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Antisense inhibition of protein phosphatase 2C acceleratescold acclimation in Arabidopsis thaliana
Sari TaÈhtiharju1 and Tapio Palva1,2*1Department of Biosciences, Division of Genetics, 2Institute of Biotechnology, PO Box 56, FIN-00014 University of
Helsinki, Finland
Received 21 December 2000; revised 13 March 2001; accepted 15 March 2001.*For correspondence (fax +35 8 9 191 59079; e-mail Tapio.Palva@helsinki.®).
Summary
Two related protein phosphatases 2C, ABI1 and AtPP2CA have been implicated as negative regulators of
ABA signalling. In this study we characterized the role of AtPP2CA in cold acclimation. The pattern of
expression of AtPP2CA and ABI1 was studied in different tissues and in response to abiotic stresses. The
expression of both AtPP2CA and ABI1 was induced by low temperature, drought, high salt and ABA. The
cold and drought-induced expression of these genes was ABA-dependent, but divergent in various ABA
signalling mutants. In addition, the two PP2C genes exhibited differences in their tissue-speci®c
expression as well as in temporal induction in response to low temperature. To elucidate the function of
AtPP2CA in cold acclimation further, the corresponding gene was silenced by antisense inhibition.
Transgenic antisense plants exhibited clearly accelerated development of freezing tolerance. Both
exposure to low temperature and application of ABA resulted in enhanced freezing tolerance in
antisense plants. These plants displayed increased sensitivity to ABA both during development of frost
tolerance and during seed germination, but not in their drought responses. Furthermore, the expression
of cold-and ABA-induced genes was enhanced in transgenic antisense plants. Our results suggest that
AtPP2CA is a negative regulator of ABA responses during cold acclimation.
Keywords: protein phosphatase 2C, cold acclimation, ABA, signalling, gene expression, Arabidopsis
thaliana.
Introduction
Low and freezing temperatures constitute one of the major
limitations of plant growth, productivity and distribution
(Boyer, 1982). Several plant species are able to cold
acclimate and acquire enhanced freezing tolerance
through exposure to low non-freezing temperatures.
Such adaptation to low temperature is associated with a
number of physiological and developmental alterations
(Sakai and Larcher, 1987). Molecular and cellular
responses to low temperature have been extensively
characterized and a large number of cold-induced genes
have been identi®ed from a number of plants (see
Thomashow, 1999; for a recent review). Yet the mechan-
isms underlying the perception and transduction of low
temperature signals manifested in development of freez-
ing tolerance are just being revealed.
ABA regulates diverse developmental and physiological
responses, including seed maturation, desiccation, dor-
mancy and germination as well as guard cell closure. ABA
also mediates adaptive responses to abiotic environmental
stresses including drought and low temperature (Leung
and Giraudat, 1998). Several types of evidence suggest that
ABA is essential for cold acclimation (Thomashow, 1999).
For example, both ABA-de®cient (aba1) and ABA-insensi-
tive (dominant negative abi1±1 allele) mutants of
Arabidopsis are impaired in development of freezing
tolerance (Heino et al., 1990; LaÊng et al., 1994; MaÈntylaÈ
et al., 1995). Mutants in ABA responsiveness have de®ned
several components of ABA-mediated signal pathways. Of
these ABI3, ABI4 and ABI5 encode transcriptional regula-
tors involved in ABA-induced gene expression (Finkelstein
and Lynch, 2000; Finkelstein et al., 1998; Giraudat et al.,
1992) and ERA1 encodes b subunit of a protein farnesyl
transferase (Cutler et al., 1996). ABI1, as well as ABI2,
encode protein phosphatases 2C (PP2C) (Leung et al., 1994;
Leung et al., 1997; Meyer et al., 1994). This far six genes
encoding PP2Cs have been characterized in Arabidopsis
The Plant Journal (2001) 26(4), 461±470
ã 2001 Blackwell Science Ltd 461
thaliana (Kumori and Yamamoto, 1994; Rodriquez et al.,
1998; Stone et al., 1994; Wang et al., 1999) and four of them
have been shown to be responsive to ABA. However,
precise role of most of these genes in ABA signalling is
unclear. Transient overexpression of ABI1 and AtPP2CA,
blocked both ABA-inducible and ABA-repressible gene
expression in maize mesophyll protoplasts, suggesting
that ABI1 and AtPP2CA function as negative regulators in
ABA signalling (Sheen, 1996; Sheen, 1998). Recent studies
have suggested a role for protein phosphatase 2C (ABI1) as
a negative regulator of ABA-mediated responses to
drought (Gosti et al., 1999). In addition to PP2Cs, also
other protein phosphatases have been implicated in stress
signalling. Protein phosphatase 2 A has been shown to
repress both cold-induced gene expression and freezing
tolerance in alfalfa as well as ABA signalling in tomato
(Monroy et al., 1998; Wu et al., 1997).
