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This is to certify that the

dissertation entitled

The role of CBF orthologs in the cold
acclimation of blooming sour cherry
(Prunus cerasus) and strawberry
(Fragaria x ananassa)

presented by

Christopher Lawrence Owens

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Horticulture

 

 

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THE ROLE OF CBF ORTHOLOGS IN THE COLD ACCLIMATION OF
BLOOMING SOUR CHERRY (PRUNUS CERASUS) AND STRAWBERRY
(FRAGARIA x ANANASSA)

By

Christopher Lawrence Owens

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOHPY
Department of Horticulture

2001

ABSTRACT
THE ROLE OF CBF ORTHOLOGS IN THE COLD ACCLIMATION OF
BLOOMING SOUR CHERRY (PRUNUS CERASUS) AND STRAWBERRY
(FRA GAR/A x ANANASSA)
BY

Christopher Lawrence Owens

Many temperate fruit crops suffer yield reductions due to freeze damage.
Several early blooming rosaceous crops, such as strawberry (Fragan'a x
ananassa), and many of the cultivated Prunus species, are particularly prone to
floral freeze damage. The pistil and young fruitlet, is often the most susceptible
to freezing injury. Many cultural techniques are employed to mitigate freeze
damage to flowers, but these techniques are often costly and ineffective. Fruit
cultivars with improved floral freezing tolerance would have a major effect on
solving this problem.

Cold acclimation is the process by which a plant exposed to low
temperature increases its freezing tolerance. Studies on cold acclimation of
Arabidopsis have identified a family of cold-inducible transcription factors, CBF1,
2, and 3, that appear to be key regulators of cold acclimation and many cold
regulated (COR) genes in a diverse number of plant families. Several COR
genes appear to not be expressed in the pistils of Arabidopsis following exposure
to low temperature despite expression in many vegetative and other reproductive

tissues. Therefore, it was hypothesized that the CBF-COR gene system will be

conserved in the Rosaceae and that the expression of these genes will be
compromised in pistils of rosaceous plants.

Following 16 days of 4°C treatment to whole plants, the freezing tolerance
of strawberry receptacles was significantly enhanced, from -3.8 to -5.3 °C for
'Honeoye', with similar increases observed for many genotypes. In contrast, sour

cherry pistils Showed no significant increase in freezing tolerance for the 3

cultivars examined.

Orthologs of CBF1 were cloned from strawberry and sour cherry with
degenerate PCR primers. The putative orthologs FaCBF and PcCBF have 48%
amino acid identity to CBF1 and mRNA levels were up—regulated in leaves of
both crops following exposure to 4° C from 15 minutes to 24 hours. mRNA
expression of FaCBF and PcCBF in pistils of strawberry and sour cherry was not
detected following 4° C exposure at any time point tested.

Agrobacterium-mediated transformation of a CaMV35S-CBF1 construct
was conducted on Fragaria x ananassa 'Honeoye' crown discs. Two transgenic
lines were regenerated that expressed the transgene at low levels in both leaves
and receptacles with pistils. Receptacles of the transgenic lines showed no
significant change in freezing tolerance when compared to wild type plants.
However, the temperature at which 50 °/o electrolyte leakage occurred in
detached leaf-discs from the two transgenic lines was -8.2° C and -10.3° C,
respectively. These freezing tolerance values were significantly greater than the

value for the wild-type ’Honeoye' leaf discs of -6.4° C.

To Fergus and Ajax

iv

ACKNOWLEDGMENTS

Foremost I wish to thank my advisors and committee members: Dr. Amy
lezzoni, Dr. Jim Hancock, Dr. Jim Flore, and Dr. Mike Thomashow. Their
support and encouragement have been unwavering. I cannot imagine a better
group of scientists to learn from. Audrey Sebolt, Pete Callow, Renate Karle, and
Sarah Gilmour have been invaluable in assisting me in my research. I wish to
thank Dr. Julie Graham for visiting MSU and sharing her expertise in strawberry
transformation. The graduate students, post-docs, and undergraduates that I
have had the pleasure to work and play with have made my time at MSU truly
memorable. Finally I wish to thank my family, Judy, Fergus, and Ajax for

supporting me for the last several years.

TABLE OF CONTENTS

LIST OF TABLES..................... vu
LIST OF FIGURES... vm
INTRODUCTION ................................................................................................... 1

CHAPTER 1
COLD ACCLIMATION IN BLOOMING STRAWBERRY (FRAGARIA X
ANANASSA) AND SOUR CHERRY (PRUNUS CERASUS) ............................... 21

CHAPTER 2
IDENTIFICATION OF CBF ORTHOLOGS IN STRAWBERRY (FRAGARIA x
ANANASSA) AND SOUR CHERRY (PRUNUS CERASUS) ............................... 40

CHAPTER 3
ENHANCEMENT OF FREEZING TOLERANCE OF STRAWBERRY BY
HETEROLOGOUS EXPRESSION OF CBF1 ...................................................... 59

LIST OF TABLES

Table 1. Geographic origin of strawberry selections utilized in freezing tolerance
experiments ......................................................................................................... 25

Table 2. Temperature at which 50% electrolyte leakage occurred for leaf discs
removed from blooming, whole plants exposed to 4° C for 0, 2, 4, 8, or 16 days
............................................................................................................................. 28

Table 3. Temperature at which 50% electrolyte leakage or half of the pistils or
receptacles plus pistils were dead occurred for strawberry receptacles, leaf discs,
and sour cherry pistils removed from blooming, whole plants exposed to 4° C

............................................................................................................................. 30
Table 4. Relative water content of strawberry receptacles/pistils from non-

acclimated and acclimated plants ........................................................................ 31
Table 5. PCR primers utilized in this study ......................................................... 47

Table 6. Temperature at which 50% electrolyte leakage occurred for wild-type
‘Honeoye’ plants and 35-1-1 and 35-1-2 plants which over-express CBF1 ......... 68

vii

LIST OF FIGURES

Figure 1. Amino acid alignment of CBF1 and conceptual translations of CBF-like
cDNAs reported in GenBank (Brassica napus DREBP AF084185, Nicotiana
tabacum AP2 containing protein AAG43548). Identical residues are indicated by
shaded boxes. Positions of Similarity used to design degenerate primers are
indicated by the boxed areas in the consensus sequence, which is the CBF1
sequence minus gaps ......................................................................................... 46

Figure 2. Amino acid sequence alignment of CBF 1, PcCBF, and FaCBF
Identical residues are shaded ............................................................................. 49

Figure 3. PcCBF and FaCBF transcript levels determined by northern analysis in
leaves of sour cherry ‘Montmorency’ and strawberry ‘Honeoye’ following 0 hour,
1 hour, 4 hours, and 24 hours of exposure to 4°C. Eukaryotic elongation factor
4a (elF4a) was used as a RNA loading control ................................................... 50

Figure 4. PcCBF and FaCBF transcript levels determined by northern analysisin
pistils of sour cherry 'Montmorency' and receptacles with pistils of strawberry
‘Honeoye’ following 0 hour, 1 hour, 4 hours, and 24 hours of exposure to 4°C.
elF4a was used as a RNA loading control .......................................................... 51

Figure 5. Southern analysis of wild-type and 35-1 -1 and 35-1-2 ‘Honeoye’
strawberry plants transformed with CBF 1. Restriction digestion conducted with
Hindlll .................................................................................................................. 66

Figure 6. CBF1 transcript levels in wild-type plants” and 35-1-1 and 35-1-2
transgenic plants which over-express CBF 1. Eukaryotic elongation factor 4a
(elF4a) was used as a RNA loading control ....................................................... 67

viii

Introduction

Low temperature is one of the most important environmental factors
limiting plant growth and development. Improved freezing tolerance is a major
goal of plant breeders of many crops. The Rosaceae is a diverse plant family,
consisting of many of the most economically important temperate fruit crops,
such as apple (Ma/us x domestica), pear (Pyrus communis), peach (Prunus
persica), sweet cherry (Prunus avium), sour cherry (Prunus cerasus), almond
(Prunus dulcis), apricot (Prunus armeniaca), plum (Prunus domestica),
strawberry (Fragaria x ananassa), and brambles (Rubus spp.). Almost all
cultivated fruit species within this family are subject to freezing damage during
bloom. Many rosaceous species bloom very early during the growing season, at
a time in which fluctuating temperatures are often capable of dipping several
degrees below freezing. In addition to the complete crop loss that can occur
following severe spring frosts, the crop fluctuations that occur cause large swings
in crop supply. Uncertain crop yields from year to year lead to insufficient crop in
some years, and over-production and thus poor prices in others.

Within rosaceous flowers, the organ with the greatest susceptibility to
freezing damage is often the pistil (Ki and Warmund, 1992; Darrow, 1966). For
example, in strawberry, both receptacles and pistils have been shown to be killed
by a temperatures as high as -2° C (Boyce and Marini, 1978). Field observations
of sour cherry flowers following a freeze event in the orchard often Show

damaged pistils while the remaining floral tissues are undamaged (Dennis and

Howell, 1974). Since the pistil is often the most freezing susceptible tissue within
the flower, breeding efforts directed at improving the physiological freezing
tolerance of these flowers needs to first be directed at improving the freezing
tolerance of the pistil.

Many cultural techniques are employed to prevent or reduce the damage
that is caused by late frosts. These techniques include the use of field heaters,
wind machines, overhead irrigation, and row-covers (Reiger, 1989). In addition,
prevention of freezing damage during bloom is a major factor in orchard site
selection. Fruit plantings are often established on hillsides where cool air can
drain down the slope, away from the planting, and also near large bodies of
water that help moderate temperatures, mitigating sudden dips in temperature.
However, floral freezing damage within fruit plantings remains a major problem.
Many of the cultural techniques employed to reduce freeze damage are either
costly or not very effective (Perry, 1998). A more lasting solution to this problem
would be the development of cultivars with enhanced floral freezing tolerance. A
breeding approach to this problem could provide valuable new tools for the
prevention of this serious problem.

Efficient breeding of fruit cultivars for improved floral freezing tolerance is
dependent on the available information on the classical and molecular genetics
of floral freezing tolerance in the Rosaceae. Freezing tolerance in rosaceous
species is complexly inherited (Cain and Andersen, 1980; Watkins and
Spangelo, 1970; Mowrey 1964; Powers, 1945; Dorsey and Bushnell, 1925).

Little information is available on the inheritance of floral freezing tolerance in fruit

crops, although variation in floral freezing tolerance has been reported for several
rosaceous crops: strawberry (Hummel and Moore, 1997; Boyce and Marini,
1978), apple (Janick and Moore, 1996), and peach (Scott and Cullinan, 1939).
More detailed genetic analyses of floral freezing tolerance would be desirable to

help provide information for the breeding of this trait in fruit cultivars.

Breeding for improved freezing tolerance:

Several approaches can be taken by plant breeders and plant scientists to
attempt to improve the freezing tolerance of cultivated plants. Classical plant
breeding has attempted to utilize naturally existing variation for freezing tolerance
in the selection of parents and in the ascertainment of the quantitative genetics of
freezing tolerance. However, for many important crops such as wheat and
barley, whole plant freezing tolerance of current cultivars has not significantly
changed in decades (Fowler and Gusta, 1979). Breeding for freezing tolerance
in fruit crops has been more successful than the other major crops. However,
most fruit crops are large in Size, take up a large amount of space, and have
long-cycling generations. As a result, even though genetic variability for floral
freezing tolerance has not been exhausted, fruit crops have the disadvantage of
taking an extremely long time to be bred through traditional methods, especially
considering that many important traits, in addition to improved floral freezing
tolerance need to be addressed by the fruit breeder.

Understanding the molecular basis of the variation in freezing tolerance in

native germplasm could lead to the development of strategies for improving

freezing tolerance in elite germplasm. This approach has received little attention
and is perhaps best suited for a crop that has sufficient naturally existing
variation for freezing tolerance, such as several fruit species. While little work
has been conducted on the genetics of freeze tolerance in fruit crops, several
studies have shown that desirable alleles for quantitative traits do exist in wild
accessions of several crop plants, even if those traits are not phenotypically
evident (Xiao et al, 1998; Bemacchi et al., 1998; Tanksley and McCouch, 1997).
For example, in tomato, QTL for fruit size and color have been identified and
back-crossed from a wild accession of Lycopersicon pimpinellifolium to the
cultivated L. esculentum, neither of which was observed in the wild selection
(Bemacchi et al., 1998). Therefore, characterization and utilization of wild
germplasm for crop improvement is likely to be another avenue for fruit breeders
interested in improving floral freezing tolerance.

