Journal of Experimental Botany, Vol. 47, No. 296, pp. 291-305, March 1996
Journal of
Experimental
Botany
REVIEW ARTICLE
The molecular biology of plant acclimation to low
temperature
Monica A. Hughes1 and M. Alison Dunn
x
Department of Biochemistry and Genetics, The University of Newcastle upon Tyne, Newcastle upon Tyne
NE24HH, UK
Received 6 June 1995; Accepted 2 November 1995
ABSTRACT
Introduction
In many temperate plant species, a period of exposure
to a low positive temperature will acclimate the plants
to withstand a subsequent freezing stress. A number
of plant genes, which are up-regulated at steady-state
mRNA levels by an acclimation treatment, have been
isolated from both monocotyledon and dicotyledon
species. Most of these genes are also responsive to a
drought treatment and/or abscisic acid. The acclimation of plants to freezing stress is a complex process
and although genetic studies in some species have
identified genes with a major effect, in general, the
inheritance of frost tolerance is multigenic. In view of
this, it is not surprising that a range of different genes
have been cloned. The precise function of the proteins
encoded by these genes is unknown. However, analysis of predicted protein products and studies of
recombinant proteins, together with detailed expression studies, are beginning to provide information
about some of the genes. Both transcriptional and
post-transcriptional controls have been shown to be
involved in the expression of these genes. Although
studies of RNA stabilizing systems are still in their
early stages, a number of low temperature responsive
promoters have been studied using reporter gene
constructs. Other approaches to the molecular
analysis of cold acclimation include the isolation of
non-acclimating mutants and the production of
transgenic plants.
Acclimation for frost tolerance
Key words: Abscisic acid, cold acclimation, drought, frost
tolerance, low temperature.
1
To whom correspondence should be addressed. Fax +44 191 222 7424.
© Oxford University Press 1996
In many overwintering temperate plant species a period
of low positive temperature will increase tolerance to a
subsequent subzero (freezing) temperature. This process
is known as cold or frost acclimation and is acknowledged
to be complex, involving a number of biochemical and
physiological changes. Cellular and metabolic changes
that occur during cold acclimation include increased levels
of sugars, soluble proteins, proline, and organic acids as
well as the appearance of new isoforms of proteins and
altered lipid membrane composition (Hughes and Dunn,
1990). There is an increasing body of evidence to show
that many of these biochemical and physiological changes
are regulated by low temperature through changes in
gene expression and in recent years a number of lowtemperature-responsive (LTR) genes have been cloned
from a range of both dicotyledon and monocotyledon
species.
The ability to acclimate to frost tolerance is under
genetic control and a low temperature of 6 °C to 2 °C,
which will cold acclimate genetically competent plants,
such as winter cereals, will damage a chill-sensitive species,
such as maize. However, not all chill-tolerant plants are
able to acclimate for frost tolerance and many spring
cereal cultivars can be classified in this group. In addition
to acclimation for frost tolerance, a low positive temperature treatment may also acclimate chill-tolerant plants
for growth at low temperatures (Hughes and Pearce,
1988). In species and cultivars which will cold acclimate,
the low temperature treatment may also alter the growth
habit and vernalize the plants. Further, there is often an
interaction between low temperature and other environmental factors, particularly daylength and water status
(Pan et cil., 1994).
292
Hughes et al.
Genetic analysis of cold acclimation
The genetic analysis of cold acclimation has been carried
out primarily in cereals. Winter hardiness of a cereal crop
is composed of a number of interacting components, frost
tolerance, vernalization requirement and photoperiod
sensitivity, each of which is complex. In the diploid cereal
barley it has been suggested that a change in the growth
habit caused by the vernalization response can contribute
directly to winter hardiness of the crop, however, Doll
et al. (1989) were able to separate vernalization requirement and good frost tolerance in a number of doubled
haploid lines produced by crossing a winter, frost hardy
cultivar (Vogelsanger Gold) with four spring, frostsensitive cultivars (Tron, Tystofte Prentice, Clipper, and
Alf). Takahashi and Yasuda (1970) concluded that there
were three major genes for vernalization requirement
located on barley chromosomes 4, 7 and 5. More recently
Hackett el al. (1992) mapped a quantitative trait locus
(QTL) for vernalization on barley chromosome 4
although this locus may strictly be a heading (flowering)
date locus (Pan et al., 1994). A 74-point linkage analysis
of a spring x winter barley (Morex x Dicktoo) cross by
Pan et al. (1994) has identified a significant QTL for
winter survival on chromosome 7.
The homeologous wheat chromosome for barley chromosome 7 is chromosome 5. In hexaploid wheat five
vernalization loci have been identified. Vrn 1, Vrn 4 and
Vrn 3 are located on chromosome 5A, 5B and 5D,
respectively (Xin et al., 1988). Vrn 2 is located on
chromosome 2B (homeologous barley chromosome 2),
and Vrn 5 is on chromosome 7B (homeologous barley
chromosome 1) (Law, 1966). In addition, the Sp\ gene
for vernalization has been mapped to the rye chromosome
5, which is also homeologous to wheat chromosome 5
(Plaschke et al., 1993). Of the chromosomes identified as
carrying genes involved in frost tolerance in wheat, those
most frequently implicated are chromosomes 5A, 7A, 4D,
and 5D (Sutka and Snape, 1989). Sutka and Snape (1989)
have reported a locus having a major effect on frost
tolerance (Fr\) on chromosome 5A which is closely linked
to the recessive vrn 1 allele (Sutka et al., 1996) and
Roberts (1990) identified two loci on wheat chromosome
5A which are involved in frost tolerance.
These genetic studies only reveal loci which are involved
in the response of cereals to low temperatures, where the
parents used in the cross contain different alleles which
segregate in the progeny of the F, hybrid or alter the
genotype of cytological substitution or addition lines. The
power of this analysis to dissect the processes of frost
acclimation depends, therefore, on the amount of variation available within the crop (species).
