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3 * LIBRARY
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This is to certify that the
dissertation entitled

TRANSCRIPTION FACTORS THAT CONFIGURE THE LOW
TEMPERATURE TRANSCRIPTOME OF ARABIDOPSIS THALIANA

presented by

Jonathan Thomas Vogel

has been accepted towards fulfillment
of the requirements for the

PhD. degree in Cell and Molecular Biology

 

 

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2/05 cszC/DmoOtnfiud-nts

TRANSCRIPTION FACTORS THAT CONFIGURE THE LOW TEMPERATURE
TRANSCRIPTOME OF ARABIDOPSIS THALIANA

By

Jonathan Thomas Vogel

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Program in Cell and Molecular Biology

2005

ABSTRACT

TRANSCRIPTION FACTORS THAT CONFIGURE THE LOW TEMPERATURE
TRANSCRIPTOME OF ARABIDOPSIS THALIANA

By
Jonathan Thomas Vogel

Many plants can sense low temperature and respond by altering gene expression.
These changes in gene expression can ultimately lead to increased freezing tolerance as
part of a dynamic physiological process known as cold acclimation. One well known
pathway that contributes to cold acclimation is the CBF/DREB] cold response pathway.
This pathway includes the action of the transcriptional activators CBF], 2, and 3 (also
known as DREBI b, c, and a, respectively) that are rapidly induced in response to low
temperature and activate the expression of a suite of target genes, known as the CBF
regulon. The CBF regulon gene products act in concert to increase plant freezing
tolerance. Work in this dissertation focused on gaining a better understanding of the
changes in gene expression that occur in response to low temperature, the extent to which
the CBF pathway contributes to these changes, and whether six other transcription factors
that were coordinately regulated with CBF 2 affected cold-responsive genes.

In this work, the Affymetrix GeneChip containing probe sets for approximately
24,000 genes was used to define a core set of cold-responsive transcripts. A total of 514
transcripts were found to have altered transcript accumulation, 302 of which were up-
regulated and 212 down-regulated, in plants grown in two commonly used growth
conditions (soil and solid media). These genes were termed the COS (gold standard)
gene set. Hierarchical clustering assigned each transcript to one of seven expression

classes and bioinformatic analysis revealed multiple novel potential cis-elements present

in the promoters of genes in each expression class. Further bioinforrnatic analysis
revealed additional motifs among one of these expression classes, cluster III, but
experiments indicated no evidence that any of the novel motifs in clusters H1 or IV were
cold-responsive. One element, GTGATCAC, conferred constitutive GUS activity when
fused as a tetramer in front of the reporter. A functional analysis of the COS transcripts
revealed how the plants might be globally altering their metabolism in order to cope with
low temperature, which has generated a number of new hypotheses that can be tested.

Plants overexpressing CBF 2 or one of six transcription factors that were
coordinately regulated with CBF 2 were also profiled using the GeneChips. The CBF2
regulon was comprised of 85 cold-induced and eight cold-repressed genes. Of the six
genes that were induced in parallel with CBF 2 only one, ZA T12, influenced cold-
responsive genes when constitutively expressed. The ZAT12 regulon contained nine
cold-induced genes and 15 cold-repressed genes. Constitutive expression of ZA T12
resulted in a small, but reproducible increase in freezing tolerance, indicating a role for
ZAT12 in cold acclimation. ZAT12 also appeared to have a role in a negative regulatory
circuit that dampened expression of the CBF genes. Constitutive expression of ZAT12
dampened CBF transcript accumulation in response to low temperature, while decreased
levels of ZA T12 resulted in higher levels of CBF transcript accumulation.

The definition of the COS genes provides a framework on which the low
temperature regulatory networks of Arabidopsis can be constructed and this dissertation
begins this process by defining two regulons, the CBF2 and ZAT12 regulons. Future
experiments will expand our knowledge of these and other networks and could eventually

lead to novel strategies for engineering plants with increased stress tolerance.

ACKNOWLEDGEMENTS

In the completion and writing of this dissertation, I would like to first and
foremost thank my wife Kelly, to whom I am indebted for endless love and support.
Besides making life worth living, her editing skills are superb. I would also like to thank
my mentor, Michael Thomashow, whose guidance made this dissertation possible. For
their helpful guidance and advice, I thank my committee; John Ohlrogge, Steve
Triezenberg, Ken Keegstra, and Tim Zacharewski. Thanks to my family, especially my
parents, Thomas and Margery Vogel, whose years of love while raising me allowed me to
grow into the person I am today.

Thanks also to all the members of the Thomahsow laboratory who provided
intellectual stimulation and created a great working environment. I truly enjoyed my
time in the lab. Thanks specifically to those members I have worked with, including
Keenan Amundsen, Corien Bakermans, Donatella Canella, Marcela Carvallo, Diane
Constan, Daniel Cook, Colleen Doherty, Sarah Fowler, Sarah Gilmour, Michael
Mikellsen, Susan Myers, Ritu Sharma, Lahong Sheng, Heather Van Buskirk, Ryan

Warner, Daniel Zarka, and Xin Zhang.

iv

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ vii

LIST OF FIGURES ......................................................................................................... viii

CHAPTER 1 Literature Review
Introduction ............................................................................................................ 1
Freezing-Induced Damage ...................................................................................... 2
Mechanisms for Freezing Tolerance ...................................................................... 5
Low Temperature Perception ............................................................................... 10
Low Temperature Signal Transduction ................................................................ 12
Cold-Responsive Gene Expression ...................................................................... 15

CHAPTER 2 Roles of the CBF2 and ZAT12 Transcription Factors in Configuring the
Low Temperature Transcriptome of Arabidopsis

Summary .............................................................................................................. 20
Introduction .......................................................................................................... 22
Results .................................................................................................................. 25
Discussion ............................................................................................................ 51
Acknowledgements .............................................................................................. 59
Materials and Methods ......................................................................................... 60
CHAPTER 3 A Functional Analysis of the Arabidopsis Cold Transcriptome
Summary .............................................................................................................. 69
Introduction .......................................................................................................... 70
Results .................................................................................................................. 73
Conclusion ............................................................................................................ 91
Acknowledgements .............................................................................................. 93
Materials and Methods ......................................................................................... 94

CHAPTER 4 The Identification of Six Novel Motifs in the Promoters of Cold-regulated
Genes and the Determination of Their Cold-Responsiveness

Summary .............................................................................................................. 95
Introduction .......................................................................................................... 97
Results ................................................................................................................ 100
Discussion .......................................................................................................... 107
Materials and Methods ....................................................................................... 111

CHAPTER 5 A Summary of the Gene Regulons that Contribute to the Arabidopsis Cold

Transcriptome ..................................................................................................... 1 l4

APPENDICES
Appendix A. Probe sets up-regulated by low temperature in the plate experiment.

............................................................................................................................ 121
Appendix B. Probe sets down- -regulated by low temperature in the plate
experiment ........................................................................................................... 142
Appendix C. Probe sets up- regulated by low temperature in the soil experiment...
............................................................................................................................ 161
Appendix D. Probe sets down-regulated by low temperature in the soil
experiment ........................................................................................................... 178
Appendix E. Probe sets up-regulated by low temperature in both the plate and soil
experiments ......................................................................................................... 190
Appendix F. Probe sets down-regulated by low temperature in both the plate and
soil experiments .................................................................................................. 203
Appendix G. Probe sets up—regulated in response to CBF 2 expression ............. 211
Appendix H. Probe sets down-regulated in response to CBF2 expression ........ 218
Appendix 1. Probe sets up-regulated in response to ZAT12 expression ............ 220
Appendix J. Probe sets down-regulated in response to ZAT12 expression ....... 223
Appendix K. The best BLAST hits for ZAT12 in Arabidopsis ......................... 229
Appendix L. Supplementary material for Chapter 4 .......................................... 230
Appendix M. PERL scripts ................................................................................ 232

LITERATURE CITED ................................................................................................... 235

vi

LIST OF TABLES
Table 2.1. Potential low temperature regulatory elements I ............................................. 30
Table 2.2. Potential low temperature regulatory elements 11 ............................................ 32

Table 2.3. Chi-square analysis of expected and observed changes in gene expression in

response to low temperature and transcription factor overexpression .............................. 36
Table 3.1. Overrepresented GO terms among the COS transcripts .................................. 76
Table 3.2. Overrepresented GO terms among COS transcript expression clusters .......... 77
Table 3.3. Overrepresented GO terms in the CBF2 regulon ............................................. 87
Table 3.4. Overrepresented GO terms in the ZAT12 regulon .......................................... 90
Table 4.1. Overrepresented motifs in cluster III ............................................................. 103
Table 4.2. Number of promoters with each pair-wise motif combination ...................... 103
Table 4.3. Overrepresented motifs in cluster IV ............................................................. 105
Table A. 1. Probe sets up-regulated by low temperature in the plate experiment ........... 121

Table B. 1. Probe sets down-regulated by low temperature in the plate experiment ...... 142
Table C. 1. Probe sets up-regulated by low temperature in the soil experiment ............. 161
Table D. 1. Probe sets down-regulated by low temperature in the soil experiment ........ 178

Table E. 1. Probe sets up-regulated by low temperature in both the plate and soil
experiments ..................................................................................................................... 190

Table F. 1. Probe sets down-regulated by low temperature in both the plate and soil

experiments ..................................................................................................................... 203
Table G. 1. Probe sets up-regulated in response to CBF2 expression ............................. 211
Table H. 1. Probe sets down-regulated in response to CBF2 expression ........................ 218
Table 1.1. Probe sets up-regulated in response to ZAT12 expression ............................ 220
Table J. 1. Probe sets down-regulated in response to ZAT12 expression ....................... 223
Table L. l. The genes which were used to create tetramers of each motif ...................... 230

vii

LIST OF FIGURES
Figure 2.1. Low temperature responsive probe sets ......................................................... 27
Figure 2.2. Hierarchical cluster and expression profiles of the 514 COS genes .............. 29

Figure 2.3. Expression profiles of the COS genes and those that comprise the CBF2 and
ZAT12 regulons ................................................................................................................ 33

Figure 2.4. CBF2 and ZAT12 induce a majority of the most highly cold-induced
transcripts .......................................................................................................................... 36

Figure 2.5. Expression of transcription factors that are candidates for configuring the low
temperature transcriptome ................................................................................................ 38

Figure 2.6. Potential regulatory sequences common to the promoter regions of CBF 2,

ZAT12, ZA T10/STZ, MYB73, CZFI, and CZFZ ................................................................ 39
Figure 2.7. ZA T12 transcript levels in transgenic Arabidopsis plants .............................. 41
Figure 2.8. Regulation of COS genes by CBF2 and ZAT12 ............................................ 42

Figure 2.9. Effect of ZAT12 expression on plant morphology and freezing tolerance ..... 43
Figure 2.10. ZAT12 overexpression dampens cold induction of CBF], 2, and 3 ............ 46
Figure 2.11. T-DNA insertion lines in the ZAT12 locus ................................................... 48
Figure 2.12. RT-PCR analysis of ZAT12 expression in the two T-DNA insertion lines .48

Figure 2.13. Decreased ZAT12 expression enhances cold induction of CBF], 2, and 3 .49

Figure 3.1. Functional categories of the 514 COS transcripts .......................................... 74
Figure 3.2. Functional categories represented in each expression class of the COS

transcripts .......................................................................................................................... 79
Figure 3.3. Functional categories of the CBF2 and ZAT12 reuglons ............................... 86
Figure 3.4. Hydropathy plots for novel COR-like peptides in the CBF2 regulon ............ 88

Figure 4.1. Overlap of the two novel motifs and the CRT/DRE element in cluster III
promoters ........................................................................................................................ 103

Figure 4.2. WebLogos depicting conserved sequences flanking the CRT/DRE, ABRE,
and two novel motifs in cluster 111 genes ........................................................................ 105

viii

Figure 4.3. The motif GTGATCAC confers constitutive activity to a GUS reporter

......................................................................................................................................... 106
Figure 5.1. The low temperature regulatory networks of Arabidopsis ........................... 119
Figure K. 1. Amino acid alignment and relationship tree of the best BLAST hits for

ZAT12 in the Arabidopsis genome ................................................................................. 229

Figure L. 1. Sequences of the tetramers created from motifs found in clusters III and IV
......................................................................................................................................... 230

ix

CHAPTER ONE
LITERATURE REVIEW
Introduction

Photosynthesis, the overall process of using light energy to synthesize organic
compounds, allows plants to maintain an autotrophic lifestyle. This autotrophic lifestyle
includes remaining rooted in place, which is fine most of the time but prevents plants
from evading environmental stress. However, evasion is not the only method of dealing
with stress. Plants have evolved a number of effective mechanisms to cope with multiple
biotic and abiotic environmental stresses while remaining sessile.

One abiotic stress plants must deal with is temperature fluctuation. Cold or
freezing temperatures can significantly limit plant growth or result in damage or death.
Plants exhibit a wide range in their ability to survive freezing temperatures. Plants from
the tropics have essentially no capacity for surviving even a mild freeze and are damaged
by chilling temperatures in the range of 0°C to 12°C. Some examples include tomato,
maize (Taylor et al., 1974), and rice (Tajima et al., 1983). Other plants, such as the
cultivated potato (Solanum tuberosum), exhibit chilling resistance, but are still injured or
killed by any ice formation (Sukumaran and Weiser, 1972). There are other plants that
are killed by temperatures in the range of -6°C to -1°C. Most Citrus is killed in this range
(-6°C to -3°C) (Yelenosky and Guy, 1989). Depending on the species, herbaceous plants
from temperate regions (wheat, canola, Arabidopsis) generally survive freezing in the
range of -5°C to -30°C (Fowler and Gusta, 1979; Gilmour et al., 1988). The record
holders for freezing tolerance include tree species from boreal forests that regularly

survive winters with temperatures below -30°C and can even survive immersion in liquid

nitrogen when fully cold-acclimated (Sakai and Larcher, 1987). The level of freezing
tolerance achieved is not constitutive, but induced in response to low, non-freezing
temperatures (below approximately 10°C), in a process known as cold acclimation (Guy,
1990; Thomashow, 1998). Cold acclimation allows plants that would normally be killed
by mild freezing conditions (-5°C) to survive at much lower temperatures (-20°C).

The study of cold acclimation in plants is important. Not only does understanding
conditional freezing tolerance (or lack of it) further science, it has practical applications
for agricultural production. Freezing temperatures negatively impact crop productivity
and limit the geographical locations where plants are grown (Thomashow, 1998).
Understanding the molecular basis of cold acclimation and freezing tolerance could lead
to new strategies of improving crop productivity through increased freezing tolerance.
Much research has focused on how freezing damages plant cells, what mechanisms exist
to protect the plant, and how plants sense and respond to low temperatures in order to

activate protective mechanisms.

F reezing-Induced Damage

Water, while essential to life, forms ice crystals spontaneously (homogeneous
nucleation) or when catalyzed by another substance (heterogeneous nucleation) that can
kill or damage a cell. At temperatures just below 0°C, homogeneous ice formation is
unlikely and nucleation is needed. In the absence of any nucleator, a single cell will
freeze only when the temperature falls below -38.5°C, the point of spontaneous
nucleation (Franks, 1985). Otherwise, water can remain in a supercooled state. Many

substances can act as nucleators including ice nucleation-active bacteria,

organic/inorganic debris, and biological molecules (Pearce, 2001). In plants, freezing
temperatures generally'first induce ice formation in intercellular spaces because these
spaces have a higher freezing point due to a lower solute concentration and contain ice
nucleating agents (Brush et al., 1994). Ice formation in intercellular spaces has the
potential to physically disrupt cells (Levitt, 1980). However, most damage results from
severe cellular dehydration associated with freezing (Levitt, 1980; Steponkus and Webb,
1992). Since the chemical potential of ice at a given temperature is lower than liquid
water, extracellular ice formation results in a decrease in water potential outside the cell
and a consequent movement of water through the plasma membrane to the extracellular
space, leading to the dehydration of the cell. The amount of dehydration is dependent on
both the temperature and the initial solute concentration of the cytoplasm. Cellular
dehydration also occurs in drought and salinity stress, which suggest that the mechanisms
that protect a plant from freezing-induced dehydration should also protect the plant from
the dehydration caused by drought or salinity stress.

Cellular dehydration during freezing can potentially have numerous deleterious
effects, including protein denaturation, the precipitation of molecules, and membrane
damage. Since 1912, the plasma membrane has been thought to be a primary site of
freeze-induced injury (Levitt, 1980). Studies done with rye protoplasts have shown that
freezing-induced dehydration can result in three main membrane lesions (Webb et al.,
1994; Uemura et al., 1995). In the range of -2°C to -4°C, expansion-induced lysis can
occur, caused by osmotic expansion and contraction cycles (during freeze/thaw cycles).
The contraction of the plasma membrane during freezing results in the endocytic

vesiculation of the plasma membrane, which decreases plasma membrane surface area. If

sufficient membrane is lost, water re-entering the cell during expansion can lyse the
membrane. Cold acclimation prevents this type of lesion from occurring, as the plasma
membrane is retained in exocytotic extrusions that allow expansion.

Between -4°C and -10°C, freezing can induce lamellar-to-hexagonal 11 phase
transitions, which involves the fusion of various membranes. In this type of damage, a
lamellar membrane, when brought into close apposition with another membrane due to
cellular dehydration, can form non-bilayer Hex 11 phase lipids. This results in a loss of
membrane integrity. The chloroplast envelope is particularly susceptible to this type of
damage (Steponkus et al., 1993). Again, protoplasts isolated from cold-acclimated leaves
generally do not suffer this type of damage.

Fracture jump lesions can occur at temperatures below -10°C with cold
acclimation. This lesion was observed as a localized deviation in the fracture plane of the
plasma membrane during cyroelectron microscopy and is likely due to localized fusion of
the plasma membrane with other cellular membranes (Webb et al., 1994). This type of
lesion has only been observed with cold acclimation and varies greatly between plant
species (Webb et al., 1994; Uemura et al., 1995). While little is known about why
variation exists, fracture jump lesions appear to be a relative measure of the potential
freezing tolerance that can be achieved.

In addition to dehydration induced damage, cold temperatures can generate
reactive oxygen species (ROS) which can contribute to membrane damage (McKersie
and Bowley, 1997). Low temperature causes a decrease in the turnover rate of
components of photosystem 11, leading to excess excitation energy and the generation of

reactive oxygen species, including hydrogen peroxide (Huner et al., 1998). ROS not only

damages membrane lipids, but can also harm carbohydrates, proteins, and nucleic acids
(McKersie and Bowley, 1997). Additionally, ROS species generated at low temperature
could be activating a programmed cell death response (Wagner et al., 2004), which
evidence suggests occurs in plants exposed to low temperature (reviewed in Kratsch and

Wise, 2000).

Mechanisms for Freezing Tolerance

The mechanisms involved in freezing tolerance are not well understood, but
numerous studies have identified physiological and biochemical changes that occur
during cold acclimation. Some of these changes include reduced growth rates, decreased
water content (Levitt, 1980), altered membrane lipid composition (Uemura and
Steponkus, 1994), the production of new proteins (including membrane stabilizers,
antifreeze proteins, and chaperones) (Smallwood and Bowles, 2002), transient increases
in abscisic acid (Chen et al., 1983), increased levels of antioxidants (McKersie and
Bowley, 1997), and the accumulation of compatible osmolytes (proline, betaine, polyols,
and soluble sugars) (Levitt, 1980; Yancey et al., 1982). Given the many changes that
occur during cold acclimation, it is hard to discern those changes that contribute to
freezing tolerance from those that simply occur in response to low temperature.
Nevertheless, the multitude of changes indicate that cold acclimation is a dynamic event,
requiring multiple biochemical, metabolic, and cellular changes to occur in order to
achieve freezing tolerance.

Given that cell membranes appear to be a primary site of freezing damage,

protecting membranes at freezing temperatures seems essential. In fact, alterations in

membrane lipid composition and structure are some of the best documented changes that
occur during cold acclimation (reviewed in Nishida and Murata, 1996). While not
completely understood, ultrastructural changes in the membrane become apparent within
6 h of cold treatment in Arabidopsis (Ristic and Ashworth, 1993). Moreover, the changes
in membrane lipid composition that occur during cold acclimation correlate with
membrane cryostability (Steponkus, 1984; Uemura et al., 1995). Direct evidence also
exists for membrane lipid composition playing a direct role in freezing tolerance.
Increased levels of mono- and di-unsaturated species of phosphotidylcholine can prevent
membranes from forming endocytotic vesicles during hyperosmotic stress, while
disaturated species of phosphotidylcholine can not (Steponkus et al., 1988).

These in vitro experiments are complemented by experiments in planta, as
alterations in lipid composition have been shown to influence chilling tolerance in
Arabidopsis and tobacco. After prolonged exposure to chilling temperatures (2°C to
5°C), Arabidopsis mutants with diminished levels of unsaturated lipids become chlorotic
(Wu et al., 1997; Vijayan and Browse, 2002). At low temperature, these mutants, fad5 ,
fad6, and the fad3-2 fad 7-2 fad8 triple mutant were found to be more susceptible to
photoinhibition than wild type Arabidopsis (V ijayan and Browse, 2002). Additionally,
experiments in tobacco revealed that some of this plant’s chilling sensitivity can be
alleviated if the level of membrane unsaturation is increased (Moon et al., 1995).

Plants increase the levels of a number of low molecular weight organic solutes
upon cold acclimation, including proline, glycine betaine, and sugars (raffinose, sucrose,
and trehalose). These molecules are known as compatible solutes or compatible

osmolytes, as they are highly soluble and their accumulation does not interfere with

cellular metabolism. While their main role in freezing tolerance is thought to be in
maintaining cellular osmotic balance, this is not the only role they play. Some
compatible solutes stabilize membranes (Hincha and Crowe, 1998; Hincha et al., 1999),
while others serve as enzyme cryoprotectants (reviewed in Sakamoto and Murata, 2001 ).
Many compatible solutes accumulate to high levels during cold acclimation, which
correlates well with increased freezing tolerance. For example, increased levels of
proline (30-fold higher) have been found in the constitutively freezing tolerant mutant
eskz'mol (Xin and Browse, 1998). Conversely, a lack of sugar accumulation during cold
acclimation in the Arabidopsis sfr4 mutant correlates with its sensitivity to freezing
(Warren et al., 1996). Still, the specific role of each osmolyte in freezing tolerance is
unclear. Dissection of osmolyte function has been complicated by their metabolic roles,
which are generally essential.

Freezing tolerant plants can withstand ice formation in their apoplast (Griffith and
Yaish, 2004), thought to be due in part to the production of a number of anti-freeze
proteins (AFPs) that prevent or alter ice formation in this compartment (Urrutia et al.,
1992; Duman, 1994; Hon et al., 1995; Hincha et al., 1997; Worrall et al., 1998;
Smallwood et al., 1999; Yeh et al., 2000). Antifreeze proteins interact with ice crystals
and inhibit their growth. They do so through two mechanisms. First, they can prevent
the accretion of water molecules to the growing face of an ice crystal. Secondly, AF Ps
can depress the freezing point of water through binding to the surface of ice crystals,
without affecting the melting point of the solution (this is known as thermal hysteresis
activity). Most plant AF Ps are homologous to pathogenesis-related proteins and also

provide protection against psychrophilic pathogens (Griffith and Yaish, 2004). Given the

difficulty of mounting a defense response in sub-zero conditions, accumulating proteins
during cold acclimation that serve a dual purpose could be advantageous for the plant.
Unfortunately, there is no consensus sequence for identifying an AF P through a database
search, so they must be identified experimentally. While in vitro data firmly confirms the
thermal hysteresis activity of AF Ps identified in plants, in vivo data for their function is
limited (Griffith and Yaish, 2004). Still, their widespread occurrence and expression at
low temperature in diverse plant species suggest they play a role in freezing tolerance.
Plants produce numerous small, highly hydrophilic polypeptides during cold
acclimation. These proteins have a relatively simple amino acid composition, have
repeated amino acid motifs, are generally predicted to form amphipathic a-helices, and
remain soluble upon boiling in a dilute aqueous buffer (reviewed in Thomashow, 1999).
These proteins have been identified by a number of researchers and hence possess
multiple designations, including COR (c_old-regulated), LTI (low temperature induced),
KIN (gold-inducible), RD (responsive to desiccation), and ERD (early dehydration-
inducible). Additionally, some of these proteins are homologs of the LEA (late
gmbryogenesis abundant) proteins, which are produced just before seed desiccation
during embryogenesis (Dure etal., 1981). The exact function of these proteins is
unknown. They are, however, also expressed in response to dehydration and ABA.
Given the similarities between freezing and dehydration injuries and the similar
biochemical properties of these proteins, one hypothesis is that they function in
mitigating dehydration-induced damage. This hypothesis is supported by work on the

COR15a protein from Arabidopsis.