A third approach that is receiving attention is to attempt to understand how
individual plants protect themselves from freezing temperatures. Many plants
have the ability to cold acclimate, or develop increased freezing tolerance
following exposure to low temperature. By understanding the physiological and
molecular nature of cold acclimation it may be possible to develop novel
strategies for improving plant cold hardiness. For some of our important
agronomic. crops in which freezing tolerance in new cultivars has plateaued, this
approach is of significant interest. In Arabidopsis, manipulation of one regulatory

gene, identified through this avenue of inquiry has lead to a whole plant

improvement in freezing tolerance through transgenic approaches (Jaglo-Ottosen
etal,1998)

Although variation for whole plant freezing tolerance exists within the
available germplasm for many rosaceous species, variability for floral freezing
tolerance appears to be much more limited. No major advances in floral freezing
tolerance have been made in many fruit cultivars over the last several decades.
Despite some variation, the floral freezing tolerance of strawberry cultivars
introduced in the 19705 is little better than cultivars introduced in the 19205
(Boyce and Marini, 1978). Therefore, an approach attempting to determine if
manipulation of the cold acclimation machinery is capable of improving the floral

freezing tolerance of these plants is warranted.

Cold acclimation in giants:

Sensing of cold temperature:

There are still many gaps in our understanding of how plants sense low
temperature, translate this information into alterations of gene expression, plant
biochemistry and physiology, and eventually change plant phenotype (ie. cold
acclimation). The specific nature of how plants first sense low temperatures is
poorly understood. It has been postulated that changes in membrane fluidity
during exposure to low temperature may lead to conformational changes in
membrane bound proteins (Murata and L05, 1997; Monroy and Dhindsa, 1995;
Minorsky, 1989). Changes in protein conformation could lead to changes in

protein activity, binding, or perhaps even localization. One specific change could

be in the activity of membrane bound Ca2+ channels. Ca2+ has been implicated
as an important signal transduction pathway component in response to low
temperature. Transient increases in cytosolic Ca2+ caused by influxes of Ca2+
from extracellular locations have been demonstrated in response to low
temperatures (Torrecilla et al., 2000; Monroy and Dhindsa, 1995; Knight et al.,
1991 ). Experiments preventing this influx of Ca2+, through the use of Ca2+
chelators or Ca2+ channel blockers, has been shown to prevent expression of
low-temperature induced genes and to reduce the plant's ability to cold acclimate
(Monroy and Dhindsa, 1995).

A full description of the events both up and downstream of these Ca2+
increases waits to be determined, but additional experiments have suggested a
Signal transduction cascade involving protein phosphorylation (Monroy et al.,
1998), Ca-dependent protein kinases (Sheen, 1996), mitogen-activated protein
kinases (Jonak et al., 1996) and a calcineurin B-like protein (Kudla et al., 1999).
The likely end result of such a cascade is the activation of transcription factors

necessary for the induction of genes conferring freezing tolerance to the plant.

Regulation of cold-induced gene expression:

Many genes have been identified that are regulated in response to low
temperature. Our knowledge of how these genes are regulated and their exact
role in freezing tolerance is still incomplete. Two main approaches have been
taken to better understand the factors that regulate this large number of cold-

induced genes. One approach, based on the differential expression of either

proteins or mRNA transcripts has either directly or indirectly lead to the
identification of regulatory proteins involved in the regulation of cold-induced
gene expression: CBF 1, 2, and 3 in Arabidopsis thaliana (Stockinger et al., 1997,
Gilmour et al., 1998) and SCOF1 in Glycine soja (Kim et al., 2001). A second
approach has been to identify mutants with altered cold acclimation or freezing
tolerance phenotypes. In Arabidopsis, this mutational approach has identified:
sensitivity to freezing (sfr) mutants that do not cold acclimate (Warren et al.,
1996), eskimo1 that shows constitutive freezing tolerance even at warm
temperatures (Xin and Browse, 1998), and H081 which was identified by
possessing heightened expression of several cold-regulated genes following cold
treatment (lshitani et al., 1998).

The combined data from both of these approaches is beginning to provide
a framework for understanding plant gene expression in response to cold. Cold
signaling pathways appear to be complex, and have many points in parallel as
well as many interactions (lshitani et al., 1997). However, there appear to be at
least 3 partially distinct pathways leading to the induction of cold regulated gene
expression: ABA-independent and ABA-dependent pathways that ultimately up-

regulate many of the same genes, and a separate pathway defined by eskimo1.

_BF-COR genes/sfr6/hos1MBA-independent pathway:
Perhaps the best characterized cold-induced genes are the Arabidopsis
cor genes. In Arabidopsis, studies of the molecular basis of cold acclimation

have identified a common cis-acting regulatory element, the C-

repeat/Dehydration Responsive Element (CRT/DRE) which is defined as a
CCGAC, within the promoters of several cold induced (COR) genes (Stockinger
et al., 1997). In turn, all of the currently characterized Arabidopsis COR genes
are coordinately up-regulated by CBF 1, CBF2, or CBF3, a family of cold and
drought inducible transcriptional activators that bind to promoters containing a
CRT/DRE (Thomashow et al., 2001; Kasuga et al., 1999; Liu et al., 1998; Jaglo—
Ottosen et al., 1998). Even though freezing tolerance is a complexly inherited
trait, manipulation of just one gene, CBF 1, has been shown to improve the whole
plant freezing tolerance of Arabidopsis and Brassica napus (Jaglo, 2001; Jaglo—
Ottosen, et al., 1998). Additionally, it is known that these COR genes can be up-
regulated by additional transcription factors DREBZA and DREB2B, which are
important in the response to dehydration (Liu et al., 1998).

The CBF family of transcriptional activators are up-regulated in response
to cold and drought stress, but not by exogenous ABA (Gilmour et al., 1998).
CBF 1, 2, and 3 are AP2/EREBP domain containing proteins. How CBF is
regulated and how it is connected to upstream signaling components is not
known.

SFR6 is a protein that may interact with CBF1 in the cold-induced
regulation of the COR genes (Knight et al,, 1999). Evidence for this comes from
the 8er mutant in Arabidopsis which is deficient in freezing tolerance following
cold acclimation, and does not show expression of COR6. 6, COR15a, or COR78
in response to cold temperature, even though CBF3 expression following cold

exposure is normal (McCown et al., 1996; Warren et al., 1996; Knight et al.,

1999). It has been postulated that SFR6 is required for cold-induced activation of
the CBF3 protein. The exact nature of the interaction between SFR6 and CBF3
is not known, nor has the SF R6 protein been characterized.

Another mutant, hos1, exhibits enhanced expression of several COR
genes in response to cold temperature (lshitanit et al., 1998). In addition, CBF2,
CBF3, but not CBF1 were shown to have enhanced gene expression in the hos1
mutants. HOSl encodes a novel ring-finger protein that is constitutively
expressed in Arabidopsis, and has been shown to be targeted to the nucleus
following cold temperature exposure (Lee et al., 2001). The native HOS1
transcript is transiently expressed in response to cold, but quickly recovers and is
expressed for up to 2 days before declining. Ring-fingers are small, zinc-binding
domains found in many proteins with diverse function, and may be involved in
ubiquitination and subsequent degradation of proteins. It has been speculated
that HOSI may be a regulator of the up-stream signaling components leading to

cold-induced CBF expression (Lee et al., 2001).

SCOF1/ABA-dependent pathway

SCOF1 is a soybean regulatory protein that defines a related cold signal
transduction pathway that appears to be ABA-dependent (Kim et al., 2001).
SCOF1 is a cold and ABA-inducible zinc-finger protein, that has been shown to
interact with a G-box protein in soybean to up-regulate genes that contain G-

boxes in their promoter regions (Kim et al., 2001). G-boxes are a common

promoter element which share the same core sequence as ABA-responsive-
elements (ABRE) - ACGT. Many characterized COR genes, such as COR15a
and COR78 possess both ABRE and CRT/DRE in their promoters (Stockinger et
al., 1997; Baker et al., 1994). Expression of SCOF1 in Arabidopsis leads to COR
gene expression in the absence of a cold stimulus and enhanced freezing
tolerance when compared to non-transgenic controls (Kim et al., 2001).

However, SCOF1 does not appear to regulate COR gene expression through the
CRT/DRE as is the case with CBF1, 2, and 3. SCOF1 appears to define a cold-
sensing pathway that leads to the induction of many genes similar to those on
the CBF-ABA independent pathway, but does so through a different set of

intermediaries, possibly involving ABA.

21km

At least one additional cold signaling pathway may be associated with
eskimo1. eskimo1 is an Arabidopsis mutant that shows constitutive freezing
tolerance in the absence of a prior cold temperature stimulus (Xin and Browse,
1998). Non-acclimated eskimo1 plants possess approximately 70% of the
freezing tolerance of acclimated wild type plants, and acclimated eskimo1 plants
possess freezing tolerance greater than acclimated wild type plants.
Significantly, 4 COR genes are not expressed in the non-acclimated eskimo1
plants, suggesting that these mutants achieve increased freezing tolerance
without up—regulating the genes in the CBF-COR gene pathway. Eskimo1

mutants show enhanced levels of proline and soluble sugars, known compatible

10

osmolytes involved in providing increased freezing tolerance, increased
expression of A‘-pyrroline-5-carboxylate synthetase (P5CS), the key regulator of
proline biosynthesis, and decreased expression of proline oxidase (AtPOX), a

key enzyme of proline degradation (Xin and Browse, 1998).

Integrating gene expression to physiologfl responses to cold;

Many physiological and biochemical changes are known to occur in plants
in response to low temperature. These changes include: alterations in lipid
metabolism, changes in the properties of membranes (Murata and Los, 1997),
shifts in carbohydrate production and localization, accumulation of proline,
betaine, and other compatible osmolytes (Levitt, 1980) , and Shifts in plant
secondary metabolism (Guy, 1990). However, very few of the cold-induced
genes characterized in plants have been directly tied to a known physiological
function. Linking the large number of cold-induced physiological and biochemical
changes to the molecular genetics of cold acclimation is a major future challenge.

Cold induced genes fall into many categories including: lipid desaturases,
antifreeze proteins, and superoxide dismutases; the largest number having
unknown functions. A large subset of the genes of unknown function share
similarity with a class of proteins known as LEA proteins, which were first
described in cereals and are transcribed late in embryogenesis during seed
desiccation (Thomashow, 1999). Even though some of these cold-induced
genes share homology with LEA proteins and some do not, many of them share

several common characteristics. Common traits include: proteins that are highly

11

hydrophilic, possess simple amino acid compositions, remain soluble upon
boiling in aqueous buffer, and have many simple repeated amino acid motifs
(Thomashow, 1999). More detailed studies of one of these genes, cor15a from
Arabidopsis, suggest that the COR15am protein has the ability to shift downward
the temperature at which the lamellar to hexagonal Il phase transition occurs in
plant membranes (Steponkus et al., 1998). This phase transition is known to
lead to extensive cellular damage upon return of the cells to non-freezing
temperatures. This temperature shift allows the cells to withstand lower freezing
temperatures without suffering damage. In the case of COR15am, the inner
membrane of the chloroplast is thought to be the binding target, and it is
hypothesized that this protein has the ability to alter the intrinsic curvature of the
membrane leading to the temperature shift of the phase transition (Steponkus et
al., 1998). Since many of the cold-induced genes share properties with COR15a
it is possible that membrane stability is a common mode of freezing tolerance in
plants. However, little is known about the physiological role of most cold-induced

plant genes.

CBF as a key regulator of cold acclimation:

Considering that CBF genes are some of the best characterized cold
acclimation related genes, appear to be key regulators of cold acclimation, and
their over-expression is capable of improving the freezing tolerance of

Arabidopsis, there is significant interest in determining if orthologs of the CBF

12

gene family exist and function in a similar fashion within cultivated plants,
including the Rosaceae.

Indeed, CBF-like proteins have been identified in Brassica napus, Triticum
aestivum, and Secale cereals, and the chilling-sensitive Lycopersicon
esculentum (Jaglo et al., 2001). In these crops, these CBF-like proteins are cold-
regulated in a fashion similar to CBF1, 2, and 3 of Arabidopsis, and possess high
levels of amino acid identity. Additionally, overexpression of CBF1, 2, or 3 in
Brassica napus increases its freezing tolerance and up-regulates Bn115 and
Bn28, homologs of COR15a and COR 6.6 of Arabidopsis. These findings
suggest the CBF-COR system is highly conserved in diverse plant families, and
may be a key regulator of cold temperature stress tolerance in many plants. If
this set of genes is conserved in the Rosaceae, perhaps these genes can be
manipulated to improve floral freezing tolerance.

Cold induced gene expression in the Rosaceae has not received much
attention. However, a small number of cold induced genes have been identified
through analyses of differential expression of proteins and mRNA in response to
cold. A cold-induced dehydrin gene, ppdhn1, has been found to be expressed in
the bark and xylem parenchyma cells of peach, shows increased expression
during winter months and is associated with differences in cold hardiness in
sibling genotypes of evergreen and deciduous peach (Artlip et al., 1997;
Wisniewski et al., 1999). Three cold induced genes have also been identified
from leaves of strawberry, Fcor1, 2, and 3 (Ndong et al., 1997). Fcor1 appears

homologous to LTI6b, a low-temperature induced gene of Arabidopsis. Fcor2

13

appears to code for an hydroxyproline rich glycoprotein, and Fcor3 which Shows
reduced transcript expression in response to low temperature has some similarity
with subunit V from spinach photosystem I (Ndong et al,, 1997).

