putative ABA responsive element (ABRE) is
underlined with a dashed line.

49

Control CBF1.'GUS CBF2.'GUS CBF3.'GUS

 

‘ J
‘9

Cold‘v ($ng

-

 

 

 

 

 

 

 

 

Figure 2.7. Staining of transgenic plants containing whole CBF promoter/GUS
fusions. Representative plants containing promoter fusions from CBF1, CBF2,
and CBF3 are shown after growth at warm temperatures and after 3 days of low
temperature (4°C) treatment. Blue color is seen in the leaves and stems of the
plants and indicates B-glucuronidase (GUS) activity resulting from the CBF
promoter activated GUS expression. The control contains the GUS gene but no
active promoter and therefore no blue color.

lines containing the whole promoter CBF/GUS fusion, the GUS reporter was
induced to high levels by low temperature and was not present or at very low
levels at warm temperatures in unstressed plants (Figure 2.8). This suggested
that the original staining information from warm grown plants may have resulted
from a transiently stress activated promoter orland developmentally or circadian
rhythm induced expression and stability of the GUS protein. In fact [3-
glucuronidase activity resulting from GUS gene activation was visible by staining

warm grown seedlings within a few days after germination. Seedlings continued

50

CBF1 promoter CBF2 promoter CBF3 promoter
W C W C W C W C W C W C

' v

   

GUS

  

rRNA

Figure 2.8. Whole promoter CBF/GUS fusions demonstrate low temperature
induced promoter activity. All three CBF promoters accumulated little or no
transcript under warm growth conditions, (W), but had increased levels of GUS
marker transcript within 90 minutes of low temperature treatment, (C), as
determined by RNA blot hybridization. The rRNA panel shows an ethidium
bromide stained gel picture of ribosomal RNA representative of the loading for

the blots.

Days after germination

8 10
.1, .4, /' 2\ J 'I'IH/
v k I o ' c k

Figure 2.9. Seedling staining indicating CBF promoter/GUS gene activity.
Transgenic plants containing a CBF2 promoter/GUS transgene were monitored
for GUS activity at various time points during development at warm
temperatures. By 4 days after germination, blue staining was clearly visible in
the hypocotyls. Between 8 and 10 days after germination cotyledons and true
leaves were also staining blue.

 

 

 

 

 

 

 

 

51

to stain blue at warm temperatures for at least 14 days (Figure 2.9. Charles
Herman, unpublished). Whether the staining at later time points is due to
continuing activation of the CBF promoters or GUS protein stability is unclear.
Certainly the CBF gene mRNA transcript is not present at the times used in RNA
blots and so the disparity may simply be due to GUS protein stability.
Since all three CBF promoters seemed to be functioning similarly at an RNA level
in regards to low temperature induced expression, only the promoter from the
gene CBF2 was chosen for further deletion analysis. For 5’ deletion promoter
fusions, the transformants containing deletions to -419, -369, -319, and -189
relative to the presumptive start of transcription showed similar levels of induction
(Figure 2.10). The deletion to -94 had elevated transcript levels at low
temperature in several lines. However, accumulation was much lower than the
longer promoter fusions. Variation in expression levels between lines of a single
deletion type may have been caused by populations that were not homozygous
for the transgene or by insertion of a transgene in regions within the genome that
were more or less active. However, the line carrying a promoter deletion to -68
exhibited no low temperature induced transcript accumulation (Figure 2.10).
These observations indicated that the region between -189 and -68 included a
cis-acting element(s) involved in the low temperature induced expression of
CBF2.

The 3’ deletion lines were then analyzed. The transformants containing
the CBF2 promoter deleted 3’ to -35 relative to the start of transcription

demonstrated low temperature induced transcript accumulation. The

52

transformants containing a CBF2 promoter fragment deleted to -244 or -145
showed very low levels of induction or even loss of low temperature induced
transcript accumulation (Figure 2.10). This again indicated loss of an element(s)
necessary for promoter activity between -145 and -35 and also that the
apparently inducible expression was not due to a transcript stability function
associated with the 5’ untranslated region since this region is not present in the
3’deletion plasmids.

Based on this information, a plasmid was constructed which contained the
promoter fragment from -189 to -35 fused to GUS. A tandem repeat or dimer of
this fragment was also fused to GUS. Both plasmids were independently
transformed into Arabidopsis plants. For each plasmid construct, RNA blots
were examined for expression of GUS reporter transcripts from several lines of
plants. While a low level of expression was seen for the monomer construct,
strong induction was observed when the dimer construct was transformed into
plants (Figure 2.11). This confirmed the notion of a low temperature responsive
element(s) located in this 155 base pair region of the CBF2 promoter.

Expression of CBF genes in response to mechanical agitation, ABA,
dehydration, osmotic stress and cycloheximide.

Earlier work had shown the CBF genes to be exquisitely sensitive to
mechanical agitation, which resulted in abundant transcript accumulation. This
response affected our ability to monitor the activity of CBF in plants that were
subjected to ABA treatments or drought stress because a confounding

mechanical agitation response precipitated by the method of treatment obscured

53

A CBF2 promoter 5' deletions

 

 

 

flfl -319 -189 -94 -68
, "a t ,
GUS d ._ , ’1 3 E - a?
cane-.0000... 11.. .
rRNA ' T TT’T‘ "

 

CBF2 promoter 3’ deletions
'35 -145 -244
WC WC WC WC WC WC WCWCWC WC WC WC WCWCWCWCWC WC

GUS ouuw- .

CBF1........... IO...

 

 

 

 

 

  
 

   

 

 

rRNA

 

Figure 2.10. Expression pattern of deleted CBF promoter/GUS fusions.
Results from an RNA blot hybridization showing expression of genes from
transgenic plants containing the CBF2 promoter deleted 5’ (A) or 3' (B) to
indicated lengths and fused to the GUS gene. The level of GUS and CBF1
transcript after warm (W) or cold (C) treatment is shown. Each W/C pair
represents an independent transgenic line. The rRNA panel shows an ethidium
bromide stained gel picture of ribosomal RNA representative of the loading for

the blots.

the response. The ABA response may also have been obscured by an osmotic
stress response since plants were treated with a hypotonic solution with or
without ABA. Subsequently, procedures were developed that minimized
mechanical stimulation and osmotic stress and enhanced the visualization of
transcripts so that the effects of ABA and drought stresses on CBF expression
could be better observed. Plants containing whole promoter GUS fusion were

grown on agar medium and treated with 100uM ABA over a 24h period, or

54

dehydrated over a 6h period, and expression of the endogenous CBF genes and
the GUS reporter were observed. All three genes and the GUS gene fused to
the CBF promoters responded to both ABA and dehydration treatments (Figure
2.12). While the transcript levels due decrease rapidly after ABA treatment, they
initially appear higher and remain higher than controls for about two hours. The
decrease in transcript may reflect a decrease in active ABA levels as light has a
tendency to racemerize the ABA to an inactive form. Alternatively the initially
high levels of transcript in ABA treatment samples may be a result of an
augmentation of the mechanical agitation response and the presence of
transcript at later time points simple reflects decay from a higher level. This
alternative does not appear to be the case for drought treatments since transcript
levels are maintained through the treatment time and are not maintained in the
controls. Endogenous CBF transcripts were not induced to as high a level as
when the plants experienced a cold stress. This could result if rapid turnover of
the CBF RNA was retarded by low temperature. In fact turnover rate of the CBF
transcript was very rapid. Within 90 minutes of low temperature treatment, CBF
transcript accumulation reached a peak but in less than 90 minutes after stopping
treatment, transcript was no longer visible by RNA blot hybridization (Figure
2.13). The turnover rate for COR153 was less than 8 hours (Hajela, et al., 1990)
but is not visible in figure 2.13 because sample times were not extended beyond
90 minutes. Additional evidence suggesting rapid turnover of CBF transcripts
and a comparison of the relative activity of a CBF promoter after cold, ABA and

drought treatment is shown in Figure 2.14. The relative level of endogenous

55

A Monomer

 

 

 

 

 

 

Figure 2.11. Monomer and dimer promoter fragment expression patterns.
Expression of the GUS transgene was examined in transgenic plants containing
the monomer 155bp fragment fused to GUS and the dimer of the 155bp fragment
fused to GUS. Expression from the endogenous CBF gene is shown as well.
Each pair of lanes (W= warm, 0: cold) represents an independently transformed
plant line. The rRNA panel shows an ethidium bromide stained gel picture of
ribosomal RNA representative of the loading for the blots.

CBF transcript is high at cold temperature but low within 2h after treatment with
ABA or drought. The level of GUS transcript when fused to the CBF2 promoter
however, is relatively high for ABA treatment but less so for drought. The
difference between the level of transcripts for figure 2.12 and 2.14 are due in part
to the length of time the blots was exposed to film. Blots from figure 2.12 were

exposed over three times longer to help visualize the transcripts.

56

ABA Drouoht
0.25h1h 2h 4h 24h 0.25h 1h 2h 4h 6h

 
 
  
 

CBF1 sp.
CBF1/GUS O.
rRNA
CBF2 sp.
CBF2/GUS
rRNA
CBF3 sp.
CBF3/GUS 1..
rRNA

Figure 2.12. Effects of ABA and drought treatments on CBF expression. RNA
blot hybridization of CBFpromoter/ GUS fusions after treatment with ABA or
drought stress for indicated times. Each blot row pair represents a different CBF
gene promoter fused to GUS and probed with either a gene specific probe (sp.)
or a GUS probe. ABA treatment required floating plants on water (-) and a
solution with ABA (+). Drought treatment included dehydrating whole plants in a
desiccator (+) while controls H were handled the same as drought stressed
plants but kept sealed in Petri dishes. Hybridization blots were exposed for ten
days to visualize bands. The rRNA panel shows an ethidium bromide stained gel
picture of ribosomal RNA representative of the loading for the blots.