COR15a is a 15 kDa polypeptide that is targeted to chloroplasts, where it is
processed to a mature 9.4 kDa protein (COR15am). Regions of the polypeptide are
predicted to form amphipathic 111-helices. In order to determine its role in freezing
tolerance, Artus et a1. (1996) created transgenic plants constitutively expressing COR15a.
The chloroplasts of nonacclimated transgenic plants were 1°C to 2°C more freezing
tolerant than wild type chloroplasts in the range of -4°C to -8°C. Additionally,
protoplasts isolated from the leaves of nonacclimated transgenic plants were 1°C more
freezing tolerant in the same temperature range (Steponkus et al., 1998).

One hypothesis concerning how CORISa brings about increased chloroplast
freezing tolerance is that COR15a stabilizes membranes. Steponkus et a1. (1998) showed
that in the temperature range of -4.5°C to -7°C, COR15am decreased the incidence of
HexII phase lipid formation in regions where the plasma membrane was brought into
close apposition with the chloroplast envelope during freezing-induced dehydration.
COR15am is thought to decrease the incidence of HexII lipid formation by altering the
intrinsic curvature of the inner membrane of the chloroplast envelope, thereby lowering
the temperature at which HexII lipids form. In vitro data supports this hypothesis, as
COR15am increased the lamellar-to-hexagonal 11 phase transition temperature of
dioleoylphosphatidylethanolamine (this is a sensitive test for an effect on monolayer
curvature) and promoted formation of the lamellar phase in a lipid mixture composed of
the major lipid species of the chloroplast envelope (Steponkus et al., 1998). Given that
other COR proteins are predicted to form similar structures (amphipathic a-helices), the
possibility exists that these proteins also help stabilize membranes against freezing-

induced dehydration injury by altering the intrinsic curvature of membranes. Indeed,

work on a LEA 11 protein (CAP85) and a novel COR-like protein (CAP160) from spinach
indicate they may also play a role in stabilizing membranes (Kaye et al., 1998), as might
the dehydrins (members of the LEA II protein family) RAB 1 8, COR47/RD17,

LT129/ERD10, and LTI30/XER02 (Puhakainen et al., 2004).

Low Temperature Perception

The mechanism by which plants sense low temperature is unknown. While the
nature of this “thermometer” is a mystery, low temperature alters a number of
physiological processes that could act as the initial transducer of a cold stimulus. Low
temperature can alter RNA and DNA secondary structure and the enzymatic activity of
proteins. Changes such as these could function as the sensor. The photosynthetic
apparatus itself may act as the sensor, detecting changes in temperature through increased
energy imbalances and photoinhibition (Huner et al., 1998).

One of the earliest and most direct effects of low temperature on a cell is a
decrease in membrane fluidity (Levitt, 1980). The idea that changes in membrane
fluidity could act as the initial cold sensor is supported by a number of studies. Initial
observations in Synechocystis revealed that rigidification of membranes could induce the
expression of cold-regulated genes (Vigh etal., 1993). Murata and L05 (1997)
hypothesized that the sensor is found in microdomains of the plasma membrane and
senses physical alterations in membrane phase transitions, leading to conformational
changes or alterations in the phosphorylation state of the sensor. Studies in alfalfa also
support the hypothesis that the sensor works through interpreting membrane fluidity

(Orvar et al., 2000). Treatment of alfalfa cells at warm temperatures with benzyl alcohol

10

increases membrane fluidity and inhibits the accumulation of the cold-responsive
transcript CAS30 in response to low temperature. Conversely, treatment of cells with
dimethyl sulfoxide, which reduces membrane fluidity, increased CAS30 levels at warm
temperatures. The authors also showed that actin microfilament reorganization and an
influx of calcium was required for cold-responsive expression of CAS30. This work
provided a model where upon cold treatment membrane fluidity decreases, followed by a
downstream reorganization of actin microfilaments, and finally by an influx of calcium.
A number of studies have shown a rapid and transient increase in cytosolic
calcium upon cold treatment (Knight et al., 1991; Monroy et al., 1993; Knight and
Knight, 2000), that comes both from apoplastic and vacuolar stores (Knight et al., 1996).
Calcium is required for maximal expression of the cold-responsive transcripts KIN] and
CAS30 (Knight et al., 1996; Tahtihatju et al., 1997; Orvar et al., 2000) and the acquisition
of freezing tolerance in response to low temperature (Monroy et al., 1993; Monroy and
Dhindsa, 1995). Plants that have been cold-acclimated display an altered calcium
transient upon subsequent cold treatment (Knight et al., 1996; Plieth et al., 1999; Knight
and Knight, 2000). Knight et al. (1996) has shown that the peak levels and duration of
the calcium transient becomes diminished in magnitude and prolonged in length upon
cold acclimation, indicating that the sensor likely possesses the ability to “remember” the
cold. Due to such evidence, the suggestion has been made that the sensor is one or more
of the calcium channels located in the plasma membrane. In fact, some membrane bound
mechano-sensitive calcium channels are activated by low temperature (Ding and Pickard,

1993).

ll

The thermometer could also resemble low temperature sensors found in other
species. In Synechocystis, evidence points towards the role of two-component regulators
as temperature sensors. Mutations in the Hik33 or Hik19 (histidine kinase) genes greatly
reduced the expression of a number of low temperature inducible genes (Suzuki et al.,
2001; Mikami et al., 2002). Further, microarray analysis on the AHik33 mutant reveal
that at least one additional cold sensor exists in Synechocystis (Mikami et al., 2002; Inaba
et al., 2003). Two-component systems exist in plants, but these two Hik genes have no
plant homologs and, to date, there is no evidence that such a system functions as the
temperature sensor in plants.

In mammalian systems, two TRP (_transient receptor potential) nonselective cation
channels have been identified that are responsive to cold (Peier et al., 2002; Story et al.,
2003). One, TRPM8, is a methanol and cold-responsive TRPM-class channel that was
discovered in sensory neurons (Peier et al., 2002). The other, ANKTMl, is distantly
related to TRP channels and possesses very little amino acid similarity to TRPM8 (Peier
et al., 2002). ANKTMl was cold-responsive and also found in sensory neurons, but was

not responsive to methanol.

Low Temperature Signal Transduction

The initial temperature sensing event has to be transmitted to downstream
effectors in order for the plant to respond. Cold-responsive signaling cascades are poorly
understood in plants. Only recently have studies elucidated some of these signaling

events, but most of the players have not been placed into coherent pathways.

12

As stated earlier, low temperature signaling involves transient calcium
accumulation. A number of proteins can be involved in detecting and responding to
calcium, including calmodulin (CaM), calcium dependent protein kinases (CDPKs), and
calcium-regulated phosphatases (Knight and Knight, 2001). Overexpression of a
calmodulin gene in Arabidopsis resulted in decreased levels of expression of the cold and
ABA responsive COR genes, suggesting that CaM plays a role as a negative regulator
(Townley and Knight, 2002). Calmodulin is not the only player to be implicated in
calcium mediated cold signaling. In Arabidopsis, there are approximately 40 CDPKs,
some of which have been proposed to play a role in ABA, drought, cold, and salinity
responses (Sheen, 1996). Serine/threonine phosphatases (PPases) can also interact with
calcium and regulate signaling pathways. The activity of protein phosphatase 2A
increases in alfalfa in response to cold and this activity depends on calcium influx.
Additionally, inhibiting protein kinases with staurosporine inhibits the low temperature
induction of the CAS15 gene, while inhibiting protein phosphatase activity with okadaic
acid induces CAS15 at 25°C (Monroy et al., 1998). Type-2C PPases include a sub-group
of proteins with properties similar to the calcium sensor protein calcineurin B. The
expression of one calcineurin B-like (CBLs) calcium sensor, CBLI , is induced by
drought, salt, ABA, and cold (Albrecht et al., 2003; Cheong et al., 2003). Studies on
CBLl reveal that it plays a role in drought, salt, and cold stress signaling. A calcium
sensor-associated protein kinase, CIPK3, was also found to modulate cold and salinity
gene expression, but not drought-induced gene expression (Kim et al., 2003). The
authors propose that CIPK3 functions as a cross-talk node between ABA and abiotic

stress signaling.

13

The levels of phosphatidic acid (PtdOH) and inositol triphosphate(1nsP3) increase
in plants upon cold shock, along with activation of phospholipase C and D activity
(Ruelland et al., 2002). These events are dependent upon calcium, as chemically
blocking calcium influx inhibited PtdOH and Inng formation. These results indicate that
cold signaling may involve one or more phosphoinositide signaling pathways. Further
support comes from a study where the plasma membrane bound phospholipase D5
(PLDS) was either knocked out or overexpressed in Arabidopsis (Li et al., 2004). Plants
with no PLD5 activity displayed sensitivity to freezing at -7°C at the whole plant level,
while overexpression of PLD8 resulted in plants that were more freezing tolerant (at -
10°C ) than wild type plants. These two mutant plants did not impact COR proteins,
soluble sugar levels, or proline accumulation. This suggests that the alteration in freezing
tolerance brought about by altering PLD6 represents a separate signaling pathway from
the pathway that activates COR genes, proline, and soluble sugars.

MAPK cascades have also been implicated in cold signal transduction. In these
cascades, a MAPK kinase kinase (MAPKKK) phosphorylates a MAPK kinase,
(MAPK), which in turn phosphorylates a MAPK. These MAPK’s then activate or
inactivate a transcription factor through phosphorylation. Introduction of a tobacco
MAPKKK (NPKl) into maize enhanced cold tolerance by 2°C (Shou et al., 2004),
indicating it may play a role in cold signaling pathways. Cold activated MAP kinases
have also been found in alfalfa (Jonak et al., 1996) and Arabidopsis (Mizoguchi et al.,
1996). Recently, a more detailed study on the MAPKK MKK2 in Arabidopsis revealed
its role in cold and salt stress signaling. Teige et al. (2004) reported that MKK2 is

activated by salt and cold stress, which in turn activates MPK2 and MPK4 through

14

phosphorylation. MEKKl (a MAPKKK) was found to regulate MKK2. A null mutation
of MKK2 resulted in plants that were hypersensitive to cold shock or germination on
media containing salt, while MKK2 overexpression lead to increased tolerance to cold
shock and increased germination on media containing salt. RT-PCR analysis revealed
altered levels of several cold— and salt-responsive transcripts in the MKK2 constitutive
expression lines. The study by Teige et al. (2004) was the first report of an entire MAPK

cascade involved in both cold and salt signaling.

Cold-Responsive Gene Expression

Signal transduction cascades generally result in the activation of transcriptional
regulators that influence the expression of multiple genes in response to a given stimulus.
During cold acclimation, multiple changes in gene expression occur. The transcripts for
COR proteins, antioxidants, enzymes involved in cryoprotectant production, and many
other gene products are induced in response to low temperature (Thomashow, 1998).
While few transcriptional regulators involved in modifying cold-responsive genes are
known, one pathway is now well established.

The previously mentioned COR proteins were first identified by their high
transcript accumulation in response to cold (Hajela et al., 1990). Studies on the
regulation of COR15a transcription resulted in the isolation of a fragment of the COR15a
promoter that could drive expression of a reporter gene in response to low temperature
(Baker et al., 1994; Yamaguchi-Shinozaki and Shinozaki, 1994). One sequence of
interest in this promoter fragment was the C-repeat (TGGCCGAC), also known as the

DRE (dehydration responsive element). The core sequence of the C-repeat, CCGAC,

15

was also found in the promoters of other COR genes. Using a yeast one-hybrid approach,
Stockinger et al. (1997) isolated a protein that could bind to the C-repeat and activate
transcription. This protein was named CBF 1 (Q-repeat binding factor 1). Since
overexpression of COR15a led to only a modest improvement in chloroplast freezing
tolerance, a question arose as to whether the simultaneous expression of multiple COR
genes could alter the freezing tolerance of Arabidopsis. The discovery of CBF 1 provided
a tool to answer this question. Arabidopsis overexpressing CBF] were found to
accumulate levels of COR transcripts similar to cold-acclimated plants and were
constitutively freezing tolerant without a period of cold acclimation (J aglo-Ottosen et al.,
1998). This and other studies (Liu et al., 1998; Kasuga et al., 1999; Gilmour et al., 2000;
Gilmour et al., 2004) have revealed that CBF expression has a significant impact on plant
freezing and chilling tolerance (Gong et al., 2002).

The Arabidopsis genome encodes three cold-responsive CBF transcriptional
activators CBF], CBF2, and CBF3 (Stockinger et al., 1997; Gilmour et al., 1998;
Medina et al., 1999), which are also known as DREBIb, DREBlc, and DREBla (Liu et
al., 1998; Kasuga et al., 1999), respectively. The CBF transcription factors are members
of the AP2/EREBP family of DNA-binding proteins (Riechmann and Meyerowitz, 1998).
The CBF proteins bind to the CRT (C-repeat)/DRE (dehydration responsive element)
(Baker etal., 1994; Yamaguchi-Shinozaki and Shinozaki, 1994). In vitro studies on the
binding of CBF to the CRT/DRE element defined (A/G)CCGAC as the consensus
binding sequence (Sakuma et al., 2002). The CBFI-3 genes are induced within 15 min of
plants being exposed to cold temperatures followed at about 2 h by induction of a group

of genes that contain the CRT/DRE-regulatory element, known as the “CBF regulon”

l6

(Gilmour et al., 1988; Liu et al., 1998). The level of freezing tolerance observed by
expression of the CBF regulon is substantial; constitutive expression of the CBF1-3 genes
increases the freezing tolerance of nonacclimated plants 3.5-7.5°C (Gilmour et al., 2000;
Gilmour et al., 2004). While the exact mechanisms by which the CBF regulon enhances
freezing and chilling tolerance are not known, it is clear that multiple mechanisms are
involved. This includes the synthesis of low molecular weight cryoprotective molecules
such as proline, sucrose, and raffinose (Gilmour et al., 2000; Taji et al., 2002) and the
production of proteins with cryoprotective properties such as COR15a (Artus et al., 1996;
Steponkus et al., 1998).

The CBF cold response pathway is conserved across plant species (Jaglo et al.,
2001). Cold-responsive CBF orthologs have been found in freezing tolerant and chilling-
sensitive plants, including wheat, rye, barley, oat, Brassica napus, rice, maize, tomato,
and others (Jaglo et al., 2001; Choi et al., 2002; Chen et al., 2003; Dubouzet et al., 2003;
Shen et al., 2003; Qin et al., 2004; Zhang et al., 2004). The existence of cold-responsive
CBFs in chilling-sensitive species leads to fundamental questions on the nature of cold
acclimation and chilling-sensitivity. Do these chilling-sensitive plants contain a complete
CBF cold response pathway? Are they deficient in an important component(s) of the CBF
pathway? Work in chilling-sensitive tomato indicates that tomato has a functional CBF
cold response pathway, but the CBF regulon of tomato differs dramatically from
Arabidopsis (Zhang et al., 2004). This study suggests that tomato either lacks a co-
activator activity required for CBF function that is present in Arabidopsis or lacks

functional CRT/DRE elements within the genome.

17

While the CBF cold response pathway is conserved across plant species and
increases the freezing tolerance of some plants, it is unknown whether additional cold
response pathways exist in Arabidopsis. Studies with Arabidopsis mutants suggest that
additional pathways do exist. The eskimol mutant of Arabidopsis is constitutively more
freezing tolerant than wild type plants, but COR genes are not constitutively expressed
indicating that the mutation activated a freezing tolerance pathway outside the CBF
system (Xin and Browse, 1998). Similarly, ada2 mutants of Arabidopsis (ADA2 encodes
a transcriptional adaptor protein) are constitutively more freezing tolerant than wild type
plants, but COR genes are not constitutively induced suggesting that the ADA2 protein is
involved in inhibiting expression of a freezing tolerance pathway that is distinct from the
CBF cold response pathway (Vlachonasios et al., 2003). Microarray studies profiling
roughly one-third of Arabidopsis genes in response to low temperature also indicate the
existence of additional low temperature pathways (Seki et al., 2001; Fowler and
Thomashow, 2002; Kreps et al., 2002; Seki et al., 2002).

No study has yet defined the low temperature transcriptome for Arabidopsis on a
whole genome level, nor the extent to which the CBF transcription factors influence these
genes. Also unknown are the identities of additional transcription factors that configure
the low temperature transcriptome. To further understand the roles of the CBF proteins
in configuring the low temperature transcriptome and to identify additional transcription
factors with roles in cold acclimation, work in this dissertation aimed to define a low
temperature transcriptome for Arabidopsis, to determine the contribution of the CBF
transcription factors to the cold transcriptome, and to determine the regulons of other

cold-responsive transcription factors.

18

CHAPTER TWO

ROLES OF THE CBF2 AND ZAT12 TRANSCRIPTION FACTORS IN
CONFIGURING THE LOW TEMPERATURE TRANSCRIPTOME OF
ARABIDOPSIS

The majority of work in this chapter was published in the Plant Journal.
Vogel, Jonathan T., Zarka, Daniel G., Van Buskirk, Heather A., Fowler, Sarah G.,

and Thomashow, Michael F. (2005) Roles of the CBF2 and ZAT12 transcription factors
in configuring the low temperature transcriptome of Arabidopsis. Plant J. 41, 195-211.

19

SUMMARY

The CBF cold response pathway has a prominent role in cold acclimation. The
pathway includes action of three transcription factors, CBFl, 2, and 3 (also known as
DREBIb, c, and a, respectively), that are rapidly induced in response to low temperature
followed by expression of the CBF-targeted genes (the CBF regulon) that act in concert
to increase plant freezing tolerance. Results of transcriptome profiling and mutagenesis
experiments, however, indicate that additional cold-response pathways exist and may
have important roles in life at low temperature. To further understand the roles that the
CBF proteins play in configuring the low temperature transcriptome and to identify
additional transcription factors with roles in cold acclimation, the Affymetrix GeneChip®
containing probe sets for approximately 24,000 Arabidopsis genes was used to define a
core set of cold-responsive genes and to determine which genes were targets of CBF 2 and
six other transcription factors that appeared to be coordinately regulated with CBF 2. A
total of 514 genes were placed in the core set of cold-responsive genes, 302 of which
were up-regulated and 212 down-regulated. Hierarchical clustering and bioinformatic
analysis indicated that the 514 cold-responsive transcripts could be assigned to one of
seven distinct expression classes and identified multiple potential novel cis-acting cold-
regulatory elements. Eighty-five cold-induced genes and eight cold-repressed genes were
assigned to the CBF 2 regulon. An additional nine cold-induced genes and 15 cold-
repressed genes were assigned to a regulon controlled by ZAT12. Of the 25 core cold-
induced genes that were most highly up-regulated (induced over 15-fold), 19 genes
(84%) were induced by CBF2 and another two genes (8%) were regulated by both CBF2

and ZAT12. Thus, the large majority (92%) of the most highly induced genes belong to

20

the CBF2 and ZAT12 regulons. Constitutive expression of ZAT12 in Arabidopsis caused
a small, but reproducible, increase in freezing tolerance, indicating a role for the ZAT12
regulon in cold acclimation. In addition, ZAT12 was found to down-regulate expression
of the CBF genes indicating a role for ZAT12 in a negative regulatory circuit that

dampens expression of the CBF cold response pathway.

21

INTRODUCTION

Many plants increase in freezing tolerance in response to low nonfreezing
temperature, a phenomenon known as “cold acclimation” (Thomashow, 1999;
Smallwood and Bowles, 2002). Cold acclimation in Arabidopsis involves action of the
CBF cold response pathway (Thomashow, 2001). The pathway includes the CBF] ,
CBF 2, and CBF 3 genes (Gilmour etal., 1998; Medina et al., 1999; Jaglo et al., 2001),
also known as DREBI b, DREBIc, and DREBI a, respectively (Liu et al., 1998), which
encode transcriptional activators that bind to the CRT (C-repeat)/DRE (dehydration
response element) regulatory element present in the promoters of COR and other cold-
responsive genes (Baker et al., 1994; Yamaguchi-Shinozaki and Shinozaki, 1994;
Stockinger et al., 1997; Gilmour et al., 1998). Transcripts for CBF], 2, and 3 accumulate
rapidly (within 15 min) upon exposing plants to low temperature followed by induction
of the CBF-targeted genes known as the CBF regulon. Constitutive expression of the
CBF genes results in constitutive expression of the CBF regulon and increased freezing
tolerance without a low temperature stimulus (J aglo-Ottosen et al., 1998; Liu et al., 1998;
Gilmour et al., 2000; Gilmour et al., 2004). The freezing tolerance conferred by the CBF
regulon involves the production of cryoprotective polypeptides such as COR15a (Artus et
al., 1996; Steponkus et al., 1998) and the accumulation of compatible solutes such as
sucrose, raffinose, and proline (Nanjo et al., 1999; Gilmour et al., 2000; Gilmour et al.,
2004)

The CBF cold response pathway is currently the best understood genetic system
with a role in cold acclimation. However, it does not appear to be the sole pathway with

a role in freezing tolerance. The eskimoI mutant of Arabidopsis described by Xin and

22

Browse (1998) is constitutively more freezing tolerant than wild type plants, but the COR
genes are not constitutively expressed indicating that the mutation activated a freezing
tolerance pathway outside the CBF system. Similarly, ada2 mutants of Arabidopsis
(ADA2 encodes a transcriptional adaptor protein) are constitutively more freezing tolerant
than wild type plants, but COR genes are not constitutively induced suggesting that the
ADA2 protein is involved in inhibiting expression of a freezing tolerance pathway that is
distinct from the CBF cold response pathway (Vlachonasios et al., 2003).

To more fully understand the role of the CBF cold response pathway in cold
acclimation, investigators have examined the changes that occur in the Arabidopsis
transcriptome in response to low temperature and overexpression of the CBF
transcription factors. Fowler and Thomashow (2002) surveyed the expression of about
8,000 Arabidopsis genes in response to low temperature. The results indicated that
extensive changes occur in the transcriptome during cold acclimation. In particular, 306
(~4%) genes were found to be either up- or down-regulated at least 3-fold in response to
low temperature. However, only 12% of these genes could be assigned to the CBF
regulon (i.e., were both cold- and CBF-responsive); at least 28% of the cold-responsive
genes were not affected by expression of the CBF transcription factors, including 15
encoding known or putative transcription factors. Thus, it was concluded that cold
acclimation is associated with the activation of multiple low temperature regulatory
pathways. Similar conclusions have been reached by others studying the Arabidopsis
low temperature transcriptome (Seki et al., 2001; Kreps et al., 2002; Seki et al., 2002).

Here, the regulation of the low temperature transcriptome of Arabidopsis was

further explored. Using the Affymetrix GeneChip® containing probe sets for

23

approximately 24,000 genes, I defined a core set of cold-responsive genes and
determined whether they were targets of CBF 2 or six other transcription factors that
appeared to be coordinately regulated with CBF2. I conclude that the majority of genes
that are most highly induced in response to low temperature are part of the CBF2
regulon; that the ZAT12 transcription factor participates in the induction and repression
of cold-responsive genes; and that the ZAT12 regulon contributes to an increase in
freezing tolerance. The results also indicate that certain cold-responsive genes are
members of both the ZAT12 and CBF2 regulons and that ZAT12 has a role in a negative

regulatory circuit that dampens expression of the CBF cold response pathway.