Perhaps the most efficient approach to elucidating the molecular basis of
cold acclimation in the Rosaceae, would be to take advantage of the wealth of
information that is being generated from studies in model plant species, such as
Arabidopsis. As mentioned above, the CBF family of transcriptional activators
are important regulators of cold acclimation in Arabidopsis. When over-
expressed these genes have been shown to enhance the freezing tolerance of
both Arabidopsis and Brassica napus, and orthologs of the CBF gene family
have been identified in a diverse array of plant species, such as wheat, rye, and
tomato (Jaglo et al, 2001). Identifying and characterizing CBF orthologs in the
Rosaceae would be a good starting point to transfer the information gained from
model species to this important family of cultivated plants.

The interaction between cold temperature and photoperiod on cold
acclimation of woody fruit crops has been extensively studied (Howell and
Weiser, 1970; Weiser, 1970), and it is known that the over-wintering structures of
perennial, temperate fruit crops possess the ability to cold acclimate. However,
the ability of reproductive tissues of perennial plants to cold acclimate is largely
unknown, although there is indirect evidence that rosaceous flowers may have
that ability. Some cultivars of peach trees de-acclimated under a fluctuating
15l5° C temperature regimen have marginally better floral freezing tolerance than

those brought out of dormancy at a constant 25° C (Lu and Rieger, 1993). There

14

is anecdotal evidence that peach flowers In the orchard survive frosts better
following periods of cool temperature (Scott and Cullinan, 1939). Additionally,
flowers from day-neutral strawberries collected at the end of a two-month period
in autumn had slight improvements in floral freezing tolerance compared to the
flowers collected at the onset of the study period (Boyce and Marini, 1978).
While these studies provide circumstantial evidence that floral tissues can cold
acclimate, there have been no direct comparisons of the freezing tolerance of
non-acclimated rosaceous flowers to those exposed to low, non-lethal

temperature.

The objectives of this research were to:

1) determine if the cold acclimation response observed in Arabidopsis also
occurs in newly formed strawberry and sour cherry pistils and leaf tissue.

2) determine if expression of CBF1 increases freezing tolerance in the pistils, and
vegetative tissues of strawberries, which is transformable.

3) determine if strawberry and sour cherry possess homologs of CBF and if so:
(a) whether these genes are turned on in response to cold temperatures, (b)
whether these cold regulated genes are differentially expressed between the
pistils and the vegetative tissues, and (c) if differential gene expression will
correlate with lower frost tolerance of the pistils compared to the vegetative

fissues.

15

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20

Chapter 1 - Cold Acclimation in Blooming Strawberry (Fragaria x
ananassa) and Sour Cherry (Prunus cerasus))

Many temperate fruit crops suffer yield reductions due to freeze damage.
Early season freezes during or just prior to bloom are the main cause of low-
temperature induced crop loss (Rodrigo, 2000). Several early blooming
rosaceous crops, such as strawberry (Fragan'a x ananassa), and many of the
cultivated Prunus species, are particularly prone to floral freeze damage. The
economically viable part of the plant, the pistil and young fruitlet, is often the most
susceptible to freezing injury (Ki and Warmund, 1992; Darrow, 1966). Site
selection, row covers, overhead irrigation, and other cultural techniques are often
employed to mitigate freeze damage during bloom. However, these techniques
are often labor intensive, costly, or not very effective (Perry 1998; Anderson and
Whitworth, 1993; Rieger, 1989). Fruit cultivars with improved floral freezing
tolerance would provide lasting and more cost-effective means of preventing
damage.

Many plants have the ability to cold acclimate, or develop improved
freezing tolerance following exposure to low, but non-lethal temperatures. Cold
acclimation is associated with numerous biochemical changes in plants:
increases in proline concentration, sugars, and other compatible solutes;
changes in lipid metabolism (Thomashow, 1999; Hughes and Dunn, 1990); and,
significantly, changes in gene expression. Genes thought to be associated with

the plants adaptation to freezing temperatures have been identified in many plant

21

species, such as alfalfa (Medicago sativa), spinach (Spinacia oleracea), barley
(Hordeum vulgare), wheat (Triticum aestivum), and Arabidopsis thaliana (Monroy
et al. 1993; Hong et al. 1992; Houde et al. 1992; Guy at al., 1985).

One approach to develop cultivars with enhanced freezing tolerance is to
identify and utilize candidate genes playing an important role in this trait.
Although freezing tolerance is a complexly inherited trait, whole plant freezing
tolerance of Arabidopsis can be improved by the over-expression of a single
cold-induced transcription factor (Jaglo-Ottosen et al., 1998). A large number of
cold regulated genes have been identified from many crop plants, including a few
from rosaceous fruit crops (Wisniewski et al., 1999; NDong et al., 1997). It is
plausible that cold-induced genes exist within the Rosaceae that are orthologous
to those in Arabidopsis capable of improving freezing tolerance. Whether these
genes can be utilized, through either biotechnological manipulation or
exploitation of natural allelic variation, to improve floral freezing tolerance in
rosaceous crops is unknown. It is also unknown if the same relationship exists
between cold-induced gene expression and onset of cold-acclimation in
rosaceous flowers. Before an attempt to identify genes associated with cold-
acclimation in rosaceous flowers, it is desirable to first determine if these flowers
are capable of cold acclimation.

The interaction between cold temperature and photoperiod on cold
acclimation of woody fruit crops has been extensively studied (Howell and
Weiser, 1970; Weiser, 1970), and it is known that the over-wintering structures of

perennial, temperate fruit crops possess the ability to cold acclimate. However,

22

the ability of reproductive tissues of perennial plants to cold acclimate is largely
unknown. There is indirect evidence that rosaceous flowers may have an ability
to cold acclimate. Some cultivars of peach trees de-acclimated under a
fluctuating 15l5° C temperature regimen have marginally better floral freezing
tolerance than those brought out of dormancy at a constant 25° C (Lu and
Rieger, 1993). There is anecdotal evidence that peach flowers in the orchard
survive frosts better following periods of cool temperature (Scott and Cullinan,
1939). Additionally, flowers from day-neutral strawberries collected at the end of
a two-month period in autumn had slight improvements in floral freezing
tolerance compared to the flowers collected at the onset of the study period
(Boyce and Marini, 1978). While these studies provide circumstantial evidence
that floral tissues can cold acclimate, there have been no direct comparisons of
the freezing tolerance of non-acclimated rosaceous flowers to those exposed to
low, non-lethal temperature.

The objectives of this study were to determine if the flowers of sour cherry
(Prunus cerasus) and strawberry (Fragaria x ananassa) are able to cold
acclimate. The specific objectives of this research were to: 1) determine if the
leaves of fully blooming, non-acclimated strawberry and sour cherry grown under
long-days have the ability to cold acclimate, 2) determine if the receptacles and
pistils of blooming, non-acclimated strawberry and sour cherry, respectively,
grown under long-days have the ability to cold acclimate, and 3) determine if
there is any genotypic variability among cultivars in the ability of strawberry and

sour cherry pistils to cold acclimate.

23

Materials and Methods

Plant Material, growth conditions:

Containerized, dormant, rooted runners of ‘Honeoye’ and ‘Chandler’
Fragaria x ananassa; RH-18, EBB1, NC 95-16-6, NC 95-18-5, NC 96-29-1, and
NC 96-28-1 F. virginiana; FRA 0883, and F RA 368 F. chi/oensis; as well as 3
year old, dormant plants grown from rooted cuttings of ‘Montmorency’,
‘Rheinische Schattenmorelle’, and ‘Erdi Botermo’ Prunus cerasus were grown in
growth chambers maintained at 24 °CI18 °C with 14 h day-lengths at 800
mmol-m‘z-S'1 photosynthetically active radiation (PAR) and 8 h nights until the
onset of flowering. Four degree cold treatments were administered by
transferring plants to a cooler maintained at a constant 4 °C with 14 h day-

lengths at 200 mmol-m‘z-s‘1 PAR and 8 h nights.

24

Table 1. Geographic origin of strawberry selections
utilized in freezing tolerance experiments

 

 

Selection Geographic
Origin
Fragaria x anannasa
Honeoye New York
Chandler California
Fragaria virginiana
EBB1 Alaska
RH-18 Wyoming
NC 95-16-6 Mississippi
NC 95-18-5 Mississippi
NC 96-28-1 Alabama
NC 96-29-1 Alabama
Fragaria chiloensis
FRA 0883 British
F RA 368 Columbia
British
Columbia

 

Determination of Freezing Tolerance:

Freezing tolerance was determined on detached leaf-discs and pistils
(sour cherry) or receptacles with pistils (strawberry) from fully open flowers by
two methods : visual scoring and electrolyte leakage (Sukumaran and Weiser
1972). Detached pistils were visually scored as either dead (brown) or alive
(green). For the electrolyte leakage experiments leaf-discs, pistils, or receptacles
with pistils were detached from the whole plant, and stored in de-ionized, distilled
water until all had been placed in 16 mm diameter x 125 mm long glass culture

tubes. Three leaf discs, 5 cherry pistils, or 3 strawberry receptacles with pistils

25

were placed in each culture tube. Data from 3 tubes were pooled for each
temperature point of the controlled freezing run.

Freezing-runs were conducted by placing the tubes in freezing-water
baths containing a mixture of ethylene glycol and water. The tubes were
maintained at 0 °C for 1 h, after which a small amount of de—ionized ice was
placed in contact with the plant material in each tube to nucleate ice formation.

The tubes were kept for an additional hour at 0 °C, and the temperature was then

decreased at the rate of 4 °C-h". Every 30 minutes a set of tubes was
transferred to a separate freezing-bath kept at the same temperature as the bath
it came from, and maintained at that temperature for an additional 30 minutes (for
a total of 1 h at each temperature). After 1 hour of freezing, tubes were placed
on ice until the completion of the freezing experiment. Freezing runs were
carried out to a final temperature of —20 °C for leaf-discs, and —10 °C for pistils
and pistils/receptacle.

Following completion of the freezing run, tubes were placed in a rack on
top of ice, wrapped with plastic, and kept at 4 °C overnight. Following thawing, 3
mL of de-ionized, distilled water were added to each tube, and the tubes were
then Shaken for 3 hours. Electrical conductivity of the water was measured from
each tube with a bench-top electrical conductivity meter (Corning 441, Acton,
MA), the plant material in the tubes was placed at —80 °C for 1 hour, and the
original water was then added back to each tube for an additional 3 hours, with

agitation. A final electrical conductivity was then determined for the water in

eachtube.

26

Three sets of data points were generated for each temperature point of
the freezing run, with 3 tubes representing each data point. These data were
used to generate an electrolyte leakage curve over the range of temperatures

used in the freezing run. Three to four curves were generated per treatment.

Determination of Relative Water Content:
The fresh weight and dry weight of strawberry receptacles were
determined for ‘Honeoye’ and ‘Chandler’ following either exposure to 4° C for 16

days or 0 days to determine the relative water content of each.

Data Analysis:

The electrolyte leakage and visual damage data were fit to a logistic
model (y = al(1 + be“) with CurveExpert 1.3 (Microsoft Corp.) The temperature
at which 50 percentage electrolyte leakage (EL5°) occurred was used to compare
treatments. The EL5° was determined by solving the logistic equation fit to each
curve with y set to 50 % for each curve. (Linden et al., 2000)

Statistical Significance of treatment comparisons was made with either a
two-sample t-test or one-way analysis of variance with Minitab 13.0 (Minitab Inc.

State College, PA).

27

Results

Freezing tolerance of lea ves:

Both sour cherry and strawberry leaf discs from fully blooming plants showed a
significant increase in freezing tolerance following 8 to 16 days of exposure to 4°
C (Table 2). The highest freezing tolerance was observed after 16 days of

exposure to cold temperature.

Table 2. Temperature at which 50% electrolyte leakage occurred for leaf discs
removed from bloominwhole plants exposed to 4° C for 0, 2, 4, 8, or 16 days.

 

 

Days of exposure to 4° C Sour Cherrya Strawberry
0 -5.4 a -6.4 a
2 -5.5 a -6.9 a
4 -5.2 a -7.0 a
8 -7.1 b -8.5 b
16 -10.9 c -13.2 c

 

a Values within columns followed by the same letter are not Significantly different
by LSD, ot= 0.05. Cultivars used were ‘Montmorency’ sour cherry and ‘Honeoye’

strawberry.
Freezing tolerance of flowers:

Since 16 days of exposure to 4° C provided the greatest increase in
freezing tolerance in strawberry and sour cherry leaves, this treatment was
utilized to determine the effect of low-temperature exposure on pistil freezing
tolerance. ln strawberry, significant increases in receptacle freezing tolerance
were observed in about half of the selections examined when measured by
electrolyte leakage after 16 days of cold treatment (Table 3). When measured by

visual scoring of receptacle and pistil viability, all of the genotypes except RH-18

28

showed a significant increase in freezing tolerance (Table 1.3). There was no
significant correlation between the 50% electrolyte leakage values for leaf discs
and the pistil damage values, regardless of the method utilized to determine the
freezing tolerance (data not shown). No significant differences were observed
between the freezing tolerance of any sour cherry pistils exposed to cold

temperature and those that were not, regardless of genotype (Table 1.3).