Mutational and transgenic studies of frost tolerance
The plasma membrane is considered a primary site of
injury during freezing (Lyons et al., 1979) and membrane
lipid composition is widely considered to be important in
tolerance to low temperatures. Freezing of plant tissues
results in the formation and growth of extracellular ice
crystals, this inflicts a dehydrative stress on cells. Injury
due to freezing can be caused by lesions in the plasma
membrane that result in loss of osmotic responsiveness
during subsequent thawing (Steponkus, 1984). Plants that
have been grown at low temperatures show an increase
in the level of lipid unsaturation (Harwood et al., 1994),
but changes in lipid composition and the ratio of lipid to
protein in membranes may also change (Harwood et al.,
1994; Palta et al., 1993). Roughan (1985) has surveyed
the fatty acid composition of phosphatidylglycerol in 74
plant species, to show a broad correlation between chill
sensitivity and the percentage of saturated plus transmono-unsaturated phosphatidylglycerol. More direct
evidence that changes in phospholipid content affect the
cryo-behaviour of plasma membranes has been produced
by Steponkus et al. (1988) using a liposome-protoplast
fusion technique. Liposomes of different molecular species
of phosphatidylcholine were fused with protoplasts from
non-acclimated rye seedlings to selectively enrich the
plasma membrane. The modified protoplasts showed that
increased levels of mono- and di-unsaturated species of
phosphatidylcholine mimic the behaviour of protoplasts
from acclimated plants and suffer less expansion-induced
lysis during thawing.
The overwintering annual species Arabidopsis thaliana
is able to acclimate for frost tolerance (Lin et al., 1990)
and shows a low temperature vernalization response
(Chandler and Dean, 1994). Figure 1 shows a summary
of the mutations of Arabidopsis which affect lipid biosynthesis and which have been evaluated for their effect on
cold tolerance. The fad 2 mutation results in plants with
a deficiency in the production of polyunsaturated (lowmelting point) fatty acids and these plants are killed by
long exposure to low (non-freezing) temperatures (Miguel
et al., 1993). Similarly, fad 5 and fad 6 mutants are
deficient in unsaturated lipids, but these mutations affect
lipids within the chloroplasts and the mutant plants show
defects in normal chloroplast development at low temperatures (Hugly and Sommerville, 1992). All of these
mutant Arabidopsis are chill-sensitive and can, therefore,
no longer acclimate for frost tolerance. Tht fab 1 mutation, results in an increased level of 16:0 (saturated) fatty
acids compared with wild type. Recently, Wu and Browse
(1995) have shown that although fab 1 Arabidopsis mutants contain a higher level of saturated (high-meltingpoint) fatty acids in phosphatidylglycerol compared with
many chill-sensitive species, these mutants are not chillsensitive. This observation is not in agreement with the
previous results.
An outline of studies which have manipulated levels of
unsaturated fatty acids in plants by transformation is
shown in Fig. 2. Murata et al. (1992) transformed tobacco
Molecular biology of plant acclimation
Gene
Biochemical function
fadi
1
11:1
fads
11:1
18:2
18:1
16:1
I
I
18:1
Chffl sensitivity
I
—> r~
18:2
16:0
16:0 ACP
^
I
(frfastid)
16:2
—> r~
18:2
(plastid)
16:1
18:1
-
(ER)
18:2
> I" I
18:0
I
I
fab\
—> r~
I
I
fade
I
18:1
293
I
(plastid)
16:0
18:0 ACP
Fig. 1. Mutations of Arabidopsis thaliana which affect lipid biosynthesis and have been evaluated for their effect on chill tolerance.
lobacco
chilling sensitive
intermediate
SiArabidopsis
W chiding resistant
E.coli
prokaryote
\
transferase
3
Transgenic
tobacco
Arabidopsis
chilling sensitive
chilling sensitive
acyt-ACP : glycerol-3-phosphate
acyt-ACP: glycerol-3-phospriate
acyt-ACP : glycaroKH>nosphate
a>-3 fatty a d d desaturase (tad 7)
acyttransferase 1 cannot dlscrimlnata between saturated and cto-unsaturated fatty adds
acyttiaiisfaiMsa 2 has preferential activity for cfe-unsaturatad fatty adds
acyttransferase 3 cannot discriminate between 16:0-ACP and 18:OACP
converts 18 J and 16:2 to 16:3 and 16:3 respectively.
Fig. 2. Manipulation of chill-sensitivity in Nicotiana labacum and Arabidopsis thalianu by transformation.
(which was considered to have an intermediate level of
chill-sensitivity) with acyl-ACP: glycerol-3-phosphate
acyltransferase from either chill-sensitive squash or
chill-resistant Arabidopsis. This resulted in higher levels
of high-melting-point (saturated) phosphatidylglycerol in
the squash enzyme transformant and lower levels in the
Arabidopsis enzyme transformant. In addition, the squash
enzyme transgenic tobacco plants had increased chillsensitivity whilst the Arabidopsis enzyme transgenic plants
were more chill-resistant.
294
Hughes et al.
Two other groups have manipulated levels of unsaturated lipids by transformation and produced a change in
the chill-sensitivity of the resulting plants. Wolter et al.
(1992) transformed Arabidopsis with an E. coli acylACP:glycerol-3-phosphate acyltransferase which can not
discriminate between 16:0-ACP and 18:0-ACP. The
resulting transgenic plants contain more saturated phosphatidylcholine and are chill-sensitive. More recently,
Kodama et al. (1994) produced transgenic tobacco that
contain an w-3 fatty acid desaturase gene (fad 7) from
Arabidopsis, these plants contain increased levels of polyunsaturated lipids and an increase in resistance to chilling
temperatures.