An osmotic stress response as a result of treating plants with a hypotonic
solution during ABA experiments was also observed. Warm grown plants
transferred directly to water often had higher "background" levels of CBF
transcripts present when compared to plants that were transferred to liquid
growth medium. Figure 2.15 demonstrates a dramatic example of this. The

level of background transcript present when plants were placed in water was not

57

Time (min) at Temp ratpre
90 90 90 90 90 90 90
W C W C W W C

c
CBF ...*

 

 

 

       

 

COFI15a

rRNA and ‘0‘-.-"

 

Figure 2.13. Turnover rate of CBF gene transcript. Rapid turnover is seen when
plants are repeatedly moved from warm temperatures (24°C) to cold
temperatures (4°C) to warm, etc. at 90-minute intervals. CBF transcripts peak by
90 min with cold treatment (C) but is gone after an additional 90 min in the warm
(W). COR15a transcript increases steadily and does not disappear. The rRNA
panel shows an ethidium bromide stained gel picture of ribosomal RNA
representative of the loading for the blots. .

Temp. ABA Drought
W C ' + ' +

CBF

rRNA
GUS
rRNA

 

Figure 2.14. Relative promoter activity after cold, ABA and drought treatments.
Endogenous CBF gene transcript levels were determined for warm grown (W)
and 1.5h cold (C) treated plants as well as for plants treated for 1.5h with ABA or
drought stress. (-)= control for each treatment, and (+) = treatment condition.
The level of GUS transcript in transgenic plants containing a CBF2 promoter
fused to GUS was also measured using similar treatments. Hybridization blots
were exposed to film for three days. The rRNA panel shows an ethidium
bromide stained gel picture of ribosomal RNA representative of the loading for
the blots.

58

always this high.

To investigate whether a cis-acting element in the promoter of CBF2 was
also responsible for induction of gene expression by mechanical agitation, the
same lines of plants containing CBF2 promoter GUS fusions were treated by
mechanically agitating seedlings on plates. Significant levels of transcript
accumulation had been observed within 15 minutes of treatment for whole
promoter GUS fusions (Figure 2.16A). In fact, the peak level of expression
resulting from continuous mechanical agitation appeared to occur within 15 min
to 1 h and then quickly decreased (Figure 2.16B). To test for responsiveness to
mechanical stress in the promoter deletion lines, plants were agitated for 15
minutes and transcript accumulation was observed. In response to mechanical
stress, 5’ deletions to -419, -369, -319, and -189 had increased transcripts. A

much lower level of transcript accumulated for the 5’ deletion to -94, while loss of

Osmotic Stress

+ - + - +
d I2

Figure 2.15. Response of CBF promoter to hypo-osmotic shock. Three
independent lines of transgenic plants containing the CBF2 promoter fused to
GUS were treated by transferring them from solid medium to plates containing
either distilled water (-) or liquid growth medium (+). Transcript levels for GUS
were then examined. The rRNA panel shows an ethidium bromide stained gel
picture of ribosomal RNA representative of the loading for the blots.

 

 

     

GUS

rRNA

59

expression was observed in the plants containing a transgene with a promoter
deletion to -68 (Figure 2.16C). The plants containing promoter/marker fusions
with 3’ deletions to -35 had mechanical stress induced transcript accumulation
but those with promoter fragments deleted to -294 and -145 showed no activity.
The 155 base pair fragment as a monomer and dimer was also checked and
transgenic plants containing them also showed elevated expression levels of the
marker for both with much higher levels for the dimer (Figure 2.16C). These
patterns of expression were seen in at least two independent lines for each
construct.

Finally the accumulation of CBF was measured in response to the addition
of cycloheximide. Seedlings containing the 155 base pair fragment as a dimer
were treated for 90 minutes at room temperature after addition of cycloheximide
to the Petri dishes. RNA hybridization using the GUS probe demonstrated an
increase in CBF transcript while no such increase was seen in controls without
cycloheximide but handled identically (Figure 2.17).

Mutations in the promoter. Within the 3’ region of the CBF2 promoter
that is sufficient for low temperature responsive gene expression (-189 to -35,
Figure 2.6), there is a DNA sequence element that resembles an ABA responsive
element (ABRE). Site directed mutagenesis was used to alter this sequence to
determine if it played a role in the stress response. The mutated promoter
fragment responded to treatment with ABA similar to non-mutated controls
(Figure 2.18A). Three additional mutations were created elsewhere in the

promoter where conservation of sequence was found between the other three

60

A Mechanical Agitation

 

 

 

 

 

 

 

2_CBF1__ _CBF2_ _CBF3_
GUS I I
rRNA
B Time h of MechanicalA itation
0 0.25 1 2 4 6 24
GUS
rRNA
C 5' Deletion 3’ Deletion
419-36931018964 '68 3544524412? t$235
GUS
CBF2
rRNA

 

Figure 2.16. Responsiveness of CBF promoters to mechanical agitation. In A,
an RNA blot hybridization shows the response of whole CBF promoters fused to
GUS and treated by shaking seedlings, that had been grown in sealed Petri
dishes, for 15 minutes. The 0 lane is a control taken before plants were shaken.
Each lane represents an independently transformed plant containing the
promoter/GUS fusion (two lines for each promoter/GUS fusion). In B, the result
from continuous mechanical agitation over 24 hours is shown for the CBF2
promoter/GUS fusion. In C, plants containing promoter fusions with either 5’ or 3’
deletions and the monomer and dimer fragments (-189 to —35 x1, x2) were
shaken for 15 minutes and RNA hybridized to either the marker GUS or the
endogenous CBF2 gene. Similar results were seen when additional lines were
tested. The rRNA panel shows an ethidium bromide stained gel picture of
ribosomal RNA representative of the loading for the blots.

61

C cloheximide
dimer dimer
CBF2 pro 1-5 3-1

 

+

+ - + -

 

 
 
 
 

 

 

Figure 2.17. Effects of cycloheximide treatment. Warm grown plants containing
either the whole CBF2 promoter or the dimer fragment were treated with
cycloheximide (+). Blot hybridization with a GUS probe was used to determine
the amount of transcript that accumulated in response to the treatment. Control
plants were treated in a solution without cycloheximide (-). A plant line
containing the whole promoter from CBF2 fused to GUS is compared with two
independently generated lines containing a dimer of the 155bp CBF2 promoter
fragment. The rRNA panel shows an ethidium bromide stained gel picture of
ribosomal RNA representative of the loading for the blots.
CBF genes. All the mutations were then tested for responsiveness to low
temperature (Figure 2.6 and 2.18B). The genes with promoters containing
mutations at m1, m2 and m3 were induced to relatively high levels in response to
low temperature. However, only marginal levels of induction were seen for many
lines containing the mutation at m4. In addition, in some plants the level of
transcript present at warm temperatures was almost as high at that seen in the
cold.

Discussion

The discovery of CBF1 with its ability to moderate the cold acclimation process in

plants by regulating expression of multiple genes involved in freezing tolerance

62

A ABA

m2
CBF2 pro 1-6 2-1

 

GUS

rRNA

 

 

 

 

 

 

 

B Low Temperature
m1 m2 m3 m4
,1 A l A A'vl ‘1' ‘A'ql' l l A‘A l l l A l
GUS . I. A a. .4. . ” “-
' 1
CBF mmx. ' 0 0 o 0 .
rRNA 6‘ Ag; I‘AAAAO‘A A A" ALLLAAA

 

Figure 2.18. Effect of site directed mutagenesis on ABA and low temperature
responsive expression. In A, the promoter activity levels from ABA treated plants
(+) were determined by blot hybridization using the marker gene GUS as a
probe. Control plants were treated in a solution without ABA (-). A plant line
containing the whole promoter from CBF2 fused to GUS is compared with two
independently generated lines containing the CBF2 promoter fragment deleted to
-189 and mutated to remove a potential ABRE (m2). In B, plants containing
promoters with site directed mutations were treated with cold (C), or left
untreated (W) and expression of GUS and CBF were compared using an RNA
blot hybridization. Each pair of lanes represents independently transformed plant
lines. The rRNA panel shows an ethidium bromide stained gel picture of
ribosomal RNA representative of the loading for the blots.

was a major advance that has led to a better understanding of some of the early
steps in one low temperature signaling cascade. The CBF1 gene and its two
tandem homologs, CBF2 and CBF3 are all cold induced (Gilmour et al., 1998).

Here the transcript levels for all three CBF genes have been shown to increase

dramatically within 15 min of transferring plants to a low temperature

63

 

environment. Following at about 2-4h after CBF induction, the level of transcript
for the CRT/DRE containing COR genes increases. The kinetics of these
experiments suggest that low temperature stimulated increases of CRT/DRE
containing COR gene transcripts requires a signaling cascade in which CBF
transcript accumulation is one of the earliest events. Induction does not
necessarily require a large change in temperature as was demonstrated when
plants were slowly cooled over an 8h period. However, lower temperatures still
seemed to stimulate higher levels of transcript accumulation. This could be due
to a change in temperature that is still too rapid for the plant to acclimate
completely. By 24h the level of CBF transcript reaches what appears to be a
new steady state level, and this level persists indefinitely if the plant perceives a
continuous cold stimulus. If the plant desensitized to the cold stimulus, one
might expect the signal to eventually disappear. This is not the case. In fact, the
plants respond quite rapidly and to a high level when exposed repeatedly to low
temperatures.