In addition to low temperature, drought and application
of ABA also trigger cold acclimation as well as expression
of most of the cold-induced genes suggesting that the
stress-induced expression of these genes is controlled by a
multitude of signalling pathways (Nordin et al., 1991;
Nordin et al., 1993; Shinozaki and Yamaguchi-Shinozaki,
1996). This far, six different either ABA-dependent or ABA-
independent regulatory pathways have been implicated in
the transduction of information between the initial osmotic
or low temperature stimulus and target gene expression
(Shinozaki and Yamaguchi-Shinozaki, 2000). However, the
molecular mechanisms underlying the perception and
transduction of the ABA signal to the target genes in the
nucleus remain elusive and knowledge of the transduction
pathways, especially in cold stress responses, is still
incomplete. The involvement of PP2Cs in cold acclimation
is also far from clear. In this study we wanted to elucidate
the role of protein phosphatase AtPP2CA during cold
acclimation. First we characterized the expression pro®les
of AtPP2CA and ABI1 in different tissues and in response
to abiotic stresses. We show that these genes exhibit
differential expression in response to cold and drought. To
study the contribution of PP2Cs to ABA signaling we
generated transgenic AtPP2C antisense plants. The inhib-
ition of AtPP2CA expression accelerated cold acclimation
and development of freezing tolerance of Arabidopsis.
Furthermore, the transgenic plants appeared hypersensi-
tive to ABA. This enhanced responsiveness to low tem-
perature and ABA was accompanied by corresponding
enhancement of low temperature-and ABA-induced gene
expression
Results
AtPP2CA and ABI1 are expressed differentially
To elucidate the role of PP2Cs in mediating plant response
to environmental stress we ®rst characterized the pattern
of expression of the corresponding genes in different
tissues and in response to various abiotic stresses. To
characterize the spatial expression of AtPP2CA we studied
accumulation of the corresponding transcript in roots,
stems, leaves and ¯owers of Arabidopsis. The accumula-
tion of AtPP2CA transcripts was detected in all of the plant
tissues tested, with the most prominent expression in
leaves (Figure 1a). Although the Arabidopsis PP2Cs are
implicated in stress signalling (Gosti et al., 1999; Sheen,
1998) their expression in response to cold stress has not
been characterized. We thus ®rst examined the accumula-
tion of AtPP2CA and ABI1 transcripts in response to the
low non-freezing temperature of + 4°C (Figure 1b). Low
temperature exposure led to an increase in AtPP2CA
transcript levels until they reached a steady state level
after 12 hours, whereas the expression of ABI1 exhibited a
transient increase followed by a rapid decrease.
Many of the cold-induced genes are also responsive to
exogenous ABA and dehydration (Thomashow, 1999). We
thus tested if the expression of AtPP2CA and ABI1 would
be responsive to exogenous ABA and to different stress
treatments including low temperature, dehydration and
high salt. The involvement of ABA in activation of AtPP2CA
and ABI1 in response to these stresses was examined by
utilizing various ABA signalling mutants (Figure 2). All
stress treatments tested induced expression of both
AtPP2CA and ABI1. As expected ABA treatment increased
the transcript levels slightly more in the era1±1 mutant
than in the wild type. However, ABI1 expression level was
lower in particular in the abi2±1 and abi3±1 mutants while
Figure 1. Expression of AtPP2CA in different organs and during coldacclimation of Arabidopsis.(a) AtPP2CA transcript levels in different organs of Arabidopsis. Eachlane contains 10 mg total RNA isolated from roots (R), stems (S), leaves(L) and, ¯owers (F) of 5-week-old plants. (b) Comparison of AtPP2CA andABI1 expression during cold acclimation in 3-week-old plants. Total RNAwas isolated from plants grown in control conditions (0) and exposed to4°C for times indicated.