24

RESULTS
Identification of a Core Set of Cold-Responsive Genes

The first objective was to identify a core set of cold-responsive genes that could
be used to further our understanding of the low temperature regulons and regulatory
networks of Arabidopsis. The goal was not to identify all genes that were cold-
responsive, but to identify a set of genes that were reproducibly cold-responsive using
two common lab conditions, plants grown on soil in pots and plants grown on plates
containing solid culture medium. This was accomplished using the Arabidopsis
Affymetrix GeneChip® ATHl array, which contains probes for approximately 24,000
genes, to compare the transcriptomes of plants grown in soil and on plates at “warm”
temperatures (22°C soil, 24°C plates) with those of plants that had been transferred to
low temperature (4°C) for 1 h, 24 h, and 7 d. In the plate experiments both root and
shoot tissue was harvested, while in the soil experiments, shoot tissue was harvested.
Pooled RNA from multiple samples at each time-point was labeled and hybridized to the
arrays. A probe set was designated as being up-regulated if, in both biological samples
for a given time-point, the detection algorithm assigned a call of “present” in the cold-
treated sample, the change algorithm assigned a call of “increased,” and the signal log
ratio was greater than or equal to 1.3. A probe set was designated as being down-
regulated if, in both biological samples for a given time-point, the detection algorithm
assigned a call of “present” in the warm sample, the change algorithm assigned a call of
“decreased,” and the signal log ratio was less than or equal to -1.3. The signal log ratio

cutoffs used corresponded to approximately a 2.5-fold change.

25

Using these criteria, 1,295 probe sets were cold-responsive in the plate
experiments and 938 probe sets were cold-responsive in the soil experiments (the
complete “raw” data sets for these experiments are posted at the TAIR website
“www.arabidopsis.org” and the cold-responsive probe sets are listed in Appendices A-D).
In the plate experiments, 673 probe sets were up-regulated, 627 were down-regulated and
five were both up- and down-regulated at different times in the experiment. In the soil
experiments, 557 probe sets were up-regulated, 382 were down-regulated and one was
both up- and down-regulated at different times in the experiment. A comparison of the
probe sets that were cold-responsive in the plate and soil experiments indicated that 302
were up-regulated in both experiments and that 212 were down—regulated in both
experiments (Figure 2.1a; Appendices E and F), with the largest number of changes
occurring at 24 h (Figure 2. lb). These changes in gene expression were statistically
significant at a p<0.05, with 95% (490/514) statistically significant at a p<0.005 (see
Materials and Methods). Many of the probe sets that were categorized as not being
responsive in both soil- and plate-grown plants showed a corresponding increase or
decrease in both experiments, but did not meet the strict criteria used for designating
cold—responsiveness in both experiments. Some probe sets, however, were only
responsive in either the soil or plate experiments. The reason for these differences is
currently unknown, but is likely to reflect, in part, the differences in the culture
conditions and tissues harvested in the experiments. Exploring these differences will be
the subject of future study. The goal accomplished here was the identification of 514
probe sets that were cold-responsive in four experiments using two different culture

conditions; i.e., an identification of a robust set of cold-responsive genes that could be

26

(a)

Up-regulated probe sets Down-regulated probe sets

Plates
41 S

3

300

NM
OLD
CO

150
100

Cold responsive
probe sets

 

U1
OO

1 24 168
Time (h) at 4‘C

Figure 2.1. Low temperature responsive probe sets.

(a) Probe sets differentially regulated (2.5-fold cutoff, p S 0.05) at low temperature in
plants grown in soil (soil) and on solid medium (plates).

(b) Number of probe sets with altered accumulation at l h, 24 h, and 168 h after transfer
to low temperature in both soil and solid medium.

used in deciphering the low temperature regulatory network of Arabidopsis. I refer to

these genes as a “Qld standar ” (COS) set of cold-responsive genes.

Members of the COS Gene Set can be Assigned to One of Seven Expression Clusters
The 514 COS genes were subjected to hierarchical clustering based on their
relative transcript levels in plants after 1 h, 24 h, and 7 d of cold treatment (Figure 2.2a).
Each gene was assigned to one of seven expression clusters (Figure 2.2b). Expression
clusters I and II comprised down-regulated transcripts and clusters III to VII comprised

up-regulated transcripts. The major difference between clusters I and II was that the

27

transcripts in the former cluster continued to decrease in levels between 24 h and 7 (1
whereas those in cluster 11 showed some recovery over this time interval. A major
difference between the up-regulated transcripts in clusters III and IV, as compared to
those in clusters V, VI, and VII, was that those in the former two clusters were delayed in
response; i.e., there was little increase in the levels of the transcripts in clusters III and IV
after 1 h of cold treatment whereas there was an increase for the transcripts in clusters V,
VI, and VII. Also, a distinguishing feature of cluster V was that the cold-response for
these transcripts was transient in nature; transcript levels increased dramatically by 1 h,
but returned to near “nu-stressed” levels by 24 h.

The clustering of genes with similar expression patterns raises the possibility of
using bioinformatic approaches to identify potential cis-acting regulatory elements
involved in coordinate gene regulation (reviewed in Rombauts et al., 2003). In this
regard, MotifSampler (Thijs et al., 2002) was used to search for 8 bp sequences that were
significantly overrepresented in the promoter regions of the COS genes that comprised
the different expression clusters. Those elements that were enriched 5-fold or more (as
compared to all genes on the array) are listed in Table 2.1. No potential cold-regulatory
elements were identified for the genes in clusters I and II, the cold-repressed genes, but
many were identified for the up-regulated genes in clusters III to VII. Most of these
potential regulatory sequences were novel. However, an analysis of the genes comprising
cluster III lead to the identification of the motif erCGAC which contains the CRT/DRE
element, (A/G)CCGAC, the element to which the CBF transcriptional activators bind
(Sakuma et al., 2002). The sequence (A/G)CCGAC was present within lkb upstream of

123 (53%) of the 233 genes included in the cluster (as shown below, most of the genes

28

(a) Time (h) at 4°C (b) Expression profiles of clusters
0 1 24 168

 

10 !Cluster 1, 101 probe sets
1 ‘ .-.. ..
0.1 5

10 ICluster II, 111 probe sets

1 ~- ,4
0.1 i ‘ is?

 

 

1000 Cluster III. 233 probe sets

ll 100 <pé’e3m ‘— -
10 A
1 _

 
   

o Cluster IV, 13 probe sets

s; 100 i _
m 10 g ./ ’- '_- ;.
‘6 1 ‘1 _

g: 1

_l

 

 

'” 100
10

 

 

Cluster VI, 8 probe sets

 

 

'r 7 '7‘7’7 i777? * i '7'

IV 100 Cluster V11. 16 probe sets

 

 

 

1 o 3 . _7(z_‘:$'-‘ 3:“ 5‘
VI 0
VII 0 1 24 168

 

Figure 2.2. Hierarchical cluster and expression profiles of the 514 COS genes.

(a) Hierarchical cluster analysis of the 514 COS genes. Log of ratio values for each
probe set responding to low temperature at 1 h, 24 h, or 168 h was used to generate
the cluster (see Materials and Methods). Down-regulated probe sets (n=212, green)
consisted of two sub-clusters (yellow and orange side bars). Up-regulated probe sets
(n=302, red) comprised five sub-clusters (red, blue, green, navy, and light blue side
bars).

(b) Expression profiles for the genes in each cluster are presented in a graph format. The
graphs plot log of ratio values over time (h).

Images in this figure are presented in color.

29

Table 2.1. Potential low temperature regulatory elements I'

 

 

Class Motif PlantCARE Class 4%) GeneChip (%)

Class I None

Class II None

Class III erCGAC CRT/DRE 33 6

Class IV CAATGAGG Novel 23 0.8
GTGATCAC Novel 8 0.4
GnATTGAC Novel 39 4
TGTATACA Novel 23 2

Class V AGCGCGTG Novel 16 0.9
erGCGTG Novel 22 2
erGCGTk Novel 25 3
AGCCGmCC Novel 9 0.5
ACCGmGT Novel 22 3
rCCGCsT JERE 25 4

Class VI GAGAArGG Novel 25 4
CTCTCACT Novel 38 3
kTrGCTGT Novel 50 4
kaCCTCT Novel 63 12
AkaTACG Novel 38 2
yAnCTTCC Novel 63 1 1

Class Vll GsCTCGTG Novel 13 0.8
rAGnCGAC Novel 38 6
AACGCGTT Novel 13 0.5
GCTTCGTC Novel 6 1
GCyTCGTC Novel 6 1
CArAGka Novel 38 6

 

aAll 8bp motifs overrepresentedin the promoters of a given class of transcripts are shown.
The motifs were queried to PlantCARE
(http://oberon.fvms.ugent.be:8080/PlantCARE/index.html) to determine if they were
similar to any known cis-element. The percent of promoters in each class containing the
motifs and for all transcripts on the GeneChip was calculated.

assigned to the CBF regulon were members of this cluster). In addition, a potential

jasmonic acid responsive element along with three other novel elements, having in

common the sequence CGCGT, were enriched in cluster V.

The CBF 2 Regulon Comprises 93 COS Genes

Previous transcriptome profiling experiments that surveyed about one-third of the

Arabidopsis transcriptome indicated that: l) the CBF transcription factors controlled

30

expression of about 30 cold-regulated genes (Seki et al., 2001; Fowler and Thomashow,
2002); and 2), that there were no obvious differences in the genes that were affected by
overexpression of either CBF] , 2, or 3 (Gilmour et al., 2004). Here these previous
findings were extended by profiling two independent transgenic lines overexpressing
CBF 2 using the “full genome” ATHl Affymetrix array. Probe sets were designated
CBF2 up-regulated if, in both biological samples, the detection algorithm assigned a call
of “present” in the transgenic line, the change algorithm assigned a call of “increased,”
and the signal log ratio was greater than or equal to 1.3. A probe set was labeled as
down-regulated by CBF2 if, in both biological samples, the detection algorithm assigned
a call of “present” in the wild type sample, the change algorithm assigned a call of
“decreased,” and the signal log ratio was less than or equal to ~13.

Of the 302 COS genes that were up-regulated in response to low temperature, 85
were also up-regulated in response to CBF 2 expression at control temperature (Appendix
G). These genes were assigned to the CBF regulon. Analysis of the promoter regions of
these genes using Motif‘Sampler identified six versions of the CRT/DRE element (Table
2.2). Alignment of all six motifs revealed a consensus of (A/G)CCGACnT.
Additionally, when the sequence lkb upstream of these genes was examined for longer
motifs (n=10), the most conserved sequence in the CBF regulon was found to be
erCGACnTm. Further analysis indicated that of these 85 genes, 68 (80%) had at least
one potential CRT/DRE element (A/GCCGAC) within 1 kb upstream of the start of the
protein coding sequence and thus, were likely to be direct targets of the CBF transcription
factors. All but three of these 68 genes fell into expression cluster 111 (Figure 2.3), the

expression cluster for which the bioinformatic analysis described above identified the

31

Table 2.2. Potential low temperature regulatory elements 11'

 

 

Class Motif PlantCARE Class (%) GeneChip (%)
CBF2 and ACCGACrT CRT/DRE 28 1
cold up erCGAC CRT/DRE 60 6
rCCGACnT CRT/DRE 55 4
GACCGAC CRT/DRE 24 2
GCCGAC CRT/DRE 40 5
GyCGrC CRT/DRE 47 10
CCGACrT CRT/DRE 44 4
CBF2 and AGnCGnCT Novel 40 3
cold down CACmACAC Novel 25 4
CAAGTTGr Novel 50 4
CGAwCyAG Novel 25 2
CTI'TGCCT Novel 25 2
TTCnGAGT Novel 50 5
TCanTCs Novel 50 9
Zat12 and GCATTGAC Novel 44 0.8
cold up yCTCTTCA Novel 44 6
CAATGmkG Unnamed 44 3
TGAGGTCA Novel 22 0.7
rsAATGAG Novel 56 6
mCAACTTs Novel 56 8
AwGAkaC OCS 67 11
ACAwm'l'l'C Novel 67 12
Zat12 and TATCCAAA Novel 40 5
cold down GAmTAAGA Novel 33 5
TAyGCCAG Novel 1 3 0.4

 

alAll 8bp motifs overrepresentedin the promoters of a given class of transcripts are shown.
The motifs were queried to PlantCARE
(http://oberon.fvms.ugent.be:8080/P1antCARE/index.html) to determine if they were
similar to any known cis-element. The percent of promoters in each class containing the
motifs and for all transcripts on the GeneChip was calculated.

CRT/DRE element. The 17 genes that were both cold- and CBF-regulated, but did not
have potential CRT/DRE elements within 1 kb of the start of the coding sequence, might
have active CRT/DRE elements elsewhere within or surrounding the gene coding
sequence or might have been targets of transcription factors that were up-regulated in
response to CBF2; such factors include RAP2.1 (Atlg46768) (Okamuro et al., 1997), a
putative zinc finger (At5g04340), a CONSTAN S B-box zinc finger (At2g47890), and a

protein related to squamosa promoter binding protein (Atl g765 80).

32

Cold CBF2 ZAT12

 

 

 

Cluster 1, 101 probe sets ‘ 5 probe sets
2 ‘g i - l-I - _ , __ -
8 g. lywr v, _+ rv,
E g Cluster Il,111 probe sets 8 probe sets 10 probe sets
.1:

g 'o i w 1 “hw: !N
0% g Eifiifv, _, A _ »-
luster III 233 probe sets 78 probe sets 3 probe sets

1 £2...” _ "-15...“ ”(r L
, , ,9 — -7 ._
C—fizz A ,A I l-fim LEE if E __-_ I
Cluster IV, 13 probe sets :3 probe sets 6 probe sets
1M i —"-'f"" T‘;-_— —’-: - .— ' :ab‘éi—J:
.. 1 -- .
E 'g Cluster V, 32 probe sets
8 a «- l
.5 g
Q 3
D 'o
C
(0

 

 

Figure 2.3. Expression profiles of the COS genes and those that comprise the CBF2
and ZAT12 regulons.

The expression profiles for all of the COS genes are presented in the panel labeled
“Cold.” In the panels labeled “CBF2” and “ZAT12” are the cold-regulation profiles for
those genes that comprise the CBF2 and ZAT12 regulons, respectively. In each graph,
log of ratio values are plotted versus time (h). Images in this figure are presented in
color.

33

Of the 212 genes that were down-regulated in response to low temperature, eight
(4%) were down-regulated in response to CBF 2 overexpression (Appendix H). These
genes, which belong to expression cluster 11 (Figure 2.3), were also assigned to the CBF
regulon. The promoter regions of these genes did not have potential CRT/DRE elements,
but an analysis using MotifSampler identified a number of potential novel elements
(Table 2.2). These genes are not likely to be direct targets of the CBF transcription
factors, but could be repressed by the expression of genes that are members of the CBF
regulon.

The effects of CBF 2 overexpression were not limited to cold-responsive genes.
Transcripts for 66 cold non-responsive genes were up-regulated and 35 were down-
regulated by CBF 2 overexpression. Assignment of many of these genes to the cold-
nonresponsive class, however, was due to the strict criteria used to designate a gene as
being cold-responsive. In fact, many of these transcripts were increased or decreased in
the cold, but not to the same level used for selecting the COS transcripts. There were,
however, some CBF2-regulated transcripts that showed no sign of cold-responsiveness.
The altered expression of these genes likely resulted from the secondary effects that
CBF 2 overexpression has on plant growth and development, including stunted growth
and a delay in flowering (Gilmour et al., 1998; Liu et al., 1998). A question thus raised
was whether it was valid to assign a gene to the CBF regulon based on the criterion of the
gene being both cold- and CBF2-responsive. Statistical analysis supported such
assignments. In particular, the transcript levels for 151 genes were up-regulated and 43
genes down-regulated in the CBF 2 overexpressing plants which corresponded,

respectively, to 0.66% and 0.34% of the total genes assayed on the array. Thus, by

34

chance, one would expect two of the 302 cold-induced COS genes to be up-regulated in
response to CBF2 overexpression and less than one of the 212 cold-repressed COS genes
to be down-regulated in response to low temperature (Table 2.3). Instead, 85 of the cold-
induced genes and eight of the cold-repressed genes were up- and down-regulated,
respectively, in response to CBF 2 overexpression (Table 2.3). The large differences
between expected and observed genes argued strongly against the hypothesis that the
observed regulation of the COS genes by both cold and CBF2 was due to chance

(p<0.0001).

CBF 2 Regulates a Majority of Highly Induced COS Genes

The results presented above indicated that the CBF regulon included a significant
portion (28%) of the cold-induced COS genes. Further analysis indicated that of the
genes that were most highly induced in response to low temperature, the large majority
were members of the CBF regulon. Shown in Figure 2.4 is a ranking of the 302 cold-
induced transcripts ordered from the most highly induced to the least highly induced
based on the average fold-change at 24 h. All 13 transcripts that increased 25-fold or
more in response to low temperature were induced in response to CBF 2 expression. Of
those transcripts that increased more than 15-fold, 84% increased in response to CBF 2
expression. In contrast, about 90% of the transcripts that accumulated less than 5-fold in

response to low temperature fell outside of the CBF regulon.

35

Table 2.3. Chi-square analysis of expected and observed changes in gene expression
in response to low temperature and transcription factor overexpression

 

 

Total b Chi-square

Transgenic changesa Total possible Expectedc Observedd p-value
Up Down Up Down Up Down ULDown Up Down

CBF2 151 43 22746 12726 2 0.7 85 8 <0.0001 <0.0001
ZAT12 47 158 22746 13320 0.6 2.5 9 15 <0.0001 <0.0001
RAV1 83 63 22746 14884 1.1 0.9 1 0 0.7 0.7
STZ 58 11 22746 13922 0.8 0.2 2 0 0.4 5
MYB73 28 2 22746 14198 0.4 0 0 0 0.9 n/a
CZF2 36 24 22746 14198 0.5 0.4 1 1 1 0.9

 

a The number of probe sets up- or down-regulated by expression of each transcription
factor (see Materials and Methods).

b The number of probe sets that could possibly be up- or down-regulated by each
transcription factor. Every probe set on the GeneChip had an equal probability of being
up-regulated in a transgenic line, but only those probe sets “present” in the control
samples could be down-regulated and this number fluctuated between the various
controls.

c The number of probe sets expected to be regulated by a transcription factor and the cold
(see Materials and Methods).

d The actual number of probe sets that were both cold and transcription factor regulated.

     
  

    

      

(a). , (b)
218 100 N=7 N=3 N=3 N=12 N=80N=197
115 «90l-----I
90 880IIIIIII
cml I I I I I I
50 geol I I I I I I
‘5 "’ .5,- E E E E E 1
\°
$35 °40IIIIIII
525 a20lIIIIII
2 a I I I I I I I
.915 cLlolIIIIII
250-4545-35 35-25 25-1515-5 <5
5 Fold change class
5 ‘ ICBF

Cold up-regulated transcripts ranked by
average fold change at 24 h (high to
low)

Figure 2.4. CBF2 and ZAT12 induce a majority of the most highly cold-induced

transcripts.

(a) Cold up-regulated transcripts were ranked by average fold change at 24 h and colored
red if they were responsive to CBF2, green if responsive to ZAT12, and blue if
responsive to both CBF2 and ZAT12.

(b) Cold up-regulated transcripts were placed into classes defined by fold-change and the
percentage of transcripts responsive to CBF2, ZAT12, both, or neither (not assigned)
are indicated. The number of transcripts in each fold-change class is indicated above
each bar. Images in this figure are presented in color.

INot assigned

 

 

36

Candidate Transcription Factors for Regulating COS Gene Expression

The experiments described above indicated that 70% of the cold-induced COS
genes, and more than 95% of the cold-repressed COS genes, remained unassigned to a
regulon. The challenge was to determine which of the more than 1,700 transcription
factors encoded by the Arabidopsis genome (Riechmann, 2002) might have roles in
controlling expression of these genes. It was hypothesized that transcription factors that
were coordinately regulated with CBF2 might be involved in regulating expression of
these genes. Genes encoding transcription factors that were candidates for being
coordinately regulated with CBF 2 included six that were previously identified as being
up-regulated within 1 h of transferring plants to low temperature (Fowler and
Thomashow, 2002). These genes were ZA T12, RA V1, MYB 73, STZ/ZA T10, and two
genes designated CZFI (gold-induced zinc finger proteins) (At2g40140) and CZF 2
(At5g04340) [CZF 2 is likely to correspond to ZA T6 (Meissner and Michael, 1997)
designated from a partial sequence of the gene]. Northern analysis confirmed that
transcripts for these six genes increased within 1 h of exposing plants to low temperature
(Figure 2.5a). Gene fusion experiments indicated that the promoter regions (lkb
upstream of the ATG) of each of these genes, with the exception (for unknown reasons)
of RA V1, were responsive to low temperature (D. Zarka, J. Vogel, H. Van Buskirk, M.F.
Thomashow, unpublished).

Additional experiments indicated that transcripts for all six genes, like those for
CBF 2 (Zarka et al., 2003), increased in response to both mechanical agitation and

treatment with cycloheximide (Figure 2.5b). This was not true of all cold-inducible

37

(a) (b) Time of treatment 91)
Time at 4°C (hL MA CHX
00512482472168 0.25.5124824 0124824

‘ r...OOe-v CBF2

 

 

 

000000" ZAT12

 

.0".qu RAV1

4 00.04.00. MYB73

 

 

18". Ir . ZAT10

 

.. cuyucmc- CZF1
was...“ CZF2
~ It.“ RAP2.1
It aggoos RAP2.7
If'irrvq «9 1: CZF3
_, a} 6§gfi HPPBF-Za

Q9! .0111 1 eIF4a

Figure 2.5. Expression of transcription factors that are candidates for configuring

the low temperature transcriptome.

RNA was isolated from plants that were exposed to low temperature, mechanical

agitation, or treatment with cycloheximide for various times and the transcript levels for

the indicated genes was determined by RNA blot hybridization. e1F4a was used as a

loading control on each blot and a representative blot is shown.

(a) Transcript accumulation in response to low temperature. Plants were grown at 24°C
and then exposed to 4°C for the indicated times.

(b) Transcript accumulation in response to mechanical agitation (MA) and treatment with
cycloheximide (CHX). Plants were grown at 24°C and then treated with mechanical
agitation (MA) or cycloheximide (CHX) for the times indicated (see Materials and
Methods for details).

 

 

 

 

 

 

 

 

 

 

 

genes that encode transcription factors. For instance, RAP2.1, RAP2. 7 (At2g28550),
CZF 3 (At4g38960), and HPPBF—Za (At3g47500), which are induced after two hours of
cold-treatment (Figure 2.5a), were not induced in response to mechanical agitation or
treatment with cycloheximide (Figure 2.5b). The link between cold, mechanical, and
cycloheximide regulation of CBF 2 is not known. However, two regulatory regions
within the CBF 2 promoter, designated ICErl (induction of CBF _e_xpression region D and

ICEr2, have been shown to be involved in gene induction by each of these three stimuli,

38

indicating a regulatory link between them (Zarka et al., 2003). A question thus raised
was whether ICErl or ICEr2 sequences were present in the promoters of ZA T12, MYB 73 ,
ZA T10, CZF1, and CZF 2. Searches indicated that the promoters of each of these genes

had four sequences that overlapped with ICErl, and a fifth sequence that overlapped with

 

 

 

 

 

ICEr2 (Figure 2.6).
(8)
Sequence PlantCARE % Genome
ICE rggion 1
ACACGIGI G-box 3%
_____AAFGAAA_S. MPE 44%
AQAYJTQI light responsive 25%
£939ch novel 8%
I E r ion 2
QCsAAACG novel 3%
(b)
ACACGTGT
Iczrl ATGGGTCAAAQQACAQATGTQAGATTCTCAGTGA
AArGAAAs AGAwTCT
GAGrAAGG

CC SAAACG
ICEr2 AGAGACAGAAACTCCGCGTTCGACCC

 

Figure 2.6. Potential regulatory sequences common to the promoter regions of

CBF2, ZA T12, ZA T10/STZ, M YB 73, CZF1, and CZF2.

(a) Motifs that overlap the CBF 2 ICErl or ICEr2 sequences that are conserved in the
promoter regions of ZA T12, ZA T I 0/STZ, MYB 73, CZF1, and CZF2 (see Materials
and Methods for details). Base pairs of the motifs that are perfectly conserved in all
the promoters analyzed are underlined. The sequences were queried to PlantCARE
(http://oberon.fvms.ugent.be:8080/PlantCARE/index.html) to determine if they were
similar to known cis-acting regulatory elements. All promoters in the Arabidopsis
genome were examined for the presence of these motifs and the percent containing
the motif is given.

(b) Position of motifs aligned to the ICErl and ICEr2 (underlined) sequences.