CBF induction appears to be regulated at a transcriptional level as was
shown when the CBF promoters were fused to the GUS reporter gene. The
putative transcription factors involved in CBF induction though are probably not
the CBF proteins themselves as the promoters of all the CBF genes lack the
CRT/DRE sequence and overexpression of CBF1 does not cause the
accumulation of CBF3 transcript (Gilmour et al., 1998). There are however
several conserved motifs in the promoters of all three CBF genes and while most

do not seem to be required for low-temperature induction, there is still the

64

pos

09.”

he

possibility they may function to confer specificity or alter responsiveness under
certain conditions.

In this study, the three CBF promoters in whole and multiple deletion
fragments of CBF2 fused to GUS were used to characterize responsiveness to
different stresses. While the picture is still not clear as to what minimum
element(s) of the promoter is required to induce expression under all conditions,
some parts of a 155 base pair region appear to be sufficient. This region was
responsive to all the stresses tested including low temperature and mechanical .
agitation and to a lesser extent ABA and water stress. ABA and dehydration
treatments result in distinct patters of expression with drought stress presenting a
sustained elevated level of transcript while the ABA treatment resulted in a higher
initial level but rapid decay. While this could represent inactivation of ABA in the
light, it may also represent an augmentation of the response to mechanical stress
which then quickly disappears. In either case, because the responses to ABA
and dehydration stresses were low they may have been missed in earlier reports
(Liu et al., 1998, Shinwari et al., 1998) due to a relatively large induction caused
by mechanical agitation whether intentional or accidental. This difference in the
level of expression between low temperature and dehydration or ABA response
may mark a bifurcation in the signaling cascade between the two stress
responses. The CBF genes are mainly responsive to low temperature stress and
two additional CRT/DRE binding proteins, DREBZA and DREBZB (dehydration
responsive element binding proteins) identified by Liu et al. (1998) are

responsive to dehydration stress.

65

At present the receptor of the low temperature signal (or drought or
mechanical signal) is not known nor are the earliest events in the signaling
cascade that precede CBF induction. Gilmour et al. (1998) have proposed the
presence of an unknown transcriptional activator termed ICE (inducer of CBF
expression) that is present at warm temperatures. Upon exposure of a plant to
low temperatures, ICE is proposed to be modified as a result of activation of the
signaling cascade. ICE may then promote the increase orland relieve inhibition
of CBF gene expression, which leads to the cold acclimation response.

There is also growing evidence that calcium and phosphorylation play
roles as early signaling components (Knight et al., 1996, Monroy and Dhindsa,
1995, Sheen, 1996; Tahtiharju et al, 1997). Cold shock has been shown to
cause an immediate increase in cytosolic free calcium that could be partially
inhibited by EGTA or lanthanum (Knight et al., 1996). By blocking calcium
channels with lanthanum or EGTA, induction of cold-regulated gene expression
was inhibited (Monroy and Dhindsa, 1995). Further supporting the role of
calcium, at warm temperatures the addition of a calcium ionophore stimulated the
influx of calcium and induced expression of some cold regulated genes (Monroy
et al., 1998). In addition to its possible role in cold shock, calcium release may
be effecting a response from the CBF promoters as a result of circadian
oscillations. Cycles of light or temperature are known to be entraining stimuli and
calcium levels oscillate with circadian rhythm (McClung, 2001). Therefore, a
transient spike in calcium levels after a light to dark transition may cause a

transient activation of the CBF promoter similar to the increase in response after

66

a warm to cold shift. If the promoter is fused to a marker such as GUS which
produces a stable protein, accumulated activity of the GUS protein would be
visible after a few days of growth, which could explain the inability to use GUS
staining to follow patterns of gene expression (Figure 2.7).

Calcium dependent phosphorylation/ dephosphorylation processes also
seem to be involved in activating the cold acclimation process. The application
of a protein phosphatase inhibitor stimulated the induction of the cold regulated
gene CAS15 while a protein kinase inhibitor blocked cold regulated gene
expression (Monroy et al., 1998). Jonak et al., (1996) have also shown in alfalfa
that a mitogen-activated protein kinase (MAP kinase) pathway is activated within
10 min of 3 plants exposure to low temperature.

Interesting ideas for upstream activation of CBF genes are suggested if
one examines the effects associated with the application of the translation
inhibitor cycloheximide . Mahadevan and Edwards (1991), Edwards and
Mahadevan, (1992), and Cano et al., (1994), while working on the induction of c-
jun and c-fos found that cycloheximide could activate some protein kinases
involved in signal transduction pathways even if concentrations of cycloheximide
were not high enough to inhibit translation. Berberich and Kusano (1997) also
found that subinhibitory concentrations of cycloheximide induce a calcium
dependent protein kinase (ZmCDPK1) from maize. Thus a protein kinase, such
as a MAPK or CDPK, may be activated in a signal transduction pathway in
Arabidopsis by the addition of cycloheximide and substitute for low temperature

or other stress requirements such as the release of calcium.

67

An additional level of regulatory control was suggested by a brief set of
experiments described here involving treatment of plants with concentrations of
cycloheximide believed to be high enough to prevent translation of CBF
transcripts and subsequently prevent transcription of COR genes. We found in
treated Arabidopsis plants, the level of CBF transcripts were elevated without the
plants experiencing a cold treatment. The same concentration of cycloheximide
did inhibit accumulation of COR genes after placing plants in the cold for several
hours (HuanYing Qin, personal communication). One possible explanation for
that is that some other factor that acts on the CBF promoter is a negative
regulator and that rapid turnover and inhibition of new synthesis allows active
transcription of the CBF genes at warm temperatures.

Currently the specific minimal cis-acting element sufficient to induce low
temperature responsiveness to the CBF genes is not known. Additional rounds
of promoter fragment fusion studies, site directed mutagenesis or electrophoretic
mobility shift assays and DNase I footprinting would be helpful for locating such
an element. Identification of a cis-acting element would aid in the isolation of
binding factors such as the postulated ICE protein. However, the larger 155 bp
fragment already known to be sufficient for low temperature responsiveness is
possibly adequate to use for the capture of some binding factors. Such factors
would then need to be further characterized to confirm their role in low
temperature specific activation and to separate them from other stress responses

if possible.

68

Materials and Methods

Bacteria and growth conditions. Cultures of Escherichia coli DH5a and
Agrobacten'um tumefaciens GV3101 were grown aerobically at 37°C or 30°C
respectively, in LB broth (Sambrook et al., 1989) or on solid LB agar medium
containing 1.5% agar. Plasmid-containing cultures were maintained in a medium
with selective antibiotics. Plasmids typically conferred resistance to kanamycin
(Km, 50mg/L) or ampicillin (Ap, 100mg/L).

Bacterial DNA isolation and cloning. Plasmids were constructed using
PCR (polymerase chain reaction) and standard molecular biological techniques
(Sambrook, et al., 1989). Primers used to amplify fragments of the CBF
promoters contained sites for unique restriction enzymes so that after
amplification, the PCR product could be restricted and then cloned directly into
the final vector in one step.

Site directed mutations were created in regions of the specific promoter
fragments by either Promega’s Gene Editor (Madison, WI) or Stratagene’s Quick
Change (La Jolla, CA) mutagenesis kits according to the product guide.
Promoter fragments and mutations were sequenced on an Applied Biosystems
Automated Fluorescent Sequencer at the Michigan State University-DOE Plant
Research Laboratory Sequencing Facility.

Plant transformation. The DNAs were transferred to Arabidopsis via a
whole plant dipping method similar to that described by Clough and Bent (1998).
Since the binary vector used to transfer the DNA to the plant also contained a

bacterial gene which conferred resistance to Km, plants containing transferred

69

DNA were identified by germinating seeds on medium containing Km (50mg/L).
These Krn resistant seedlings were grown to maturity and the next generation
seeds (T2) were often used for experiments.

Plant growth conditions. Plants were grown in soil and on agar
medium. When grown in soil, seeds were scattered on the surface of pots of soil.
The soil was kept moist and covered with clear plastic film to retain moisture until
seeds germinated then the cover was removed. Seedlings were grown in
chambers with 24h light (~120 umol rn‘2 s") at 22°C. Low temperature
treatments were usually done in the same chamber using half of the light as
when the plants were grown warm or they were placed in a refrigerated room at
4°C with dim light (~25 umol m'2 s“). Humidity was not controlled.

Agar medium contained Gamborg’s B5 nutrients (Life Technologies Inc.,
Gaithersburg, MD), and 1% phytagar (Life Technologies Inc., Gaithersburg, MD).
Plate grown seedlings were kept at ~24°C with 16h light (~35-100 umol rn’2 s")
and 8h dark.

Treatments with ABA, cycloheximide or dehydration were performed by
growing seedlings on filter papers that had been placed on top of agar so that the
seedlings could be lifted off the plate with minimal damage or stress. For ABA
and cycloheximide treatments, the filters with 10-14 day old grown seedlings
were floated on solutions of ABA (100mM) or cycloheximide (10ug/ml) in covered
dishes for various time periods. For dehydration treatments, filters with seedlings
were removed from plates and placed in desiccation chambers for different time

penods.