These studies, where a single gene is manipulated,
provide powerful evidence for the involvement of polyunsaturated lipids in the resistance of membranes to low
positive temperatures and although this may not be
directly involved in frost acclimation, clearly plants must
be chill-tolerant in order to acclimate at the low (chilling)
temperature. The anomalous behaviour with respect to
chill-sensitivity, of the fab 1 mutants (Wu and Browse,
1995) highlights the complexity of low temperature
responses in plants. Wu and Browse (1995) comment on
the possible pleiotropic effects of some of the transgenes
and the difficulty of interpreting some of the single gene
modifications for a multigenic character, in plants with
very different genetic backgrounds.
Low-temperature-responsive (LTR) genes
Overview of LTR genes
Details of most of the LTR genes which have been studied
to date are shown in Table 1. Twenty of the gene/gene
families come from dicotyledon species and 11 from
monocotyledons. Although this list contains a wide range
of different genes there are some general points which are
demonstrated by the compilation. Most of the genes were
isolated from cDNA libraries using a differential screening
procedure where normal growth temperatures and a low
positive (acclimating) temperature were used in the generation of different pools of mRNA. This technnique
isolates genes which are up-regulated by low temperature
at steady-state mRNA levels and where the mRNA
species is present at relatively high levels in the mRNA
population. RC\ and RC2 were, however, isolated by a
technique designed to identify rare transcripts (Jarillo
et al., 1994).
A number of the genes have been isolated from the
same species independently by different groups (e.g. cor
6.6 (kin 1); Hajela et al., 1990; Kurkela and Franck,
1990). In addition, cognates of some genes have been
independently isolated from several species (e.g. /// 30
from Arabidopsis (Welin et al., 1994); cap 85 from spinach
(Neven et al., 1993); cor 39 from wheat (Guo et al..
1992)). Ten gene/gene families have been isolated from
Arabidopsis and 6 gene/gene families from barley. This
number of LTR genes in one plant species is consistent
with the acknowledged complexity of the frost acclimation
process. It is considered unlikely that this is an exhaustive
list of LTR genes since the technique employed to isolate
them will not identify genes expressed at low levels or
genes transiently expressed during the acclimation process. The nature of the material used as a source of
mRNA is also a factor in the isolation of these genes.
Thus one possible explanation for the fact that, with the
exception of Ccr 1, none of the Arabidopsis LTR genes
have been cloned in barley, may be that different material
was used as a source of the cDNA library. Five of the
six barley genes were isolated from a cDNA library made
from shoot meristems, whereas the Arabidopsis libraries
were made from mRNA extracted from whole plants,
largely made up of mature leaves.
Role of gene families
Many of the genes which have been the subject of
extensive studies are members of small multigene families.
In Arabidopsis cor 6.6 (kin 1) and kin 2 are linked in
tandem, they are expressed at different levels in response
to low temperature and they also differ in their response
to drought (Kurkela and Borg-Franck, 1992). Similarly,
cor 15A and cor 15B from Arabidopsis are linked in
tandem and, although cor 15A is responsive to both low
temperature and drought, cor 15B is only responsive to
low temperature (Lin and Thomashow, 1992; Wilhelm
and Thomashow, 1993).
The complexity of the relationship between genes in
LTR multigene families is illustrated in barley. Four
members of the bit 4 gene family have been cloned (Dunn
et al., 1991; White AJ et al., 1994). These LTR genes are
also responsive to drought and ABA, but the relative
response to these three factors varies between the individual genes. The gene bit 4.9 is strongly induced by low
temperature and only slightly induced by drought,
whereas bit 4.1 is equally responsive to both treatments
(Fig. 3). There is also a temporal control of expression
of bit 4.2, which is only expressed in seedlings (White AJ
et al., 1994).
There are only two members of the bit 101 family
present in the barley genome. In this pair of genes both
are responsive to low temperature and the cDNA
sequences are over 70% homologous. Because of the level
of homology between the two genes it was impossible to
be confident of the use of gene specific probes and a
quantitative reverse transcriptase PCR technique was
used to measure relative levels of expression. Figure 4
shows the result of using this technique to measure the
expression of bit 101.2 in low temperature-treated shoot
meristems. A dilution series of cloned genomic DNA,
Table 1. Low temperature responsive genes
DICOTYLEDONS
Species
Gene
Other members
of gene family
Cognates in
other species
Class of proteins
or gene
Other stresses
Reference
Arabidopsis lhaliana
for 6.6 {kin 1)
A-m 2
fl/i 28
AFP
Drought, ABA
cor 78
In 78, /f; 65
-
NK
Drought
ABA (salt)
Hajela el al. (1990); Kurkela and Franck
(1990); Kurkela and Borg-Franck (1992)
Horvath et al. (1993); Nordin el al
(1993); Yamaguchi-Shinozaki and
Shinozaki(1994)
Lin and Thomashow (1992); Wilhelm and
Thomashow (1993)
Lin et al. (1990); Welin el al. (1994)
Welin et al. (1994)
Nordin el al. (1991)
Lang and Palva (1992)
Carpentered al (1994)
Jarillo et al. (1994)
Dolferus et al. (1994)
Weretilnyk el al. (1993)
Orr et al (1992)
SSez-Vasquez et al. (1993)