39

Transgenic plants that constitutively expressed RA V1, M YB 73, ZAT1 0, CZF 2, or
ZAT12 under control of the CaMV 355 promoter were generated and the transcriptomes
profiled to determine whether any of these transcription factors controlled expression of
COS genes. Results with plants overexpressing RA V1, MYB73, ZAT1 0/SIZ, or CZF 2,
using one GeneChip per line, indicated that the expression of few, if any, COS genes
were affected by these transcription factors (northern analysis indicated that the transcript
levels for each of the transgenes was equal to or greater than the maximum level for the
corresponding endogenous gene attained upon cold treatment; data not shown). RAV1
up-regulated one COS transcript (At1g35 140), STZ/ZATlO up-regulated two COS
transcripts (At4g22470 and At4g12490), and CZF2 up-regulated one COS transcript
(At4g12490) and down-regulated one COS transcript (At2g40610). These effects were
confirmed by northern analysis (data not shown). However, the chance of observing up-
or down-regulation of the COS genes by this group of transcription factors was no greater
than expected by random occurrence; i.e., the extent to which COS genes were affected
by expression of the transcription factors was no greater than that observed for non-COS
genes (Table 2.3). Thus, no COS genes could be confidently assigned to regulons for
RAV1, MYB73, STZ/ZATIO, or CZF 2. As described in the next section, COS genes

could be assigned to the ZAT12 regulon.

The ZA T12 Regulon Comprises 24 COS Genes
Transgenic plants that constitutively expressed ZA T12 under control of the CaMV
3SS promoter were generated (Figure 2.7) and the transcriptome profiled using the

Affymetrix array. An examination of two independent transgenic lines indicated that 24

40

COS genes were responsive to ZAT12 overexpression (Appendices I and J). Of the 302
COS genes that were up-regulated in response to low temperature, nine were also up-
regulated in response to ZA T12 overexpression and fell into expression clusters III and IV
(Figure 2.3). Of the 212 COS genes that were down-regulated by low temperature, 15
were also down—regulated by ZAT12 overexpression and fell into expression clusters I
and 11 (Figure 2.3). Significantly, seven of the ZAT12 responsive genes were also
responsive to CBF 2 overexpression (Figure 2.8), indicating the possibility that these two

transcription factors coordinately regulate expression of these COS genes.

Time gt 4°C (h)

WT 358::ZAT12
O 2 24 V #2 #81115

mi an.

9191114....“ Q“.

  
 

 

 

 

Figure 2.7. ZA T12 transcript levels in transgenic Arabidopsis plants.

Transgenic lines expressing ZA T12 under control of the CaMV 35$ promoter (#2, #8 and
#15) or carrying an empty vector (V) were grown at 24°C and the levels of ZAT12
transcripts determined by RNA blot analysis. For comparison, wild type (WT) plants
were exposed to low temperature (4°C) for 0 h, 2 h, and 24 h and ZAT12 transcript
accumulation was determined by RNA blot analysis. eIF 4a was used as a loading
control.

41

Up-regulatod transcripts Down-regulated transcripts

    
   
   
   
   

pEARLI
pEARLl P450

pEARLI

resist.

chitinase

5

 

 

Figure 2.8. Regulation of COS genes by CBF2 and ZAT12.
Cold-responsive transcripts are indicated by boxes. Arrows indicate transcripts also
responsive to CBF2 or ZAT12 expression.

42

  

surviva N= t-test

 

wild type 6% 449 n/a

 

“do, 7% 396 0.37

 

 

 

 

 

WT 1‘55 24W ‘ ‘ " - 358::ZAT12 54% 598161110“

 

 

Figure 2.9. Effect of ZA T12 expression on plant morphology and freezing tolerance.

(a) Six week old wild type (WT) and ZAT12 overexpressing (35S::ZAT12) plants.

(b) Magnified view of six week old ZAT12 overexpressing plants.

(0) Mature eleven week old ZAT12 overexpressing plants. A US. dime (17mm) is
shown for size comparison.

((1) Effect of freezing on nonacclimated ten day old wild type and ZAT12 expressing
plants.

(6) Magnified view of nonacclimated ZAT12 plant after fieezing.

(1) Table of results of multiple fi‘eeze tests, including the percentage of plants scored as
surviving (% survival), the number of plants examined (N =), and the p-value from a
t-test.

Images in this figure are presented in color.

A number of genes that were not cold-regulated were responsive to ZAT12
expression; 38 were up-regulated and 143 were down-regulated. As with CBF2
overexpression, assignment of many of these genes to the cold-nonresponsive class was
due to the strict criteria used to designate a gene as being cold-responsive and a number
were either increased or decreased to a lower extent than the COS genes. Expression of
the remaining genes was likely a consequence of the effects that ZAT12 expression had

on growth and development (Figure 2.9). ZAT12 overexpressing plants were smaller

43

than wild type plants and had curled leaves, short petioles, short bolts, and downward
pointing siliques. As with the CBF 2 overexpression experiments, the question raised was
whether it was valid to assign a gene to the ZAT12 regulon based on the criterion of the
gene being both cold- and ZAT12-responsive. Again, statistical analysis supported such
assignments. Chi-square analysis indicated that it was highly unlikely for a given gene to
be both cold- and ZAT12-responsive by chance (p<0.0001) (Table 2.3). Thus, the 24
COS genes that were responsive to ZAT12 were assigned to the ZAT12 regulon.

The binding site for ZAT12 is unknown, and thus I was unable to tentatively
assign genes to being direct targets for the ZAT12 protein. However, bioinformatic
analysis of the promoter regions of the genes that were up-regulated in response to both
cold and ZA T12 expression identified multiple novel sequences that were
overrepresented, three of which had CATTG as a core sequence suggesting that this may
be a part of the ZAT12 binding site (Table 2.2). Three additional novel elements were

detected in the genes that were down-regulated in response to both cold and ZAT12.

The ZA T12 Regulon Contributes to Freezing Tolerance

Whole plant freeze assays indicated that ZA T12 expression resulted in a slight, but
detectable, increase in freezing tolerance. Whereas nonacclimated control plants were
killed by freezing to -5°C, the innermost and youngest leaves of the rosettes in 54% of the
ZA T12 overexpressing plants survived (Figure 2.9). No difference in freezing tolerance
was observed between cold-acclimated control and cold-acclimated ZA T12

overexpressing plants (not shown).

44

ZA T12 Negatively Regulates CBF Gene Expression

The level of CBF], 2, and 3 transcripts peaks at about 2 h after shifting plants to
low temperature and then, as the COR gene transcripts begin to accumulate, the CBF
transcripts begin to decrease. It was of interest to determine whether ZAT12 might have
a role in this down-regulation of CBF gene expression as the ZAT12 protein can function
as a transcriptional repressor (Hiratsu et al., 2002) and its induction kinetics (Figure 2.5)
were consistent with this possibility. To test this hypothesis, the kinetics of low
temperature induction of the CBF1-3 genes in wild type plants and in transgenic plants
overexpressing ZA T12 was compared. The results indicated that the CBF 1, 2, and 3
transcripts accumulated to much lower levels in response to low temperature in the
ZA T12 overexpressing plants as compared to the control plants (Figure 2.10). Transcript
levels of the CBF-targeted genes COR 78 and COR6. 6, however, were only slightly lower

in the ZAT12 transgenic plants.

45

(a) Time at 4°C (h)

 

 

 

 

 

 

 

 

 

 

WT 358::ZAT12 #2 358::ZAT12 #8 358::ZAT12 #15
0124824168012482416801248241680124 824
can Ofigea to. ~ "00::- as:
CBF2 ...*‘ ... t.'t: t!‘-..i~
car-'3 eggs: areas.» 113* 1”“
COR78 ..An 4. . . In. .w ill .
COR6.6 it . . " .13
rRNA IO...

   

 

 

 

 

 

 

m .
so: \.
C
3
E1012482416801248241680124824168
é Timeat4°C(h)
g -—~—wr
—-.—-#2
..*..#8
--—#15

 

 

 

 

0124824168 0124824168
Timeat4°C(h)

Figure 2.10. ZAT12 overexpression dampens cold induction of CBF], 2, and 3.

Wild type (WT) or ZA T12 overexpressing (358::ZA T12) plants were grown at 24°C and

then exposed to 4°C for the times indicated. RNA was isolated and CBF], 2, and 3

transcript levels were determined by RNA blot hybridization using gene specific probes.

rRNA was used as a loading control.

(a) Transcript accumulation in response to low temperature. Plants were grown at 24°C
and then exposed to 4°C for the indicated times.

(b) Quantitative transcript accumulation in response to low temperature. The highest
transcript level after normalization was set to 100 and all other values are shown
proportionally.

Since constitutive expression of ZAT12 resulted in lower CBF transcript

accumulation, loss of ZAT12 would likely result in higher CBF transcript accumulation.

46

To test this hypothesis, two independent T-DNA insertions in the ZAT12 locus were
ordered from public collections (Alonso et al., 2003; Mario et al., 2003). These lines
were then screened by PCR for plants homozygous for the insertion and sequenced to
verify the insertion site. The T-DNA in SALK line 037357 was 294 bp upstream of the
ATG and the T-DNA in GABI-Kat line 348H06 was inserted after 326 bp of the coding
region (Figure 2.11). Given the location of the insertion in the SALK line, these plants
might still express ZA T12 at low levels, while the GABI-Kat line appeared to be a null
mutation. RT-PCR results show that after 2 h at low temperature, wild type plants
exhibited increased accumulation of ZAT12, while the SALK line displayed reduced

ZA T12 accumulation (Figure 2.12). RNA blot analysis resulted in similar results (Figure
2.13). No full length ZA T12 transcript could be detected in the GABI-Kat line by RT-
PCR analysis (Figure 2.12), however, RNA blot analysis revealed high levels of two
products lower in size than the firll length ZAT12 transcript (Figure 2.13). This
discrepancy is likely due to the CaMV 355 promoter present in the T-DNA used to create
the GABI-Kat population, as the plasmid used was originally designed for activation
tagged screens (Mario et al., 2003). The sizes of the transcripts detected by RNA blot
analysis would indicate that the transcripts are truncated, so if they are being translated,
they may not be fully functional.

In response to low temperature, the accumulation of CBF], 2, and 3 transcripts
was higher in both ZAT12 insertion lines compared to wild type plants at the 2 h time-
point (Figure 2.13). CBF levels were slightly higher in the GABI-Kat line compared to
the SALK line. As in the ZA T12 overexpressing lines, expression of COR6.6 and COR 78

was little, if at all, impacted.

47

SALK_037357 GABl-Kat 348H06

 

RB LB
RB LB V
| ZAT12 I V _

 

 

 

 

 

 

 

|-— 294 bp —-‘— 326 bp i 163 bp -—-l

B DNA binding zinc finger domains

Repression motif

Figure 2.11. T-DNA insertion lines in the ZA T12 locus.

This figure depicts the two T-DNA insertion lines available from the public collections
with insertions in the ZAT12 locus. The dark line indicates the upstream and downstream
regions, while the box indicates the ZAT12 protein coding sequence. The two zinc
fingers of ZAT12 are represented by white boxes, while the EAR-like repression motif is
indicated by the dark box. T-DNA insertions are represented by the triangles, with the
right and left borders (RB, LB) of the T-DNA indicated.

WT SALK GABl-Kat

 

0 2 0 2 0 2 no RT

 

Figure 2.12. RT-PCR analysis of ZAT12 expression in the two T-DNA insertion
lines.

Total RNA was isolated from warm grown (0) plants or plants cold shocked at 4°C for
two hours (2). The plants used were Col-0 (WT), SALK_037357 (SALK), and GABI-
Kat 348H06 (GABI-Kat). PCR was performed on cDNA reverse transcribed from 5 pg
of total RNA (see Materials and Methods) with primers specific to ZAT12 or actin. A
sample of RNA which was not reversed transcribed was also used to control for
contaminating genomic DNA.

48

Tlme at 4°C (h)
(8!) WT SALK_037357 GABl-Kat 348H06
0124824 0124824 0124824

can an I a W “a, W
CBF2 ‘ G
CBF3 . ""-- «a I . .

ZAT12

COR6.6

COR78

 

(b)

 

01248240124824 0124824
Time at 4°C (h)
T—o—twri'TTTi
-—I--SALK_037357
----A- - - 'GABI-Kat 348H06 :

Normalized units

 

01248240124824
Time at 4°C (h)

Figure 2.13. Decreased ZAT12 expression enhances cold induction of CBF], 2, and

3.

Wild type (WT) or ZAT12 T-DNA insertion lines (SALK_037357 and GABI-Kat

348H06) plants were grown at 24°C and then exposed to 4°C for the times indicated.

RNA was isolated and CBF], 2, and 3 transcript levels were determined by RNA blot

hybridization using gene specific probes. Transcript accumulation for ZA T12, COR6. 6,

and COR 78 were also monitored and rRNA was used as a loading control.

(a) Transcript accumulation in response to low temperature. Plants were grown at 24°C
and then exposed to 4°C for the indicated times.

(b) Quantitative transcript accumulation in response to low temperature. The highest
transcript level after normalization was set to 100 and all other values are shown
proportionally.

49

Since the level of COR gene mRNA accumulation was little affected by loss of
ZAT12, one would not expect to see a change in the freezing tolerance of these lines. To
test this, whole plant freeze tests and electrolyte leakage assays were performed on the
two T-DNA insertion lines. No effect was seen on whole plant freezing tolerance or
electrolyte leakage for either line (both with and without cold acclimation, data not

shown).

50

DISCUSSION

The objectives of this study were to determine more completely the role of the
CBF transcriptional activators in configuring the low temperature transcriptome and to
identify additional transcription factors that participate in this process (the functional
roles of the cold-responsive genes identified in this study are considered in Chapter 3).
To begin, I used the “full genome” Affymetrix GeneChip® array to define a core set of
cold-responsive genes that were reproducibly up- or down-regulated in response to low
temperature in plants grown both in soil and on solid culture medium, two commonly
used growth conditions. The experiments resulted in the identification of 514 such cold-
responsive genes, referred to as COS (“cold standard”) genes, 302 of which were up-
regulated and 212 down-regulated at least 2.5-fold after either 1 h, 24 h, or 7 d of
exposing plants to low temperature. This gene set does not contain all cold-responsive
genes as the tissue harvested from the soil-grown plants was primarily foliar, whereas
both root and shoot tissue was harvested from the plate—grown plants. In addition, many
genes were not designated as COS genes due to the strict signal log ratio cutoff imposed
in the analysis. Over 80% of the up-regulated genes that were designated as being
“present,” “increased” and had a signal log ratio of 2 1.3 in both replicates for a given
growth condition, were also “present” and “increased” under the other growth condition.
The same situation held for approximately 70% of the down-regulated genes. However,
the objective here was not to identify all cold-responsive genes, but to generate a robust
gene set that could be used in efforts to decipher the low temperature regulatory network

of Arabidopsis. The COS gene set should have considerable utility in this regard.

51

To assess the extent to which the low temperature transcriptome falls under
control of the CBF1-3 transcription factors, I determined which cold-induced and cold-
repressed COS genes were up-regulated and down-regulated, respectively, at least 2.5—
fold in response to constitutive CBF 2 expression. Those genes that were both cold— and
CBF2-responsive were assigned to the CBF regulon. The analysis indicated that 93 of
the 514 COS genes were members of the regulon, 85 of which were cold-induced.
Significantly, these 85 genes included the majority of those genes that were the most
highly induced in response to low temperature; i.e., 84% of the genes induced more than
15-fold, and 50% of those induced between five to 10-fold, were found to belong to the
CBF regulon. In addition, greater than 50% (37/67) of the COS genes that continued to
be up-regulated after 7 d of cold-treatment were members of the CBF regulon. Taken
together, these results indicate that the CBF transcription factors have a prominent role in
regulating the expression of those cold-responsive genes that are both highly and stably
induced in response to low temperature.

In a newly published study, Maruyama et al. (2004) also presented results
identifying genes that are members of the CBF/DREBI regulon. In these experiments,
both the Affymetrix AtGenomel GeneChip and a RIKEN Arabidopsis full-length cDNA
array (Seki et al., 2001) were used. The two arrays combined represented approximately
12,000 different genes. Of these, 38 were found to be responsive to both low temperature
and DREBIA/CBF 3 overexpression. In these experiments, 22 of these 38 genes were
also assigned to the CBF regulon. Of the 16 genes that not assigned to the CBF regulon,
one was not represented on the array used (there was no probe for the KIN2 gene) and

seven others fell short of the 2.5-fold cutoff used in one or more samples from either the

52

cold-treated plants or plants overexpressing CBF 2. The remaining eight genes
(At4g14000, At4g15910, At5g17460, At1g29390, At2g43510, At1g27730, At2g31360,
and At2g33830) showed “no change” in these experiments in both transgenic lines
overexpressing CBF 2. One possibility that could potentially explain this difference in the
two studies is that CBF2 and DREBlA/CBF3 might have somewhat different target-gene
specificities. Though this possibility cannot be ruled out at present, the recent findings of
Gilmour et al. (2004) argue against it. In particular, these investigators used the
Affymetrix AtGenomel GeneChip to compare the transcriptomes of transgenic
Arabidopsis lines overexpressing either CBF], 2, or 3 and found no qualitative
differences in target gene specificity between the three transcription factors. Other
possible explanations for the observed differences include differences in the probe sets
used to represent genes in the Affymetrix AtGenomel GeneChip and the Affymetrix
Arabidopsis ATHl GeneChip, potential cross hybridization of probes on the cDNA
arrays, and differences in growth conditions.

The CBF proteins are transcriptional activators that bind to the CRT/DRE DNA
regulatory element. Thus, it was not surprising that of the 85 CBF regulon genes that
were cold-induced, 68 (80%) had at least one putative CRT/DRE element within the 1 kb
upstream region of the gene coding sequence. It is likely that most, if not all, of these
genes are direct targets of the CBF transcription factors. The other 17 cold-induced
genes that belong to the CBF regulon might have active CRT/DRE elements elsewhere
within or surrounding their coding sequences. Alternatively, these genes could be targets
of one or more of the transcription factors that belong to the CBF regulon, such as

RAP2.1 (Appendix G), or their regulation might be affected by expression of other CBF

53

regulon genes. Similarly, down-regulation of the eight CBF regulon genes is likely to
involve the action of regulatory or other proteins induced by the CBF transcription
factors as the CBFl-3 proteins have not been reported to act as repressors.

Previous transcript profiling and mutagenesis experiments suggested the existence
of cold regulatory pathways in addition to the CBF cold response pathway in Arabidopsis
and that such putative pathways might have roles in freezing tolerance (Xin and Browse,
1998; Fowler and Thomashow, 2002; Kreps et al., 2002; Seki et al., 2002; Vlachonasios
et al., 2003). This hypothesis was confirmed here. A new low temperature regulatory
pathway, the ZAT12 cold-response pathway, was defined and evidence presented
indicating that it has a role in cold acclimation. In particular, I show that ZAT12 is
induced in parallel with the CBFI-3 transcription factors; that it regulates expression of
24 COS genes, nine of which were cold-induced genes and 15 cold-repressed; and that
expression of the ZAT12 regulon results in a limited, but reproducible, increase in
freezing tolerance. Additionally, the results indicated that the ZAT12 cold response
pathway interacts in at least two fundamental ways with the CBF cold response pathway.
First, there is overlap in the genes that comprise the ZAT12 and CBF2 regulons (Figure
2.8); four genes were up-regulated and three down-regulated in response to both
transcription factors and low temperature. Thus, the ZAT12 and CBF cold response
pathways appear to coordinately regulate the expression of certain COS genes.

Moreover, constitutive ZA T12 expression was found to dampen induction of the CBF1-3
genes in response to low temperature (Figure 2.10), while decreased expression of ZAT12
increased levels of CBFI-3 after 2 h at low temperature (Figure 2.13). Thus, the ZAT12

regulon appears to be involved in negative regulation of the CBF cold response pathway.

54

Precisely how ZAT12 controls expression of the 24 COS genes and suppresses
induction of the CBF1-3 genes remains to be determined. However, ZAT12 has an EAR
(ERF-associated gmphiphilic repression)-1ike repressor domain (Ohta et al., 2001) and
can act as a repressor (Hiratsu et al., 2002). Thus, a simple scenario would be that
ZAT12 directly represses expression of CBF1-3 and the 15 down-regulated COS genes
that belong to the ZAT12 regulon, the consequences of which include induction of the
cold-induced COS genes that belong to the ZAT12 regulon. It must be stressed,
however, that this scenario is highly speculative and that its central tenet, that ZAT12
binds to a cis-acting regulatory element in promoters of the cold- and ZAT12 repressed
genes, cannot be assessed at present as the putative DNA binding site of the ZAT12
protein has not been reported.

In addition to a role for ZAT12 in cold acclimation reported here, ZAT12 has
been implicated in multiple other abiotic stress responses. Transcripts for ZAT12 have
been shown to increase in response to high light (Iida et al., 2000), wounding (Chen et
al., 2002; Cheong et al., 2002; Rizhsky et al., 2004), low-oxygen (Klok et al., 2002),
hydrogen peroxide (Desikan et al., 2001), heat, and treatment with paraquat (Rizhsky et
al., 2004). Also, overexpression of ZAT12 in transgenic Arabidopsis plants has been
shown to result in increased tolerance to high light (Iida et al., 2000) and oxidative stress
caused by application of paraquat (Rizhsky et al., 2004). As both high light
(Demmigadams and Adams, 1992) and cold (Wise and Naylor, 1987) are associated with
the formation of reactive oxygen species, the role of the ZAT12 regulon may be to help
plants cope with oxidative stress. In this regard, it is of interest that ZAT12 expression

resulted in down-regulation of transcripts encoding a putative L-ascorbate oxidase

55

(At5g21100; Appendix J). This down-regulation could account, at least in part, for the
increased levels of ascorbic acid, a potent antioxidant (Noctor and Foyer, 1998;
Sanmartin et al., 2003), observed in cold-acclimated Arabidopsis plants (Cook et al.,
2004). In addition, ZAT12 expression resulted in accumulation of transcripts encoding
arginine decarboxylase (At4g34710; Appendix I) which is involved in the production of
putrescine, a polyamine that appears to have protective roles against abiotic stress,
including oxidative stress (Ye et al., 1997; Bouchereau et al., 1999).

Transgenic Arabidopsis plants overexpressing ZAT12 have been described in two
previous studies (Iida et al., 2000; Rizhsky et al., 2004) in which different effects on plant
morphology were noted. Rizhsky et al. (2004) reported that ZA T12 overexpression had
no effect on plant growth and development while Iida et al. (2000) found that ZAT12
overexpressing plants had leaves that were thicker, rounder, and darker green than the
control plants. The ZA T12 expressing plants in this study had curled leaves, short
petioles, short bolts, and downward pointing siliques. A simple explanation for these
different observations is that the levels of ZA T12 expression attained in the three studies
were different. It is more difficult, however, to explain the differences observed in the
makeup of the ZAT12 regulon reported by Rizhsky et al. (2004) and that reported here.
Rizhsky et al. (2004) reported up-regulated expression of 67 genes using a SLR cutoff of
0.8. Of these, only two were up-regulated in these experiments (“P, “I” in both ZAT12
lines) and of these only one was up-regulated at least 2.5-fold. Moreover, of the nine
genes that were found to be up-regulated in response to both low temperature and ZAT12
expression, none were found to be induced in response to ZAT12 by Rizhsky et al.

(2004). No data are available for a comparison of down-regulated transcripts.

56

While the CBF and ZAT12 regulons include the majority of the highly and stably
expressed cold-regulated genes, most of the COS genes remain to be assigned to
regulons. Determining which of the approximately 1,700 transcription factors encoded
by the Arabidopsis genome have roles in regulating expression of these genes is now the
challenge. As hypothesized above, transcription factors that are up-regulated in parallel
with the CBFI-3 transcription factors seem to be logical candidates, and proved to be true
for ZA T12. However, I was unable to assign COS genes to regulons controlled by any of
the other genes encoding known or putative transcription factors that were up-regulated
along with the CBF genes; namely RA V], M YB 73, STZ/ZA T10, or CZF2. One
interpretation of these results is that these transcription factors simply do not have a role
in configuring the low temperature transcriptome. However, the results obtained with
DREBZa (Liu et al., 1998) suggest that it may be premature to draw this conclusion.
DREBZa is a dehydration-induced gene that encodes an AP2-domain transcription factor
that binds to the CRT/DRE element. Expression of the DREB2a protein in transient
assays activates expression of reporter genes containing the CRT/DRE sequence.
However, constitutive overexpression of the DREBZa gene in transgenic plants does not
result in constitutive expression of CRT/DRE containing genes. Thus, it has been
proposed that the DREBZa protein must undergo posttranslational modification induced
by dehydration stress to be active (Liu et al., 1998). Similarly, the RAV1, MYB73,
STZ/ZATIO, and CZF2 proteins could be activated at low temperature to regulate
expression of bona fide target genes. Another possibility is that the proteins cannot work

alone, but must function in conjunction with other cold-regulated proteins.