Variation in floral freezing tolerance:

Significant variation in strawberry receptacle freezing tolerance was
observed for all methods utilized to determine the freezing tolerance, and for both
non-acclimated and non-acclimated plants. The freezing tolerance of non-
acclimated receptacles with pistils ranged from -1.0° C to -4.2° C by visual
scoring, and acclimated receptacles with pistils ranged from -4.0° C to -7.6° C
(Table 1.3). The rankings of the genotypes for receptacle freezing tolerance was
not correlated between the two methods utilized for measuring freezing
tolerance. No discernible patterns were observed between floral freezing
tolerance and geographic origin of the strawberry genotypes or species.

When measured at the a =0.10 significance level, there were significant
genotypic differences among the three sour cherry cultivars for pistil freezing
tolerance when measured by electrolyte leakage following 16 days of 4° C
treatment (Table 3). No other significant genotypic differences in pistil freezing

tolerance were observed in sour cherry.

29

Table 3. Temperature at which 50% electrolyte leakage or half of the pistils or

receptacles plus pistils were dead occurred for strawberry receptacles, leaf discs,

and sour cherry pistils removed from blooming, whole plants exposed to 4° C.

 

 

 

 

 

 

Visual Electrolyte Leakage
Scoring
Receptacles Leaf
Receptacles Discs
Genotype 0 days 16 days 0 days 16 days 16 days
of4°C of4°C of4°C of4°C of4°C
Honeoye -3.8 -5.3 ** -3.6 -6.7 ** -13.2
Chandler -2.6 -4.5 ** -3.0 -6.5 ** -10.6
EBB1 -1.0 -4.0 ** -3.8 -4.8 "s -15.3
NC 96-28-1 -2.5 -5.1 ** -3.4 -4.7 "s -14.4
NC 95-18-5 -2.5 -6.0 ** -6.3 -6.3 "s -11.7
NC 95-16-6 -3.1 -5.3 ** -4.2 -5.3 "s -11.4
NC 96-29-1 -3.5 -6.1 ** -5.3 -4.0 "s -11.4
RH-18 -4.2 -5.0 "s -4.5 -5.7 "s -12.1
FRA 0883 -3.0 -7.6 ** -2.9 -6.0 ** -12.2
FRA 368 -3.2 -6.3 ** -3.8 -7.0 ** -11.5
F-test within ** ** ** ** **
column
Pistils Pistils Leaf
Discs
Montmorency -1.5 -2.5 "s -1.9 -2.3 "s -10.9
Rheinische -2.0 -2.8 "s -2.7 -3.4 "3 -11.4
Schattenmorelle -
Erdi Botermo -2.2 -2.7 "s -2.0 -2.9 "s -11.2
F-test within ns ns ns * nS
column

 

 

 

 

**, ns: within row represent significant at a = 0.05 or non-significant t-test for

comparisons of 0 days and 16 days of cold treatment for both electrolyte leakage

and visual scoring. *, ns: within column represent significant at a = 0.10 or non-

significant F-test.

3O

Relative Water Content (R WC) of acclimated strawberry receptacles:
No significant differences in the RWC were detected between non-

acclimated and acclimated 'Honeoye' or 'Chandler' receptacles (Table 4).

Table 4. Relative water content of strawberry receptacles/pistils from non-
acclimated and acclimated plants.

 

 

Cultivar 0 days of 4° C 16 days of 4° C
Honeoye 67.4% 678%”
Chandler 77.4% 78.6%"s

 

ns: non-significant t-test within row at or = 0.05.

Discussion

The results from this study demonstrate that fully expanded leaves of
actively growing, flowering strawberry and sour cherry possess the ability to cold
acclimate after 8 to 16 days of exposure to 4° C with a 14 hour photoperiod
(Table 1.2), with maximal cold hardiness levels attained of —10.9° C and -13.2°
C after 16 days of 4° C exposure for sour cherry and strawberry, respectively. By
comparison, Arabidopsis leaves reach a maximal cold hardiness, measured as
the 50 % electrolyte leakage point, of approximately -9.0° C following 12 days of
exposure to 4° C, with no additional increase in freezing tolerance observed
following 4° C up to 25 days (Gilmour et al., 1988).

Receptacles and their attached pistils of most of the strawberry genotypes
examined had enhanced freezing tolerance following exposure to 4° C for 16

31

days (Table1.3). After this 16 day period, the strawberry pistils/receptacles often
showed statistically significant improvement in freezing tolerance when
measured by electrolyte leakage or by visual scoring.

In contrast, the three sour cherry cultivars examined in this study showed
no statistically significant enhancement in pistil freezing tolerance under the
same conditions reported for strawberry. Although not a direct comparison,
these data are consistent with the reported literature on floral freezing tolerance
in strawberry and Prunus. Flowers of day-neutral strawberry have been
observed to have enhanced freezing tolerance in the autumn, compared to
flowers borne earlier in the season (Boyce and Marini, 1978). Conversely, peach
flowers have been reported to either not possess, or show only slight differences
in, the ability to de-acclimate when brought out of dormancy in cool conditions as
opposed to warm (Lu and Rieger, 1993). Data from field observations place the
freezing tolerance of sour cherry pistils at full bloom to be approximately -2.2° C
(Dennis and Howell, 1974), close to the values determined under controlled
conditions in this study. Taken together these findings suggest sour cherry pistils
are unlikely to be able to withstand freezing temperatures below approximately
-2 to -3°C at full bloom. Perhaps sour cherry selections have developed
adaptive changes in bloom date in order to avoid freezes instead of developing
physiological freezing tolerance per se.

Some differences were observed in the freezing tolerance of strawberry
and receptacles when measured by electrolyte leakage as opposed to visual

scoring. For many genotypes these differences were minor, often less negative

32

values being measured by visual scoring than by electrolyte leakage. However,
for two strawberry genotypes, visual scoring seemed to be a better indicator of
freezing damage. NC 95-18-5 receptacles showed no significant change in
freezing tolerance when measured by electrolyte leakage, with non-acclimated
plants Showing a surprisingly high freezing tolerance. The visual scoring of these
same samples indicate that there was a clear difference in the freezing damage
between the 0 and 16 days of exposure to 4° C. Also, NC 96-29-1 pistils,
showed a high freezing tolerance at 0 days of exposure to 4° C and low freezing
tolerance after 16 days of 4° C when measured by electrolyte leakage. The
cause of these discrepancies is not known. Since visual scoring is an un-
ambiguous method for determining the viability of these tissues, as well as being
much more rapid than electrolyte leakage, it is recommended that visual scoring
be utilized as a method for determining strawberry and sour cherry pistil damage
following controlled freezes.

Significant variation for floral freezing tolerance was observed in the
strawberry genotypes examined at either the non-acclimated or acclimated state,
approximately 3° C between the extreme genotypes. Similarly, significant
variation in the scale of freezing tolerance improvement following low
temperature exposure was seen in strawberry. These results suggest there may
be useful variation for floral freezing tolerance and floral cold acclimation in
strawberry. Variation in freezing tolerance did not appear as great in sour cherry,

but only a small number of genotypes were examined.

33

Although significant variation in floral freezing tolerance was observed
among the strawberry genotypes tested, no discernible Boo-geographical
patterns could explain this variation. The strawberry selections tested were
chosen to examine a wide range of latitudes and elevations of origin. However,
these factors may not be the best predictors of temperature during bloom.
Strawberries are often covered by snow in northern areas and at upper
elevations. The potential for an insulating snow cover and also for the variance
in spring-temperature fluctuations may be better indicators of a plants ability to
withstand freezing temperatures during bloom. Additionally, the variability
measured in these experiments may be suggestive of ecological variation during
the autumn. Strawberry flowers of short-day genotypes are initiated in the
autumn in response to decreasing day-lengths and temperature. The cold
acclimation ability observed here probably exists as part of the plant’s
mechanism for withstanding autumn and winter temperatures. AS mentioned
earlier, the length of cold treatment required to observe an increase in pistil
freezing tolerance in strawberry is longer than is likely to occur in the spring.
Such a prolonged, but non-damaging cold period is more likely to occur in the
autumn. Therefore, the variation in floral cold acclimation ability may be better
explained by exploring climatic and ecological variation of these genotypes at the
ends of the growing season as the plants are beginning to harden off.

The freezing tolerance of leaves was not significantly correlated with the
freezing tolerance of pistils. These results suggest freezing tolerance of leaves is

not a good indicator of floral freezing tolerance or of the ability for flowers to cold

34

acclimated. Probably the best way to screen for floral freezing tolerance in
breeding parents is to visually assess damage in non-acclimated plants,
considering the length of time required to observed a significant increase in floral
freezing tolerance. The plants have the ability to genetic capability to cold
acclimate so an important question is why are pistils either unable or less
proficient in the ability to acclimate?

Several studies have shown a correlation between tissue water content
and freezing tolerance (Graham and Mullin, 1976; Johnston 1923). Our results
showed no significant difference in the tissue water content of non-acclimated
and acclimated ‘Honeoye’ and ‘Chandler’ receptacles and pistils. Although, only
two cultivars were examined, this evidence suggests that genetic and
physiological factors other than water content play a role in freezing tolerance
following cold acclimation.

The reason why strawberry and sour cherry flowers’ differ in their ability to
cold acclimate is not known. One possibility is that the addition of receptacle
tissue enhances the pistil freezing tolerance in strawberries. No observations
were made of non-viable styles attached to viable receptacle tissue following a
freezing event. However, other reports of controlled freezes of strawberry floral
parts have shown no Significant difference in the freezing tolerance of pistils and
receptacles at 5 stages of floral development (Ki and Warmund, 1992). In fact,
there was a striking correspondence in the critical temperature for freezing

damage between the 2 tissues for 4 of the 5 developmental stages examined. It

35

therefore seems unlikely that the ability of strawberry pistils to acclimate is an
artifact of the inclusion of receptacle tissue in the freezing damage assessment.

Some authors have suggested that leaves are critical for the sensing of,
and attainment of cold acclimation in fruit trees (Weiser, 1970). Defoliation of
apple trees has been shown to limit the ability of these plants to cold acclimate
as they enter dormancy (Howell and Stackhouse, 1973; Weiser, 1970). One
speculation is that sour cherry pistils have a reduced ability to acclimate,
compared to strawberry, because of the reduced canopy development of these
plants compared to strawberry, which maintain a fuller canopy during flowering.
This hypothesis could be tested by examining the effects of reduced leaf area on
strawberry floral cold acclimation.

Manipulation of one key regulator of cold acclimation in Arabidopsis has
led to an enhancement of freezing tolerance of this plant (Jaglo-Ottosen et al.,
1998). Knowledge that strawberry pistils also exhibit the ability to cold acclimate
raises the possibility that these plants may have a similar molecular machinery
underlying the cold acclimation response, and that it may be active in these
tissues. Regardless of the validity of this hypothesis, there is likely to be a
molecular component underlying the cold acclimation response in strawberry. By
identifying these components, it may be possible to improve floral freezing
tolerance of these plants and these tissues. In contrast, sour cheny pistils do not
appear to acclimate, suggesting that there may be a deficiency in the Signal
transduction pathway between the sensing of cold temperature and the onset of

acclimation, a deficiency which apparently does not exist in leaves. Identifying

36

which of these components, if any, is non-functional may ultimately lead to the

ability to manipulate the underlying genes and improve floral freezing tolerance.

37

Literature Cited

Anderson JA and J Whitworth 1993. Supercooling strawberry plants inoculated
with ice-nucleation-active bacteria and treated with Frostgard. HortScience
28:828—830.

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

Boyce BR and RP Marini 1978. Cold acclimation of everbearing strawberry
blossoms. HortScience 13:543-544.

Darrow GM 1966. The strawberry. Holt, Rinehart, and Winston, New York.

Dennis FG and GS Howell 1974. Cold hardiness of tart cherry bark and flower
buds. Res. Rep. Michigan State Univ. Ag. Exp. Station 220.

Gilmour SJ, RK Hajela, and MF Thomashow 1998. Cold acclimation in
Arabidopsis thaliana. Plant Physiol. 87:745-750.

Graham PR and R Mullin 1976. A study of flower bud hardiness in azalea. J.
Amer. Soc. Hort. Sci. 10127-10.

Guy CL, KJ Niemi, and R Brambi 1985. Altered gene expression during cold
acclimation of spinach. Proc. Natl. Acad. Sci. USA 82:3673-3677.

Hong B, R Barg, T-HD Ho 1992. Developmental and organ-specific expression
of an ABA- and stress-induced protein in barley. Plant Mol. Biol. 18:663-674.

Horvath DP, BK McLamey, and MF Thomashow1993. Regulation of
Arabidopsis thaliana L. (Heyn) cor78 in response to low temperature. Plant
Physiol. 103:1047-1053.

Houde M, RS Dhindas, and F Sarhan 1992. A molecular marker to select for
freezing tolerance in the Gramineae. Mol. Gen. Genet. 234:43-48.

Howell GS and SS Stackhouse 1973. The effect of defoliation time on
acclimation and dehardening of tart cherry (Prunus cerasus L.). J. Amer. Soc.
Hort. Sci. 98:132-136.