462 Sari TaÈhtiharju and Tapio Palva
ã Blackwell Science Ltd, The Plant Journal, (2001), 26, 461±470
expression of AtPP2CA was not affected. Dehydration
stress-induced accumulation of AtPP2CA and ABI1 tran-
scripts was reduced or inhibited in aba1±1 and abi1±1
mutants. Surprisingly, dehydration-induced expression of
AtPP2CA was also reduced in era1±1. In contrast to other
treatments, high salinity-induced expression of both PP2C
genes was ABA-independent, as indicated by accumula-
tion of the mRNA also in the aba1±1 mutants. The most
striking difference between AtPP2CA and ABI1 expression
was in their response to cold stress. While expression of
AtPP2CA was slightly enhanced in the era1±1 mutant, the
cold-induced expression of ABI1 decreased in aba1±1,
abi2±1, abi3±1 and era1±1 mutants. AtPP2CA and ABI1
transcript levels did not increase in the aba1±1 mutant in
response to low temperature.
Generation of transgenic AtPP2CA-antisense plants
Since no potent inhibitor of PP2C has been identi®ed, the
contribution of AtPP2CA to cold acclimation cannot be
investigated by pharmacological approaches (Hunter,
1995). We thus decided to use antisense technology to
silence the AtPP2CA gene. To generate transgenic
Arabidopsis plants, which show antisense inhibition of
AtPP2CA, the full-length cDNA sequence derived from the
Ler ecotype was placed under the control of the constitu-
tive CaMV 35S promoter in antisense orientation. This
construct was introduced into plants and kanamycin
resistant transformants were selected. Independent trans-
genic lines were screened for antisense inhibition of
AtPP2CA expression at the transcriptional level. Two
lines harbouring one copy of antisense AtPP2CA gene
were selected for further experiments and they showed
similar responses. The expression of AtPP2CA was
inhibited in both control and cold-induced plants showing
that the antisense inhibition of this gene is effective even
under inducing conditions (Figure 3). The speci®city of this
antisense inhibition was demonstrated by assessing
expression of the related PP2C, ABI1.
Transgenic antisense plants cold acclimate faster than
wild-type plants but are not affected in drought tolerance
To determine whether the inhibition of AtPP2CA had any
effect on cold acclimation and freezing tolerance, we
tested the freezing tolerance of wild type and antisense
plants during a seven-day acclimation period (Figure 4a).
Figure 3. Expression of AtPP2CA and ABI1 in wild type and transgenicantisense plants.The expression of AtPP2CA and ABI1 was compared in wild type (Ler)and transgenic AtPP2CA-antisense plants (A115) exposed to eithercontrol conditions (NA) or to 4°C for 24 h (CA). AtPP2CA antisense RNAprobe was used to detect the expression of endogenous AtPP2CA mRNA.
Figure 2. Abiotic stress-induced expression of AtPP2CA and ABI1 indifferent ABA signalling mutants.(a) The expression pro®les of the AtPP2CA gene in response to ABA andabiotic stresses. Total RNA was isolated from control plants grown at20°C and from plants exposed to different stress treatments; 60 mM ABAfor 6 h, dehydration for 4 h, 300 mM NaCl (salt) for 24 h and 4°C for 24 h(cold). 3 week-old wild type plants (Ler), ABA-de®cient (aba) and ABA-insensitive (abi) mutants and mutants having enhanced response to ABA(era) were used.(b) The expression pro®les of the ABI1 gene in responseto ABA and abiotic stresses. Membrane hybrized with AtPP2CA speci®cprobe was stripped and rehybrized with ABI1 speci®c probe.