70

Mapping. Mapping was performed using the recombinant inbred lines of
Lister and Dean (1993) derived from the ecotypes Lansberg erecta and Columbia
(obtained from the Arabidopsis Biological Resource Center, Columbus, OH).
DNA was extracted (Rogers and Bendich, 1988) from 99 recombinant inbred
lines and used to map the position of the CBF gene and the four COR genes
using the protocols described by Lister and Dean (1993) (obtained from the
Nottingham Arabidopsis Stock Centre website
http://nasc.nott.ac.uk/Rl_data/how-to-man.html). Enzymes used to generate
RFLPs for the genes CBF2, COR6. 6, COR15a, COR47 and COR78 included
Dral, EcoRl, Aflll, EcoRV and Dral, respectively. Segregation data was
submitted to the Nottingham Arabidopsis Stock Center and analyzed using the
MAPMAKER program (Lander et al., 1987).

Staining for B—glucuronidase acitivty. In order to stain plant tissue to
determine location of B—glucuronidase activity, experimentally treated plants
were immersed in a GUS staining solution as described by Jefferson (1987) and
incubated overnight at 37°C. Tissue was cleared with several rinses of 70%
ethanol to aid in the localization of the staining. Images in this dissertation are
presented in color.

RNA Isolation. Total RNA was extracted from plant material with the use
of the Qiagen RNeasy Plant Mini Kits (Qiagen lnc., Valencia, CA) with
modifications. To obtain adequate and consistent yields with the kit, the amount

of starting plant tissue was doubled. Subsequently the amount of extraction

71

buffer (RLT) was also doubled. The remaining procedure was performed as
described in the Qiagen manual.

RNA Blots. Prepared RNA samples were mixed with formaldehyde
loading buffer containing ethidium bromide (~25ng/mL) and electrophoresed on
formaldehyde agarose gels according to standard procedures (Sambrook et al.,
1989). RNA was transferred to nylon membrane in 20X SSPE according to
standard protocols (Sambrook et al., 1989). After transfer, the RNA was cross-
linked to the membrane with UV light from a UV Stratalinker 2400 (Stratagene,
La Jolla, CA). The membrane was prehybridized in a Robbins hybridization
incubator model 400 (Robbins Scientific Corp., Sunnyvale, CA) in a solution
containing 50% formamide, 5X SSPE, 5X Denhardt’s solution, 0.5% sodium

dodecyl sulfate (SDS) and 100ug/ml sheared, denatured salmon sperm DNA.
After 3-4 hours of prehybridization, 32P-Iabeled probe was added to the solution
to hybridize overnight at 42°C. The probe was prepared from a gel-isolated
fragment by a random priming method (Feinberg et al., 1983). After
hybridization, the membrane was rinsed briefly in a solution of 2X SSPE and
0.5% SDS at room temperature followed by two washes at room temperature or

42°C for 10—30 minutes. The membrane was then washed 2-3 times for 15

minutes each at 50°C with a solution containing 0.1X SSPE and 0.5% SDS.

72

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76

Chapter 3

SCREEN FOR MUTATIONS IN THE SIGNAL TRANSDUCTION
PATHWAY LEADING TO INDUCTION OF COR GENES
Summary
An experimental approach was designed for the identification of elements

important in the low temperature signal transduction pathway leading to the
induction of cold regulated (COR) genes. Several plasmids were constructed
that contained a known cold responsive cis-acting element in the context of either
the COR153 or COR78 gene promoters from Arabidopsis and fused to several
different marker genes. These plasmids were used to integrate the marker gene
into plants. Plants were then chemically mutagenize then selected or screened
for aberrantly regulated expression of the marker. Aberrant expression, either
up—regulation of the marker under non-inducing conditions or loss of expression
under inducing conditions, may indicate a mutation in the signal transduction
pathway. Several plants were identified, which had diminished expression levels
of at least one of the COR genes, and these mutant were further analyzed. RNA
blot hybridization demonstrated however that in most cases when the transcript
for a COR gene was absent or diminished, transcripts of the marker gene were
also reduced. This indicated that some form of gene silencing was likely taking
place and that the mutation was not in the signal transduction pathway. Means
for improving the mutagenesis strategy to help identify signaling mutants are

discussed.

77

 

Introduction

Because plants are generally sessile and cannot avoid environments that
may be harmful to their survival, they have developed mechanisms to adapt to
their environment as it continuously changes around them. Freezing
temperature is one common adverse environmental factor that affects survival,
productivity and plant distribution. Plants from temperate regions can generally
be acclimated to freezing temperatures by first treating them with low nonfreezing
temperatures. This cold acclimation process results from biochemical and
physiological changes occurring in the plant. Some changes may include altered
composition of membranes and proteins, changes in activity of some enzymes or
accumulation of certain cryoprotective compounds such as sugars or
polyamines. The expression of many genes is also up regulated in response to
cold stress. One group of genes, the COR genes, consists of four stress
responsive gene families, (COR6. 6, COR15, COR47, and COR78), which are
differentially regulated but with at least one gene in each family being induced by
low temperature. These genes maintain many other common properties. The
mRNA transcript levels increase within two hours of plants being transferred to
cold (4°C) and remain elevated as long as the plants are maintained at low
temperature. The transcript levels decrease rapidly when the plants are returned
to normal growth temperature (Thomashow, 1994). The genes are also induced
by dehydration stress orland by the exogenous application of abscisic acid.

Proteins encoded by the genes remain soluble upon boiling due to their high

78

hydrophilicity (Hajela et al., 1990), which may be important during periods of
stress that result in membrane-membrane interaction such as during freezing or
dehydration stresses. One of the more thoroughly studied genes, COR15a,
encodes a polypeptide that is targeted to the stromal compartment of the
chloroplasts and can increase their freezing tolerance (Artus et al., 1996). The
increase in freezing tolerance appears to result from an interaction with the
membrane that reduces the formation of deleterious hexagonal ll phase lipids
upon freezing-induced dehydration (Steponkus et al., 1998).

The incentives for studying the mechanisms by which the COR genes are
regulated include a desire to understand the basic strategies used in low
temperature and other stress biology and also to use the information obtained to
potentially bear against the cost of feeding an ever increasing population reliant
upon a small group of crops forced to grow in environments that are less than
ideal for producing their maximum yield. At the start of this work, a low-
temperature and dehydration responsive cis-element had been identified in the
promoter of the COR genes (Yamaguchi-Shinozaki and Shinozaki, 1993, Baker
et al., 1994) but no direct trans-acting factors nor any other upstream
components had been identified that played a direct role in the their regulation.

Therefore, an attempt was made to identify some of the regulatory factors
associated with the low temperature signal transduction pathway using several
different cold responsive gene promoter fragments. Because there is no easily
detectable phenotype specific for the induction of any of the COR genes, several

marker genes were tested. The markers that were used included a herbicide

79

selection (BAR) which confers resistance to glufosinate ammonium, a negative
selection marker (codA), which when induced can convert the innocuous 5-
fluorocytosine (5-FC) into a toxic compound, and a phenotypic screen based on
production of anthocyanin in mutant Arabidopsis plants that did not normally
produce anthocyanin. A fourth marker gene encoding beta-glucuronidase (GUS)
had been used in a similar study prior to this work (Wilhelm, 1996). The first
three markers had potential benefits over GUS including use of either selective
methods or the ability to nondestructively screen large populations of plants
through isolation of phenotypic variation. All of these also had drawbacks that
are discussed later.

A fifth marker system used by lshitani et al. (1997) relied on the cold
regulated COR78 promoter fused to the Iuciferase gene. This screen has
already identified many mutants affected in their response to low temperature
and other stresses. The advantage of using Iuciferase is the ability to rapidly
screen large populations of young seedlings with a high degree of sensitivity.
Their success buoyed our hopes for finding some low temperature stress
signaling genes.

It was hoped that new mutants could help identify components of the
signal transduction pathway, especially if mutants altered in their response to a
low temperature stimulus were also be altered in their response to other
stresses. In addition, the number of mutants that could be isolated might have
given a sense of the complexity of the signaling pathways involved. Several

mutant lines isolated were identified with lower levels of transcript from at least

80

one COR gene in response to low temperature stress. These plants were further
analyzed. In the end, however, no genes associated with low temperature signal
transduction were isolated from any of the marker fusions and mutagenesis
screens. Some information gleaned from experiments using these plants may

benefit future experiments.