Monroy et al. (1993)
Wolfraim el al. (1993 a, b)
Labergeera/. (1993); Luo el al. (1991)
Castonguay el al (1994)
Neven et al. (1993)
Anderson et al. (1994)
Zhu elal. (1993)
rd 29A, rrf 29B
Brassica napus
(oil seed rape)
Medicago saliva
subspp falcata
(alalfa)
cor 15A
for 15B
fin 115
NK
Drought (ABA)
for 47
In 30
Ili 140
rab 18
Ccr 1
RCI 1
Adh
BN 115
//; 45
Cfr 2
RCI 2
BN 19, BN 26
BnC 24B
COJ 18
SAY 2075
-
cap 85, for 39
bit 801
for 15A
for 6.6
bbc 1
Iti 30, for 39
-
D-ll LEA
D-ll LEA
NK
D-ll LEA
RNABP
14-3-3
ADH
NK
AFP
NK
NK
D-ll LEA
NK
proline rich
D-ll LEA
HSP
Osmotin
Drought, ABA
Drought, ABA
Drought, ABA
Drought, ABA
(Drought)
_
Drought, hypoxia
NK
_
Drought, ABA
Drought
Drought, ABA
rlt 1412; rlt 1421
NK
_
Wl4
6// 14.2; bit 14.1
A 086
6// 4.2; 6// 4.6; bit
-
LTP
Drought, ABA
bh 101
6/f 63
WM 1
bit 801
r/( 1412
Wcs 120
bit 101.2
_
rlt 1421
Wcs 200, Wcs 66
ESI 2,
Ccr 1
bit 14
_
NK
EF-la
D-7 LEA
RNABP
NK
D-ll LEA
_
NK
ABA
-
Wcs 19
for 39
Wfor 410
_
-
In 30, fa/> 85
-
NK
D-ll LEA
D-ll LEA
Drought, ABA
Drought, ABA
fijV 28
BnC 24A
cas 15
cas 17
Msaci A
HKWCIC
Spinacia oleracea
(spinach)
Solatium commersonii
cap 85
ER-HSC 70
A 13
MONOCOTYLEDONS
Hordeum vulgare
bit 14
(barley)
Secale cereale (rye)
Triticum aestivum
ABA
(wheat)
Dunn el al. (1990);
Cativelli and Bartels (1990)
Dunn el al. (1991); White et al. (1994)
Goddard et al (1993)
Dunn et al. (1993)
Sutton et al. (1992)
Dunn et al. (1996)
Zhang et at. (1992)
Houde et al. (1992); Ouellet et al. (1993);
Chauvin et al. (1994)
Chauvin et al. (1993)
Guo et al. (1992)
Danyluk el al. (1994)
Key to abbreviations: AFP = antifreeze proteins; ABA = abscisic acid; LEA = late embyogenesis abundant proteins; RNABP = RNA binding protein; N K = n o t known; bbc 1 —breast basic
conserved gene; HSP»heat shock protein; ADH = alcohol dehydrogenase; 14-3-3 kinase regulatory protein; LTP = hpid transfer protein; EF-1 a°= elongation factor-la; ESI 3 = salt stress
induced gene from wheatgrass; - = none; ( ) bracket indicates stress response of other members of gene family.
s
2.
o
12"
o
I
oo
I"
6'
0)
296
Hughes et al.
WM.1
gWM.B
MM.9
D Low Temperature E3 Drought
Fig. 3. Comparative Northern blot analysis of the relative abundance
of bit 4.1, bit 4.6 and bit 4.9 transcripts in barley (cv. Igri) shoot
meristems. Histogram of integral of densitometric peaks. Relative
response to drought expressed as a percentage of induction by low
temperature (6°C day/2°C night) (White AJ et al., 1994).
ng/ulplasmid
QQQ6
cloned from barley and these genes show a pattern of
regulation similar to that found in the cognate gene family
in rye (Zhang et al., 1993). That is, all of the genes are
responsive to low temperature and not drought or ABA,
but the organ distribution of gene expression is different
for each gene (Phillips and Hughes, unpublished).
Smith (1990) suggested that phenotypic plasticity of
plants in response to different environmental factors may
be due to the differential expression of different members
of multigene families. This can be extended to the cellular
or metabolic level where different environmental
conditions require modification of cell structure and/or
metabolism in order to maintain vital processes. These
modifications may require different protein isoforms
under different environments. This hypothesis provides
an explanation for the pattern seen in the bit 4 gene
family. However, the production of different bit 14 isoforms in different parts of the plant is consistent with the
suggestion that the modification of cells in different tissues
must be modulated during the low temperature acclimation process.
R
Response to other stresses
P = plasmid PCR product
R = RT PCR product
Fig. 4. Quantitative reverse transcriptase (RT) PCR: expression of the
barley gene bit 101.2 in shoot meristems given a 7 day treatment at
6°C day/2 °C night (10 h day). The PCR product from cloned genomic
DNA is 450 bp and includes the intron, the RT PCR product from
identical aliquots of a mRNA extract is 378 bp. The dilution series of
cloned genomic DNA used was 4.0 ng j j ' 1 to 0.006 ng ^ l 1
which contains an intron, is added to PCR of a constant
level of cDNA produced from idential aliquots of an
mRNA extract. The primers anneal to sites either side of
the intron and, therefore, produce different sized products
from the two templates. The point of equivalence of
quantity of the two PCR products is a measure of the
level of cDNA (mRNA) in the extract. This reaction is
repeated using mRNA from control temperature plants
and with genomic DNA and specific primers for bit 101.
This study has shown that bit 101 is expressed at 10 times
the level of bit 101.2 at both control and low temperature.
Three members of the bit 14 gene family have also been
Seventeen of the 30 LTR gene/gene families tested are
also responsive to drought and/or abscisic acid (Table 1).
In addition, two of the genes have cognates which are
responsive to salt stress. At least 3 of the bit 4 gene family
are also induced by pathogen infection (Table 2; Molina
and Garcia-Olmedo, 1993). Because, during freezing, ice
crystals form outside the cells, both freezing and drought
are considered dehydrative stresses (Steponkus, 1984). It
is reasonable, therefore, to suggest that similar modifications to the cell may be required for tolerance to both
stresses and the production of common gene products
may therefore be induced. A rapid transfer to a low
positive temperature will cause a reduction in root water
uptake and a transient 'locking-open' of stomata causing
transient loss of plant turgor (Pardossi et al., 1994).