57

A final point regards the potential cis-acting regulatory elements that were
identified for the COS genes. In using MotifSampler (Thijs et al., 2002) to analyze the
promoter regions of COS genes included in the seven cold-regulated expression clusters
(Figure 2.2), I was able to identify a number of potential cis-acting regulatory elements
involved in cold-regulated gene expression. Most of these potential elements were novel.
However, the fact that the CRT/DRE element was identified as a potential element for
genes in expression cluster III, which includes the large majority of CBF regulon genes
(Figure 2.3), lends credence to the hypothesis that at least some of the sequences are
active. If this can be verified, the elements could then be used in biochemical and genetic
approaches to identify additional transcription factors involved in shaping the low

temperature transcriptome.

58

ACKNOWLEDGEMENTS

Since this work is part of my dissertation, I would like to acknowledge Dr. Sarah
Fowler and Dr. Heather Van Buskirk, who performed the soil timecourse experiment
which I analyzed. Heather also helped with some of the northern blots shown in Figure
2.5. Dr. Daniel Zarka performed a number of the whole plant freeze tests for Figure 2.9,
northern blots for Figure 2.5, and created and analyzed the promoter-GUS fusion
constructs mentioned in this chapter. I would like thank Dr. Rebecca Grumet, Dr.
Marianne Huebner, and Dr. Vince Melfi for their help with statistical analysis and Dr.
Gert Thijs for helpful advice with MotifSampler. GeneChip hybridization and scanning
was performed by Dr. Annette Thelen in the Genomics Technology Support Facility at
Michigan State University. Thanks to Kurt Stepnitz and Marlene Cameron for help with
photography and graphics and Dr. Sarah Gilmour for critical reading of the manuscript
submitted to the Plant Journal. ESTs and seeds for SALK__037357 were obtained from
the Arabidopsis Biological Resource Center (ABRC). I was a recipient of a US.
Department of Education Graduate Assistantship in Areas of National Need (GAANN)
fellowship that helped fund my studies. This study was supported in part by a grant from
the NSF Plant Genome Project (DBI 0110124). A project description, databases, and
other information regarding the project can be found on the web at
http://aztec.stanford.edu/cold/index.html. Research in the Thomashow laboratory was
also funded in part by grants from the DOE (DEFG0291ER20021) and the Michigan

Agricultural Experiment Station.

59

MATERIALS AND METHODS
Constructs and Plant Transformation

Plasmids were constructed using PCR (polymerase chain reaction) and standard
molecular biological techniques (Sambrook and Russell, 2001). Primers used to amplify
the coding regions of each transcription factor from either genomic DNA or cDNA
contained sites for unique restriction enzymes. PCR fragments were cloned into the
pGEM-T easy vector (Promega, Madison, WI). The inserts were excised using
restriction enzymes for the sites present in the primers and then cloned into a plant
expression vector. The following primers and restriction enzymes were used: ZAT12
Spe I 5’-ACTAGTCAGAAGAAAAATGGTTGCGATA-3’, BamH I 5’-
GGATCCGAAAAATTCAAAGAATGAGAGAAACA-3’; RAV1 Xba I 5’-
CTAGACAGATTAAATGGAATCGAGTAGC-3’, BamH I 5’-
GGATCCGAGTTGTTACGAGGCGTGAA-3’; CZF2 Xba I 5’-
TCTAGATCTTCAAGATAATGGCACTTGAAA-3’, BamH I 5’-
GGATCCTCCTAGGTTTATGTTTAGGGTTTCTC-3’; MYB73 Xba I 5’-
TCTAGATAAAAAGATCCGGCGATGTC-3’, BamH I 5’-
GGATCCCACTCTACTCCATCTTCCCAAT-3’; STZ/ZATlO Xba I 5’-
TCTAGACGAGAGACAAGAAATCCTCAGAA-3’, BamH I 5’-
GGATCCTTTCCTTAAAGTTGAAGTTTGACC-3 ’ .

The DNAs were transferred to Arabidopsis thaliana (L.) Heynh. ecotype
Columbia (Col-0 or glI Col-PRL) via a whole plant dipping method similar to that
described by Clough and Bent (1998). Seed germination on medium containing

kanamycin (50 mg/L) (Sigma-Aldrich, St. Louis, MO) was used to identify plants

60

containing transferred DNA. Expression of the transgene was monitored via RNA blot

analysis and T3 lines homozygous for the transferred DNA were used in all experiments.

Plant Growth and Experimental Treatments

Arabidopsis thaliana constitutively expressing ZA T12, RA V], M YB 73, CZF 2, or
SIZ/ZA T10 (this study) or CBF2 (Gilmour et al., 2004) were grown in controlled
environment chambers at 22°C under constant illumination from cool-white fluorescent
lights (~100 umol m'zs'l) in Baccto planting mix (Michigan Peat, Houston). Pots were
subirrigated with deionized water as needed. Plants were also grown on solid agar
medium which contained Gamborg’s B5 nutrients (Caisson Laboratories, Inc., Rexburg,
ID) and 0.8% phytagar (Life Technologies Inc., Gaithersburg, MD) at 24°C under
constant illumination from cool-white fluorescent lights (~100 umol m'zs‘l). Seeds were
stratified four days at 4°C before being grown at warm temperatures.

Experiments were performed on seedlings that were 10-12 days old. Treatments
with cycloheximide (Sigma-Aldrich, St. Louis, M0) were performed by growing
seedlings on filter papers that had been placed on top of the agar so that the seedlings
could be lifted off the plate with minimal damage or mechanical stress. The seedlings
were then floated on a solution of cycloheximide (10 ug/ml) in covered dishes for various
times. Mechanical treatment involved tapping plates on a bench top for 15 minutes
before harvesting tissue. All transgenic lines for the transcriptome analysis were grown
in plates on solid media. Cold shock treatments involved transfer of plants (in the plate
experiment, 10 day old plants were used with ~100 per plate; in the soil experiment 18

day old plants had been grown one plant per pct, 50 per flat) to a 4°C chamber with

61

constant dim light (~25 umol rn'2 3"). Two biological duplicates of both the plate and

soil experiments were performed for profiling.

Affymetrix GeneChip Hybridization and Data Collection

RNA isolation, probe labeling, hybridization, and data collection were performed
as described (Fowler and Thomashow, 2002) with the following modifications. For the
plate grown transgenic and wild type Arabidopsis, approximately 100 plants grown on a
single plate were collected and total RNA was extracted using the Qiagen RNeasy Plant
Mini Kits (Qiagen Inc., 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 buffer (RLT) was also doubled. The remaining
procedure was performed as described in the Qiagen manual. Biotinylated target RNA
was prepared from 16 pg of total RNA equally pooled from two to four plates. For the
soil grown plants, aerial parts of twelve plants were harvested from various parts of a flat
for each time-point. Total RNA was extracted from two seedlings at once as described
above. All samples for a given time-point were then pooled and precipitated.
Biotinylated target RNA was prepared from 16 pg of total RNA. The samples were
hybridized to the Affymetrix Arabidopsis ATHl GeneChip (Affymetrix, Santa Clara,
CA) representing ~24,000 Arabidopsis genes. Two biological replicates were analyzed
per growth condition, for a total of four samples per time-point.

The results from the microarray experiments have been submitted to TAIR
(www.arabidopsis.org) according to MAIME guidelines under the accession numbers

ME00320, ME00321, ME00322, and ME00323.

62

A flymetrix GeneChip Data Analysis

The Affymetrix Microarray Suite 5.0 statistical algorithms were used for
microarray data analysis. Output from all GeneChip hybridizations was scaled globally
such that its average intensity was equal to an arbitrary target intensity of 500 allowing
comparisons between GeneChips. Signal (gene expression) and Signal Log Ratios (SLR)
were calculated from the GeneChip fluorescence intensity data. The software was also
used to determine whether each gene was present or absent (Detection) and whether the
SLR represented a genuine change in mRNA accumulation (Change). SLRs and Change
calls were determined for each transgenic line or cold sample compared to its
corresponding wild type sample. Microsoft Access was used as the database
management software and to filter and query the data.

Probe sets that met the following criteria were selected for further analysis. Those
determined to be up-regulated by the cold treatment were selected as having, at any time-
point, an absolute call of present in both cold samples (soil or plates), difference calls of
increase, and SLRs of at least 1.3 for both change comparisons. Those determined to be
down-regulated by the cold treatment were selected as having, at any time-point, an
absolute call of present in both warm samples, difference calls of decrease, and SLRs of
at least -1.3 for both change comparisons. Those probe sets meeting the above criteria in
both the soil and plate experiments at any time-point were analyzed. Statistically
significant changes in mRNA abundance were determined using the statistical package
with GeneSpring 6.0 (Silicon Genetics, Redwood City, CA). The GeneChip data was

imported into GeneSpring and normalized to respective control samples as specified by

63

the manufacturer for single color array data. The 3,178 transcripts with reliably altered
levels (probe sets with an absolute call of “present” in all four cold samples and four
difference calls of “increase” for a given timepoint or an absolute call of “present” in all
four warm samples and four difference calls of “decrease” for a given timepoint) were
analyzed using ANOVA (p-value cutoff 0.05 or 0.005) and the Benjamini and Hochberg
False Discovery Rate multiple testing correction.

Genes up-regulated by CBF 2 (lines E2 and E24) or ZAT12 (lines p15-8 and p15-
15) expression were selected as having an absolute call of present in both transgenic
samples, difference calls of increase, and a SLR of at least 1.3 for both change
comparisons between wild type and transgenic lines. Those determined to be down-
regulated were selected as having an absolute call of present in both wild type samples,
difference calls of decrease, and a SLR of at least -1.3 for both change comparisons
between wild type and CBF transgenic lines. Probe sets changing in the RA V1, MYB 73,
CZF 2, or SIZ/ZA T10 expressing lines were selected in the same manner, but with data
from only one GeneChip experiment per line.

Statistical analysis of the probe sets changing in a given overexpression line
compared to the cold-responsive transcripts was performed by chi-square analysis using
Yate’s correction, x 2 = 2(lO-El-0.5) 2/ E. The expected number of cold-responsive probe
sets was calculated by dividing the number of probe sets changing in a transgenic line by
the total number of possible probe sets that could change in the cold and multiplying by
the number of cold-responsive probe sets (302 or 212). Every probe set on the

GeneChip had an equal probability of being up-regulated in a transgenic line, but only

64

those probe sets “present” in the control samples for each transgenic line could be down-
regulated hence this number could fluctuate between various controls.

Hierarchical clustering was performed with GeneSpring 6.0, using data
normalized as described previously. Hierarchical clustering was done using the log of
ratios (i.e., the ratio of the signal to the control, not their logs) of both the soil and plate
experiment data using a standard correlation (separation ratio of 1 and a minimum

distance of 0.001).

Whole Plant Freeze Test

Arabidopsis plants were grown on plates as described above. Cold- and non-
acclimated plants were placed at -2°C in the dark for three hours after which freezing of
the plates was nucleated with ice chips. The plants were incubated an additional 21 h at -
2°C, followed by -5°C for 24b. The temperature was then raised to 4°C for 24 h. The
plants were allowed to recover at 24°C for 48 h in continuous light and then scored for
survival. A t-test was used to assess difference in the mean survival between control and
experimental plants. In most experiments, the control and test plants were grown on
separate plates (data presented in Figure 2.91), but similar results were obtained when

control and test plants were grown on the same plate (Figure 2.9d).
RNA Blot Analysis

RNA blots were prepared (10 ug of total RNA was loaded) and hybridized as

described (Hajela et al., 1990) using normal high stringency wash conditions (Stockinger

65

et al., 1997). Quantitation of mRNA abundance was performed with a Molecular Irnager
FX Pro multi-imager system (Bio-Rad Laboratories, Hercules, CA).

Probes for CBF (Gilmour et al., 1998), COR6. 6, COR78 (Hajela et al., 1990),
eIF 4a (Taylor et al., 1993), ZA T12 (Fowler and Thomashow, 2002), 25S rRNA (Delseny
et al., 1983), RAP2. I and RAP2. 7 (Okamuro et al., 1997) were prepared as previously
described. Probes for M YB 73, SIZ/ZA T10, CZF1, CZF 2, CZF 3, and HPPBF-Za were
PCR amplified from cDNAs using the primer pairs used in “Constructs and Plant
Transformation” except for the following: CZF1 (At2g40140) 5’-
TCTAGACACTTTTGAATCACAGGCAAGA-3’, 5 ’-
GGATCCGCTTCTTATGCCACAATCTGC-3’; CZF 3 (At4g3 8960) 5’-
TCTAGAGAAAAAGCAAGATGCGGATTJfl53
GGATCCTTATCACTTCTCAGACTCTCG—3 ’; HPPBF-2a 5 ’-
TCTAGAAAAATGATGATGGAGACTAGAGA8Z53
AGATCTCATATGTAACTCTAAATCTGTTCATGG-3’. The RA V 1 probe was isolated
from an A011 and BamHI digest using an AclI site present in the RA V1 coding region and
the BamHI site in the PCR primer used for amplifying the coding region. Probes for
genes up- or down-regulated in the cold and in one of the single GeneChip experiments
profiling transgenic lines were performed with restriction enzyme digests of ESTs
available from the Arabidopsis Biological Resource Center at Ohio State University
(Columbus). Probes were labeled with 32P by random priming (Feinberg and Vogelstein,

1983).

66

R T -PCR

Total RNA was extracted using the Qiagen RNeasy Plant Mini Kits (Qiagen Inc.,
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
buffer (RLT) was also doubled. The remaining procedure was performed as described in
the Qiagen manual. The reverse transcription reaction was performed with 5 ug of total
RNA which was first treated with 10 U of RNase-free DNase I (Roche, Indianapolis, IN)
for 15 min at room temperature in 1x RT buffer (Invitrogen, Carlsbad, CA), and heat
inactivated for 10 min at 70°C. The rest of the reverse transcription procedure was
carried out using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen,
Carlsbad, CA) as described in the manual (primed with random hexamers). PCR was
performed using Actin3 primers (forward 5’~GGTCGTACTACCGGTATTGTGCT-3’,
reverse 5’-TGACAA'ITTCACGCTCTGCT-3’) to standardize the amount of DNA to add
to each reaction and ZAT12 transcript accumulation was monitored using the following
primers (forward 5’-CCTTAGGAGGTCACCGTGC-3’, reverse 5’-

CAAGCCACTCTCTTCCCACT-3’).

Motif A nalysis

Motif searches were performed with the command line version of MotifSampler
v3.0 (http://www.esat.kuleuven.ac.be/~thijs/Work/MotifSampler.html). The dataset
searched was the 500 bp locus upstream sequence downloaded from TAIR
(http://www.arabidopsis.org) on March 17, 2003. Arabidopsis intergenic regions were

used as a background model with the order set to 3. The motif length was set to 8bp,

67

with the prior probability of finding 1 motif instance set to 0.3. No limit was set for the
number of motif instances that could be found per sequence, the maximum overlap
between different motifs was set to l, the number of motifs per run to be found was set to
3, and the total number of runs was set to 100. The highest scoring motifs were then
ranked by Motimanking using Arabidopsis intergenic sequence as a background model
and the Kullback-Lieber distance to score the top ten motifs. Those motifs greater than
5-fold overrepresented in a class compared to the rest of the promoters on the GeneChip
were analyzed. The number of motifs present in the upstream regions of Arabidopsis
genes was determined using Patrnatch at TAIR or a set of custom PERL scripts
(Appendix M). All PERL scripts are available upon request. All motifs were queried at
PlantCARE (Lescot et al., 2002) to determine if they were similar to any known
sequence.

MotifSampler was also used to identify motifs present in the 1000 bp locus
upstream sequence (downloaded from TAIR) of CZF1, CZF 2, MYB73, ZAT12,
STZ/ZATIO and the ICE regions (including flanking sequences) of the CBF2 promoter.
The ICE regions searched were ICErl: atgggtcaaaGGACACATGTCAGAttctcagtga and
ICEr2: agagacagaaACTCCGcgttcgaccc (Zarka et al., 2003). The defined ICE regions are

capitalized. Only motifs common among all the promoters were analyzed.

68

CHAPTER THREE

A FUNCTIONAL ANALYSIS OF THE ARABIDOPSIS COLD TRANSCRIPTOME

SUMMARY

The experiments described in Chapter 2 resulted in the identification of a set of
transcripts that change in plants grown under two common laboratory conditions (solid
medium and soil) at low temperature. This set was designated the COS (991d standard)
transcripts. The experiments in Chapter 2 also indicated that the transcription factor
CBF2 affected the levels of a majority of the most highly cold-induced COS transcripts.
Another transcription factor, ZAT12, was found to contribute to the low temperature
transcriptome and alter the stress tolerance of Arabidopsis. Work presented here furthers
our understanding of Arabidopsis cold acclimation through a functional analysis of the
COS transcripts and the CBF2 and ZAT12 regulons. The COS gene set contained
transcripts of diverse biological functions, which changed with time at low temperature.
Several categories of transcripts were found to be overrepresented among the COS genes
compared to all transcripts in the genome. Transcripts encoding four new COR-like
proteins were identified, along with transcripts that are likely involved in protecting cells
from the damage associated with cold and freezing stress. Analysis of the CBF 2 and
ZAT12 regulons revealed enrichment for stress—related transcripts. Additionally, the
ZAT12 regulon contained transcripts likely involved in protecting the plant from

oxidative stress and putative antifreeze proteins that could alter ice formation.

69

INTRODUCTION

Temperature fluctuation is a significant stress experienced by plants, including
agricultural crops that are grown in temperate climates. Many plants increase in freezing
tolerance in response to low nonfreezing temperature, a phenomenon known as “cold
acclimation”. Cold acclimation involves the action of the CBF/DREBl cold response
pathway (Thomashow, 1999; Smallwood and Bowles, 2002). Transcripts for CBF],
CBF 2, and CBF 3 (Gilmour et al., 1998; Jaglo-Ottosen et al., 1998; Medina et al., 1999),
also known as DREBI b, DREBIc, and DREBIa, respectively (Liu et al., 1998),
accumulate rapidly in response to low temperature and encode transcriptional activators
that bind to the CRT (C-repeat)/DRE (dehydration response element) regulatory element
present in the promoters of COR and other cold-responsive genes (Baker et al., 1994;
Yamaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997; Gilmour et al.,
1998). Constitutive expression of the CBF regulon results in increased freezing tolerance
without a low temperature stimulus (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Gilmour
et al., 2000; Gilmour et al., 2004). The CBF regulon increases freezing tolerance in part
through the production of cryoprotective polypeptides such as COR158(Artus et al.,
1996; Steponkus et al., 1998) and the accumulation of compatible solutes such as sucrose,
raffinose, and proline (Nanjo et al., 1999; Gilmour et al., 2000; Gilmour et al., 2004).

While best understood, the CBF pathway does not appear to be solely responsible
for freezing tolerance. The mutants eskimoI (Xin and Browse, 1998) and adaZ (ADA2
encodes a transcriptional adaptor protein) (Vlachonasios et al., 2003) are both
constitutively more freezing tolerant than wild type plants, but the COR genes are not

constitutively expressed. This suggests that the mutations activated freezing tolerance

70

pathways outside the CBF system, indicating that other pathways may be active at low
temperature.

Changes in the Arabidopsis transcriptome and metabolome at low temperature
also indicate the existence of additional pathways for cold acclimation. Studies
investigating changes in transcript abundance during cold acclimation and with
overexpression of the CBF transcription factors have revealed: 1), that cold acclimation
involves many changes in gene expression; and 2), that the CBF pathway does not
mediate all of these changes (Seki et al., 2001; Fowler and Thomashow, 2002; Kreps et
al., 2002; Seki et al., 2002; Maruyama et al., 2004). Besides altering gene expression,
low temperature significantly reconfigures the metabolome (Cook et al., 2004; Kaplan et
al., 2004). The CBF cold response pathway was found to have a prominent role in
configuring these metabolic changes, but at least 20% of the alterations could not be
attributed to CBF (Cook et al., 2004).

In Chapter 2, a more global survey of the low temperature transcriptome was
conducted using the Affymetrix GeneChip® containing probe sets for approximately
24,000 genes. In this work, a core set of cold-responsive genes was defined, as were the
regulons of the cold-responsive transcription factors CBF 2 and ZAT12. The goal of the
study was not to define every cold-responsive transcript, but to define a set of transcripts
that could be used to understand low temperature regulatory networks of Arabidopsis. To
this end, a total of 514 transcripts were found to be altered in accumulation after 1 h, 24
h, or 7 d at low temperature in two growth conditions (soil or solid media, 2.5-fold cutoff,

p<0.05), defined as COS (Qld standard) transcripts (Chapter 2).

71

In order to gain a better understanding of what types of transcripts are altered by
low temperature and how those changes could influence freezing tolerance, a functional
analysis of the COS transcripts, including the CBF2 and ZAT12 regulons, was
performed. This analysis revealed that the COS genes have diverse biological functions
and that the types of functions represented are different after various times at low
temperature. Transcripts encoding four new COR-like genes were identified, along with
transcripts likely involved in protecting cells from the damage associated with cold and
freezing stress. Analyses of the CBF2 and ZAT12 regulons revealed they were enriched
for stress-related transcripts and provided insight into how the ZAT12 regulon might
impact freezing tolerance. Finally, the co-regulation of transcripts by CBF2, ZAT12, and
other transcription factors reveals the complexity of low temperature regulatory

networks.

72

RESULTS
The COS Gene Set Comprise Diverse Biological Functions

The 514 COS transcripts were manually classified into exclusive functional
categories and sub-roles using annotation available at TAIR, MIPS, and TIGR
(Appendices E and F). This allowed each transcript to be placed into a single functional
category. Examination of the 302 up-regulated and 212 down-regulated transcripts
revealed that they encoded proteins of diverse biological function (Figures 3.1). In
comparing percentages, more stress-related, RNA processing and translation, protein
processing and fate, and transcription transcripts were up-regulated than down-regulated
and more metabolism and energy transcripts were down-regulated than up-regulated
(Figure 3.1). While this analysis provides a simple and understandable summary of the
data, transcripts can fit into multiple functional categories.

In order to address the issue of transcripts fitting multiple categories, a second
classification method was also used. The COS transcripts were classified into gene
ontology (GO) functional categories using the GO functional categorization tool available
at TAIR (http://www.arabidopsis.org/tools/bu1k/go/). The gene ontology system divides
transcripts into multiple non-exclusive categories under three organizing principles
(molecular function, biological process, and cellular component). GeneMerge (Castillo-
Davis and Hartl, 2003) was then used to determine if any GO categories were statistically
overrepresented compared to the rest of the genome. One caveat of this analysis is that
all the transcripts analyzed were cold-responsive, yet the GO ontology only classified 15

transcripts as cold-responsive. One problem with relying on automated annotation is that

73

302 ukggulated 212 down-@gulated

         

0.3%
10%
13%
39%

5% 5%
4% 5192‘
2%

"r 0.5% 7%

1% 6% 13% 2‘2 3% “4% 5*

 

El Metabolism .Transcription I RNA processing

I Signaling B Energy & translation

I Transport El Stress related I Cellular biogenesis

U Cellular I DNA replication I Protein processing
organization 8. cell division 81 fare

I Unknown

 

 

Figure 3.1. Functional categories of the 514 COS transcripts.

Each of the COS transcripts was placed into an exclusive functional category based on
annotations available at TAIR, TIGR, MIPS, and in published literature (see Materials
and Methods). Images in this figure are presented in color.

the annotation for each transcript has not been examined individually by a person hence
they are not always up—to—date. Among the up-regulated transcripts, transcription factors
and several categories of stress responsive genes were over-represented, i.e. had an E-
score S 0.05 (Table 3.1). Among the down-regulated genes, water transporters,
oligopeptide transporters, oxidorcductases, carbonate dehyrdratases, and
phosphoethanolamine N-methyltransfcresases were overrepresented (Table 3.1). The
manual and automated analysis revealed that the COS gene set contains transcripts of

diverse biological functions and is enriched for stress responsive transcripts.