Results

 

Regulation of marker gene expression by low temperature. The
choice of an appropriate promoter and marker gene is important for developing a
screen for gene regulation mutants in the low temperature signal transduction
pathway. Prior to this work a COR153 promoter fused to a GUS reporter was
used to screen over 10,000 mutagenized plants without successfully identifying a
mutant in the low temperature signal pathway (Wilhelm 1996). Subsequent to
this screen, several promoter or promoter fragments and marker combinations
were tested to identify the most useful. The first marker tested was for the
bacterial gene BAR that encodes a protein which, when expressed in plants,
confers resistance to herbicides with the active ingredient glufosinate ammonium
(De Block et al., 1987, Thompson et al., 1987). Initially multiple copies or
“multimers” of a COR15a promoter fragment containing the C-repeat (CRT) was
fused to BAR. Earlier work with promoter/GUS fusions had shown that multimers
of this CRT gave low temperature regulated expression in tobacco (Figure 3.1A).
However, when this multimer was fused to BAR (or GUS) and transformed into
Arabidopsis, expression was constitutive (Figure 3.1B). The multimeric form of

the CRT was used because a smaller fragment of the COR15a promoter

81

containing only a single CRT did not result in any detectable transcript on an
RNA blot (Figure 3.1B) or GUS protein as determined by colorimetric assays
(Jefferson, 1987). A larger (approximately 1kb) fragment of the COR15a
promoter was then fused to the BAR gene and also to a negative selection
marker codA gene (Stougaard. 1993). The codA gene encodes a protein that
can convert the normally innocuous compound 5-FC to the toxic compound 5-
fluorouracll. These promoter-marker fusions were individually inserted into a

binary vector that also contained the COR158 promoter fused to GUS

A Whole 7mer Monomer
W C W C W C

Gusll ,. m

B 7mer Monomer
WC WC WC WC WC WC

COR15a . . . . .

 

 

 

  
  

 

 

Figure 3.1. Expression pattern of COR15a promoter/GUS fusions. RNA blots
demonstrate expression patterns of a GUS gene fused to a whole COR15a
promoter, a multimer (7mer) CRT and a monomer CRT. Tobacco plants
containing promoter fusions have cold induced transcript accumulation (A), while
transgenic Arabidopsis plants high level of expression in warm and cold for the
multimer and no transcript accumulation if the monomer is used (B). Each pair of
samples from Arabidopsis represents an individual transgenic line. The
endogenous COR15a gene transcript is also shown in B for comparison of
expression at warm (W) and cold (C) treatment conditions.

82

resulting in a double marker vector. The constructs were then used to transform
Arabidopsis. From the over 50 independent transformants generated for each
construct, eight lines were chosen for further tests. These lines appeared normal
when grown on agar medium and in soil and segregated 3:1 for kanamycin-
resistant to kanamycin-sensitive in the T1 population suggesting a functional
transgene insertion at a single locus for each line. For each line, plants
homozygous for the transgene were identified and used to bulk seeds for later
experiments.

Before mutagenizing plants, seedlings were grown under standard growth
conditions to determine the suitability of the selection methods for isolation of
mutants. To test the GUS marker, ten day old seedlings grown on solid agar
medium were exposed to low temperature (4°C) for four days and stained
according to Jefferson (1987). For all the lines, warm controls did not stain, and
for all but one of the BAR containing lines when placed at low temperature, the
plants stained blue indicating a functional GUS marker. These same lines were
then grown and treated with either a solution containing glufosinate ammonium
or 5-FC.

The plants containing the BAR gene marker were grown on either agar
medium, sand or potting mix for 10-21 days before treatment with glufosinate
ammonium. Glufosinate ammonium was then either sprayed on leaves of plants
until run off or added to the growth medium. Concentrations ranging from 25uM
to 2.5mM quickly killed non-transgenic control plants but in all cases some warm

grown and cold treated transgenic plants containing the BAR gene survived.

83

Several different treatment regimes including changes in pretreatment growth
conditions, age of treated plants and glufosinate ammonium concentrations
never resulted in consistent killing of all warm grown transgenic plants.

The plants containing the codA gene were grown on solid agar medium
with 5-FC either added to the medium or flooded onto the surface after plants
were 10-14 days old. The 5-FC was added at either 1 mg/ml or 3 mg/ml. After
growing in the warm, plates were transferred to a low temperature growth
chamber. Several temperature regimes were tested since plants grew so slowly

at 0-4°C that no effect of the 5-FC was observed. At temperatures above 12°C,
the activity of the COR153 promoter is diminished and plants were less

consistently affected by the 5-FC. A cycling of temperatures between 8-10°C for
16h per day and 22°C for 8h per day under continuous light was chosen because

it was low enough to induce COR15a promoter driven expression of the marker
and the warm period allowed faster growth. Greater than 90% of seedlings
would eventually die after 2-4 weeks under this growth regime. However,
seedlings had to be placed sparsely (<100 seedlings per 10 cm Petri plate) onto
agar plates in order to achieve this level of selection. This would have required
large amounts of space and considerable labor to screen through the number of
seedlings expected to obtain mutants. And even at a 90% mortality level if the
thousands of mutagenized seedlings had been analyzed, they would have
generated too many false positives to make this selection method suitable for the

large-scale testing required.

84

Finally a third marker, the R gene was fused to the COR78 promoter (the
plasmid was a gift from Mark Alfinito, Stanford University). The R gene encodes
an anthocyanin specific transcriptional activator from maize, which is involved in
the anthocyanin biosynthetic pathway and rescues a transparent testa glabrous
(ttg) mutation in Arabidopsis (Lloyd et al., 1992). The ttg mutant plants do not
produce anthocyanin. However, when the R gene is introduced into the plants
and driven by the COR78 promoter (herein termed COR78pR plants), then plants
start to produce red/purple colored anthocyanins in the leaves when grown at low
temperature. Grown at warm temperature the plants appear light green in color.

Plants containing the R gene were grown for about 14 days in potting mix
before initiation of cycling low temperature treatments of 10°C (16h) and 22°C
(8h). The cycling temperature allowed faster growth and seemed to shorten the
time for color to develop in leaves. Even so at least 12-14 days of this treatment
was required for significant and even color development if a large population of
seeds was planted in a small area.

From the numerous independent transformants, two lines were chosen for
further experiments. One line contained a single transgene insertion and the
other line contained two independent insertions as determined by DNA blot
analysis (Figure 3.2). RNA was also isolated from these lines and their
expression pattern from RNA blot analysis matched that of other COR genes
(Figure 3.3 and Fig 3.4 parent lines).

Isolation of mutants with altered expression of the marker gene after

low temperature treatments. About one gram of transgenic seeds from each

85

 

of the two COR78pR plant lines was mutagenized by EMS (ethyl
methansulfonate) treatment essentially as described by Haughn et al. (1986),
divided into 30 pools, germinated and the M1 plants were allowed to self-
pollinate. The M2 seeds from each pool (about 0.59 each) were collected
independently. M2 seeds from the different pools were scattered onto the
surface of flats of potting mix and grown for about 10 days before starting low

temperature treatments. During the screening it became evident that shading

1.6 3.4
B H B H

 

 

 

 

 

Figure 3.2. Determination of transgene copy number in transformed plants.

DNA blot hybridization pattern observed for restricted genomic DNA isolated from
the parent plant lines 1.6 and 3.4, which were subsequently used in mutagenesis
experiments. The probe used for the hybridization was a 3 P labeled R gene
fragment. Lanes labeled B contain DNA restricted with the endonuclease 39! II,
which cuts twice within the R gene but outside of the sequence used to make the
probe. Lanes labeled H contain DNA restricted with the endonuclease Hind III,
which cuts one time within the R gene but 5’ to the fragment used to make the
probe.

86

R gene
COR15a

 

 

Figure 3.3. Gene expression pattern in transgenic plants. RNA blot hybridization
demonstrating the normal expression pattern for the cold regulated COR15a
gene and the cold regulated marker R gene. NT is a nontransgenic plant, 1.6
and 3.4 are transgenic plants containing the R gene regulated by the COR78
promoter. W= warm grown plants, C= one day cold (4°C) treated plants.

from neighboring plants could cause a phenotype similar to what was expected
for mutants therefore putative mutants lacking anthocyanin after a set length of
time were transplanted out of the flats to individual cell pots and left in the
treatment chamber an additional week. Those putative mutants that did not

produce anthocyanin were transferred to normal 22°C growth chambers and

allowed to set seed.

Approximately 150,000 M2 seedlings were screened and about 100
COR78pR plants were selected as putative mutants and grown for seed. This
seed was then used to confirm the mutant phenotype. Of these, about 65 were
confirmed mutants lacking anthocyanin when grown at low temperature and were
used for further characterization by examining the expression of other COR
genes and observing the effect on freezing tolerance.

Characterization of mutants. The effect of the mutation on expression
levels of other COR genes was examined by RNA blot analysis. RNA was
isolated from low temperature treated plants for each mutant and the parent non-

mutagenized lines. The samples were electrophoresed, blotted and hybridized

87

with probes specific for the COR genes, (COR 6.6, COR15a, COR47, and
COR78) as well as the R gene and a constitutively expressed Arabidopsis elF4A
(eukaryotic initiation factor 4A) gene (Metz et al., 1992). The majority of mutants
had no change in the level of expression for any gene except for the marker that
was not expressed in several lines. A few had slightly decreased or complete
loss of expression for COR78 only but none had consistently lowered expression
levels for all other COR gene (Figure 3.4).

Three of the most severe mutants with diminution of expression for
COR78 were tested for diminution of freezing tolerance as determined by the use
of an ion leakage assay. Several independent ion leakage assays were
performed and two assays are shown in figure 3.5. Each mutant sample and
parent is represented by three replicates at each temperature. The variation in
percentage of ion leakage between samples from a given temperature was large
within a single assay and between the independent assays. This variability,
inherent in the assay, may be due to handling differences between samples and
non-uniformity in the tissues used for each sample. For example, no attempts
were made to account for leaf age or size. Taking into account the sample
errors, no significant change was observed in the temperature at which a 50%
leakage point was reached between those plants that expressed the COR78
gene normally and those in which the COR78 gene expression had been
reduced or eliminated. In both types of plants, 50% ion leakage had been

surpassed at about -6°C.