However, the low positive temperature treatment which
induces the expression of the LTR genes is not primarily
a dehydrative stress and the extent to which different
signal transduction pathways are involved in controlling
the expression of genes by drought and low positive
temperature remains to be determined.
Function of gene products
Including the D-11 (dehydrin) class of late embryogenesis
abundant (LEA) related genes, there is no known biochemical function for 26 of the 31 LTR gene/gene families
listed in Table 1. A number of studies have investigated
both freezing tolerance and levels of gene expression in
the same genotype under a range of acclimating treat-
Molecular biology of plant acclimation
297
Table 2. Barley lipid transfer protein genes
Gene
Chromosome
Presence
of introns
Itp 1
Itp 3
W/4.1 (Itp 2)
bit 4.2 (Itp 4)
bit 4.6
bit 4.9
7
5
3
3
3
3
y
y
NK
—
—
—
3 amino acid
deletions at
R82
—
y
y
y
y
Response
NK
—
LT
LT
LT
LT
Tissue
distribution
NK
NK
Drought
NK
Drought
Drought
NK,
Pathogen
Pathogen
Pathogen
NK
Pathogen
NK
ABA
ABA
ABA
NK
ABA
Aleurone layer
NK
Leaf
Coleoptile
NK
Shoot meristem
— = None; NK = not known
ments. Monroy et al. (1993a) showed that changes in
freezing tolerance of alfalfa cv. Apica are paralleled by
changes in the transcript levels of cas 15. In barley, the
steady-state mRNA levels of three LTR genes were measured at a range of temperatures during acclimation and
deacclimation. In this study the level of frost tolerance
(LT J0 in a regrowth test) was shown to correlate with the
level of gene expression (Pearce et al., 1996).
The relationship between levels of gene expression and
the ability of different genotypes to acclimate for frost
tolerance has also been investigated. Danyluk et al. (1994)
have shown that Wcor 410 is only expressed in freezingtolerant gramineae. Houde et al. (1992) have shown that
the accumulation kinetics of Wcs 120 mRNA during
acclimation is positively correlated with the capacity of
each wheat genotype to develop frost tolerance. Similarly,
Zhang et al. (1992) showed that the low temperature
induced steady-state mRNA levels of two rye LTR genes
(rlt 1412, rlt 1421) were lower in the relatively frostsensitive rye cultivar, Rhayader, compared with the frosthardy cultivar, Puma. These comparative studies support
the proposal that these LTR genes have a function in
frost acclimation.
Low temperature isoforms
Biochemical studies have suggested that growth at low
temperature will result in the production of low temperature isoforms of enzymes involved in 'housekeeping'
functions. With the exception of the barley protein synthesis elongation factor-la (EF-la, Dunn et al., 1993),
this type of gene is not present in Table 1. The lack of
these genes may be due to the cloning strategy commonly
used. Many of the low-temperature isoforms of proteins
can be expected to have similar structures and, consequently, the coding regions of the mRNA will be highly
homologous and cross hybridize. This means that such
clones would not be detected easily in a differential screen.
The barley EF-la is a highly conserved protein, it is a
member of a multigene family in the barley genome and
the low temperature induction of bit 63 can only be seen
in Northern blot analysis if the 3' untranslated sequence
of the mRNA is used as a probe.
Cryo-protection
A number (8) of the LTR genes in Table 1 are predicted
to encode proteins with the characteristics of the D-l 1 or
dehydrin class of LEA proteins. These proteins accumulate during the late stages of seed development and are
predicted to have a role in dehydration of the seed. The
dehydrins are characterized by a repeated consensus 15
amino acid sequence near the C terminus of the protein
and, in addition, many have a tract of serine residues
upstream of these domains (Close et al., 1993). The
proteins are very hydrophilic and are 'boiling stable'.
They were originally thought to act as osmoprotectants
although it has recently been suggested that they may
have a role in nuclear protein transport (Close et al.,
1993).
The chloroplast targeted protein product of cor 15 A in
Arabidopsis is also hydrophilic and 'boiling stable' (Lin
and Thomashow, 1992) and this protein has been shown
to have cryoprotective activity in vitro against the freezelabile enzyme L-lactate dehydrogenase. It is difficult to
extrapolate from this in vitro assay for cryoprotective
activity, to the in vivo function, but the common occurrence of 'boiling stable' hydrophilic proteins amongst the
LTR genes makes cryoprotection an attractive role for
them. Two other genes (cor 6.6 from Arabidopsis and BN
28 from oil seed rape) have homology to antifreeze
proteins, thus cor 6.6 {kin 1) bears some compositional
similarity to the fish alanine-rich antifreeze proteins
(Kurkela and Franck, 1990). These clones support the
suggestion that a significant proportion of the LTR genes
cloned have a cryoprotective function. Hincha et al.
(1990) have used an in vitro test for thylakoid membrane
cryoprotection against mechanical freeze-thaw damage,
to study protein extracts from leaves of cabbage and
spinach. Their results show that proteins with significant
cryoprotective activity are only isolated from frost-
298
Hughes et al.
acclimated plants. It will be interesting to see the structure
of these proteins when they have been purified.
Membrane/lipid
modifications
It is perhaps surprising, given the importance of membrane lipid composition in chill-tolerance and frost acclimation (outlined in 'Mutational and transgenic studies of
frost tolerance'), that none of the genes in Table 1 has a
direct role in the synthesis of these compounds. The only
gene family for which a lipid related function is proposed
is the bit 4 gene family in barley, which is predicted to
encode a group of non-specific lipid transfer proteins
(White AJ et al., 1994).
A summary of the lipid transfer protein (LTP) genes
which have been cloned in barley is shown in Table 2.