74

The Functional Classes of COS Genes Differ Over Time Indicating that Changes Early in
the Cold Response Influence Later Changes

A question that arose was whether the types of cold-responsive transcripts
changed over time or whether the overall composition of the up- or down—regulated
transcripts remained constant. Hierarchical clustering of C08 transcript accumulation
after 1 h, 24 h, and 7 d of cold treatment resulted in the assignment of the transcripts to
one of seven expression clusters (Vogel et al., 2005). These clusters were used to
examine the functional categories of the transcripts over time.

The seven expression clusters were comprised of COS transcripts with different
functional profiles (Figure 3.2 and Table 3.2). The overall picture which emerged from
this analysis was that early in the cold response (clusters V-VII) there was considerable
enrichment for transcription factors and signaling molecules compared to the overall
composition of the COS gene set. At later times (clusters I-IV and VII), the composition
of the COS transcripts was more diverse. These later changes included transcripts whose
function is thought to be involved in protecting the plant from stress (COR proteins,
sucrose synthase, raffinose synthase, etc.). The is consistent with the current model of
how the CBF cold response pathway functions, where the CBF transcription factors are
induced quickly upon cold stress (within 15 min) and then regulate the expression of
genes later in the cold response. The analysis presented below reveals that this paradigm

occurs on a genome-wide level.

75

Table 3.1. Overrepresented GO terms among the COS transcripts

 

 

 

 

 

 

 

Frequency in Frequency
Class GO Ontology genome in class E-score GO Term
302 up- Biological 63/2762? 15/302 8.14E-14 response to cold
regulated Process 109/2762? 15/302 4.32 E1 0 response to temperature
genes 66/27627 12/302 2.71 E-09 response to water
59/27627 11/302 1.55E-08 response to water
depnvafion
590/27627 26/302 8.71 E—07 response to abiotic
stimulus
930/27627 33/302 1.32E-06 response to external
stimulus
889/27627 31/302 6.1 1 E-06 response to stress
11/27627 5/302 2.63E-05 cold acclimation
39/27627 7/302 7.89E-05 salinity response
64/27627 8/302 1.93E-04 response to abscisic acid
stimulus
54/27627 7/302 7.90E-04 response to osmotic stress
1841 /2?627 40/302 1.06E-02 response to stimulus
13743/27627 1841302 2.22E-02 physiological processes
22/27627 4/302 3.49E-02 hyperosmotic salinity
response
23/27627 4/302 4.195-02 hyperosmotic response
344/27627 13/302 4.71 E-02 response to hormone
stimulus
Molecular 1847/27627 39/302 1.60E-02 transcription regulator
activity
Function 42/27627 5/302 2.35E-02 transcriptional activator
activity
1734/27627 36/302 4.35E-02 transcription factor activity
212 Biological 65/27627 5/212 4.49E-02 oligopeptide transport
down- Process
regulated Cellular 6642/27627 78/212 9.96E-04 membrane
genes Component 4520/27627 57/212 3.17E-03 endomembrane system
18/27627 3/212 1.57E-02 vacuole (sensu
Streptophyta)
15445/27627 143/212 1.?5E-02 cell
Molecular 35/2762? 5/212 1.39E-03 water channel activity
Function 17/27627 4/212 1 .51 E-03 oligopeptide transporter
activity
1237/27627 25/212 2.11 E-03 oxidoreductase activity
22/27627 4/212 4.51 E-03 peptide transporter activity
141/27627 7/212 2.29E-02 oxidoreductase activity
14/27627 3/212 3.11E-02 carbonate dehydratase
activity
3/27627 2/212 3.57E-02 phosphoethanolamine N-

methyltransferase activity

 

The 514 COS transcripts (Appendices E and F) were classified into gene ontology (GO)
categories using the functional categorization tool at TAIR. The categories represented
among the COS transcripts were then compared to those for all genes in the Arabidopsis
genome using GeneMerge. Overrepresented terms among the up- or down-regulated
COS genes as compared to the rest of the genome (E S 0.05) are shown. Each term is
associated with an organizing principle (biological process, cellular component, or
molecular function) listed under “GO ontology” in the table.

76

Table 3.2. Overrepresented GO terms among COS transcript expression clusters

 

 

Frequency in Frequency

 

 

 

 

 

 

 

 

 

77

Class GO Ontolongenome in class E-score GO Term
1 Cellular 18/2762? 3/101 1 .12E-03 vacuole (sensu Streptophyta)
Component 36/27627 3/101 9.31 E-03 vacuole
15/2762? 2/101 4.04E-02 vacuolar membrane (sensu
Streptophyta)
Molecular 35/27627 5/101 2.43E-05 water channel activity
Function 3/27627 2/101 5.4?E-03 phosphoethanolamine N-
methyltransferase activity
141/27627 5/101 2.38E-02 oxidoreductase activity
154/27627 5/101 3.58E-02 alpha-type channel activity
7/2762? 2/101 3.79E-02 squalene monooxygenase activity
158/27627 5/101 4.03E-02 channel/pore class transporter
activity
8/2762? 2/101 5.04E-02 hydrolase activity\acting on acid
halide bonds\in C-halide
compounds
2 Cellular 15445/27627 79/111 1.94E-02 cell
Component 6642/2762? 42/111 2.35E-02 membrane
3 Biological 63/2762? 12/233 6.05E-11 response to cold
Process 66/2762? 11/233 2.94E-09 response to water
59/2762? 10/233 2.23E-08 response to water deprivation
109/2762? 12/233 5.05E-08 response to temperature
11/2762? 5/233 6.16E-06 cold acclimation
590/27627 21/233 1.19E-05 response to abiotic stimulus
64/2762? 8/233 2.27E-05 response to abscisic acid
stimulus
39/2762? 6/233 2.9?E-04 salinity response
930/2762? 24/233 4.48E-04 response to external stimulus
889/27627 23/233 7.23E-04 response to stress
54/2762? 6/233 2.11E-03 response to osmotic stress
22/2762? 4/233 1.09E-02 hyperosmotic salinity response
23/27627 4/233 1.31 E-02 hygerosmotic response
Cellular 80/2762? 5/233 3.11E-02 heterotrimeric G-protein complex
Component 82/2762? 5/233 3.49E-02 extrinsic to plasma membrane
86/2762? 5/233 4.33E-02 extrinsic to membrane
4 Biological 104/2762? 4/13 7.00E-06 lipid transport
Process
Cellular 4520/2762? 8/13 3.61 E-03 endomembrane system
Component
Molecular 143/2762? 4/13 1.09E-05 lipid binding
Function 55/2762? 2/13 6.88E-03 structural constituent of cell wall
144/2762? 2/13 4.66E-02 rhodopsin-Iike receptor activitL
5 Biological 1412/2762? 9/32 2.28E-03 regulation of transcription\DNA-
Process dependent
1437/2762? 9/32 2.62E-03 transcription\DNA-dependent
2303/2762? 11/32 3.33E-03 regulation of biological process
1957/2762? 10/32 4.82E-03 regulation of transcription
1967/2762? 10/32 5.03E-03 regulation of nucleobase

\nucleoside \nucleotide and
nucleic acid metabolism

 

 

 

2032/2762? 10/32 6.63E-03 regulation of physiological
process
2032/2762? 10/32 6.63E-03 regulation of metabolism
2035/2762? 10/32 6.?1 E-03 transcripiion
Molecular 1847/27627 12/32 1.92E—05 transcription regulator activity
Function 1734/2762? 11/32 8.49E-05 transcription factor activity
293/2762? 4/32 1.38E-02 calcium ion binding
3025/2762? 11/32 1.50E-02 DNA bindigg
6 Biological 63/2762? 2/8 4.83E-03 response to cold
Process 109/27627 2/8 1.45E-02 response to temperature
1957/2762? 4/8 4.?4E-02 regulation of transcription
1967/2762? 4/8 4.83E-02 regulation of
nucleobase\nucleoside\nucleotide
and nucleic acid metabolism
Molecular 1734/27627 5/8 9.70 E—04 transcription factor activity
Function 1847/27627 5/8 1.32E-03 transcription regulator activity
42/2762? 2/8 1.32E-03 transcriptional activator activity
3025/2762? 5/8 1.39E-02 DNA binding

 

The 514 COS transcripts were binned into their respective expression classes (see
Chapter 2, Appendices E and F) and classified into gene ontology (GO) categories using
the functional categorization tool at TAIR (http://www.arabidopsis.org/tools/bulk/gol).
The categories represented among the various classes of COS transcripts were then
compared to those for all genes in the Arabidopsis genome using GeneMerge.
Overrepresented terms among the classes of COS genes as compared to the rest of the
genome (E S 0.05) are shown. Each term is associated with an organizing principle
(biological process, cellular component, or molecular function) listed under “GO
ontology” in the table.

78

 

Cluster l 101 probe sets

20%

41%

    

1% 7%
71-41% 4%

Cluster ll 111 probe sets
I

 

    

°/.
4% 5% 47.5%

 

 

 

 

 

Cluster III 233 probe sets Cluster IV 13 probe sets
1 -’\~—_-~ a if,
i / c ' j _ f ,, .2 ,
1 T H/ 3”— “! J
3 i
31%
54%
15%

Cluster V Cluster VI 8 probe sets

a .

’ A ~ \ i /"—E a I
, 7’ x «x < l,/ ‘— a
.z’ a I
1

 

 

 

 

 

Cluster VII 16 probe sets
I ., -
:1
3

25% 32%

8%
6%
25% 6%

 

gay
Metabolism

I Transcription

I RNA processing & translation
I Signaling

I Energy

I Transport

I] Stress related

I Cellular biogenesis

El Cellular organization

I DNA replication 8- cell division
. Protein processing 8. fate

I Unknom

 

79

 

Figure 3.2. Functional categories represented in each expression class of the COS
transcripts.

The 514 COS transcripts were analyzed with hierarchical clustering which allowed
visualization of transcripts with expression profiles (see Chapter 2). Each box shows the
expression class, the number of transcripts in that class, and a pie chart summarizing the
functional categories present. The average expression profile for all transcripts in a
particular cluster is shown in each graph (log of ratio values versus time). Each of the
COS transcripts in an expression profile were placed into an exclusive functional
category based on annotations available at TAIR, TIGR, MIPS, and in published
literature (see Materials and Methods). Images in this figure are presented in color.

The transcripts that were rapidly up-regulated in response to low temperature
(clusters V-VII) were highly enriched in genes with roles in transcription; 50% of the
transcripts in these three clusters encoded proteins involved in the production of mRNA
(Figure 3.2, Table 3.2). Cluster VII contained many transcription factors, but unlike
clusters V and VI, analysis with GeneMerge did not find enrichment in any given
category in this cluster (possibly due to the small sample size for this group of genes). In
addition to the enrichment for transcription factors in clusters V and VI, transcripts with
potential roles in signaling (e.g. calcium ion binding) were also enriched.

The classes of transcripts present at later times during the cold response were
more diverse. Cluster III contained the most transcripts overall and the most functional
categories of overrepresented transcripts (Figure 3.2, Table 3.2). This group of genes,
which peaked in expression afler 24 h, contained enrichment for stress-related transcripts,
five transcripts annotated as components of heterotrimeric G-protein complexes, and
transcripts that were membrane associated. Cluster III contained all of the previously
described COR genes except for COR28 (Fowler and Thomashow, 2002) and COR35

(this paper, described in a later section). Class III also contained all of the cold-induced

8O

transcripts for enzymes involved in the production of known cryoprotectants (ex: sucrose,
proline, and raffinose). All of this is consistent with the idea that the transcripts in this
class function in protecting the plant from freezing stress.

Cluster IV contained transcripts whose accumulation only increased with time and
was enriched for lipid transport and lipid binding proteins (Figure 3.2, Table 3.2). Four
protease inhibitor/seed storage/lipid transfer protein (LTP) family members were in this
cluster. Given that these transcripts increase with time, one might expect the gene
products perform important protective roles during cold acclimation. One attractive
hypothesis for their function would be that of membrane stabilization. Overexpression of
pEARLIl, an LTP family member, does not increase whole plant freezing tolerance, but
does protect cells from freezing damage as measured by a decrease in electrolyte leakage
in the range of -2 to -4°C (Bubier and Schlappi, 2004). Recent work on cryoprotectin, a
lipid-transfer protein family member isolated from cold-acclimated cabbage (Brassica
oleracea), revealed that it stabilized thylakoid membranes during freezing (Hincha et al.,
2001; Hincha, 2002). It is possible that the pEARLI-like proteins could be playing a
similar role in Arabidopsis.

Cluster IV contained other transcripts with potential roles in cold acclimation,
including a thaumatin-like transcript, polygalacturonase inhibitors, a LEA, and a
pectinesterase (Figure 3.2, Table 3.2). The accumulation of these transcripts increases
with time. One hypothesis concerning the function of these transcripts is that they
encode antifreeze proteins. A number of studies have identified potential antifreeze
proteins from different plant species that contain similarity to B-l ,3-glucanases,

thaumatin like proteins, polygalacturonase inhibitor proteins, and endochitinases (Hon et

81

al., 1995; Hincha et al., 1997; Worrall et al., 1998; Meyer et al., 1999; Smallwood et al.,
1999; Yeh et al., 2000). Cold up-regulated transcripts in cluster IV and in other clusters
contained thaumatin like proteins (Atl g20030, cluster VII; At1g75040, cluster IV), a
polygalacturonase inhibiting protein (At5g06860, cluster IV), an endochitinase
(At2g43620, cluster III), and B-1,3-glucanases (At1g32860, At3g04010, At5g55180,
cluster III).

A number of categories of transcripts were enriched among the down-regulated
genes (clusters I and 11, Figure 3.2, Table 3.2) inclduing products located in the vacuole,
water channels, oxidoreductases, peptide transporters, carbonate dehyrdratases, and
phosphoethanolamine N-methyltransferesases. One hypothesis for why transcripts are
down-regulated at low temperature would be that they are simply not needed during cold
acclimation. Another hypothesis would be that they are inherently problematic for the
plant at low temperatures and need to be down-regulated.

Transcripts for five water channels (At2g36830, At2g37180, At3g16240,
At4g17340, and At4g23400) were down-regulated in the cold and enriched in cluster 1.
During freezing stress, intracellular ice formation results in a water gradient and water
will move down that chemical gradient and out of the cell (Thomashow, 1999).
Aquaporins are thought to allow water flow in either direction, down its potential
gradient (Maurel, 1997). Down-regulation of these water channels may help regulate the
cell’s osmotic homeostasis.

Among the down-regulated transcripts, oxidoreductases (involved in a variety of
metabolic pathways), and carbonate dehydratases (involved in photosynthetic carbon

assimilation) were identified as overrepresented. Plants need to adjust their energy

82

production and metabolism to cope with low temperature. This is consistent with the
number of metabolites that change in response to low temperature, including those
involved in the TCA cycle (Stitt and Hurry, 2002; Cook et al., 2004). The decrease in
energy related transcripts mainly concerned transcripts encoding proteins involved in
photosynthesis. This has been observed in previous microarray experiments (Fowler and
Thomashow, 2002; Kreps et al., 2002; Seki et al., 2002) and elsewhere (reviewed in
Huner et al., 1998). Such a decrease may be a result of the lower light levels used during
the cold treatment, but other studies have shown that exposure to low temperature leads
to inhibition of photosynthesis and a concurrent decrease in transcript levels for
photosynthetic components (Krapp and Stitt, 1995; Strand et al., 1997).

Also enriched among the down-regulated transcripts were peptide/oligopeptide
transporters and phosphoethanolamine N—methyltransferesases. Peptide transporters
could be involved in either signaling or transporting small peptides that are to be
metabolized and used as a source of amino acids, nitrogen, or carbon (Koh et al., 2002).
Phosphoethanolamine N-methyltransferase is the key step in the production of
phosphocholine and then choline, which is the precursor of the osmoprotectant glycine
betaine (McNeil et al., 2001). Since the transcripts for this enzyme decreased, it seems
unlikely they are involved in producing higher levels of glycine betaine. Rather, the
enzyme may be involved in changing the phosphocholine pool available for producing
lipids, such as phosphotidylcholine, that are then incorporated into cellular membranes
(Lynch and Steponkus, 1987). Changes such as these may be important for signaling or

altering the membrane lipid composition.

83

Given the enrichment for stress-related transcripts in clusters III and IV, and the
number of transcription factors that are up—regulated early in the cold response, the
picture emerges that those changes that occur later in the cold response are involved in
protecting the plant from possible future freezing stress or coping with life at low
temperature, while those that occur early are involved in signaling or gene regulation to
bring about those changes. Since transcriptome profiling only measures steady state
transcript levels, one does not know which transcripts are regulated at the transcriptional
level, post-transcriptional level, or both. However, the above analysis supports the idea
that the initial increase in transcriptional regulators influences downstream changes in
gene expression. The idea that changes in gene expression that occur early in the cold
response impact the expression of later changes in gene expression is not new (Gilmour
et al., 1998), but this analysis demonstrates the cold transcriptome behaves in a similar

manner at the whole-genome level.

The Early Cold-Induced Transcription Factors CBF 2 and ZAT12 Control Regulons
Enriched in Stress-Related Transcripts, Including Novel COR-Like Genes

Examination of the regulons of cold-inducible transcription factors, such as
CBF], 2, and 3 and ZAT12, that are up-regulated quickly in response to low temperature,
further strengthens the idea that early changes in the transcriptome influence later
changes during cold acclimation. The CBF2 and ZAT12 regulons have been shown to
regulate 94 and 24 COS transcripts, respectively (Chapter 2) and most of the cold-
responsive genes that CBF 2 and ZAT12 affect are cold-responsive at later times in the

cold (24 h or 7 d). A more thorough understanding of the functions of downstream genes

84

that are controlled by CBF2 and ZAT12 could provide insight into how each regulon
alters freezing tolerance.

The CBF 2 regulon was determined by profiling changes in plants constitutively
expressing CBF 2 and comparing these genes to the COS transcripts (see Chapter 2 for
details). The functional roles of the CBF regulon were diverse (including cyroprotectant
production, lipid and cell wall modification, and transcription), with many of the up-
regulated transcripts involved in stress responses and the down-regulated transcripts
enriched for transcripts annotated with oxygen binding activity or membrane localization
(Figure 3.3a and Table 3.3). Interestingly, 91% of the up-regulated transcripts fell into
cluster III, reinforcing the idea that the early cold-responsive CBF transcription factors
play important roles in regulating the expression of genes that are induced at later times.
Since the functional roles of CBF 2 and cold up-regulated transcripts revealed significant
enrichment for transcripts encoding stress-related proteins (Table 3.3) which fall into
cluster 111, this again strengthens the idea that changes occurring later in the cold are
responsible for increased freezing tolerance. Additionally, at least 26% of the up-
regulated transcripts had unknown roles.

The unknown transcripts in the CBF 2 regulon were examined to determine if any
could be novel COR genes (transcripts encoding small, highly hydrophilic polypeptides).
A search of the unclassified highly up-regulated transcripts revealed four new COR/LEA-
like proteins based on their highly hydrophilic character and were designated COR35
(Atlg11210), COR17 (At1g16850), COR33.5 (Atlg80130), and COR42 (At4g30830)

(Figure 3.4). COR35 and COR17 both possess predicted target peptides based on

85

(a)
CBF2 and cold uo—reoulated CBF2 and cold down-reaulated

   
   

37%

 

13%

30% 2%
85 probe sets 8 probe sets

6))
ZAT12 and cold uo-reoulated ZAT12 and cold down-reoulated

11%

,/

 

m 13%
9 probe sets 15 probe sets

 

 

D Metabolism ITranscription I RNA processing

I Signaling I Energy & translation

I Transport E] Stress-related ICellular biogenesis

D Cellular I DNA replication I Protein processing
organization 8. cell division & fae

I Unknown

 

 

Figure 3.3. Functional categories of the CBF2 and ZAT12 regulons.

Members of the CBF2 and ZAT12 regulons were was placed into an exclusive functional
category based on annotations available at TAIR, TIGR, MIPS, and in published
literature (see Materials and Methods). Images in this figure are presented in color.

86

Table 3.3. Overrepresented GO terms in the CBF2 regulon

 

 

 

 

 

Frequency Frequency
Class GO Ontology in genome in class E-score GO Term
CBF2 & Biological 63/27627 11/85 1.01 E-14 response to cold
cold up Process 109/27627 11/85 5.64E-12 response to temperature
66/27627 9/85 8.05E-11 response to water
59/27627 8/85 1.76E-09 response to water
depnvafion
590/27627 16/85 4.85E-09 response to abiotic
stimulus
11/27627 5/85 1.75E-08 cold acclimation
889/27627 18/85 2.88E-08 response to stress
930/27627 18/85 5.93E-08 response to external
stimulus
64/27627 7/85 1.72E-07 response to abscisic acid
stimulus
39/27627 5/85 2.045-05 salinity response
22/27627 4/85 9.195-05 hyperosmotic salinity
response
54/27627 5/85 1.08E-04 response to osmotic
stress
23/27627 4/85 1.11E-04 hyperosmotic response
1841/27627 19/85 3.90E-04 response to stimulus
3127627 2/85 4.4OE-03 cellular response to water
depnvafion
344/27627 7/85 1.47E-02 response to hormone
stimulus
Cellular 6642/27627 33/85 4.26E-02 membrane
Component
CBF2 & Cellular 6642/27627 6/8 1.70E-02 membrane
cold down Component
Molecular 229/27627 2/8 4.45E-02 oxygen binding
Funcfion

 

CBF2 regulon members (Appendices G and H) were classified into gene ontology (GO)
categories using the functional categorization tool at TAIR
(http://www.arabidopsis.org/tools/bulk/go/). Overrepresented terms among transcripts in
the CBF2 regulon as compared to the rest of the genome (E S 0.05) are shown. Each
term is associated with an organizing principle (biological process, cellular component,

or molecular function) listed under “GO ontology” in the table.

87

I I I I I I I ‘1 I ff I I I
80 100 120 140 160 180 200 220 240 260 280 300

' 2'0 ‘ 1
“-5 At1gt 1210
o (COR35)
-45

I I I I I I I I I I I
40 50 60 70 80 90 100 110 120 130 140 150

I
I T f I
10
4 5 1 A11916850
o (COR17)
-45

I I I I I r I I I I I I I I
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300

4.5 At1980130
0 (0012335)
-45

I I I I I I T I I I I T I I I I I II
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360

 

 

 

 

 

“5 At4930830
o (COR42)
-45
[’— "F‘_['I'__—“TT“ ”T‘“ I I I fi T l I 'T’T‘TW—j
5 10 20 30 4O 50 60 70 80 90 100 110 120 130
4
COR15a

-45

Figure 3.4. Hydropathy plots for novel COR-like peptides in the CBF2 regulon.
The hydropathic character of the COR-like proteins was analyzed using the methods of
Kyte and Doolittle (1982). The x-axis of each plot represents regions with hydrophilic or
hydrophobic character; values > 0 represent hydrophilic regions, while values < O are
hydrophobic. The length of the protein (number of amino acids) is indicated along the
top of each plot. TargetP (http://www.cbs.dtu.dk/services/TargetP/) was used to deduce
potential signal peptides and the predicted cleavage sites are indicated by arrows.
COR15a is shown for comparison.

sequence analysis using TargetP (Nielsen et al., 1997; Emanuelsson et al., 2000),
suggesting they are targeted to cellular organelles. Of these four, COR] 7 was the most
highly up-regulated (>375 fold) by CBF2 expression and contained 3 CRT/DRE elements
within 500 bp of the 5’ UTR, likely making it a direct target of the CBFs. COR17
contained a group 3 LEA domain (Dure et al., 1989) and the mature peptide was rich in
Ser, Val, Thr, Ala, Glu, and Lys (51% of the amino acid residues). COR17 was also the
most highly up-regulated transcript at any time (>390-fold at 24 h) in the experiment

examining cold-responsive genes in plate grown plants. COR 1 7 lies on chromosome 1

88

next to another gene (Atl gl6840), also predicted to encode a highly hydrophilic peptide
possessing a targeting peptide (Atl g16840 was not cold-regulated). This type of
chromosomal arrangement is similar to that described for other COR gene pairs
(Thomashow et al., 1997). Given the high level of expression and predicted domains in
the protein, it seems likely that COR17 plays a protective role during freezing stress,
perhaps in a fashion similar to COR15a (Artus et al., 1996). Future experiments will be
needed to confirm this hypothesis.