88

 

 

 

COR 78 0' ’fiwu art-.0 t.-

C at. O I
t

 

 

  
  

 

 

 

 

 

'11....“ ..,”:’Q 1

 

 
 

 

Figure 3.4. Gene expression in mutagenized plants. RNA blot hybridization
expression pattern for COR genes, the R gene and elF4A in different lines of
plants that have been mutagenized. Parent lines 1.6 and 3.4 accumulate little
transcript after growth at warm (W) temperatures, however abundant transcripts
accumulate after cold (C) treatments. Putative mutants (1-65) selected by
phenotypic loss of anthocyanin deposition in leaves show varying levels of
transcript accumulation after cold treatment. Lines identified from the
mutagenesis of parent 1.6 are shown in A. Lines identified from the mutagenesis
of parent 3.4 are shown in B.

89

 

         

 

 

 

 

 

                   

         

   
  

 

          
  

  

    

 

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Figure 3.5. lon leakage assays to determine cellular fitness or loss of fitness after

a three day cold acclimation period. Two independent assays were performed (A

and B) using three replicate tubes at each temperature. In both experiments,

injury due to freezing resulted in cells losing 50% of their relative ion content at

about 6°C for the control parent line, prnt1-6, and the three mutant lines.

90

Discussion
A set of plasmids was constructed containing the promoter from a cold

up-regulated gene (either COR15a or COR78) fused to a marker gene. These
plasmids were integrated into the genome of the model plant Arabidopsis
thaliana in order to perform mutagenesis studies which could be used for
identifying factors in the low temperature signal transduction pathway leading to
cold induced expression of the known COR genes and other genes that share
the same regulatory pathway.

Initially plasmids were constructed which contained multiple copies of the
CRTs from COR158 fused to a marker. A single copy of the CRT still containing
about 180 bases of context from the COR15a promoter had not given detectable
expression in earlier tests and therefore the multimer was designed. A plasmid
containing a multimer promoter sequence fused to a marker gene was initially
transformed into tobacco, which resulted in cold up-regulated expression of the
marker gene (Leonard Bloksberg, unpublished). When a similar plasmid was
transformed into Arabidopsis, however, expression of the marker gene was
elevated in both warm and cold to fairly high levels. The marker in Arabidopsis
was the BAR gene which confers resistance to herbicides containing the active
ingredient glufosinate ammonium. Several attempts were made to adjust the
concentration of the glufosinate ammonium and change the growth medium and
other environmental conditions including changing from agar medium to soil and
increasing or decreasing light intensity and day length. Because at the time we

could not explain the unexpected high level constitutive activity of the multimer

91

design in Arabidopsis, the decision was made to use a larger piece of the
promoter which had been shown with the GUS marker to be dramatically up-
regulated by cold (Wilhelm, 1996). Again, however, when the promoter was
fused to the BAR gene, enough expression was observed in both warm and cold
grown plants to confer resistance to the herbicide. Initially we were not able to
account for the apparently constitutive low level expression of the promoter that
resulted in resistance since at the level of detection by RNA blots, transcripts
were generally absent in warm grown plants. In light of information obtained later
while working with the CBF genes which are mechanically activated and possibly
developmentally induced as well, we believe it is possible that input from
additional stresses may have resulted in enough expression from the BAR gene
to produce an active enzyme, which would be stable enough to disable the
selectivity of the herbicide and prevent the use of the BAR gene as an effective
tool. Alternatively, the glufosinate ammonium may have been directly stimulating
expression of the COR genes and therefore the selection method would never be
usable. This was not tested. These plants could not be used for a negative
selection, that is, to identify genes in the cold signal pathway through loss of their
activity, because loss of BAR gene activity would result in plants being killed
when treated with the herbicide. Use of a later generational subset of seeds was
not practical for the experiment. Also, the inconsistent killing of plants that had
not been mutagenized indicated that there would have been a large number of

false negative mutants to screen.

92

A second marker system based on the codA gene from the bacterium E.
coli was then examined. Expression of this bacterial gene for cytosine
deaminase should allow identification of candidate genes in the plant through a
negative selection method. The method is based on the idea that since
Arabidopsis does not normally contain the enzyme cytosine deaminase and does
not metabolize cytosine to uracil, it should not be able to metabolize the
innocuous compound 5-FC to the toxic compound 5-fluorouracil. While the
selection method did not appear to have the problems with a constitutively active
promoter since plants could be grown on selective medium containing the
chemical 5-FC and survive in the warm, the killing in the cold was inconsistent.
Here it appeared that mechanism for selection could work but not under the
growth conditions needed to get adequate levels of expression from the COR15a
promoter. Several attempts were made to modify the growth regimes and a
minimally successful method was developed which relied in part, on cycling
between warm and cold temperatures but the level of false negatives seen in
non-mutagenized seedlings again would have been a hindrance to identification
of plants containing real mutations in the cold signal transduction pathway. A
more consistent method with the ability to screen through larger numbers of
seedlings was needed. Such a method appeared to present itself in a system
containing a group of Arabidopsis plants which completely lack anthocyanin and
are devoid of trichomes (ttg, transparent testa glabrous mutants) and a marker
gene R from the monocot maize which rescues the Arabidopsis mutation. When

a cold inducible promoter regulates the R gene, anthocyanin is produced when

93

 

the plants are grown at low temperature. Because the method is a non-lethal
screen, the R gene marker could possibly be used for both a positive and a
negative screen. As a positive screen, plants would not produce anthocyanin in
the cold if a mutation resulted in loss of expression of one of the genes in the low

temperature signal transduction pathway. As a negative screen, plants would

 

produce anthocyanin at warm growth temperature if a mutation resulted in h
increased expression of one of the pathway genes. Of course, one of the AI

drawbacks to this screen is the potential for apparent loss or gain of R gene

expression due to mutations not in the cold signal pathway but rather in the b

anthocyanin production pathway. Additionally, a single mutation in the promoter
for the R gene may also cause a lack of anthocyanin production. The only
method to identify such alternative mutants would be through RNA blot analysis
of COR gene expression. Since the R gene is regulated by the same promoter
as COR78, a decrease in expression affecting both genes is likely due to a
mutation in the COR gene regulation pathway and not just in the promoter of the
R gene or the anthocyanin pathway.

In the end the negative screen was not extensively utilized. However, by
using the positive screen several plants were found that displayed loss of
anthocyanin when grown in the cold and RNA blots showed a decrease in both
the R gene and COR78 gene expression. The expression levels of the other
COR genes were not affected or only minimally affected. This result was not
expected since we had predicted that the low temperature regulation of COR

gene expression was taking place through the C-repeat which is present in all the

94

 

COR genes examined. This result suggested either a second pathway is being
utilized for COR78 regulation or possibly a form of gene silencing is taking place.
Silencing caused by transcriptional inactivation may result from promoter
homology between the endogenous COR78 gene promoter and the introduced
COR78pR transgene (Park et al., 1996, Jakowitsch et al., 1999). Neither of
these hypotheses was tested.

Since no mutants were identified which affected all the COR genes, the
plants with diminished COR78 expression were analyzed alone for diminished
tolerance to freezing. The ion leakage test is a rapid, albeit crude, measure for
estimating freezing tolerance by examining the extent of cell damage resulting
from freezing. Plant leaves are subjected to decreasing temperatures and later
analyzed for damage relative to the amount of ions that have leaked from cells.
While COR78 is but one of a large group of cold induced proteins its significance
may have been estimated if mutants had shown a reduced capacity to cold
acclimate. Unfortunately the difference between mutants and controls was not
significant and so the relative importance of COR78 cannot be determined.
Further characterization of these mutants was determined to be of minimal
benefit due to lack of an effect on cold acclimation in mutants with reduced
COR78 expression, and the high possibility that the loss of expression was due
to a silencing mechanism not associated with a mutation in the low temperature
signal transduction pathway. Also due to the fact that lshitani et al. (1997) had
already identified several hundred potential mutants using a COR78 promoter

fused to the luciferase gene, this line of research was terminated. Their success

95

 

 

 

resulted mainly from their choice to use Iuciferase as a screenable marker
because it allowed them to screen thousands of small seedlings on a single agar
plate quickly and efficiently. Types of mutants they identified included those that
lost the ability to activate expression of the COR genes after cold treatment as
well as those that constitutively expressed COR genes at both warm and cold
temperatures. I
It is still possible that subtle differences in the regulation pathways of I

COR78 and the other COR genes exist and there are a number of measures that t .

 

could be taken to improve the chances of finding desired mutants in the signal
transduction pathway. One would be to repeat an experiment similar to lshitani
et al., (1997) and use the luciferase gene as a screening marker. This marker
would allow for rapid screens of large numbers of seedlings using several
different stress treatments. T-DNA tagging to knock out genes and activation
tagging methods to activate expression could be used in addition to EMS
mutagenesis. A benefit of the tagging methods is the efficiency with which
mutant genes can be identified, since sequences in the tag are known.