This table, which includes the LTP gene (Up 1) expressed
in aleurone cells during germination (Mundy and Rogers,
1986), a pathogen induced LTP gene (Up 3) and two
cognates {Up 2, Up 4) of the bit 4 gene family isolated
from the spring cultivar, Bomi (Molina and GarciaOlmedo, 1993) as well as the winter barley bit 4 LTP
genes (White AJ et al., 1994), shows the complexity of
lipid transfer proteins produced by a single species. On
the basis of gene structure these genes may be divided
into two groups Up 1 and Up 3 being distinguished from
the bit 4 genes by their chromosomal location, the presence of an intron in the gene and an insertion of nine
bases in the mRNA coding sequence, which leads to the
addition of 3 amino acids at residue position 85 in the
polypeptide. Molina and Garcia-Olmedo (1993) have
shown that Up 3, bit 4.1 (Up 2) and bit 4.2 (Up 4) are
responsive to pathogen stress, but not low temperature,
in cv. Bomi. The low temperature, drought and ABA
response of the bit 4 gene family (White AJ et al., 1994,
Hughes et al., 1992) and the pathogen response of bit 4.9
(Hughes and O'Hara, unpublished) have been measured
in the winter cv. Igri. In common with other plant LTPs,
none of these genes is expressed in roots and they show
some tissue specificity. LTP have been defined by their
ability to transfer lipids between membranes (typically
mitochondria and liposomes) in vitro, however, their
function in vivo has been questioned. In barley bit 4.9 has
recently been shown to be expressed in epidermal cells of
the leaf sheath bases (Hughes et al., unpublished). All of
the bit 4 gene family possess a consensus N-terminal
signal sequence for extracellular transport (White AJ
et al., 1994) and this may not be consistent with a lipid
transfer function related to changes in membrane lipids
during frost-acclimation. Recently, an extracellular, waxassociated LTP has been isolated from broccoli (Brassica
oleracea) leaves, this is a member of a LTP gene family
which is expressed in leaf epidermal cells (Pyee et al.,
1994; Pyee and Kolattukudy, 1995). These results suggest
that the LTPs may be involved in wax synthesis and/or
secretion. A role in leaf surface biochemistry is consistent
with the response of barley LTPs to a wide range of
factors and must be investigated as an alternative to the
lipid transfer function.
Low temperature control of gene expression
Signal transduction
Abscisic acid (ABA) is implicated in the plant response
to low-temperature by a number of observations: (1) An
acclimating low-temperature treatment (4 C C/2°C) of
Arabidopsis lhaliana results in a transient, 3-fold increase
in endogenous ABA levels (Lang et al., 1994). This
increase is seen in both axenically grown and soil-grown
plants and has also been observed in other species (Lalk
and Ddrfliing, 1985). (2) Application of ABA can induce
an increase in freezing tolerance in Arabidopsis plants
grown at a control temperature (22°C) (MSntylS et al.,
1995). (3) ABA-null mutants of Arabidopsis (aba-\) are
impaired in their ability to cold acclimate with lowtemperature treatment (MantylS et al., 1995, Heino et al.,
1990). (4) Application of ABA to the ABA-insensitive
mutant (abi-l) does not induce cold acclimation (Mantyla'
et al., 1995). However, several studies have suggested
that there is more than one signal pathway for lowtemperature responses (Nordin et al., 1991; Gilmour and
Thomashow, 1991, Jarillo et al., 1993) and this is illustrated by the ability of abi-\ mutants to cold acclimate
to some extent. Table 3 shows a schematic representation
of the response of six Arabidopsis genes to low temperature, drought and ABA in 'wild type' (Lansberg erecta
ecotype) and aba-\ and abi-\ mutants (Mantyla et al.,
1995; Welin et al, 1994; Nordin et al, 1991; Jarillo et al.,
1993; Gilmour and Thomashow, 1991). All of the genes
are responsive to ABA, drought and low-temperature
although the relative level of the response to these treatments varies between the genes. With the exception of
rab 18 (whose reponse to low-temperature is relatively
weak), all of these genes are up-regulated by low temperature in both the aba-] and the abi-\ mutants. However,
although the ABA response of these genes is similar to
wild type in the ABA-null mutant (aba-\), ABA treatment
does not induce gene expression in the ABA-insensitive
mutant (abi-\). These results indicate that these genes
are up-regulated independently by low-temperature and
ABA.
The abi-\ mutation used in these studies is a semidominant mutation and this is consistent with the gene
having a regulatory role. The gene has recently been
cloned (Leung et al, 1994; Meyer et al, 1994) and the
protein encoded by this gene is predicted to have a
C-terminal domain which is homologous to type 2C
serine/threonine protein phosphatase and an N-terminal
Molecular biology of plant acclimation
299
Table 3. Response of Arabidopsis low-temperature-responsive genes in ABA-null (aba-l) and ABA insensitive (abi-1) mutants
Gene
Treatment
Low temperature
Drought
Genotype
Genotype
hi 140
In 30
hi 45
hi 78
rab 18
adh
LT^^.OX)
Genotype
wt
aba-1
abi-1
wt
aba-1
abi-1
wt
aba-1
abi-1
+++
+++
+++
+++
++
+++
+++
+++
+++
+++
NK
+
+ ++
+
+
++
+
+
+
±
0
0
0
0
±
0
+ ++
+++
++
+
+++
0
0
0
0
+++
++
+
+
+++
+
-3.0 X
-3.5 X
-6.5 X
-7.5 X
+++
+
+++
-7.4 X
++
++
-3.8 3C
-5.0 >C
-7.0 X
domain with a Ca2+ binding site. Figure 5 shows a model
for ABA signalling in plants (Bowler and Chua, 1994)
which incorporates a plasma membrane-localized ABA
receptor (Gilroy and Jones, 1994) and ABA-mediated
increases in Ca2+ levels in the cytosol. There is evidence
for the involvement of Ca2+ in both ABA (Gehring et ah,
1990) and cold (Knight etal, 1991; Monroy etai, 1993ft)
responses and the deduced structure of the abi-l gene
product suggests that Ca2+ may either activate or repress
phosphatase activity leading to a change in the phosphorylation status of another protein further down the
+
+
+
±
0
-3.0 X
signal transduction pathway. This, in turn, implicates at
least one kinase in the signal transduction pathway. The
current models for ABI 1 functions do not, however,
suggest an explanation for the independent lowtemperature and ABA cold acclimation responses. A total
of five genes which have ABA-insensitive mutations have
been isolated (Rock and Quatrano, 1994) in Arabidopsis.