The ZAT12 regulon was enriched for transcripts annotated with the biological
processes of lipid transport and binding, transport, and cell growth (Figure 3.3b and Table
3.4). Among the down-regulated transcripts, there was no enrichment for any biological
processes or molecular function, but further examination showed that six transcripts
encoded proteins of unknown function and five encoded proteins potentially involved in
metabolism. The other four transcripts were classified as signaling, transport, and stress.

ZA T12 overexpression results in altered freezing tolerance, but to a much lesser
degree than overexpression of CBF. The mechanism for this change in freezing tolerance
is not known, but the putative functions of the proteins encoded by transcripts altered by
ZAT12 reveal some possibilities. I previously speculated on the role of two transcripts,
L-ascorbate oxidase (At5g21100) and arginine decarboxylase (At4g34710), in protecting
the plant from damage during cold stress (Chapter 2). Additional transcripts influenced
by ZAT12 have stress-related roles. ZAT12 up-regulated five members of the protease
inhibitor/seed storage/ lipid transfer protein family, which may stabilize or remodel
membranes, as hypothesized by Wilkosz and Schlappi (2000). Transcripts for potential

antifreeze proteins (At2g43620 and At5g06860) were also up-regulated by ZAT12.

89

Table 3.4. Overrepresented GO terms in the ZAT12 regulon

 

 

 

 

 

Frequency in Frequency
Class GO Ontology genome in class E-score GO Term
ZAT12 & Cellular 4520/27627 8/9 4.32E-05 endomembrane
cold up Component system
6642/27627 8/9 8.66E-04 membrane
Biological 104/27627 5/9 2.31 E-09 lipid transport
Process 4687/2762? 7/9 2.84E-03 cellular process
3237/27627 6/9 4.26E-03 cell growth and/or
maintenance
1987/27627 5/9 5.10E-03 transport
3592/27627 6/9 7.67E-03 cellular physiological
Jrocess
Molecular 143/27627 5/9 1.12E-08 lipid binding
Function 1 15/27627 2/9 1 .58E-02 nutrient reservoir
activity
ZAT12 & Cellular 6642/27627 10/15 3.96E-03 membrane
cold down Component 4520/27627 7/15 4.15E-02 endomembrane
system

 

ZAT12 regulon members (Appendices G and H) were classified into gene ontology (GO)
categories using the functional categorization tool at TAIR
(http://www.arabidopsis.org/tools/bulk/go/). Overrepresented terms among transcripts in
the ZAT12 regulon as compared to the rest of the genome (E S 0.05) are shown. Each
term is associated with an organizing principle (biological process, cellular component,
or molecular function) listed under “GO ontology” in the table.

Transcripts for proteins likely involved in protecting the plant from oxidative stress were
up-regulated, including a putative anthocyanidin synthase (At4g22870) and a
dihydroflavanol 4-reductase (At5g42800), both of which are involved in anthocyanin

production, but neither is a COS transcript.

The Co-Regulation of Transcripts by Multiple Transcription Factors Indicates Many of

the Early Cold-Responsive Transcription Factors May Co-Regulate Cold-Responsive

Transcripts

9O

The co-regulation of transcripts by CBF2 and ZAT12 (Chapter 2) is of interest, as
it reveals the complexity of the networks of gene expression that occur at low
temperature. The pEARLI transcripts are a case in point. These proteins of unknown
function are members of the protease inhibitor/seed storage/lipid transfer protein family
and were first identified as transcripts responding to toxic levels of aluminum (Richards
and Gardner, 1995) and, more recently, vernalization (Wilkosz and Schlappi, 2000). Of
the five pEARLI-like genes identified as COS genes, four occur in tandem on
chromosome 4. Interestingly, all of the cold-regulated transcripts belonging to this
family were placed into either the CBF 2 or ZAT12 regulons. In addition to being
affected by expression of CBF2 or ZAT12 (Figure 2.8, Chapter 2), the transcription
factors STZ/ZA T10 and CZF2 (At5g04340) also altered the expression of one or more of
the pEARLIs (J .T. Vogel, unpublished data). Even though STZ/ZA T10 and CZF 2 did not
contribute significantly to the cold transcriptome (Chapter 2), it seems unlikely that these
cold inducible transcription factors would influence pEARLI expression by chance. A
question that arises is why such redundancy would be needed. These proteins could be
playing a critical role in freezing tolerance, vernalization, or life at low temperature.
Whatever their role, the identification of cold-regulated genes that appear to be
influenced by multiple transcription factors reveals the complexity of signaling events at

low temperature and may denote factors with critical roles in cold acclimation.

CONCLUSION

The Arabidopsis cold transcriptome is composed of transcripts encoding myriad

functions, as one would expect given the number of physiological changes that occur

91

during cold acclimation. As the work here demonstrates, those transcripts induced
quickly in the cold are enriched for signaling molecules and transcription factors, which
presumably influence downstream changes in gene expression and, ultimately, freezing
tolerance. Examination of the CBF 2 and ZAT12 regulons also supports this model. The
CBF cold response pathway regulates genes with altered accumulation at later times in
the cold, but that picture has been expanded upon here. The CBF regulon is enriched in
stress-related transcripts, the majority of whcih fall into cluster III. The ZAT12 regulon
is also composed of transcripts that are highly up-regulated later in the cold response (24
h or 7 d). The co-regulation of a number of transcripts by multiple transcription factors
only adds to the apparent complexity of cold signaling pathways.

The analysis presented in this work generates new hypotheses that can be tested
experimentally. The need exists to continue studying the functions of both the
downstream genes in order to assess their roles in cold acclimation and the upstream
factors that regulate them. This will include biochemical and physiological studies of
gene products such as the COR peptides and molecular genetic studies on the
transcription factors and cis-elements responsible for cold-regulated gene expression.
This will result in a greater understanding of Arabidopsis low temperature regulatory

networks and how plants increase their freezing tolerance during cold acclimation.

92

AKNOWLEDGEMENTS

In the completion of the work in this chapter, I would like to thank our
collaborator Dr. Seung Yon Rhee at TAIR for helpful suggestions and analyzing the data
with the GeneMerge program. Thanks also to Dr. Sarah Fowler for critical reading of

this chapter.

93

MATERIALS AND METHODS
Functional Classification of Transcripts

COS probe sets were assigned AGI locus identities derived from TAIR’s
annotation of the ATHl GeneChip using the TIGR v 5.0 annotation of the Arabidopsis
genome. COS genes were placed into functional categories manually using annotation
available at TAIR (The Arabidopsis Information Resource, www.arabidopsis.org), TIGR
(The Institute for Genomic Research, www.tigr.org), and MIPS (Munich Information
Center for Protein Sequences, http://mips.gsf.de). The transcripts were then placed into
sub-roles based primarily on this annotation and on published literature. A full
description of the microarray datasets and tables of the COS genes can be found in
Chapter 2 and Appendices E and F. The COS genes were also automatically classified
into gene ontology (GO) functional categories using the fimctional categorization tool
available at TAIR (http://www.arabidopsis.org/tools/bulk/gol). GeneMerge was then
used to find categories of transcripts statistically (E—score S 0.05) overrepresented in a
given class compared to the rest of the genome as described by Castillo-Davis and Hart]

(2003)

94

CHAPTER FOUR

THE IDENTIFICATION OF SIX NOVEL MOTIFS IN THE PROMOTERS OF COLD-

REGULATED GENES AND THE DETERMINATION OF THEIR COLD-

RESPONSIVENESS
SUMMARY
The definition of the COS (991d standards) gene set (Chapter 2), transcripts that

change in plants grown in either soil or solid media in response to low temperature,
provides the framework with which the low temperature regulatory networks of
Arabidopsis can be constructed. Understanding Arabidopsis low temperature regulatory
networks will lead to a greater understanding of how plants cold acclimate and may
ultimately result in genetic methods of increasing the freezing tolerance of agricultural
crops. One component of these regulatory networks comprises the cis-elements present
in the promoters of cold-responsive genes. Studies on the COS gene set has led to the
identification of several motifs in the promoters of genes with similar expression profiles
(Chapter 2). These motifs could be binding sites for transcription factors that regulate
transcription in a cold dependent manner. The studies presented here aimed to identify
additional motifs present in the promoters of the COS genes and to determine their cold-
responsiveness. To this end, two new novel motifs were identified in the promoters of
COS genes with the highest expression after 24 h at low temperature and that were
outside of the CBF regulon. These two novel motifs, along with four motifs previously
found to be overrepresented in the promoters of COS genes (Chapter 2), were each fused
as a tetramer in front of the GUS reporter gene, transformed into Arabidopsis, and
assayed for the activity of the reporter. None of the six motifs were found to confer cold-

responsiveness to the reporter. However, fusion of a tetramer of the sequence

95

GTGATCAC in front of the GUS reporter gene resulted in GUS activity in both warm

and cold treated plants.

96

INTRODUCTION

Cold acclimation is the process by which plants increase their freezing tolerance
through exposure to low non-freezing temperatures (Thomashow, 1999; Smallwood and
Bowles, 2002). Understanding how plants cold acclimate not only furthers science, but
may ultimately lead to novel methods of engineering increased freezing tolerance in crop
species. Such an understanding includes deciphering the low temperature transcriptome,
those genes whose expression is altered in response to low temperature.

In order to more fully understand the gene networks involved in regulating the
low temperature transcriptome, it is necessary to identify transcription factors that
regulate cold-responsive transcripts and identify the cis-elements they bind. Given that
the Arabidopsis genome encodes more than 1,700 transcription factors (Riechmann,
2002), the question becomes which regulate cold-responsive transcripts. One approach is
to constitutively express transcription factors whose mRNA levels accumulate in
response to low temperature and determine if the expression of any cold-responsive
transcripts is altered. This approach led to limited success, as one out of five
transcription factors whose mRNA accumulated in response to low temperature was
found to influence a significant number of cold-responsive transcripts (Chapter 2). This
approach is limited to only those transcription factors whose transcripts increase in the
cold and that can function independently of other cold-responsive factors and/or
modifications. Alternatively, a mutational approach can be taken. Studies using the
RD29a/C OR 78 or CBF 3 promoter fused to luciferase have identified transcription factors
such as H059 and ICE 1, whose expression is not altered by low temperatures, which

influence cold-responsive genes (Chinnusamy et al., 2003; Zhu et al., 2004). This

97

mutational approach has the drawback of taking longer to isolate, characterize, and clone
the mutated gene which may or may not be involved in cold-responsive transcription.
Additional approaches are needed in order to define novel cold response pathways in
Arabidopsis.

A more direct approach to define novel pathways is to first determine the cold-
responsive elements present in the promoters of cold-regulated genes and then isolate the
factor(s) that bind them. Such an approach was successfully used to identify the
CRT/DRE element in the promoters of certain cold-responsive genes (Baker et al., 1994;
Yamaguchi-Shinozaki and Shinozaki, 1994) and the CBF/DREBl transcription factors
that bind to this element (Stockinger et al., 1997). Constitutive expression of CBF], 2, or
3 in plants leads to activation of genes containing the CRT/DRE element in their
promoter and increases freezing tolerance (Jaglo-Ottosen et al., 1998; Gilmour et al.,
2004). The success of these experiments may have lain, in part, with the gene used in the
promoter-deletion experiments (COR15a), which has a high level of expression in
response to low temperature.

Current work suggests that had global gene expression data and computational
approaches been available when experiments studying the COR15a promoter began, the
CRT/DRE element would have quickly been identified as it has recently (Kreps et al.,
2003; Vogel et al., 2005). Fusion of the CRT/DRE motif as a tetramer in front of a
reporter gene such as GUS or LUC would have resulted in the reporter’s expression after
cold treatment. This computational approach would have saved time and effort put into

creating multiple promoter-reporter deletion constructs in stably transformed transgenic

98

plants. In fact, this exact type of bioinformatic approach was used to rapidly identify the
circadian responsive evening element in Arabidopsis (Harmer et al., 2000).

Here, I apply the above approach to the study of cold-regulated gene expression to
identify non-CBF cold response pathways. Transcriptome profiling experiments
examining transcripts with altered accumulation after 1 h, 24 h, or 7 d at low temperature
in two growth conditions (soil or solid media, 2.5-fold cutoff, p<0.05), resulted in the
identification of 514 transcripts changing in both culture conditions, defined as COS
(c_qld standard) transcripts (Chapter 2). Hierarchical clustering revealed seven expression
classes among the COS genes. Additionally, bioinformatics analysis revealed DNA
motifs that were overrepresented in the promoters of each cluster of up-regulated genes.
Elements identified in the promoters of genes whose transcripts increased the most after
24 h or 7 d of exposure to low temperature should be good candidates for testing the
motifs identified, as these motifs might confer a high level of cold-responsiveness to a
reporter gene. Among those genes that increased to the highest levels after 24 h at low
temperature, clusters III and IV, the CRT/DRE motif was identified in cluster III and four
novel motifs were found in cluster IV.

In this study, cluster III promoters were found to contain the evening element,
ABREs, and two novel motifs. The cold-responsiveness of these novel motifs, along
with the motifs previously identified in cluster IV, was tested by fusing a tetramer of each
motif in front of the GUS reporter gene and monitoring for cold induction of the reporter.
When placed as a tetramer in front of GUS, none of the motifs conferred cold-responsive
GUS activity. One element, GTGATCAC, resulted in constitutive activation of the

reporter gene. Future work that could reveal the function of these elements is discussed.

99

RESULTS
The Circadian Evening Element and the ABRE are Present in the Promoters of the Most
Highly Cold-Induced Transcripts Outside of the CBF Regulon

In an effort to decipher Arabidopsis low temperature regulatory networks, the
promoters of the 25 transcripts with the highest average fold change after 24 h of cold
treatment that were not CBF regulon members were examined. Should a cold-responsive
element be present in this group of genes, one might expect such an element to be easier
to discover if the high expression level is due to multiple copies of that element. The 500
bp upstream regions of these transcripts was searched for overrepresented motifs using
MotifSampler (Thijs et al., 2002). MotifSampler is a statistical program based on Gibbs
sampling (Thijs et al., 2001; Thijs et al., 2002). Gibbs sampling is a stochastic variant of
the expectation-maximization (EM) method (reviewed in Rombauts et al., 2003). Due to
the stochastic nature of Gibbs sampling, a detected motif can be replaced by another with
a higher score. This results in different outputs from the algorithm each time it is run.
However, motifs that are more conserved and overrepresented in a group of sequences
will be retrieved more frequently over different runs. Statistically analyzing the output of
multiple runs allows one to find the highest scoring motifs. Here, the program was run
100 times and the results were then searched for the highest scoring motifs.

Two motifs, CACGTGM and AAATATCT, were identified that were statistically
overrepresented and approximately 2-fold enriched in the promoters of these genes
compared to the rest of the promoters for genes on the GeneChip. Each is a known cis-
element, CACGTGM is an ABRE (abscisic acid response element) (Guiltinan et al.,

1990) and AAATATCT is the evening element (Harmer et al., 2000). Both elements

100

have been previously identified in the promoters of cold-regulated genes (Baker et al.,
1994; Yamaguchi-Shinozaki and Shinozaki, 1994; Kreps et al., 2002; Kreps et al., 2003),
but this is the first report of their occurrence in the promoters of the most highly cold-
induced genes not part of the CBF regulon. MotifSampler previously found the
CRT/DRE element in the promoters of cold-regulated genes (Chapter 2). The program
has now successfully found two other cis-elements present in the promoters of cold-

_ responsive genes. This indicates that the program can find overrepresented motifs which

can play a role in gene expression.

Novel Potential cis-Elements are Revealed in Cluster III Promoters when CBF Regulon
Members are Removed

In order to find additional overrepresented motifs, another approach was taken to
analyze the promoters of cold-responsive genes. Cluster 111 represents a group of good
candidate genes for identifying cold-responsive cis-elements, as their expression is high
at 24 h and elements found in these promoters might impart high levels of cold-
responsiveness to a reporter gene. Since the CRT/DRE element was the only motif
identified as 2 S-fold enriched in cluster 111, these genes were further analyzed to
determine if the presence of the CRT/DRE could be masking additional motifs that were
not as greatly enriched. By removing all the CBF regulon members and genes containing
an (A/G)CCGAC within lkb upstream among the 233 cluster III genes, 96 remained.
Analysis of these 96 promoters (500 bp upstream) with MotifSampler uncovered one

known motif, WNGMCACGTG (ABRE), and one novel element, AGGCCCAWNA, that

101

were both approximately 2-fold enriched in cluster 111 (Table 4.1). Each motif was
present in over 20 of the promoters of cluster 111 genes.

Since removal of CRT/DRE and CBF regulon member genes from the analysis
allowed the identification of a new overrepresented motif, cluster HI promoters that
contained WNGMCACGTG or AGGCCCAWNA were removed and the remaining
promoters analyzed. Among these promoters, one novel motif, AAAACCCTA, was
discovered as overrepresented and 2-fold enriched (Table 4.1). Again, this motif was
present in over 20 promoters of cluster 111 genes.

A question arose as to whether these novel elements occurred in promoters with
CRT/DRE elements, by themselves, or in some combination. To address this question,
the promoter region queried was expanded to lkb upstream and cluster III transcripts
were examined for promoters with each of the motifs (Table 4.2). Besides examining
promoters with at least two motifs, Figure 4.1 depicts the overlap between cluster 111
genes with the CRT/DRE and either of the two novel elements. The results indicate that
no cluster III gene promoter possesses all four of the motifs. Promoters with the two
novel elements also contain a CRT/DRE element more ofien than each other or
WNGMCACGTG. Given the number of promoters containing one or more of these
different motifs, multiple transcription factors are likely acting on the promoters of

cluster 111 genes.

Some of the Base Pairs Flanking the Motifs in Cluster 1]] Appear to be Conserved

When MotifSampler searches for motifs among a group of sequences, the length

of the motif to be searched for must be pre-set. To determine whether any of the novel

102

Table 4.1. Overrepresented motifs in cluster III

 

 

500bp upstream 1000bp upstream
Motif PlantCARE Class (%) GeneChip (%) Class (%) GeneChip (%)
AGGCCCAWNA novel 21/233(9%) 1159l22746 (5%) 261233 (11%) 1483/22746(6.5%)
WNGMCACGTG ABRE 27/233 (11.6%) 88822746 (3.9%) 30I233 (13%) 1245722746 (5.5%)
AAAACCCTA novel 21/233 (9%) 10360274644.5%) 28l233 (12%) 1486/22746 (6.5%)

 

Overrepresented motifs discovered in the promoters of cluster III genes that do not have
CRT/DRE elements (lkb upstream) and are not CBF regulon members. Each motif was
queried to the PlantCARE database to determine if it was a known or novel element. The
numbers of promoters in cluster III or for promoters from genes on the ATHl GeneChip
that contain the element (in the 500 bp or 1000 bp upstream regions) are shown. ABRE:
abscisic acid responsive element.

Table 4.2. Number of promoters with each pair-wise motif combination

 

 

Element CRT/DRE WNGMCACGTG AGGC_——CCAWNA AAAAC_CC_TA
—CRT/DRE 124

WNGMCACGTG 18 30

AGGCCCAWNA 12 3 26

AAAACCCTA 8 1 5 28

 

This table depicts the number of promoters of the 233 cluster III (lkb upstream) genes
that contain each motif in any given pair-wise combination. To be identified in the
promoter of a gene, the element had to be present exactly as shown.

(AlGICCGAC

VAV
V

AAAACCCTA AGGCCCAWNA

Figure 4.1. Overlap of the two novel motifs and the CRT/DRE element in cluster III

promoters.
This figure depicts the number of promoters (lkb upstream sequence) of the 233 cluster

111 genes with the CRT/DRE element and the two novel motifs. To be identified in the
promoter of a gene, the element had to be present exactly as shown.

103

motifs identified might be part of a larger consensus, a custom PERL script was written
and used to extract the 4 bp flanking sequence around each motif present within 500 bp
of the start of transcription for cluster III genes. The conservation at each position in the
aligned sequences was visualized using WebLogo (Crooks et al., 2004), which can reveal
significant features of an alignment (Figure 4.2). The alignments revealed no conserved
base pairs flanking AGGCCCAWNA. The motif WNGMCACGTG had a conserved T/G
(indicated by the arrow in Figure 4.2) and AAAACCCTA had three positions outside of
the core motif that appear to be conserved. As a comparison, the flanking sequences of
CBF regulon members containing (A/G)CCGAC were analyzed in the same fashion,
revealing a previously identified conserved T (Maruyama et al., 2004; Vogel et al., 2005).
Since flanking base pairs might be important for the binding of a transcription factor, any

analysis of these motifs should include flanking sequence.

Cluster I V Contains Novel Potential cis-elements, Including Palindromic Sequences

Cluster IV represents a second cluster of good candidate genes for discovering
and testing promoter elements, as the expression of genes in this class continues to
increase the entire time the plants are at low temperature (7 d in this experiment). Work
presented in Chapter 2 identified four novel motifs among cluster IV genes, which are 1
listed in Table 4.3. Two of the sequences, TGTATACA and GTGATCAC, are

palindromes.

104

bitsN
bitsN

illliCCGACigii]
iiiilllilllilillilfii
illilliiiillllilii
lilAAAACCCTAiill

NO

bits

1

NO

1

bits
bitsN

A

bitsN

1

bitsN

1

0 0

, "_sCCGAC.iH_
tutu-
.. “Milli-

 

. iAAAACCCTh

Figure 4.2. WebLogos depicting conserved sequences flanking the CRT/DRE,
ABRE, and two novel motifs in cluster III genes.

Each element from all cluster III promoters (500 bp upstream) was extracted, along with
4bp of flanking sequence, and aligned. Weblogo (http://weblogo.berkeley.edu/) was used
to visualize these alignments. The left side of the figure depicts each alignment with a
uniform logo height, while the right side depicts the conservation of each position by
height (the height of the y-axis is the maximum entropy for DNA, log; 4 = 2 bits).
Residues that appear to be conserved outside of the core motifs are indicated by arrows.

Table 4.3. Overrepresented motifs in cluster IV

 

 

 

500bp upstream 1000bp upstream
Motif PlantCARE Class (%) GeneChip (%) Class (%) GeneChip (%)
CAATGAGG Novel 3/13(23%) 176/22746(0.8%) 3/13(23%) 379/22746 (1.6%)
GTGATCAC Novel 1/13(8%) 96/22746 (0.4%) 1/13(8%) 199/22746 (0.9%)
GnATTGAC Novel 5/13 (38%) 937/22746 (4.1%) 5/13 (38%) 1808/22746 (8%)
TGTATACA Novel 3/13 (23%) 327/22746 (1.4%) 4/13 (31%) 647/22746 (2.8%)

 

Overrepresented motifs discovered in the promoters of cluster IV genes. Each motif was
queried to the PlantCARE database to determine if it was a known or novel element. The
numbers of promoters in cluster IV or for promoters from genes on the ATHl GeneChip

that contain the element (in the 500 bp or 1000

105

bp upstream regions) are shown.

A T etramer of the Novel Motif GT GA TCAC Fused in Front of the GUS Reporter Gene
Results in GUS Activity in both Warm and Cold Temperatures

In order to determine whether any of the newly identified novel elements from
cluster III and those previously found in cluster IV (Table 4.3) were cold-responsive, a
tetramer of each motif was fiised in fi'ont of the fl-glucuronidase (GUS) reporter gene, and
transformed into Arabidopsis. Each tetramer contained 6 bp of flanking sequence, since
some of the motifs in cluster III possessed conserved residues outside of the identified
motif. The motifs, along with flanking sequence, were taken from the context of the
promoter of the cold-responsive gene in either cluster III or IV with the highest level of
cold-regulated expression (Appendix L). T] plants were assayed for expression of GUS
in response to low temperature by histochemical staining. After 48 h or 7 d at 4°C, none
of the lines stained blue for cold-responsive GUS activity (data not shown, >20 T1 plants
screened per condition). However, plants expressing the motif GTGATCAC fused in
front of GUS stained blue in both the warm and the cold (Figure 4.3). GUS activity was
seen with both plate and soil grown plants. These preliminary results indicate that this

motif is likely a cis-acting element involved in transcriptional activation.

cold

 

Figure 4.3. The motif GTGATCAC confers activity to a GUS reporter.