It is also possible that experiments could be developed using promoter
fragments that are only inducible by low temperature to aid in the identification of
components in the cold specific signal transduction pathway. The commonality
of proposed functions for many of the cold, drought and salt tolerance genes
makes it challenging to think which regulatory genes may or may not be specific
to each stress tolerance pathway. Already Liu et al. (1998) have suggested a

bifurcation in the pathways starting with CBF/DREB1 and DREBZ. Both are

96

transcriptional activators that bind to the C-repeat/dehydration responsive
element but DREBZ is specifically induced by drought and CBF/DREB1 has been
suggested to be up regulated specifically by low temperature (Liu et al., 1998,
Medina et al., 1999). Although more recently the specificity of the regulatory
mechanisms working on CBF/DREB1 has been questioned. The CBF/DREB1
genes do not appear to be solely responsive to low temperature and in fact
respond to stresses like mechanical stimulation, ABA, drought and the chemical
cycloheximide. Nevertheless, if studies using promoter fragments regulated by
specific stresses could be developed, one might gain insights into how plants are
able to distinguish between the different stresses and then use that information to
more finely regulate responses of plants to changing environments.

Materials and Methods

Bacteria and growth conditions. Cultures of Escherichia coli DH5a and
Agrobacterium tumefaciens GV3101 were grown aerobically at 37°C or 30°C
respectively, in LB broth (Sambrook et al., 1989) or on solid LB agar medium
containing 1.5% agar. Plasmid containing cultures were maintained in a medium
with selective antibiotics specific for the plasmids.

Bacterial DNA isolation and cloning. Standard molecular biological
techniques were used for construction of all plasmids used in experiments
(Sambrook et al., 1989). Binary vectors containing the 7xCRTzGUS fusion and
the COR15a promoterzGUS fusion were used in the basic constructs to which the
6xCRTzBAR and COR158 promoterzBAR or COR15a promoterzcodA fusion

fragments were added.

97

 

 

Plant transformation. The DNAs were transferred to Arabidopsis via a
root transformation method (Valvekens et al., 1988) or vacuum infiltration
(Bechtold et al., 1993).

Plant growth conditions. Plants were initially grown in Petri dishes on
solid agar medium containing Gamborg’s B5 (Life Technologies Inc.,
Gaithersburg, MD) and with kanamycin (50mg/L) to select for T1 lines containing
the transgenes. Experimentally developed lines were grown under conditions,
which would allow for selection with glufosinate ammonium (BAR gene
containing plants) or 5-fluorocytosine (codA gene containing plants) or inducing
conditions to screen plants by the R gene. Plants grown on plates were kept at
about 24°C with 16h light (~35-85 umol m'2 s") and 8h dark. When grown in soil,
seeds were scattered on the surface of pots of soil. The soil was kept moist and
covered until seeds germinated then the cover was removed. Seedlings were
grown in chambers with 24h light (~120 pmol m‘2 s") at 22°C. Low temperature
treatments were usually done in the same chamber using half the intensity of the
light as when the plants were grown warm or they were placed in a refrigerated
room at 4°C with dim light (~25 umol rn‘2 s“). Humidity was not controlled.

RNA Isolation. Total RNA was extracted from plant material using a
lithium chloride precipitation procedure as described by Gilmour et al. (1988) or
with the use of Qiagen RNeasy Plant Kits (Qiagen lnc., Valencia, CA) with
modifications (Gilmour et al., 1998). To obtain adequate and consistent yields

with the kit, the amount of starting plant tissue was doubled. Subsequently the

98

amount of extraction buffer (RLT) was also doubled. The remaining procedure
was performed as described in the Qiagen manual.

RNA Blots. Prepared RNA samples were mixed with formaldehyde
loading buffer containing ethidium bromide (~25ng/mL) and electrophoresed on
formaldehyde agarose gels according to standard procedures (Sambrook et al.,
1989). RNA was transferred to nylon membrane in 20X SSPE according to
standard protocols (Sambrook et al., 1989). After transfer, the RNA was cross-
linked to the membrane with UV light from a UV Stratalinker 2400 (Stratagene,
La Jolla, CA). The membrane was prehybridized in 3 Robbins hybridization
incubator model 400 (Robbins Scientific Corp., Sunnyvale, CA) in a solution
containing 50% formamide, 5X SSPE, 5X Denhardt’s solution, 0.5% sodium
dodecyl sulfate and 100pg/ml sheared, denatured DNA. After 3-4 hours of
prehybridization, 32P-labeled probe was added to the solution to hybridize
overnight at 42°C. The probe was prepared from a gel-isolated fragment by a
random priming method (Feinberg et al., 1983). After hybridization, the
membrane was rinsed briefly in a solution of 2X SSPE and 0.5% SDS at room
temperature followed by two washes at room temperature or 42°C for 10-30
minutes. The membrane was then washed 2-3 times for 15 minutes each at
50°C.

Arabidopsis DNA Isolation and DNA blot analysis. DNA was isolated
from whole plants using a CTAB (hexadecyltrimethylammonium bromide) method
(Doyle and Doyle, 1987). One gram of fresh tissue was ground in a mortar with a

pestle after freezing in liquid nitrogen and placed in a plastic tube to which 3mL

99

 

of a 2X CTAB buffer (2% CTAB, 1.4M NaCI, 0.02MEDTA, 0.1M Tris pH8) were

added. The tube was incubated for 30 min at 60°C. After cooling, the sample

was extracted 2-3 times with chloroform then the DNA was precipitated with 2M
NaCl and 0.75 volumes isopropanol. After pelleting the DNA by centrifugation,
the DNA was suspended in 0.01M Tris/0.001M EDTA and precipitated again with
1/10 volume of 3M sodium acetate and 1 volume 95% ethanol. After
centrifugation the pellet of DNA was suspended in water.

Total DNA was digested with various restriction enzymes and the
fragments separated by agarose gel electrophoresis. The DNA was then
transferred to nylon membranes according to standard protocols (Sambrook et
al., 1989). The DNA was UV cross-linked to the membrane and hybridized in a
manner identical to that described for RNA blot hybridization.

Ion leakage. The percent of ions leaked from plant leaf tissue at various
temperatures was determined using an ion leakage test described by Jaglo-

Ottosen, et al., (1998).

100

 

Literature cited

Artus NN, Uemura M, Steponkus PL, Gilmour SJ, Lin C, Thomashow MF.
1996. Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a
gene affects both chloroplast and protoplast freezing tolerance. Proc Natl Acad
Sci USA. 93:13404-13409.

Baker SS, Wilhelm KS, Thomashow MF. 1994. The 5’-region of
Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought-
and ABA-regulated gene expression. Plant Mol Biol. 24:701-713.

Bechtold N, Ellis J, Pelletier G. 1993. In Planta Agrobacterium mediated
gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci
Paris, Life Sciences. 316:1194-1199.

Boyer JS. 1982. Plant productivity and environment. Science. 2182443-
448.

De Block M, Botterman J, Vandewiele M, Dockx J, Thoen C, Gossele B,
Movya NR, Thompson C, Van Montagu M, Leemans J. 1987. Engineering
herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J. 6:
2513-2518.

Doyle JJ, and Doyle JL. 1987. A rapid DNA isolation procedure for small
amounts of fresh leaf tissue. Phytochem. BU". 19:11-15.

Feinberg AP, Vogelstein B. 1983. A technique for radiolabeling restriction
endonuclease fragments to high specific activity. Anal Biochem. 132: 6-13.

Gilmour SJ, Hajela RK, Thomashow MF. 1988. Cold acclimation in
Arabidopsis thaliana. Plant Physiol. 87:745-750.

Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM,
Thomashow MF. 1998. Low temperature regulation of the Arabidopsis CBF
family of AP2 transcriptional activators as an early step in cold-induced COR
gene expression. Plant J. 16:433-442.

Hajela RK, Horvath DP, Gilmour SJ, Thomashow MF. 1990. Molecular
cloning and expression of cor (Cold-regulated genes in Arabidopsis thaliana.
Plant Physiol. 93: 1 246-1 252.

Haughn GW, Somerville CR. 1986. Sulfonurea-resistant mutants of
Arabidopsis thaliana. Mol Gen Genet. 204:430-434.

lshitani M, Xiong L, Stevenson B, Zhu JK. 1997. Genetic analysis of
osmotic and cold stress signal transduction in Arabidopsis: interactions and

101

 

convergence of abscisic acid-dependent and abscisic acid-independent
pathways. Plant Cell. 9:1935-1949.

Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow
MF. 1998. Arabidopsis CBF1 overexpression induces COR genes and enhances
freezing tolerance. Science. 208:104-106.

Jakowitsch J, Papp l Moscone EA, van der Winden J, Matzke M, Matzke
AJM. 1999. Molecular and cytogenetic characterization of a transgene locus that
induces silencing and methylation of homologous promoters in trans. Plant J.
17:131-140.

Jefferson RA. 1987. Assaying chimeric genes in plants: the GUS gene
fusion system. Plant Mol Biol Rep. 52387-405.

Liu Q, Kauga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K,
Shinozaki K. 1998. Two transcription factors, DREB1 and DREBZ, with an
EREBP/AP2 DNA binding domain separate two cellular signal transduction
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respectively, in Arabidopsis. Plant Cell. 10:1-17.

Lloyd AM, Walbot V, Davis RW. 1992. Arabidopsis and Nicotiana
anthocyanin production activated by maize regulators R and C1. Science.
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Medina J, Bargues M, Terol J, Perez-alonso M, Salinas J. 1999. The
Arabidopsis CBF gene family is composed of three genes encoding AP2 domain-
containing proteins whose expression is regulated by low temperature but not by
abscisic acid or dehydration. Plant Physiol. 119:463—469.