Three of these (abi-3, abi-4, abi-5) are seed specific but
mutations of the abi-2 gene are epistatic to abi-\ suggesting that abi-\ and abi-2 may lie on the same signal
pathway. Gilmour and Thomashow (1991) have shown
abi 1 (dominant)
ABA,
j£ drought,(LT)
aba
drought,:
LT
ABA
:• m m m-m» m iat m.a:
F?
• response
ABRE
.•£. ,«• j * r; -
ABI-1
calcium-dependent
phosphatase
2+
„>*•
• response
\
- ABA (LT)
abi 2
Fig. 5. Model for the involvement of ABA in acclimation for frost resistance. Interrupted arrow (-//-•) shows lack of frost acclimation with the
factors indicated (low temperature, LT; abscisic acid, ABA; drought) in the Arabidopsis mutants aba 1 (abscisic acid null); abi 1 (abscisic acidinsensitive) and abi 2 (abscisic acid-insensitive). Proteins and the ABA receptor are shown as shaded blocks. ABRE, abscisic response element in
ABA responsive genes.
300
Hughes et al.
that abi-2 mutants of Arabidopsis are able to coldacclimate with a low temperature treatment. But unlike
the abi-l mutant, ABA treatment of the abi-2 mutant
plants leads to induction of the low-temperatureresponsive genes tested. Although there is progress in our
understanding of the ABA signal transduction pathway
there is essentially no information about the independent
low-temperature signal pathway.
Of the LTR cloned genes listed in Table 1, the candidates for a role in signal transduction are limited. The
Arabidopsis gene RCI 1, which was isolated as a rare
transcript (Jarillo et al., 1994), has homology to a family
of mammalian proteins, known as 14-3-3 proteins, which
are thought to be involved in the regulation of multifunctional protein kinase activity.
was part of a region shown not to be important in the
low temperature response. The role of the A/GCCGAC
motif in low temperature control of transcription clearly
requires further investigation.
In common with a number of environmentally controlled genes several of the putative LTR gene promoters
contain G-Box elements (CACGTG) (Williams et al.,
1992). This element is the core motif of a number of exacting regulatory sequences including the ABA-responsive-element (ABRE). To date, insufficient data exist for
the LTR gene promoters to allow an analysis of which
aspects of the G-Box flanking sequences are important
either in low temperature regulation per se or in the
interaction of low temperature with other factors such
as ABA.
Transcriptional control of gene expression
Post-transcriptional control of gene expression
The promoter regions of a number of LTR genes have
been cloned and there are several recent functional studies
of LTR gene promoters including the regulation of BN
115 from oil seed rape (White TC et al., 1994), cor 15A
(Baker et al., 1994), rd 29A (hi 78) (Nordin et al., 1993;
Yamaguchi-Shinozaki and Shinozaki, 1994) and adh
(Dolferus el al., 1994) from Arabidopsis. These functional
studies are based on the ability of promoter constructs to
drive the low temperature expression of a reporter gene
either in transgenic plants or in a transient expression
assay using a particle delivery system (PDS). White TC
et al. (1994) point out that the core sequence A/GCCGAC
of an 8-bp TGGCCGAC repeated motif from BN 115 is
also present in those regions of the promoter conferring
low temperature induction of rd 29A (hi 78) and cor 15A.
Table 4 also shows that the sequence (ACCGAC) is
present twice in the putative barley (monocot) promoter
of bit 4.6 and the related sequence motif (ACCG/CAC/A)
is repeated in the putative promoter of bit 4.9. Nordin
et al. (1993) noted that it was also present in the putative
Arabidopsis promoter regions of cor 6.6 (kin 1), kin 2 and
rab 18. It has been proposed that this core sequence motif
is part of a low-temperature-responsive-element (LTRE).
However, this motif is not present in the low temperatureresponsive promoter of bit 101 which can confer a low
temperature reponse to the GUS (glucuronidase) reporter
gene in a transient expression assay using PDS and barley
shoot meristems (Hughes et al., unpublished). Further,
analysis of the low temperature response of the
Arabidopsis adh gene promoter by Dolferus et al. (1994)
demonstrates that the anaerobic response element (ARE)
is also important in the low temperature response of this
gene. The other element or motif identified as important
in the adJi low temperature response is a G-Box element
CCACGTGG between 218 and -209 from the transcription start site. The adJi gene does have an A/GCCGAClike motif (ACCGAT) at -337, but this putative LTRE
Nuclear run-on transcription was used to analyse the low
temperature regulation of nine barley LTR genes (Dunn
et al., 1994) in shoot meristems. This experiment showed
that six of the genes (including bit 4.9, bit 101 and bit
63) are primarily transcriptionally regulated whilst three
of the genes (including bit 14 and bit 801) are primarily
post-transcriptionally regulated. The involvement of both
forms of regulation was also reported by Hajela et al.
(1990) for four Arabidopsis LTR genes, where one gene
was found to be transcriptionally controlled and 3 were
found to be post-transcriptionally controlled. In alfalfa,
a combination of the techniques of nuclear run-on transcription and transcription inhibition by cordycepin
during deacclimation, has shown that cas 18 is regulated
by low temperature at both the transcriptional and the
post-transcriptional level (Wolfraim et al., 1993). The
demonstration of post-transcriptional controls in the low
temperature regulation of gene expression in three species
indicates that this form of regulation may be of general
importance in frost acclimation.