Howell GS and CJ Weiser 1970. The environmental control of cold acclimation
in apple. Plant Physiol. 45:390-394.

Hughes MA, and MA Dunn 1990. The effect of temperature on plant growth and
development. Biotechnology and Genetic Engineering Reviews 82161-88.

38

Jaglo-Ottosen KR, SJ Gilmour, DG Zarka, O Schabenberger, and MF
Thomashow1998. Arabidopsis CBF1 overexpression induces COR genes and
enhances freezing tolerance. Science 280: 1 04-1 06.

Johnston ES 1923. Moisture relation of peach buds during winter and spring.
Md. Agr. Expt. Sta. Bul. 255. p. 59-86.

Ki WK and MR Warmund 1992. Low-temperature injury to strawberry floral
organs at several stages of development. HortScience 27:1302-1304.

Linden L, P Palonen, and M Linden 2000. Relating freeze-induced electrolyte
leakage measurements to lethal temperature in red raspberry. J. Amer. Soc.
Hort. Sci. 125:429-435.

Lu 8 and M Rieger1993. Effect of temperature preconditioning on ovary
freezing tolerance of fully opened peach flowers. J. Hort. Sci. 68:343-347.

Monroy AF, Y Castonguay, S Laberge, F Sarhan, LP Vezina, and RS Dhindsa
1993. A new cold-induced alfalfa gene is associated with enhanced hardening at
subzero temperature. Plant Physiol. 102:873-879.

Ndong C, F Ouellet, M Houde, and F Sarhan 1997. Gene-expression during cold
acclimation in strawberry. Plant Cell Physiol. 38:863-870.

Perry KB 1998. Basics of frost and freeze protection for horticultural crops.
HortTechnology 8:10-15.

Rieger M 1989. Freeze protection for horticultural crops. Hort. Rev. 11245-109.

Scott DH and FF Cullinan 1939. Peach variety resistance to cold injury at
blossom time. Proc. Amer. Soc. Hort. Sci. 37:209-214.

Sukumaran NP and CJ weiser 1972. HortScience 7:467

Thomashow MF 1999. Plant cold acclimation: freezing tolerance genes and
regulatory mechanisms. Ann. Rev. Plant Physiol. Plant Mol. Biol. 50:571-599.

Weiser CJ 1970. Cold resistance and injury in woody plants. Science 169:1269-
1278.

Wisniewski M, R Webb, R Balsamo, TJ Close, XM Yu, and M Griffith 1999.
Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60:
A dehydrin from peach (Prunus persica). Physiologia Plantarum 105:600-608.

39

Chapter 2 - Identification of CBF orthologs in strawberry (Fragaria x
ananassa) and sour cherry (Prunus cerasus)

Low temperature is one of the most important environmental factors
limiting crop plant growth, distribution, and productivity (Sakai and Larcher,
1987). Plant species differ greatly in their ability to develop freezing tolerance
through a process known as cold acclimation (Thomashow, 1999). Traditional
breeding of many major crops has met with only limited success in improving
freezing tolerance. For example, the freezing tolerance of wheat cultivars has
not been substantially improved in the past 50 years (Fowler and Gusta, 1979).
As a result, considerable effort has been spent to understand the nature of cold
acclimation, in the hopes that novel strategies for improving crop freezing
tolerance may be developed.

Many physiological and biochemical changes occur in plants in response
to cold temperature, including changes in gene expression (Alberdi and
Corcuera, 1990; Guy, 1990). Many cold-induced genes have been identified in
several crop species, including alfalfa (Medicago sativa), spinach (Spinacia
oleracea), barley (Hordeum vulgare), and peach (Prunus persica) (Wisniewski et
al., 1999; Monroy et al., 1993; Hong et al., 1992; Guy et al., 1985). In
Arabidopsis, studies of the molecular basis of cold acclimation have identified a
common cis-acting regulatory element, the C-repeatlDehydration Responsive
Element (CRT/DRE) which has the conserved core sequence CCGAC, and

found within the promoters of several cold induced (COR) genes (Stockinger et

40

al., 1997). This element is present in one to multiple copies in the promoters of
many cold-induced plant genes, including COR6.6, COR15a, COR47, and
COR78 of Arabidopsis and BN115 of Brassica napus (Stockinger et al., 1997;
Yamaguchi-Shinozaki and Shinozaki 1994; Baker et al., 1994; Jiang et al., 1996).
In turn, all of the currently characterized Arabidopsis COR genes are coordinately
up-regulated by CBF 1, CBF2, or CBF3, a family of cold and drought inducible
transcriptional activators that bind to promoters containing a CRT/DRE
(Thomashow et al., 2001; Kasuga et al., 1999; Liu et al., 1998; Jaglo-Ottosen et
al., 1997). Even though freezing tolerance is a complexly inherited trait,
manipulation of just one gene, CBF 1, has been shown to improve the whole plant
freezing tolerance of Arabidopsis (Jaglo-Ottosen, et al., 1997). Because CBF
genes are key regulators of cold acclimation and over-expression of CBF1, 2, or
3 is capable of improving the freezing tolerance of Arabidopsis, there is
Significant interest in determining if orthologs of the CBF gene family exist in
cultivated species and function in a similar fashion to improve freeze tolerance.

Many temperate fruit crops are in the Rosaceae and their distribution is
restricted by low temperature stress. Floral damage is often the main form of
freeze damage, particularly in several of the early blooming members of the
Rosaceae (e.g. strawberry, cherry, peach, almond, apricot) (Rodrigo, 2000).
Many cultural techniques are employed to mitigate the damage caused by
freezes during bloom (Rieger, 1989). However, improved cultivars with

enhanced floral freezing tolerance would be of considerable interest to help

41

prevent crop loss caused by freeze damage and to help stabilize yearly yield
fluctuations.

One approach to develop cultivars with enhanced freezing tolerance is to
identify and utilize candidate genes playing an important role in this trait. It is
plausible that cold-induced genes exist within the Rosaceae that are orthologous
to those in Arabidopsis capable of improving freezing tolerance. Whether these
genes can be utilized, through either biotechnological manipulation or
exploitation of natural variation, to improve floral freezing tolerance in rosaceous
crops is unknown. It is also unknown if the same relationship exists between
cold-induced gene expression and onset of cold-acclimation in rosaceous
flowers.

There is evidence from Arabidopsis suggesting that some of the genes
involved in cold acclimation may not be expressed in pistils. In transgenic
Arabidopsis containing reporter genes driven by the cor15a and cor78 promoters,
no expression of the reporter was observed in ovaries following whole-plant
exposure to 4 °C despite reporter expression in many vegetative and
reproductive tissues (Baker et al., 1994; Horvath et al., 1993). It is unknown
what effect the lack of expression of these genes has on the ability to cold
acclimate or develop freezing tolerance in Arabidopsis flowers. Considering the
extreme freezing susceptibility of rosaceous and other species’ pistils, it is
tempting to speculate that reduced expression of cold-acclimation genes is partly

responsible for the high sensitivity of these organs to freezing.

42

The interaction between cold temperature and photoperiod on cold
acclimation of woody fruit crops has been extensively studied (Howell and
Weiser, 1970; Weiser, 1970), and it is known that the over-wintering structures of
perennial, temperate fruit crops possess the ability to cold acclimate. When
tested under controlled conditions, detached pistils from sour cherry (Prunus
cerasus) do not appear to have the ability to cold acclimate (Owens, Chapter 1).
In contrast, receptacles with attached pistils of strawberry show an improvement
in freezing tolerance of approximately 3° C following whole plant exposure to 4°
C for 16 days (Owens, Chapter 1). If orthologs of the CBF gene family can be
identified in strawberry and sour cherry, the relationship between expression of
these CBF orthologs and cold acclimation in pistils of both these crops can be
determined.

The objectives of this study were to identify orthologs of the CBF gene
family in strawberry (Fragaria x ananassa) and sour cherry (Prunus cerasus),
and to determine if any putative orthologs were expressed in response to low
temperature, and if so, were there any differences in expression levels in leaves

and pistils of these two crops.

Materials and Methods
Plant Material and Growing Conditions:

Containerized, dormant, rooted runners of ‘Honeoye’ Fragaria x
ananassa and 3 year old, dormant plants from rooted cuttings of Prunus cerasus

‘Montmorency’ were grown in growth chambers maintained at 24 °Cl18 °C with

43

14 h day-lengths at 800 mmol-m'2~s'1 photosynthetically active radiation (PAR)
and 10 h nights until the onset of flowering. Four degree cold treatments were

administered by transferring plants to a cooler maintained at a constant 4 °C with

14 h day-lengths at 200 mmol-m’z-s'1 PAR and 10 h nights.

RNA extractions, northerns:

RNA extractions followed the protocol reported by Tao et al. (1999). Total
RNA was extracted from both young, developing sour cherry and strawberry
leaflets, pistils and receptacles from fully open flowers by grinding fresh tissue
under liquid nitrogen, homogenized in phenolzchloroformzisoamyl alcohol
(25:24:1 vlvlv), mercaptoehtanol, and 0.1 M Tris (pH 9.0), 0.1 M LiCI, 1% SDS,
and 10 mM EDTA. The aqueous phase was extracted with phenol: chloroform:
isoamyl alcohol 3 times, and then precipitated with LiCI at a final concentration of
4 M overnight at -20° C. The RNA was recovered, re-suspended, and
precipitated with sodium acetate and isopropanol, re-suspended and precipitated
a second time with sodium acetate and ethanol.

Northern analyses were carried out according to standard molecular
biology methodology texts (Sambrook et al. 2001) Twenty micrograms total RNA
was separated on agarose under denaturing conditions, transferred to a nylon
filter, and probed with randomly-primed 32P-labeled probes. The final stringency

of washes was 60° C without formamide, and 0.2% SSC.

44

Degenerate primer PCR, iPCR, sequencing:

Degenerate primers for CBF were designed by aligning CBF or putative
CBF orthologs from Arabidopsis thaliana (Stockinger et al., 1997), Brassica
napus (Zhou et al., unpublished submission to GenBank), and Nicotiana tabacum
(Durrant et al., unpublished submission to GenBank) (Figure 1). Three regions of
similarity were found between these sequences and used to design nested
primers for PCR. The degenerate primers used to amplify partial genomic
fragments of putative CBF orthologs in strawberry and sour cherry are listed

below (Table 1).

45

CBF1 (1)

 

BnDREBP (1)
Ni cotiana (1)
Consensus (1)
CBF1 (29)
BnDREBP (32)
Nicotiana (41)
Consensus (41)
CBF1 (69)
BnDREBP (72)
Nicotiana (81)
Consensus (81) ‘ .
121 160
CBF1 (109) ;/;E” ~ :m;,ssessgs..y

 
 
 
 

BnDREBP (112) QAE -INNTTTDHGLDM
Nicotiana (121) ,, :3 - _ "~" i ”9 S-EAENMPEY---S
Consensus (121) RLRIPESTCAKDIQKAAAEAAIAFQDETCDTTTTDHGLDM

CBF1 (149)
BnDREBP (151)
Nicotiana (157)
Consensus (161)

CBF1 (189)
BnDREBP (190)
Nicotiana (194)
Consensus (201)

 

SVQWNHNYDGBGDGDVSLwSY

Figure 1. Amino acid alignment of CBF1 and conceptual translations of CBF-like
cDNAs reported in GenBank (Brassica napus DREBP AF084185, Nicotiana
tabacum AP2 containing protein AAG43548). Identical residues are indicated by
shaded boxes. Positions of similarity used to design degenerate primers are
indicated by the boxed areas in the consensus sequence, which is the CBF1
sequence minus gaps.

46

Table 5. PCR primers utilized in this study.

 

 

Forwarda Reverse
CBF CCNAARAARCCNGCNGGNAG GGNARNARCATNCCYTCNGCC
Degenerate (43) (194)
CBF CCNAARAARCCNGCNGGNAG TCNGCRAARTTYAARCA (112)
Degenerate (43)
nested
iPC R cherry TCGCGCTGAGAGGTCGGTCT AGTCTCCTI'GAAATTC'ITCC
iPCR TI‘GCC'ITGAGGGGCCGGTCT AGTCTCC'ITGAAA’ITCTTCC
strawberry

iPCR nested GCKTGCTTRAACTI‘CGCMGAA CCTACCAGCAGGYTI‘CTTAGG
a Degenerate nucleotides were N = (AGTC), R = (AG), Y = (CT), K = (GT), M =
(AC). Numbers in parentheses indicate amino acid start position in Figure 1.

The first round of amplification was run with the first set of primers
reported in Table 1. Reactions were carried out in 25 pL volumes at final
concentrations of: buffer (10 mM Tris, 50 mM KCI, 0.001% gelatin, pH8.3), 0.05
mM dNTPs, 0.4 pM each primer, 3.0 mM MgCl2, 0.04 U-pL" Taq polymerase,
and 100 ng genomic DNA as template. The reactions were run at 94°C for 5
min, followed by 35 cycles of 94° for 1 min, 50° C for 1 min, and 72° C for 1 min,
with a final elongation of 72° C for 5 min. One microliter of these reaction
products were then utilized as template for a nested PCR reaction possessing
the same conditions as listed above, but utilizing the nested primers.