A tetramer of GTGATCAC confers activity to a GUS reporter in both warm (24°C)
grown and cold (7 d at 4°C) treated plants. Approximately 10 plants were screened in the
warm and in the cold. Bar = 5mm. Images in this figure are presented in color.

106

DISCUSSION

In an effort to identify non-CBF cold response pathways, the objective of this
study was to use a bioinformatics approach to discover novel potential cis-elements in the
promoters of highly cold-inducible genes. This goal was accomplished, as use of
MotifSampler on non—CBF regulon COS genes revealed the presence of two novel motifs
among cluster III promoters, those transcripts induced to the highest levels after 24 h at
low temperature. In addition to these two novel motifs, two known motifs, the ABRE
and the evening element, were found in the promoters of the 25 non-CBF regulon genes
with the highest fold-change at 24 h.

It was then hypothesized that these two novel elements and those identified
previously in cluster IV were cold-responsive elements. To address this question,
constructs were created where a tetramer of each motif along with flanking sequence was
fused in front of the GUS reporter gene. Plants containing these constructs were tested
for cold-inducible reporter activity. While the motif GTGATCAC conferred activity on
the GUS reporter gene, none of the other constructs tested displayed any GUS activity in
the warm or after 48 h at 4°C. This result could be due to a number of possibilities. One
explanation is that none of these elements play a role in cold-responsive gene expression.
They may instead respond to other stimuli (hormones, circadian rhythm, light, etc.) or
may not be involved at all in transcriptional regulation. Alternatively, these elements
may indeed play a role in the cold, but may need to work in combination with additional
elements to function properly. As evidence for this hypothesis, each novel element was
found in a number of promoters containing CRT/DRE elements. While none of the

elements were sufficient for cold-responsiveness, fiirther experiments would be needed to

107

test each of the above possibilities and to determine if any of the elements were necessary
for cold-responsiveness. Furthermore, the use of RT-PCR or RNA blot analysis might
reveal changes in GUS transcript accumulation which might not be reflected by the
intensity of the staining. An additional possibility is that the program used to discover
these novel motifs may have not worked optimally, resulting in the identification of
motifs that were not the most overrpresented. This seems unlikely since MotifSampler
found three motifs known to be present in the promoters of cold-responsive genes,
including the CRT/DRE.

Since the initial work done here to discover the motifs in clusters HI and IV, a
study assessing the ability of 13 motif discovery tools to find statistically overrepresented
motifs was published (Tompa et al., 2005). This study revealed a number of interesting
findings. No program worked very well on all the datasets or conditions tested and the
longer the binding site, the less likely a program was to find it. Based on their results, the
authors recommend that researchers pursue the top several motifs, not just the “best” one
and to use other data (such as chromatin IP, global expression studies, etc.) or multiple
programs when available. MotifSampler, the program used here, was found to perform
the best of all 13 programs on a dataset consisting of actual data. While the work
presented here used global expression studies and focused on the top several motifs,
future motif searches should be performed using a second program, such as Weeder
(Pavesi et al., 2004), to confirm the results of the first algorithm.

There is only one other published study that identified novel motifs in the
promoters of cold-responsive genes (Kreps et al., 2003). The study by Kreps et al. did

not use a statistical algorithm, but pattern enumeration. This method takes longer and

108

needs more computing power than statistical methods, but its advantage lies in that it
requires no statistical assumptions to be made. Kreps et al. (2003) searched the promoter
regions of cold-responsive transcripts identified in experiments using the 8K GeneChip
(Kreps et al., 2002) and found the CRT/DRE element and ABRE as the top scoring
motifs. As found in this study, their work also identified the evening element as
overrepresented among cold-responsive promoters. Kreps et al. (2003) speculate the
evening element could possess a dual role, functioning both in circadian rhythms and the
cold response. Additionally, Kreps et al. (2003) found numerous novel motifs, none of
which were the same as those analyzed in this paper. It will be interesting to see if future
experimental evidence points towards a role in cold-responsive gene expression for any
of the novel motifs they identified.

While the motif GTGATCAC did not confer cold-responsiveness to the GUS
reporter gene, fusion of this element in front of GUS resulted in GUS activity in both
warm and cold treated plants. The level of staining observed was similar to plants
expressing GUS under control of the CMV 35S promoter. This preliminary data indicates
that this motif likely constitutively stimulates transcription. These results need to be
confirmed in the T2 generation and by testing a mutated version of the element. This
element is present within 500 bp of the start of transcription for 1 14/27,186 Arabidopsis
genes. Only two of the COS genes have this element in their promoters (500 bp
upstream), the up-regulated gene At4g22470 and the down-regulated gene At2g33480.
While it may or may not play role in cold-responsive gene expression, this element could
be used in a yeast one-hybrid to identify the transcription factor which binds to it and

stimulates expression of the reporter gene.

109

While no study has yet identified a new cold-responsive cis-element through
bioinformatic means, this event is still a strong possibility. The use of additional
computer programs and more extensive global expression datasets could lead to their
discovery. Chromatin IP experiments could also be used to reveal promoters that contain
acetylated histones, revealing which genes are most likely to be transcriptionally active,
which would help to refine any bioinformatic analysis. Additionally, a number of motifs
were identified among the cold-responsive genes that have not yet been tested
experimentally (Chapter 2, Table 2.1). In the study presented here, one of the elements
discovered was constitutively active and this element could play a role in the regulation
of genes possessing the motif in its promoter. The computational discovery of motifs
among co-regulated genes holds the possibility of greatly reducing the time and effort
currently involved in uncovering cis-elements through traditional promoter bashing

experiments.

”0

MATERIALS AND METHODS
Constructs and Plant Transformation

Plasmids were constructed using standard molecular biological techniques
(Sambrook and Russell, 2001). Synthetic double stranded tetramers of each motif (along
with mutated versions of each motif) were created by annealing single stranded oligos
synthesized at the Michigan State University Macromolecular Synthesis Facility (E.
Lansing, MI) by heating at 94°C for 5 min and cooling to room temperature. Each
double stranded oligo had a 5’ EcoR I overhang, followed by an Xma I site, followed by
four copies of the motif taken from the context of a promoter containing that motif with 6
bp of sequence flanking either side, and a 3’ Xma I site that would be destroyed by
ligation into an Xma I site. Mutant versions of each motif retained the 6 bp flanking
sequence as was found in the context of the actual promoter. Oligos were then ligated
into the pBS SK' vector (Clontech, Palo Alto, CA) cut with EcoR I and Xma I. The oligo
was then cut from pBS SK‘ using Hind III and Xba I and ligated into the plant binary
transformation vector pBIlOl plus (Clontech, Palo Alto, CA) cut with the same enzymes.
The genes from which the motifs were taken and the sequence of each tetramer are listed
in Appendix L.

The DNAs were transferred to Arabidopsis thaliana (L.) Heynh. ecotype
Wassilewskija-2 (WS-2) via a whole plant dipping method similar to that described by
Clough and Bent (1998). Seed germination on medium containing kanamycin (50 mg/L)
(Sigma-Aldrich, St. Louis, MO) was used to identify plants containing transferred DNA.

Kanamycin resisitant seedlings were grown and T1 plants were used in all experiments.

111

Plant Growth and Experimental Treatments

All Arabidopsis plants were grown in controlled environmental chambers at 22°C
under constant illumination from cool—white fluorescent lights (100 umol m"2 s'l) in
Baccto planting mix (Michigan Peat, Houston). Pots were subirrigated with deionized
water. After four days stratification at 4°C, plants were also grown for 10-12 days in
Petri plates containing Gamborg’s BS nutrients (Caisson Laboratories, Inc., Rexburg, ID)
and 0.8% phytagar (Life Technologies Inc., Gaithersburg, MD) at 22°C under constant
illumination from cool-white fluorescent lights (approximately 100 umol m'2 s"). The
plates of plants were then transferred to 4°C under constant illumination from cool-white

fluorescent lights (approximately 30 umol m'2 s") and harvested at the indicated times.

Staining for GUS Activity

Staining of plant tissue for GUS activity was performed on plants grown at 22°C
and those transferred to 4°C for 24 h, 48 h, or 7 (1. Plants were immersed in a GUS
staining solution (Jefferson et al., 1987) and incubated overnight at 37°C. The tissue was

then cleared by several rinses of 70% (v/v) ethanol to assist in revealing the staining.

Motif Analysis

Motif searches were performed using the command line version of MotifSampler
v3.0 (http://www.esat.kuleuven.ac.be/~thijs/Work/Motif‘Samplenhtml). Motifs were
identified from various sub-groups of the 500 bp locus upstream sequence (TIGR v5.0

annotation of the Arabidopsis genome) downloaded from TAIR

112

(http://www.arabidopsis.org) on February 28, 2004. Arabidopsis intergenic regions were
used as a background model with the order set to 3. The motif length was set to 8 or
10bp, with the prior probability of finding 1 motif instance set to 0.3. No limit was set
for the number of motif instances that could be found per sequence, the maximum
overlap between different motifs was set to l, the number of motifs to be found per run
was set to 4, and the total number of runs was set to 100. The highest scoring motifs
were then ranked by the command line version of MotifRanking v3.0
(http://www.esat.kuleuven.ac.be/~thijs/Work/MotifSamplerhtml) using Arabidopsis
intergenic sequence as a background model and the Kullback-Lieber distance to score the
top ten motifs. The Patmatch program at TAIR or a PERL script (Appendix M) was used
to identify all promoters in the genome that contained each motif. Those motifs
approximately 2-fold enriched compared to all promoters in the genome were chosen for
further analysis. Motifs were queried at PlantCARE (Lescot et al., 2002) to determine if
they were similar to any known cis-element.

The 4 bp flanking either side of the identified motifs was extracted from the 500
bp locus upstream sequence for cluster 111 genes (Appendix E) using a custom PERL
script (Appendix M). The alignment of these sequences was then used to identify
potentially conserved residues flanking a motif through the creation of WebLogos

(http://weblogo.berkeley.edu/).

113

CHAPTER FIVE
A SUMMARY OF THE GENE REGULONS THAT CONTRIBUTE TO THE
ARABIDOPSIS COLD TRANSCRIPTOME

The goal of identifying cold-responsive genes, the cis-elements within their
promoters, and the transcription factors that bind to those elements, is to understand the
gene networks contributing to cold acclimation in Arabidopsis. Such knowledge not only
furthers science, but could provide the genetic means of improving the freezing tolerance
of agriculturally important crops. The identification of the COS gene set, as described in
this dissertation, should serve as a robust resource to begin to decipher the low
temperature regulatory network of Arabidopsis. Indeed, the work described in this
dissertation represents the start of this work. Using the COS gene set as a guide, this
chapter summarizes what is currently known about the low temperature regulatory
networks of Arabidopsis.

While transcriptome profiling experiments have led to the definition of the COS
gene set, the regulatory networks cannot be properly explained without knowledge of the
cis-elements present in the promoters of the COS genes and the transcription factors that
bind to those elements. The CBF transcription factors, which bind to the CRT/DRE
element, play a major role in configuring the low temperature transcriptome of
Arabidopsis. The CBF transcription factors each appear to regulate the same suite of
target genes (Gilmour et al., 2004). Of the 514 COS genes, 93 were CBF2 regulon
members and of these 93, the large majority, 85 (91%), were cold-induced. The
promoters of 68 (80%) of these 85 genes had one or more CRT/DRE elements,

(A/G)CCGAC, present within 1 kb upstream of the start of the protein coding sequence.

114

Thus, these genes were likely to be direct targets of CBF2. CBF2-regulated COS genes
without CRT/DRE elements in their promoters were presumably regulated by other genes
controlled by CBF 2 though this remains to be established.

The 85 cold-induced CBF regulon members represented a diverse range of
functional roles, including metabolism, transcription, intercellular communication and
signaling, transport, energy, protein processing, and cellular biogenesis. The largest
group of transcripts (30%) in the CBF2 up-regulated regulon were stress-related
transcripts. A further distinguishing characteristic of these genes was that they comprised
the majority of genes that were most highly-induced in response to low temperature. Of
the 25 COS genes that were up-regulated at least lS-fold at 24 h, 21 (84%) were
members of the CBF regulon and nearly half of those up-regulated 5- to lO-fold, 32/66
(49%), were also assigned to the CBF regulon. Conversely, approximately 90% of the
COS genes that were induced less than S-fold were not assigned to the CBF regulon.
Finally, an additional distinguishing feature of the CBF regulon COS genes was the
enrichment for genes that remained up-regulated at 7 (1. Of these 67 “long-term” up-
regulated COS genes (22.5-fold increase at 7 d), 37 (55%) were members of the CBF
regulon.

Taken together, the above results indicate that the CBF transcription factors have
a prominent role in regulating the expression of those cold-responsive COS genes that are
both highly-induced and long-term up-regulated in response to low temperature.
However, it is also clear that additional cold-response pathways participate in configuring
the low temperature transcriptome, indicated by the finding that the transcript levels for

112 (22%) of the total 302 up-regulated COS genes showed no detectible change in either

115

of the CBF 2 overexpressing transgenic lines profiled. Thus, it would appear that these
cold-regulated genes fall outside of the CBF regulon. While most of these genes are
induced less than 5-fold in response to low temperature, 33 were induced more than 5-
fold. Identifying the transcription factors that regulate the expression of these genes is
required to more completely define the low temperature regulatory network of
Arabidopsis.

ZAT12, a C2H2 zinc finger transcription factor (Meissner and Michael, 1997),
was found to define an additional cold response pathway. ZAT12 is cold-induced in
parallel with CBF 2 and shares two additional features with CBF2; it is responsive to
mechanical agitation and treatment with cycloheximide. Analysis of the ZAT12 promoter
indicates that this regulation may occur at a transcriptional level, as two regions were
found to be similar to the ICErl and ICEr2 cold-, mechanical-, and cycloheximide-
responsive regions in the CBF2 promoter (Zarka et al., 2003). Constitutive expression of
ZA T12 influenced a statistically significant number of COS genes (24) and caused a
limited, but reproducible, increase in the freezing tolerance of Arabidopsis. In addition,
ZAT12 interacted with the CBF cold response pathway in two significant ways. First,
constitutive expression of ZAT12 influenced some of the same genes as CBF2. Secondly,
constitutive expression of ZAT12 resulted in lower cold-induced levels of CBF
transcripts, while two independent T-DNA insertions in the ZAT12 locus resulted in
higher levels of CBF transcripts in response to low temperature. However, constitutive
expression of ZAT12 or lower levels of ZAT12 expression did not impact the levels of the
COR genes as severely as the CBFs. In summary, ZAT12 represents a new cold response

pathway in Arabidopsis that has a negative regulatory role in CBF expression.

116

Another transcription factor has also recently been shown to alter the expression
of cold-regulated genes outside of the CBF cold response pathway. The hos9-I mutant
displays hyperactivation of an RD29A promoter luciferase fusion in response to low
temperature, but not in response to ABA treatment or salinity stress. Additionally, this
mutation altered freezing tolerance (Zhu et al., 2004). HOS9 was found to encode a
putative homeodomain transcription factor that was constitutively expressed and did not
increase in response to low temperature. CBF transcripts were not affected by hos9-I,
but since the promoter from the reporter gene construct contained CRT/DRE elements,
this gene might be affected by the CBF pathway in some manner. Microarray
experiments performed on cold treated hos9-1 plants by Zhu et al. (2004) revealed that a
number of cold-responsive genes were affected in their expression, but only one
GeneChip experiment was performed, so these results should be viewed as preliminary.
In comparing these genes to the COS gene set, 13 COS genes were found to be cold up-
regulated and hos9-I up-regulated, one gene was cold down-regulated and hos9-1 up-
regulated, and one COS gene was cold down-regulated and hos9-1 down—regulated and
none were members of the CBF regulon. Interestingly, all 13 of the up-regulated COS
genes influenced by hos9-1 fell into expression class V, those genes up-regulated
transiently in the cold. The majority of these 13 transcripts encoded either transcription
factors or enzymes for signaling molecules. This indicated that HOS9 may play a role in
regulating some of the earliest events during cold acclimation.

In addition to ZAT12 and H089, ICE] (a MYC-like basic helix loop helix
transcription factor) impacts the expression of cold-regulated genes (Chinnusamy et al.,

2003). A dominant mutation of ICEl, ice], results in loss of CBF 3 transcript

117

accumulation in response to low temperature, but does not impact accumulation of CBF]
or CBF 2 transcripts. The mutation also alters freezing tolerance and a preliminary
microarray experiment performed without replication revealed that the expression of a
number of cold-responsive transcripts was altered in response to low temperature. A
comparison of these transcripts to the COS genes revealed that none of the down-
regulated COS genes were impacted by ice], but 78 of the up—regulated genes displayed
lower cold induction in the mutant than in wild type plants (13 were in class V, 5 in class
VI, 9 in class VII, and 51 in class III). It is interesting to note that ice] affected some, but
not all of the CBF regulon members (51 of the 85 CBF regulon members displayed
altered cold-induction in ice] ). This could possibly be due to the fold-change cutoff used
by Chinnusamy et al (2003). Alternatively, this could indicate that some genes are more
greatly impacted by CBF 3 expression, while others are more responsive to CBF] or 2.
Finally, this result could be due to the action of additional transcription factors acting on
these CBF regulon members. Regardless of the reason, ICEl seems to play a role in
regulating cold-responsive genes.

In order to complete the low temperature regulatory network of Arabidopsis
(Figure 5.1), approximately 70% of the cold up-regulated COS genes still need to be
assigned to a regulon. In this study (Chapter 2) RA VI, MYB 73, SIZ/ZA T10, and CZF 2
were overexpressed in Arabidopsis, but were not found to affect the expression of a
statistically significant number of cold-regulated genes. It is possible that these
transcription factors may need to act in concert with other factors or be modified
posttranslationally in the cold, similar to the mechanism proposed for DREBZ (Liu et al.,

1998), in order to function properly. Chromatin immunoprecipitation experiments could

118

Low temperature

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Figure 5.1. The low temperature regulatory networks of Arabidopsis.

Shown above is a summary of the transcriptional regulators currently known to be
involved in the configuring the low temperature regulatory networks of Arabidopsis
thaliana. HOS] is an E3 ubiquitin conjugating enzyme (Ishitani et al., 1998). The rest of
the proteins listed are described in the text. Other TFs stands for other transcription
factors. Boxes with lines behind them represent genes with specific cis regulatory
elements, while the ovals represent proteins.

119

be used to test whether any of these transcription factors actually bind to the promoters of
COS genes. Work on RA V1, MYB 73, STZ/ZA T10, CZF1, CZF2 and other cold-induced
transcription factors will be needed to determine if they play a role in cold—responsive
gene expression. Other transcription factors that regulate the COS genes could be found
by first identifying the cis-elements in the promoters of these genes, either through the
use of bioinformatic means or promoter bashing experiments, and then isolating the
transcription factors that bind to these elements. Regardless of the methods used, much
work still needs to be done in order to define all the regulons that comprise the
Arabidopsis low temperature transcriptome. The work presented in this dissertation
provides both a set of genes which can be used to characterize these regulons and defines

two of these regulons, the CBF2 and ZAT12 regulons.

120

 

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APPENDIX K

THE BEST BLAST HITS FOR ZAT12 IN ARABIDOPSIS

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Figure K.1. Amino acid alignment and relationship tree of the best BLAST hits for

ZAT12 in the Arabidopsis genome.

(a) The ZAT12 (At5g59820) protein sequence was used in a BLAST search against all
Arabidopsis proteins. The best BLAST hits are shown in an amino acid alignment
created with ClustalW v1.82 (add webpage). The C2H2 zinc fingers are indicated by
astericks C“). The EAR-like domain is indicated by colored circles (o), The three
best BLAST hits for ZAT12 are located one after the other on chromosome 3 and are
the only C2H2 zinc fingers in the Arabidopsis genome with this chromosomal
arrangement.

(b) A relationship tree that depicts the relative relatedness and distance of each sequence
is shown. The tree was generated by ClustalW v1.82.

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APPENDIX M

PERL SCRIPTS

Unwrap_fasta.pl PERL script
#!/usr/bin/perl -w
#This script is from Rob Halgren, MSU-GTSF. It makes fasta files usable for PERL scripts.

use strict;

$/=">";

0;

while (<>){
chomp;
my ($name,@array)=split("\n",$_);
my $sequence=join(",@array);
print uc">$name\t$sequence\n";

}

CRT_counter.pl PERL script
#l/usr/bin/perl -w

#This script takes as input a fasta sequence file in the form of “>AT1G01010 ATCTGTG. . .”, after it has
been run through the unwrap_fasta.pl script. It then counts the number of CRT/DRE elements present in
the DNA sequence and returns the AGI number followed by the number of CRT/DRE elements present.

use strict;

print "AGI (A/G)CCGAC\n";

while (<>){

chomp;

my ($acc,$seq) = split "\t";

(Sacc) = Sacc =~ />(AT[\d|\w]\w\d{5})/;
my $count=0;

while (Sseq =~ /([A|G]CCGAC)|(GTCGG[C|T])/g){
Scount++;

}

print "Sacc $count\n"

}

232

CRT_distance_counter.pl PERL script
#l/usr/bin/perl -w

#This script finds the distances between CRTs in all promoters that contain one, even overlapping ones, in
either orientation. It does not return a value if there is no CRT in that promoter. The sequences used as
input must go through unwrap_fasta.pl first.

use strict;

print "AGI\tdistance_between_CRTs\n";

while (<>){

chomp;

my ($agi,$seq) = split "\t";

#This finds all the AGI numbers and makes a list of them.
(Sagi) = Sagi =~ />(AT[\d|\w]\w\d{5})/;

#This finds all sequences between CRTs in the sequence (in both orientations) and makes an array of them.

my (@seq8)=($seq =~
/(?=(?:(?:[AIG]CCGAC)|(?:GTCGG[C|T]))(.+?)(?:(?:[AIG]CCGAC)|(?:GTCGG[C|T])))/g);

#This takes each element in the above array and determines its length. I am replacing the sequences in the
@seqs array with the length of each sequence. The foreach loop allows me to do this for each element in
the array.

foreach $_ (@seqs){

$_ = length $_;
}

#This will print out the AGI followed by the lengths of any sequences between CRTs, seperate by tabs.

print "Sagi\t",join("\t",@SeqS);
print "\n";

l

233

CRT_to_start_counter.pl PERL script
#l/usr/bin/perl -w

#This script finds the distance from the hp after the last C in (AIG)CCGAC to the end of the sequence.
When using the lkb upstream dataset from TAIR, this corresponds to the beginning to the 5' UTR. Some
genes don't have annotated UTRs, in which case the script finds the distance to the ATG. Transcripts
without annotated UTRs can be flagged manually.

use strict;

print "AGI\tdistance_to_start\n";
while (<>){

chomp;

my ($agi,$seq) = split "\t";

#This finds all the AGI numbers and makes a list of them.
(Sagi) = Sagi =~ />(AT[\d|\w]\w\d{5})/;

#This finds all sequences from the end of one CRT (starts after last "C" of (AIG)CCGAC) to the end of the
sequence (on either strand) and makes an array of them.
my (@seqs)=$seq =~ /(?=(?:[AIG]CCGAC|GTCGG[ClT])(.+))/g;

#This takes each element in the above array and determines its length. I am replacing the sequences in the
@seqs array with the length of each sequence. The foreach loop allows me to do this for each element in
the array.

foreach $_ (@seqs){

$_ = length $_;

}

#This will print out the AGI followed by the lengths of any sequences between CRTs, seperate by tabs.
print "$agi\t",join("\t",@seqs);

print "\n";

}

Motif_extractor.pl PERL script
#l/usr/bin/perl -w

#This script takes as input a fasta file that has been run through unwrap_fasta.pl. It searches for a given
element, CCGAC, and extracts the pr upstream and downstream of the element, if it is there. The script
takes the element from either strand of the DNA.

use strict;

while (<>){

chomp;

my (Sagi) = />(AT[\d|\w]\w\d{5})/;

my (@watson) = /(\w{0,4} [AlG]CCGAC\w{0,4})/g;
my (@crick) = /(\w{0,4}GTCGG[C|T]\w{0,4})/g;
my Sreverse = reverse ("@crick");

Sreverse =~ tr/ATGC/TACG/;

print "Sagi @watson $reverse\n";

}

234

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