PP2C regulates cold acclimation 463
ã Blackwell Science Ltd, The Plant Journal, (2001), 26, 461±470
Comparison of the freezing tolerance of nonacclimated
transgenic antisense plants and wild-type plants did not
reveal any differences. Neither was there any obvious
effect on plant growth and development in transgenic
antisense plants under normal growth conditions. In
contrast, the inhibition of AtPP2CA expression accelerated
cold acclimation in transgenic antisense plants. The freez-
ing tolerance of antisense plants increased by 3°C already
after one-day exposure to low temperature and the freez-
ing tolerance stayed higher than in wild type until the
plants achieved the maximum freezing tolerance after ®ve
days. Even 16 h of low temperature exposure (but not
12 h) was suf®cient to trigger a signi®cant increase (1.5°C)
in freezing tolerance.
To elucidate the involvement of AtPP2CA in dehydration
stress response we tested if the antisense plants were
more tolerant to desiccation-induced water loss. In con-
trast to the clear phenotypic difference in abi1±1 (Parcy &
Giraudat 1997), the kinetics of water loss in aerial parts of
each of the AtPP2CA antisense plants was similar to that of
wild-type plants (data not shown). When whole plants
were subjected to progressive drought stress, the anti-
sense plants tested displayed a similar drought tolerance
as wild-type plants (data not shown).
Transgenic antisense plants exhibit enhanced sensitivity
to ABA
Treatment of transgenic antisense plants and wild-type
plants with exogenous ABA gave a similar result as
Figure 5. Expression of cold- and ABA-inducible genes in wild type andAtPP2CA-antisense plants.Total RNA was isolated from wild type (Ler) and transgenic AtPP2CA-antisense plants (A115) after exposure to 4°C (for times indicated) or toexogenous ABA for 4 h. RNA was hybridized sequentially with probescorresponding to genes indicated on the left.
Figure 4. Effect of low temperature acclimation and exogenous ABA onfreezing tolerance of wild type and transgenic AtPP2CA antisense plants.(a) Freezing tolerance of 2-week-old wild type (Ler) and AtPP2CAantisense plants (A115) after cold acclimation for times indicated. Controlplants were grown at 20°C. (b) Freezing tolerance of 2-week-old wild type(Ler) and AtPP2CA antisense plants (A115) after exposure to 0, 1, 3, 10and 60 mM ABA for 24 h. Data is a mean 6 secD of three to fourindependent measurements.
464 Sari TaÈhtiharju and Tapio Palva
ã Blackwell Science Ltd, The Plant Journal, (2001), 26, 461±470
obtained for cold acclimated plants (data not shown)
suggesting the involvement of ABA in the enhanced cold
acclimation of transgenic plants. To further analyse if the
increase in freezing tolerance was due to enhanced
responsiveness to ABA, we tested the freezing tolerance
of plants exposed to different concentrations of ABA for
one day (Figure 4b). The antisense plants appeared more
responsive to ABA, as indicated by their ability to increase
their freezing tolerance already after exposure to as low as
1 mM ABA. This enhanced responsiveness was also evident
even at higher ABA concentrations. It was manifested in
the consistently higher tolerance achieved at every ABA
concentration tested in the AtPP2CA antisense plants.
The dose±response analyses described above showed
that the antisense plants were more sensitive to applied
ABA than the wild-type plants. Indeed, antisense seeds
displayed an increased sensitivity to ABA during germin-
ation as compared to the wild-type seeds (data not shown).
Even as low as 0.1 mM ABA already inhibited germination of
transgenic seeds suggesting that AtPP2CA might be
involved in several ABA responses. This is supported by
the expression pro®le of the AtPP2CA gene, which was
induced by several stresses including dehydration.