Metz AM, Timmer RT, Browning KS. 1992. Sequences for two cDNAs
encoding Arabidopsis than/iana eudaryotic protein sysnthesis initiation factor 4A.
Gene. 120:313—314.

Park YD, Papp l, Moscone EA, lglesias VA, Vaucheret H, Matzke AJM,
Matzke MA. 1996. Gene silencing mediated by promoter homology occurs at the
level of transcription and results in meiotically heritable alterations in methylation
and gene activity. Plant J. 92183-194.

Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning, A laboratory
Manual, 2nd ed. Cold Spring Harbor. Cold Spring Harbor Laboratory Press.

Steponkus PL, Uemura M, Joseph RA, Gilmour SJ, Thomashow MF.
1998. Mode of action of the COR15a gene on the freezing tolerance of
Arabidopsis thaliana. Proc Natl Acad Sci USA. 95:14570-14575.

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Stougaard Jens. 1993. Substrate-dependent negative selection in plants
using a bacterial cytosine deaminase gene. Plant J. 32755-761.

Thomashow, MF. 1994. Arabidopsis as a model for studying mechanisms
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Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp807-
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Thompson CJ, Movya NR, Tizard R, Crameri R, Davies JE, Lauwereys M,
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Streptomyces hygroscopicus. EMBO J. 622519-2523.

Valvekens D, Montague MV, Lijsbettens MV. 1988. Agrobacterium
tumefaciens-mediated transformation of Arabidopsis thaliana root explants by
using kanamycin selection. Proc Natl Acad Sci USA. 85:5536-5540.

Wilhelm KS. 1996. Studies on the regulation and protein products of the
COR15 gene family in Arabidopsis thaliana. Ph.D. Thesis. Michigan State
University. East Lansing, Michigan.

Yamaguchi-Shinozaki K, Shinozaki K. 1993. Characterization of the

expression of the desiccation-responsive rd29 gene of Arabidopsis thaliana and
the analysis of its promoter in transgenic plants. Mol Gen Genet. 236:331-340.

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Chapter 4

PERSPECTIVES AND FUTURE DIRECTIONS

Introduction

Average crop yields in the United States are three to seven times lower than that
of record yields and this large discrepancy is thought to be caused by
unfavorable environmental conditions affecting plant growth (Boyer, 1982). A
significant environmental factor contributing to the loss is low temperature stress.
This is part of the reason why the study of low temperature stresses has become
such an important area of research. In addition, our expectations for plants
productivity and growth often extend beyond evolutions initial design. Many of
the major crops grown in the temperate zones of the world such as maize, rice,
some of the bean crops, tomatoes, and cucurbits evolved in semi-tropical regions
of the world where development or maintenance of tolerance to freezing
temperature was not necessary. Accordingly, many of these plants are
susceptible to low temperature stresses.

The early spring plantings and late fall harvests required by the extended
growing season of many crops lead to unfavorable growth environments and
yield loss. Thus the goal of many plant breeders has been to increase the
tolerance of crops to all manner of environmental stresses including freezing
stress. Traditional breeding has had limited success however and there has
been little improvement in the freezing tolerance of many important crop species

over the last several decades (Thomashow 1990; Sarhan et al., 1998).

104

 

 

Frequently wild relatives of cultivated varieties of plants contain the genetic
information to increase freezing tolerance. However these same wild relatives
often also contain traits that are undesirable which adds to the difficulty of
traditional breeding for improved stress tolerance. And because freezing
tolerance is a quantitative trait, expression of several genes need to be
integrated and regulated in any crop plant that is being modified and this is not 3
easily done using traditional breeding technology. With advances in I

biotechnology and genomic information available from several different crop

 

plants and the model plant Arabidopsis, the use of molecular biological L}
techniques may now afford researchers the ability to selectively manipulate
specific aspects of physiological/biochemical pathways to increase a plants cold
tolerance. In order to do this, one still needs to understand the mechanisms of
regulation and identity of factors that are associated with low temperature
responsive gene expression.

Future directions

Many cold-responsive genes are likely to be found over the next few
years. With the use of gene chip technology and full genome Arabidopsis (and
other plant) arrays, genes involved in signal transduction pathways may begin to
appear more frequently in these lists. Transcription factors such as the CBFs
have proven themselves to be valuable tools for increasing stress tolerance
(JagIo-Ottosen et al., 1998), however high levels of these factors constitutively
expressed can have negative impact on plant productivity as well (Gilmour et al.,

2000)

105

 

 

In the present study, a 155bp fragment of the CBF2 promoter was found
to be sufficient to confer low temperature regulated expression to a marker gene.
This region was also responsive to mechanical agitation, ABA and
cycloheximide. Mutations created in several locations were unable to completely
eliminate the regulated response to stresses. This led to the conclusion that more
than one cis-element may be present and that multiple trans-acting factors
including an inducer and possibly an inhibitor of CBF expression may be at work.
Attempts to use computer software to scan the promoters of the three known
CBF promoters for likely cis-elements has had limited success to date. One of
the next steps may include using gel shift and competition assays to narrow the
region in the 155bp fragment that binds a specific protein. Foot printing
experiments may also be useful in this regard. If the region(s) of binding can be
narrowed, mutations can be made in the locations and transgenic plants can be
generated to test the in vivo effects of the loss of an element. This work would
still only give a cis-element but it would greatly enhance the ability to use
technologies like the yeast one-hybrid and southwesterns to identify trans-acting
factors. These techniques to date have been unsuccessful largely due to high
background from nonspecific binding (Sarah Fowler and Sarah Gilmour,
unpublished).

An alternative mutagenesis approach to identify trans-acting factors may
still benefit the research. Similar approaches using GUS as a marker were
unsuccessful because of problems with high protein stability. However, the use of

luciferase as a marker may be a key. Initial work with Iuciferase was similar to

106

 

GUS with high background at warm temperatures but new techniques in which
the background activity is first eliminated by the addition of high concentrations of
substrate (luciferin) have been developed. Only new Iuciferase production is
then quantified. A CBF2 promoter/Iuciferase construct is being tested in plants
and may hold some promise (Daniel Cook, unpublished).

A similar mutagenesis approach could be developed using a COR gene
promoter fused to luciferase. lshitani et al., (1997) used the COR78 promoter
fused to luciferase and found several classes of mutants affecting COR gene
expression. A COR15 promoter fusion would likely find similar classes of genes.
These mutants could also be screened by RNA blot hybridization to determine
the specific effects on CBF expression. Some of these mutants would
presumably contain defects in the signal transduction pathway and would be
interesting to study.

Mutants that do not affect CBF yet still inhibit COR gene expression would
also be interesting to study. Knight et al., (1999) have shown that one mutant,
sfr6, suppresses the low temperature induced COR genes but does not effect
CBF expression. The function of sfr6 is not known but it may play a role in either
activating CBF proteins at low temperature or assisting with binding specificity.
Other proteins with similar functions may be identified with a mutagenesis
screen.

Conclusions

Finally, many issues concerning the CBF genes have been presented

since their initial discovery and many remain mysteries. For example, why is

107

 

there such redundancy, are there specific tissues in which individual CBF genes
are more likely to respond or are there specific conditions like cold versus
drought when one of the CBF genes is more responsive? Three copies of the
CBF genes are located in direct repeats in the Arabidopsis genome (Gilmour et
al., 1998) and more recently an additional three CBF-like genes have been found
elsewhere. At present little is known about the function of the other CBF-like
genes. An additional class of CRT/DRE binding factors, the DREB2s, are
involved in increasing drought and ABA responsive expression of CRT/DRE
containing genes (Liu et al, 1998). These redundancies may just be due to the
importance of the need to cope with multiple stresses in a natural environmental
condition with a rapid and strong response. There may also be subtle
differences in types of stress or tissues in which individual genes function and we
have just not yet teased apart the differences.

Of the many cold responsive genes known in Arabidopsis and other
plants, their role in acclimating plants to low temperature is known for only a few.
This leaves the challenge of trying to assign roles for individual gene products
and determining their significance in the cold acclimation response. Very few
regulatory/signaling components are known and this is an important group.
Genes like CBF whose products govern the activation of multiple components of
the cold acclimation response pathway can have profound effects on freezing
tolerance. The discovery of more regulatory genes in other non-CBF pathways
may have additional importance for designing schemes to help crop plants cope

with environments less favorable for growth and productivity.

108

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448.

Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM,
Thomashow MF. 1998. Low temperature regulation of the Arabidopsis CBF
family of AP2 transcriptional activators as an early step in cold-induced COR
gene expression. Plant J. 16:433-442.

Gilmour SJ, Sebolt AM, Salazar MP, Everard JD,Thomashow MF. 2000.
Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple

biochemical changes associated with cold acclimation. Plant Physiol. 124: 1854-
1865.

lshitani M, Xiong L, Stevenson B, Zhu JK. 1997. Genetic analysis of
osmotic and cold stress signal transduction in Arabidopsis: interactions and
convergence of abscisic acid-dependent and abscisic acid-independent
pathways. Plant Cell. 921935-1949.

Jaglo—Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow
MF. 1998. Arabidopsis CBF1 overexpression induces COR genes and enhances
freezing tolerance. Science. 280: 1 05-1 06.

Knight H, Veale EL, Warren GJ, Knight MR. 1999. The sfr6 mutation in
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Liu Q, Kauga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K,
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EREBP/AP2 DNA binding domain separate two cellular signal transduction
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109