PCR products of interest were excised from 1.0% agarose, and purified
with Qiagen's Gel Extraction Kit (Qiagen Corp., Valencia, CA). Purified PCR
products were then cloned into the vector pGEM-T Easy (Promega Corp.,
Madison, WI) and sequenced by the Michigan State University DNA sequencing
facility.

The resultant sequence was then used to design inverse PCR (iPCR)

primers for obtaining flanking genomic sequence of the fragment already

47

obtained. The iPCR protocol followed a modified version of the protocol reported
by Willis et al. (1997). One microgram of total genomic DNA was restriction
digested with 5 U Hindlll in 10 pL overnight at 37° C, phenol:chlorofrom (1:1)
extracted, precipitated in sodium acetate and ethanol, and re-suspended in
water. Three hundred micrograms of digested DNA were self-ligated at a
concentration of 0.5 pg-mL'1 and 0.02 U- pL“ overnight at 15° C. PCR was then
conducted with the primers listed in Table 1. A nested PCR reaction was then
run with 1 pL of product from the first reaction as template for the second, using
the nested iPCR primers reported in Table 1. Nested PCR products were cloned

and sequenced as described above.

Results

Nested, degenerate primer-PCR yielded small, 225 bp fragments from
strawberry 'Honeoye' and sour cherry 'Montmorency' genomic DNA that were the
same size as a fragment from Arabidopsis genomic DNA. This fragment was
predicted to contain the entire AP2 domain plus a small 5' portion of the gene
based on the position of the nested PCR primers. Sequencing of the cloned
fragment revealed that the degenerate PCR products had predicted
approximately 95% amino acid identity to the AP2 domain of CBF1 with a small
portion 5' to the AP2 domain (data not shown). Nested inverse PCR (iPCR)
primers were designed from this sequence and iPCR reactions yielded fragments
ranging from 1 to 1.5 kb in length. Sequencing of these fragments yielded ORFS

that contained the full predicted amino acid coding region of these genes (Figure

48

2). Primers from this full length sequence were designed and used to amplify
and Clone full-length genomic clones of the putative CBF orthologs from
strawberry and sour cherry. These putative orthologs were named Prunus

cerasus CBF (PcCBF) and Fragaria x ananassa CBF (FaCBF).

CBF1
PcCBF
FaCBF

CBF1
PcCBF
FaCBF

CBF1
PcCBF
FaCBF

 

 
  
  
 

CBF1 (141) TTTTDHGLDMEETMVEAIYTPEQSE
PcCBF (135) SDEASG ----- A TAVPASE FTSASEQ ~ Iiiéffj
FaCBF (135) SEWSK -------- ATEAVEAESEQ ‘

CBF1 ( 1 8 8 ) g‘r‘ Aime: \:

PcCBF (177) S.

\. ...... .-:;:. ....

FaCBF ( 1 7 4 ) g \ I'. ..,.;.::..,.>5‘/, '

Figure 2. Amino acid sequence alignment of CBF1, PcCBF, and FaCBF.
Identical residues are shaded.

Northern analyses were conducted to determine the effects of 4° C
treatment on the mRNA expression of PcCBF and FaCBF. No expression of
these two genes was evident in non-acclimated leaf tissue of either species

(Figure 3). Following whole plant exposure to 4° C for 1 hour, faint expression of

49

PcCBF was observed in sour cherry leaves, and strong expression of FaCBF in
strawberry leaves. Of the time points measured in this experiment, 4 hours of 4°
C treatment yielded the highest level of expression for both PcCBF and FaCBF in
sour cherry and strawberry leaves, respectively. Expression appeared to begin
to taper off after 4 hours of cold treatment, as the mRNA expression was reduced
after 24 hours of exposure to 4° C in both strawberry and sour cherry leaves.

No PcCBF or FaCBF expression was detected by northern analysis in

either sour cherry pistils or strawberry receptacle tissue in any of the treatments

 

    

 

of 4°C (Figure 4).
0 1 hr 4 hr 24 hr
PcCBF
elF4a
0 1 hr 4 hr 24 hr
FaCBF
eIF4a

 

Figure 3. PcCBF and FaCBF transcript levels determined by northern
analysis in leaves of sour cherry ‘Montmorency’ and strawberry ‘Honeoye’
following 0 hour, 1 hour, 4 hours, and 24 hours of exposure to 4°C.
Eukaryotic elongation factor 4a (elF4a) was used as a RNA loading
control.

50

0 1hr 4hr 24hr

 

eIF4a

 

FaCBF

eIF4a

 

Figure 4. PcCBF and FaCBF transcript levels determined

by northern analysisin pistils of sour cherry 'Montmorency'
and receptacles with pistils of strawberry ‘Honeoye’ following
0 hour, 1 hour, 4 hours, and 24 hours of exposure to 4°C.
e|F4a was used as a RNA loading control.

Discussion

The putative CBF orthologs identified in this study have 48% deduced
amino acid sequence identity with the characterized CBF 1, 2, and 3 genes from
Arabidopsis thaliana. These proteins have very high similarity through the entire
AP2 domain. There are several additional conserved stretches: a PKKPAGRK 5'

to the AP2 domain, a AWRL 3' to the AP2 domain, and a NMAEGMLLPP near

51

the carboxy-terrninus. Similar conserved regions have been reported for CBF-
like proteins from Secale cereale, Brassica napus, Tn'ticum aestivum, and
Lycopersicon esculentum (Jaglo et al., 2001). The mRNA expression profile in
response to cold appears to be very similar to that observed in Arabidopsis. The
CBF gene family of Arabidopsis is up-regulated very quickly in response to cold
temperature, with mRNA expression observed after 15 min of exposure to 4° C
(Gilmour et al, 1998). mRNA expression hits a peak approximately 4 hours after

constant 4° C and then gradually tails off, often being low, but present after 24 h

of constant 4° C treatment (Gilmour et al., 1998). The strawberry and sour
cherry CBF orthologs described here have a similar mRNA expression profile, at
least in vegetative tissues. FaCBF and PcCBF are up-regulated within 1 hour of
exposure to 4° C, appear to have peaked at 4 h, and then decrease substantially,
but are still present after 24 h. Gene expression at further time points, beyond 24
hours of exposure to 4° C was not measured in these experiments.

Arabidopsis, an ancient polyploid, has 3 characterized cold-inducible CBF
genes (Gilmour et al., 1999). Only one putative CBF ortholog was identified from
both strawberry and sour Cherry. Considering that CBF exists as a multi-gene
family in Arabidopsis, strawberry, an octoploid, and sour cherry, a tetraploid, may
have additional, functional, cold-inducible CBFS within their genomes. Southern
analysis of the sour cherry and strawberry genome with the CBF orthologs
characterized in this study reveal complex banding patterns of varying intensities
(data not shown). Considering the polyploid nature of these crops it may be

difficult to determine the total number of functional CBF or CBF-like proteins

52

operating in these species. Whether or not the 3 cold-inducible CBF gene family
members present in Arabidopsis possess unique or redundant functions is not
known. Determining whether or not there are functional redundancies between
theoretical CBF gene family members within rosaceous species will be
challenging. One of the theoretical advantages of polyploidy is the relaxation of
selection pressure on duplicated genes (Soltis and Soltis, 1993). The possibility
exists that sour cherry and strawberry may possess several CBF-like pseudo-
genes or paralogs with diverged function.

Despite the lack of FaCBF expression in strawberry receptacles with
pistils, these organs are capable of cold acclimation (Owens, Chapter 1). One
fundamental question is: are the CBF gene family members required for cold
acclimation in higher plants? Arabidopsis has 3 cold-induced CBF genes that
are linked in tandem, have high sequence identity with each other, and thus far
appear to have redundant functions (Medina et al., 1999; Gilmour et al, 1998). It
is currently unknown what effect the silencing or the mutation of these genes will
have on the capability of Arabidopsis to acclimate. Strawberry receptacles may
be a system in which CBF expression is not evident, but cold acclimation does
occur. However, it is unknown what effect CBF expression would have on the
ability of strawberry receptacles to cold acclimate.

The molecular mechanisms underlying the cold acclimation response in
strawberry receptacles are not known. There are many characterized cold-
induced genes that do not contain CRT/DRE and are not directly regulated by

CBF. Several cold-induced genes possess ABA-responsive elements (ABRE)

53

within their promoters, and are up-regulated in an ABA dependent fashion.
Sixteen days of 4° C treatment are required to observe a significant increase in
freezing tolerance in strawberry receptacles when compared to non-acclimated
plants. Open strawbeny flowers are not normally subjected to this extensive of a
cold treatment under field conditions. Perhaps a general stress response,
including an increase in ABA production, leads to the induction of many ABRE
containing genes or other genes responsible for providing increased freezing
tolerance in a CBF-independent fashion. Information on the expression of both
CRT/DRE and non-CRT/DRE containing, cold-inducible genes within the
Rosaceae should provide additional information for testing some of these
alternative hypotheses.

Several general approaches could be taken to utilize the information
obtained in this study to ultimately improve the floral freezing tolerance of
rosaceous fruit cultivars. One fundamental question is, why do strawberry and
sour cherry pistils acclimate less so, or not at all, while the leaves acclimate to a
greater extent? Differential expression of CBF orthologs may help explain the
difference between these two tissues. Putative cold-inducible CBF orthologs
exist within the Rosaceae, but do not appear to be expressed in pistils of either
strawberry or sour cherry. However, strawberry receptacles and pistils appear to
have the ability to cold acclimate in the absence of CBF expression. If the ability
of these organs to acclimate is not due to CBF expression, an elucidation of the
molecular mechanisms leading to improved floral freezing tolerance of strawberry

following exposure to cold temperature could lead to novel methods for improving

54

floral freezing tolerance in this crop. In contrast, in sour cherry, in which pistil
cold acclimation is not evident, and interesting question is what differs between
strawberry and sour cherry that prevents sour cherry pistils from acclimating in
response to cold? An additional approach would be to observe the effects of CBF
over-expression on pistil freezing tolerance. In short, studies on the basis of the
cold acclimation ability in strawberry receptacles, or the basis of the differences
observed between strawberry and sour cherry pistils may provide information

useful for the development of cultivars with enhanced floral freezing tolerance.

55

Literature Cited

Alberdi M, and LJ Corcuera 1991. Cold acclimation in plants. Phytochemistry
30:3177-3184.

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

Fowler DB, and LV Gusta 1979.Selection for winterhardiness in wheat. I.
Identification of genotypic variability. Crop Sci. 19:769-772.

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

Gilmour SJ, AM Sebolt, MP Salazar, JD Everard, and MF Thomashow 2000.
Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple
biochemical changes associated with cold acclimation. Plant Physiol. 12421854-
1865.

Graham J, RJ McNicol, K Grieg 1995. Towards genetic based insect resistance
in strawberry using the cowpea trypsin inhibitor gene. Ann. Appl. Biol. 127:163-
173.

Guy CL, KJ Niemi, and R Brambi 1985. Altered gene expression during cold
acclimation of spinach. Proc. Natl. Acad. Sci. USA 82:3673-3677.

Guy CL 1990. Cold acclimation and freezing stress tolerance: role of protein
metabolism. Ann. Rev. Plant Physiol. Plant Mol. Biol. 41:187-223.

Hong B, R Barg, T-HD Ho 1992. Developmental and organ-specific expression
of an ABA- and stress-induced protein in barley. Plant Mol. Biol. 18:663-674.

Horvath DP, BK McLamey, and MF Thomashow 1993. Regulation of

Arabidopsis thaliana L. (Heyn) cor78 in response to low temperature. Plant
Physiol. 103:1047-1053.

Howell GS and SS Stackhouse 1973. The effect of defoliation time on
acclimation and dehardening of tart cherry (Prunus cerasus L.). J. Amer. Soc.
Hort. Sci. 98:132-136.

Howell GS and CJ Weiser 1970. The environmental control of cold acclimation
in apple. Plant Physiol. 45:390-394.

56

Jaglo KR, S Kleff, KL Amundsen, X Zhang, V Haake, JZ Zhang, T Deits, and MF
Thomashow 2001. Components of the Arabidopsis C-repeatldehydration-
responsive element binding factor cold-response pathway are conserved in
Brassica napus and other plant species. Plant Physiol. 127:910-917.

Jaglo-Ottosen KR, SJ Gilmour, DG Zarka, O Schabenberger, and MF
Thomashow 1998. Arabidopsis CBF1 overexpression induces COR genes and
enhances freezing tolerance. Science 280: 1 04-1 06.

Kasuga M, Q LiU, S Miura, K Yamaguchi-Shinozaki, and K Shinozaki 1999.
Improving plant drought, salt, and freezing tolerance by gene transfer of a single
stress-inducible transcription factor. Nature Biotech. 172287-291.