A further finding which may be significant for possible
post-transcriptional control mechanisms, is the isolation
of two LTR genes which encode RNA-binding proteins,
Ccr 1 and Ccr 2 from Arabidopsis (Carpenter et al., 1994)
and bit 801 from barley (Dunn et al., 1996). These genes
encode proteins of 16 000-17 000 Mr with two distinct
domains, the amino-terminal domain contains consensus
RNP1 and RNP2 motifs, which together with a number
of other highly conserved residues comprise a consensus
RNA-binding domain (RRM) of about 80 amino acids.
This RRM is found in many RNA binding proteins of
the nucleus, cytoplasm and cytoplasmic organelles
(Dreyfuss et al., 1993). The carboxy-terminal domain
consists of repeating glycine residues interspersed with
tyrosine and arginine. The domain structure of bit 801 is
shown in Fig. 6.
There have been a number of other reports of stress
Molecular biology of plant acclimation
301
Table 4. Low temperature response elements (LTREJ
Species
Gene
Other members
of gene family
Cognates in
other species
Putative LTRE
Experimental
evidence for
LT
response
Oilseed rape
BN 115
BN
Arabidopsis
cor 15A
cor 15B
hi 78
In 65, rd 29A
Barley
19, B J V 2 6
Reference
Drought
response
cor 15A
g«g
GCCGAC
gtata
•J
gatg
gttg
GCCGAC
GCCGAC
ctgtt
ataca
J
fl/V 115
catg
tgg
GCCGAC
ACCGAC
ctget
tact
•J
teat
ACCGAC
atcag
V
•J (rd 29A)
ACCGAC
GCCGAC
ACCGAC
atgag
aaaa
taaa
y
V
</ (rd 2 9 A )
hi 65
hi 78
tatt
caa
tgg
bit 4 6
bit 4.2, bit 4.9
atgc
ACCGAC
aggt
bit 4.9
bit 4 2, bit 4.6
atgc
agca
agca
ACCGAC
ACCCAC
ACCGAA
agtt
agat
atta
White TC et al
(1994)
Baker et al.
(1994)
•J
•J
Nordin et al.
(1993)
Yamaguchi and
Shinozaki (1994)
Nordin et al.
(1993)
White AJ et al.
(1994)
•j
DOMAIN STRUCTURE OF BLT801 PROTEIN
RNPII
1
8 13
RNPI
151152 161 aa
47 54
t
t
(87)
(126)
RGG Box
glycine rich domain
cyclic AMP-dependent protein kinase (PKA)
phosphorylation consensus motif
t
Phosphorylated
serine
octomer RNPI
t
Putative proteolytic
site
RNA recognition motif
V
hexamer RNPII .
Fig. 6. Domain structure of the RNA-binding protein encoded by the barley low temperature responsive gene, bit 801
responsive plant genes which encode glycine rich-RNA
binding proteins (GR-RNPs): Zea mays, drought, ABA
(Ludevid et al., 1992); Daucus carota, wounding (Sturm,
1992); Z. mays, heavy metals (Didierjean et al., 1992);
Brassica napus, ABA (Bergeron et al., 1993). Although
this group of genes are all predicted to encode RNA
binding proteins only three have had nucleic acid binding
properties experimentally verified, namely; MA 16 from
Z. mays (Ludevid et al., 1992); RGP-lb, a GR-RNP
with no reported environmental response from Nicotiana
302
Hughes et al.
silvestris (Hirose et al., 1993) and BLT 801 from barley
(Dunn et al., 1996). MA16 and RGP-lb show in vitro
binding preference for poly (U) and poly (G) homoribopolymers and BLT 801 binds to poly (G), poly (A)
and poly (U) homo-ribopolymers, but not poly (C). BLT
801 and RGP-lb also bind single-stranded DNA. The
two-domain organization of plant GR-RNPs has
similarity to the Al and A2/B1 groups of heterogeneous
nuclear RNA binding proteins (hnRNP) found in animals.
These hnRNPs are approximately 34 000 A/r (cf. plant
GR-RNPs 17 000 Mr), and are comprised of two domains,
an amino-terminal domain containing two RRMs and a
glycine rich carboxy-terminal domain of similar composition to plant GR-RNPs. Each domain is approximately
twice the size of those found in plants (Dreyfus et al.,
1993). These animal proteins are located in the nucleus
and have been implicated in pre-mRNA splicing including
alternative splicing. The GR-RNPs from Z. mays (Alba
et al., 1994) has been localized in the nucleolus and that
of S. alba (Heintzen et al., 1994) to the nucleus, both
were shown to be most strongly expressed in shoot
meristems.
In common with animal hnRNPAl, BLT 801 contains
a consensus phosphorylation site for cyclic AMP dependent protein kinase (PKA) at the junction between the
RNA binding and glycine rich domains. Co-operative
RNA binding and DNA annealing properties, independent of the RRM, have been demonstrated for hnRNPAl
in vitro (Cobianchi et al., 1993) with the latter DNA
annealing function removed by phosphorylation. In vitro
phosphorylation of BLT 801 by PKA has been demonstrated (Dunn et al., 1996) but the biological significance
of this remains to be determined. However, the analogy
to the animal heteronuclear proteins is clear and it is
tempting to speculate that these GR-RNPs represent the
plant equivalent involved in alternative splicing of transcripts in response to stress or other environmental cues.
Summary
Table 1 documents the considerable activity in the field
of the molecular biology of plant acclimation to low
temperature, which has been reported in the past few
years. As yet, however, there is still no coherent explanation of the acclimation response of plants to low temperature at a molecular or biochemical level. Progress has
been made in the study of low temperature responsive
promoters, some interesting results on post-transcriptional controls are emerging and there is work in the
area of signal transduction. Perhaps surprisingly, a major
gap in the molecular work is that it has not yielded much
information about the function of the low temperature
responsive genes and this must be a major challenge for
the future.
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