Liu O, M Kasuga, Y Sakuma, H Abe, S Miura, K Yamaguchi-Shinozaki, K
Shinozaki 1998. Two transcription factors, DREB1 and DREBZ, with and
EREBP/AP2 DNA binding domain separate two cellular signal transduction
pathways in drought- and Iow- temperature-responsive gene expression,
respectively, in Arabidopsis. Plant Cell 10:1391-1406.

McKown R, G Kuroki, and G Warren 1996. Cold responses of Arabidopsis
mutants impaired in freezing tolerance. J. Exp. Bot. 47:1919—1925.

Monroy AF, Y Castonguay, S Laberge, F Sarhan, LP Vezina, and RS Dhindsa
1993. A new cold-induced alfalfa gene is associated with enhanced hardening at
subzero temperature. Plant Physiol. 102:873-879.

Rieger M 1989. Freeze protection for horticultural crops. Hort. Rev. 11:45-109.

Rodrigo J, 2000. Spring frosts in deciduous fruit trees - morphological damage
and flower hardiness. Scientia Horticulturae 85:155-173.

Sakai A, and W Larcher 1987. Frost survival in plants: responses and adaptation
to freezing stress. Springer-Verlag, Berlin.

Soltis DE and PS Soltis, 1993. Molecular data-and the dynamic nature of
polyploidy. Crit Rev Plant Sci 12:243-273.

Stockinger EJ, SJ Gilmour, MF Thomashow 1997. Arabidopsis thaliana CBF1
encodes an AP2 domain-containing transcriptional activator that binds to the C-
repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in
response to low temperature and water deficit. Proc. Natl. Acad. Sci. USA
94:1035-1040.

Tao R, H Yamane H, A Sugiura, H Murayama, H Sassa, and H Mori 1999.
Molecular typing of S-alleles through identification, characterization and cDNA
cloning for S-RNases in sweet cherry. J. Amer. Soc. Hort. Sci. 124:224-233.

57

Thomashow MF 1999. Plant cold acclimation: freezing tolerance genes and
regulatory mechanisms. Ann. Rev. Plant Physiol. Plant Mol. Biol. 50:571-599.

Thomashow MF, SJ Gilmour, EJ Stockinger 2001. Role of the Arabidopsis CBF

transcriptional activators in cold acclimation. Physiologia Plantarum 112:171-
175.

Warren G, R McKwon, A Mario, and R Teutonico 1996. Isolation of mutations
affecting the development of freezing tolerance in Arabidopsis thaliana (L.)
Heynh. Plant Physiol. 111:1011-1019.

Willis TG, DM Jadayel, LJA Coignet, M AbdulRauf, JG Treleaven, D Catovsky,
MJS Dyer 1997. Rapid molecular cloning of rearrangements of the IGHJ locus
using long-distance inverse polymerase chain reaction. Blood 90:2456-2464.

Wisniewski M, R Webb, R Balsamo, TJ Close, XM Yu, and M Griffith 1999.

Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60:
A dehydrin from peach (Prunus persica). Physiologia Plantarum 105:600-608.

58

Chapter 3 - Enhancement of freezing tolerance of strawberry by

heterologous expression of CBF1

Many temperate fruit crops suffer yield reductions due to freeze damage.
Early season freezes during or just prior to bloom are the main cause of low-
temperature induced crop loss (Rodrigo, 2000). Several early blooming
rosaceous crops, such as strawberry (Fragaria x ananassa), and many of the
cultivated Prunus species, are particularly prone to floral freeze damage. Plant
species differ greatly in their ability to develop freezing tolerance through a
process known as cold acclimation (Thomashow, 1999). Many physiological and
biochemical changes occur in plants in response to cold temperature, including
changes in gene expression (Alberdi and Corcuera, 1990; Guy, 1990). In
Arabidopsis, studies of the molecular basis of cold acclimation have identified a
common cis-acting regulatory element, the C-repeatlDehydration Responsive
Element (CRT/DRE) which is defined as a CCGAC, and found within the
promoters of several cold induced (COR) genes (Stockinger et al., 1997). In
turn, all of the currently characterized Arabidopsis COR genes are coordinately
up-regulated by CBF1, CBF2, or CBF3, a family of cold and drought inducible
transcriptional activators that bind to promoters containing a CRT/DRE
(Thomashow et al., 2001; Kasuga et al., 1999; Liu et al., 1998; Jaglo-Ottosen et

al., 1998).

59

CBF1 orthologs have been identified in two cultivated members of the
Rosaceae: sour cherry (Prunus cerasus) and strawbeny (Fragaria x ananassa),
suggesting the CBF-COR gene system may be conserved in this diverse family
(Owens, Chapter 2). The putative orthologs, Prunus cerasus CBF (PcCBF) and
Fragaria x ananassa CBF (FaCBF), share 48% amino acid identity with
Arabidopsis CBF1 and are up-regulated in leaves in response to cold
temperature. The goal of this study was to provide additional support for the
hypothesis that the CBF-COR gene system is conserved in the Rosaceae by the
heterologous expression of CBF1. The expected downstream events would be
a) an increase in freezing tolerance, and b) the up-regulation of CRT/DRE
containing, cold-inducible genes. Even though freezing tolerance is a complexly
inherited trait, manipulation of just one gene, CBF 1, has been shown to improve
the whole plant freezing tolerance of Arabidopsis (Jaglo-Ottosen, et al., 1997).
Because CBF genes are key regulators of cold acclimation and over-expression
of CBF1, 2, or 3 is capable of improving the freezing tolerance of Arabidopsis,
there is significant interest in determining if orthologs of the CBF gene family
exist in cultivated species and function in a similar fashion to improve freeze
tolerance.

Knowledge of the effects of CBF1 manipulation on the floral or whole plant
freezing tolerance of rosaceous plants is of significant interest. Many temperate
fruit crops are in the Rosaceae and their distribution is restricted by low
temperature stress. Commercially, however, floral damage is often the main

form of freeze damage, particularly in several of the early blooming members of

60

the Rosaceae (e.g. strawberry, cherry, peach, almond, apricot) (Rodrigo, 2000).
Many cultural techniques are employed to mitigate the damage caused by
freezes during bloom (Rieger, 1989). However, Improved cultivars with
enhanced floral freezing tolerance would be of considerable interest to help
prevent crop loss caused by freeze damage and to help stabilize yearly yield
fluctuations.

The objectives of this study were to determine the effects of over-
expression of CBF1 on the freezing tolerance of cultivated strawbeny vegetative

and reproductive tissues.

Materials and Methods
Plant Material/Conditions:

Transformation was conducted on crowns from tissue culture stocks of
'Honeoye' Fragaria x ananassa plants that had been maintained on a Murashige
and Skoog (MS) salts media with vitamins, but no hormones for approximately 20
weeks (4.3 g-L" MS salts (GIBCO), 0.5 mg-L“ nicotinic acid, 0.5 mg-L'1
pyridoxine, 0.1 mg-L’1 thiamine, 2 mg-L"1 glycine, 100 mg-L‘1 myo-inositol, 20 g-L'
‘ sucrose, 8 g-L‘1 agar, pH 5.6) (Graham et al., 1995). Following transformation
and regeneration, rooted explants were transferred to 10 x 10 x 20 cm plastic
pots containing sterile, soil-less potting media enclosed in a plastic bag, and
gradually hardened off by opening the bags slightly each day. Tissue culture

plants, regenerants, and rooted plants were maintained at 24°C, 16 h

photoperiods.

61

Plants were induced to flower by placing them under an 8 h photoperiod at
constant 18° C for 2 weeks. After the 8 h photoperiod treatment, the plants were

transferred back to a 16 h photoperiod.

Transformation:

The transformation protocol was that of Graham et al.(1995). Briefly, 1 to
2 mm transverse crown cross-sections were taken from tissue cultured 'Honeoye'
plants, inoculated for 24 hours with Agrobacterium tumefaciens isolate LBA4404
containing the binary vector with CaMV35S-CBF1 which contains the NPTII
marker, and placed on regeneration media (4.3 g-L" MS salts, 0.2 mg- L"
benzylamino purine, 0.2 mg- L" 2,4-D, 20 g-L" sucrose, 8 g-L" agar, 250 mg - L"
ticarcillin, and 250 mg-L" kanamycin sulfate, pH 5.6). The crown discs were
transferred to fresh regeneration media every 7 days with selection for 12 weeks.
Surviving healthy shoot tips were transferred to MS media with vitamins.

Control, wild-type plants used in these experiments, were non-transformed

‘Honeoye’ plants that had been regenerated from crown-discs in a similar fashion

as the transgenics.

DNA extraction, RNA extraction, Southerns, northerns:
Genomic DNA was extracted from young strawberry leaflets by grinding
fresh tissue under liquid nitrogen, homogenizing in buffer containing 2% CTAB,

100mM Tris, 1.4 M NaCl, 20 mM EDTA, 0.1 % DTT, 1% PVPP, extracting with

62

chlorofonnllAA, and precipitating once with isopropanol, and a second time with
70% ethanol (Graham et al., 1994).

Total RNA was extracted from both young, developing strawberry leaflets,
and receptacles with pistils from fully open flowers by grinding fresh tissue under
liquid nitrogen, and homogenizing in phenolzchlorofonnzisoamyl alcohol (25:24:1
vlvlv), mercaptoehtanol, and 0.1 M Tris (pH 9.0), 0.1 M LiCI, 1% SDS, and 10
mM EDTA. The aqueous phase was extracted with phenol: chloroform: isoamyl
alcohol 3 times, and then precipitated with LiCI at a final concentration of 4 M
overnight at -20° C. The RNA was recovered, resuspended, and precipitated
with sodium acetate and isopropanol, resuspended and precipitated a second
time with sodium acetate and ethanol (Tao et al., 1999)

Northern transfers were made by fractionating 20 pg of total RNA in 1%
agarose formaldehyde gels (Sambrook et al. 2001) and transferring it to nylon
membranes by capillary action. The filters were hybridized with randomly-primed
32P-labeled probes. Probes were made from full-length cDNA of CBF 1. elF4a
was used to probe the northern membranes to determine even loading of RNA.
The final stringency of washes was 60° C without formamide, and 0.2% SSC.

Southern transfers were made by fractionating 6 pg of genomic DNA
digested overnight at 37°C with Hindlll in 1% agarose gels (Sambrook et al.
2001) and transferring it to nylon membranes. The filters were hybridized with
randomly-primed 32P-labeled probes. Probes were made from full-length CBF1

cDNA.

63

Determination of freezing tolerance/Data analysis:

Freezing tolerance was determined on detached leaf-discs and receptacle
tissue. The leaf discs were detached from the whole plant, and stored in de-
ionized, distilled water until all had been placed in 16 mm diameter x 125 mm
long glass culture tubes. Three leaf discs or receptacles plus pistils were placed
in each culture tube. Data from 3 tubes were pooled for each temperature point
of the controlled freezing run (Sukumaran and Weiser, 1972).

F reezing-runs were conducted by placing the tubes in freezing-water
baths containing a mixture of ethylene glycol and water. The tubes were
maintained at 0 °C for 1 h, after which a small amount of de-ionized ice was
placed in contact with the plant material in each tube to nucleate ice formation.

The tubes were kept for an additional hour at 0 °C, and the temperature was then

decreased at the rate of 4 °C-h". Every 30 minutes a set of tubes was
transferred to a separate freezing-bath kept at the same temperature as the bath
it came from, and maintained at that temperature for an additional 30 minutes (for
a total of 1 h at each temperature). After 1 hour of freezing, tubes were placed
on ice until the completion of the freezing experiment. Freezing runs were
carried out to a final temperature of -20° C.

Following completion of the freezing run, tubes were placed in a rack on
top of ice, wrapped with plastic, and kept at 4 °C overnight. Following thawing, 3
mL of de-ionized, distilled water were added to each tube, and the tubes were
then shaken for 3 hours. Electrical conductivity of the water was measured from

each tube, the plant material in the tubes was placed at —80 °C for 1 hour, and

the original water was then added back to each tube for an additional 3 hours,
with agitation. A final electrical conductivity was then determined for the water in
eachtube.

Three sets of data points were generated for each temperature point of
the freezing run, with 3 tubes representing each data point. These data were
used to generate an electrolyte leakage curve over the range of temperatures
used in the freezing run. Three curves were generated per treatment.

Statistical significance of treatments was determined with a two-sample t-

test within each tissue type using Minitab 13.0 (Minitab Inc., State College, PA)

Results

Two transgenic strawberry lines transformed with a CaMV 35S-CBF1
construct were recovered from the selection media, designated 35-1-1 and 35-1-
2. PCR results confirmed the presence of the transgene in these two lines (data
not Shown). Southern analysis with a full-length CBF1 cDNA as probe revealed

a single insertion of the transgene in each of the two lines (Figure 1).

65

35-1 -1 35-1-2 wt

     

Figure 5. Southern analysis of wild-type and 35-1-1
and 35-1-2 ‘Honeoye’ strawberry plants transformed
with CBF1. Restriction digestion conducted with Hindlll

Northern analysis of the two transgenic lines probed with a full-length
CBF1 cDNA showed expression of the transgene in both actively growing leaf

tissue as well as receptacle tissue of fully open flowers (Figure 2).

66

Leaves Receptacles/Pistils
35-1-135-1-2 wt 35-1-135-1-2 wt

CBF1

elF4a

 

Figure 6. CBF1 transcript levels in wild-type plants and 35-1-1

and 35-1-2 transgenic plants which over—express CBF 1. Eukaryotic

elongation factor 4a (elF4a) was used as a RNA loading control.

The freezing tolerance of leaf-discs and receptacle tissues was
determined for the two transgenic lines. Both lines showed a significant increase
in freezing tolerance of leaf-discs compared to the wild type plants when
measured by electrolyte leakage (Table 1). The temperature at which 50%
electrolyte leakage occurred was —8.3°C and —10.4° C for non-acclimated lines
35-1-1 and 35-1-2, respectively. These values were approximately 4-5° C below
the freezing tolerance of wild type leaf discs. Leaf-discs taken from transgenic
whole plants exposed to 4° C for 16 days showed a less significant increase in
freezing tolerance when compared to wild type plants. All plants showed a
significant increase in freezing tolerance following 16 days of exposure to 4° C,
when compared to non-acclimated plants.

Freezing tolerance values of the receptacle and pistils of the transgenic
lines were not significantly different from those of wild type plants acclimated for
16 days or for 0 days (Table 1), although the freezing tolerance of receptacles

did increase following cold acclimation.

67

Table 6. Temperature at which 50% electrolyte leakage occurred for wild-type
‘Honeoye’ plants and 35-1-1 and 35-1-2 plants which over-express CBF1.

 

 

Non-Acclimated Acclimateda
Receptacles Receptacles
Leaf Discs + Pistils Leaf Discs + Pistils
wild type -4.8 a -2.0 -12.3 a -5.1
35-1-1 -8.3 b -2.6 -13.4 b -5.6
35-1-2 -10.4 c -2.1 -13.8 b -5.4
F-test within ** ns * ns

column

 

**, *, ns within column represent significant at or = 0.05, 0.10 or non-significant
F-test. a acclimated whole plants exposed to 4° C for 16 days. Values within
column followed by the same letter are not significantly different at a = 0.05 with
an LSD.

Discussion

The level of freezing tolerance enhancement observed in this study
between strawberry plants expressing CBF1 and the wild-type, non-transgenic
regenerated ‘Honeoye’ plants is of a similar order of magnitude as that observed
between transgenic and wild type Arabidopsis (Jaglo-Ottosen et al., 1997;
Gilmour et al., 2000). A range of freezing tolerance values has been observed
among different transgenic Arabidopsis lines often showing approximately a 3 to
4 °C increase in the 50% electrolyte leakage point of detached leaves between
non-acclimated transgenic and wild type plants. Although data from a large
number of transgenic lines has not been reported, there appears to often be a
positive correlation between transgene expression and degree of freezing

tolerance enhancement (Jaglo-Ottosen et al., 1997; Gilmour et al., 2000). In this

68

study, only 2 transgenic lines with similar transgene expression levels were
identified and they had similar freezing tolerances.

ln Arabidopsis, high CBF3 transgene expression appears to be negatively
correlated with plant vigor (Gilmour et al., 2000). Several lines expressing CBF3
at high levels have shown a marked increase in freezing tolerance and a
significant reduction in plant size compared to the wild type plants. Among our
transgenic strawberry lines, no apparent phenotypic differences were observed,
although the generation of additional strawberry transforrnants expressing higher
levels of CBF1, 2 or 3 might lead to a wider range of phenotypes.

No enhancement in freezing tolerance was observed in the receptacle
tissue, despite expression of the transgene in the receptacles at similar levels as
was observed in developing leaves. The reason for the lack of freezing tolerance
improvement in receptacle tissue, despite a significant improvement in leaves is
not known. However, there is evidence from Arabidopsis suggesting that some
of the genes involved in cold acclimation are not expressed in pistils. In
transgenic Arabidopsis containing reporter genes driven by the cor15a and cor78
promoters, no expression of the reporter was observed in ovaries following
whole-plant exposure to 4 °C despite reporter expression in many vegetative and
reproductive tissues (Baker et al., 1994; Horvath et al., 1993). Freezing
tolerance of Arabidopsis flowers has not been directly measured, but it is
possible that the signal transduction steps or components between CBF
induction and COR gene activation are not functional. Evidence for this comes

from the 3er mutant in Arabidopsis which is deficient in freezing tolerance

69

following cold acclimation, does not show expression of COR6. 6, COR15a, or
COR78 in response to cold temperature, even though CBF3 expression following
cold exposure is normal (McCown et al., 1996; Warren et al., 1996; Knight et al.,
1999). It has been postulated that SF R6 is required for cold-induced activation of
the CBF3 protein. It is possible SF R6 or some as yet unidentified component of
the cold acclimation molecular response is functioning differently in pistils and
leaves. However, strawberry receptacles appear to be able to acclimate in the
absence of expression of a strawberry CBF ortholog (Owens, Chapter 1 and 2).
This raises the possibility that there is either some defect in the Signal
transduction pathway leading to the induction of CBF in strawberry pistils and
receptacles, but that cold acclimation is still possible through a non-CBF
mediated pathway.

The data reported in this study support the hypothesis that the CBF-COR
gene system is conserved in the Rosaceae. CBF1 is a cold-inducible
transcriptional activator that has been shown to increase the freezing tolerance of
Arabidopsis when over-expressed by up-regulating a battery of genes containing
CRT/DRE in their promoters (Jaglo-Ottosen, et al., 1997). Although, a rosaceous
CRT/DRE containing gene was not identified, the enhancement in freezing
tolerance suggests there may be an intermediary step in which several CRT/DRE
containing proteins are up-regulated leading to the observed enhancement in
freezing tolerance. Alternatively, the freezing tolerance improvement in the

transgenic plants could be do to a positional effect of the transgene. Considering

7O

that two transgenic lines were generated that both Showed an improvement in
freezing tolerance, this seems unlikely.

The probability that the CBF-COR gene system is conserved in the
Rosaceae, is further supported by the identification of putative orthologs of CBF
in both strawbeny and sour cherry that have similar mRNA expression profiles as
the CBF gene family in Arabidopsis in response to cold temperature, in
vegetative tissues. Additional support for this hypothesis could be gained by the
identification of COR gene orthologs that possess CRT/DRE in their promoters, a
demonstration of up-regulation of the COR gene orthologs by over-expression of
either Arabidopsis CBF1, 2, or 3, or over-expression of a native CBF from a
rosaceous plant. Also, a demonstration of the direct binding of the rosaceous
CBF orthologs to the promoters of COR gene orthologs would provide support for
conservation of a similar molecular mechanism of these genes.

The demonstration of an enhancement in freezing tolerance of an
Important perennial fruit crop is of major practical significance. In the case of
strawberry, freezing damage can occur both to flowers, due to its early bloom,
and also to the crown tissue of the plant due to mid-winter temperatures below its
critical threshold for survival. Strawberries have a wide cultivated range, and in
colder climates plantings are often mulched to protect them from damaging
winter temperatures where snow cover is unpredictable. Additional physiological
protection from mid-winter temperatures would allow a wider range of cultivars to

be grown in cool climates.

71

Although flowers of the transgenic plants did not show an improvement in
freezing tolerance, it remains important to know that the CBF genes are
conserved in the Rosaceae. Further study is required to determine if
manipulation of the CBF system in strawberry, or other fruit crops, is capable of
improvement the freezing tolerance of pistils. Additional experiments, attempting
to develop lines with higher transgene expression levels, or targeted expression
directly to the pistils may provide some additional information to answer this
question. Significantly, if the CBF-COR gene system is closely conserved
between Arabidopsis and rosaceous species, than Arabidopsis can be used as a
model for elucidating the nature of the cold acclimation molecular signal
transduction pathway in these important cultivated plants. Progress will be more
rapid in Arabidopsis and utilization of the information generated in this model
species will be of major utility to horticultural scientists and breeders of

horticultural plants.

72

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Baker SS, KS Wilhelm and MF Thomashow 1994. The 5’-region of Arabidopsis
thaliana cor153 has cis-acting elements that confer cold-, drought-, and ABA-
regulated gene expression. Plant Mol. Biol. 24:701-713.

Fowler DB, and LV Gusta 1979.Selection for winterhardiness in wheat. I.
Identification of genotypic variability. Crop Sci. 19:769-772.

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

biochemical changes associated with cold acclimation. Plant Physiol. 12421854-
1865.

Graham J, RJ McNicol, K Grieg 1995. Towards genetic based insect resistance
in strawberry using the cowpea trypsin inhibitor gene. Ann. Appl. Biol. 127:163-
173.

Graham J, RJ McNicol, K Greig, and Wl’G Vandeven 1994. Identification or red
raspberry cultivars and an assessment of their relatedness using fingerprints
produced by random primers. J. Hort. Sci. 69:123-130.

Guy CL, KJ Niemi, and R Brambi 1985. Altered gene expression during cold
acclimation of spinach. Proc. Natl. Acad. Sci. USA 82:3673-3677.

Guy CL 1990. Cold acclimation and freezing stress tolerance: role of protein
metabolism. Ann. Rev. Plant Physiol. Plant Mol. Biol. 41:187-223.

Hong B, R Barg, T-HD Ho 1992. Developmental and organ-specific expression
of an ABA- and stress-induced protein in barley. Plant Mol. Biol. 18:663-674.

Horvath DP, BK McLamey, and MF Thomashow1993. Regulation of
Arabidopsis thaliana L. (Heyn) cor78 in response to low temperature. Plant
Physiol. 103:1047-1053.

Jaglo KR, S Kleff, KL Amundsen, X Zhang, V Haake, JZ Zhang, T Deits, and MF
Thomashow 2001. Components of the Arabidopsis C-repeatldehydration-
responsive element binding factor cold-response pathway are conserved in
Brassica napus and other plant species. Plant Physiol. 127:910-917.

73

Jaglo-Ottosen KR, SJ Gilmour, DG Zarka, O Schabenberger, and MF
Thomashow1998. Arabidopsis CBF1 overexpression induces COR genes and
enhances freezing tolerance. Science 280: 1 04-1 06.

Kasuga M, Q Liu, S Miura, K Yamaguchi-Shinozaki, and K Shinozaki 1999.
Improving plant drought, salt, and freezing tolerance by gene transfer of a single
stress-inducible transcription factor. Nature Biotech. 17:287-291.

Liu O, M Kasuga, Y Sakuma, H Abe, S Miura, K Yamaguchi-Shinozaki, K
Shinozaki 1998. Two transcription factors, DREB1 and DREB2, with and
EREBP/AP2 DNA binding domain separate two cellular signal transduction
pathways in drought- and Iow- temperature-responsive gene expression,
respectively, in Arabidopsis. Plant Cell 10:1391-1406.

McKown R, G Kuroki, and G Warren 1996. Cold responses of Arabidopsis
mutants impaired in freezing tolerance. J. Exp. Bot. 47:1919-1925.

Monroy AF, Y Castonguay, S Laberge, F Sarhan, LP Vezina, and RS Dhindsa
1993. A new cold-induced alfalfa gene is associated with enhanced hardening at
subzero temperature. Plant Physiol. 102:873-879.

Rieger M 1989. Freeze protection for horticultural crops. Hort. Rev. 11245-109.

Rodrigo J, 2000. Spring frosts in deciduous fruit trees - morphological damage
and flowerhardiness. Scientia Horticulturae 85:155-173.

Sakai A, and W Larcher 1987. Frost survival in plants: responses and adaptation
to freezing stress. Springer-Verlag, Berlin.

Stockinger EJ, SJ Gilmour, MF Thomashow1997. Arabidopsis thaliana CBF1
encodes an AP2 domain-containing transcriptional activator that binds to the C-
repeatlDRE, a cis-acting DNA regulatory element that stimulates transcription in
response to low temperature and water deficit. Proc. Natl. Acad. Sci. USA
94:1035-1040.

Sukumaran NP and CJ Weiser 1972. An excised leaflet test for evaluating
potato frost tolerance. HortScience 7:467-468.

Tao R, H Yamane H, A Sugiura, H Murayama, H Sassa, and H Mori 1999.
Molecular typing of S-alleles through identification, Characterization and cDNA
cloning for S—RNases in sweet cherry. J. Amer. Soc. Hort. Sci. 124:224-233.

Thomashow MF 1999. Plant cold acclimation: freezing tolerance genes and
regulatory mechanisms. Ann. Rev. Plant Physiol. Plant Mol. Biol. 50:571-599.

74

Thomashow MF, SJ Gilmour, EJ Stockinger 2001. Role of the Arabidopsis CBF

transcriptional activators in cold acclimation. Physiologia Plantarum 112:171-
175.

Warren G, R McKwon, A Mario, and R Teutonico 1996. Isolation of mutations
affecting the development of freezing tolerance in Arabidopsis thaliana (L.)
Heynh. Plant Physiol. 111:1011-1019.

Wisniewski M, R Webb, R Balsamo, TJ Close, XM Yu, and M Griffith 1999.

Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60:
A dehydrin from peach (Prunus persica). Physiologia Plantarum 105:600-608.

75