Expression of cold-and ABA-induced genes is enhanced
in transgenic antisense plants
To further elucidate the role of AtPP2CA in cold acclima-
tion we examined if the increase in freezing tolerance in
antisense plants was accompanied by altered expression
of cold-induced genes. The expression of cold and ABA-
inducible genes RAB18 (LaÊng and Palva, 1992), RCI2A/LTI6
(Capel et al., 1997; Nylander et al. 2001) and LTI78 (Nordin
et al., 1991, 1993) was characterized (Figure 5). The cold
induction of these genes was enhanced in AtPP2CA
antisense plants compared to wild-type plants. LTI6 and
LTI78 expression was already evident after one-hour
exposure to low temperature in antisense plants while in
wild-type plants they were expressed later. RAB18 was
even more sensitive to low temperature since its expres-
sion was induced already after half-hour exposure to cold.
This is in contrast to the modest increase of RAB18
transcript level after 16 hours exposure to low temperature
observed in wild-type plants. Only RAB18 displayed a real
increase in sensitivity to ABA in antisense plants since
even 1 mM ABA was suf®cient to trigger RAB18 expression.
Notably, the above stress-inducible genes were not
expressed under the normal growth conditions in the
transgenic antisense plants.
To determine if the enhanced expression of stress-
inducible genes in transgenic antisense plants was due to
enhanced expression of transcriptional regulators we
analysed also the expression of CBF1/DREB1B (Figure 5)
(Liu et al., 1998; Stockinger et al., 1997). However, we could
not detect any difference in the expression of CBF1
between wild type and transgenic antisense plants.
Similarly, no differences were observed in the expression
of CBF2/DREB1C, CBF3/DERB1A (Gilmour et al., 1998; Liu
et al., 1998) and DREB2A (Liu et al., 1998) genes (data not
shown).
Discussion
Differential expression of AtPP2CA and ABI1
Our study clearly shows for the ®rst time that protein
phosphatases AtPP2CA and ABI1 are cold inducible. We
also provide a detailed kinetic analysis of expression of
these genes during Arabidopsis cold acclimation.
According to this analysis both AtPP2CA and ABI1 are
triggered at the onset of cold acclimation but only
AtPP2CA is strongly expressed during the whole acclima-
tion process. These differences in expression pro®le
suggest that these PP2Cs have separate roles in cold
acclimation. In addition to these temporal differences, the
two PP2Cs also exhibit some difference in their spatial
expression patterns. The AtPP2CA gene appeared to be
more intensively expressed in leaves, unlike ABI2, which is
mainly expressed in stems and roots, or ABI1, which is
strongly expressed in leaves, roots and stems (Leung et al.,
1997) suggesting some speci®city in function.
In addition to differences in spatial and temporal
expression, a detailed analysis of AtPP2CA and ABI1
expression during abiotic stresses also showed that
genes are expressed differentially. Both PP2Cs are, in
addition to low temperature, also induced by salt and
dehydration stress, and by exogenous ABA. The cold-and
dehydration-but not salt-induced expression of AtPP2CA
and ABI1 is ABA-dependent since both mRNAs were
reduced in the ABA-de®cient aba1±1 mutant as has been
previously shown for ABI1 and ABI2 (Leung et al., 1997).
Expression patterns of AtPP2CA and ABI1 were clearly
divergent during dehydration and in particular during cold
stress. The ABI-and ERA-dependent expression of ABI1
suggested the existence of a complex regulation of ABI1
expression in cold stress while the expression of AtPP2CA
was only ABI1-dependent. Surprisingly, dehydration-
induction of AtPP2CA, as well as cold-induction of ABI1,
was reduced in era1 mutant, which is hypersensitive to
ABA (Cutler et al., 1996). ERA1 has been shown to function
as a negative regulator of ABA signalling in guard cells (Pei
et al., 1998) as well as in seeds (Cutler et al., 1996). We thus
suggest that ERA1 may function as a regulator of AtPP2CA
and ABI1 expression. However, further studies are needed
since the era1 mutant is in Columbia background and not
in Ler as aba-and abi-mutants. The apparent involvement
of ABI3, a seed-speci®c transcriptional activator (Giraudat
et al., 1992), in the regulation of AtPP2CA and ABI1
expression was surprising. However, Rohde et al. (2000)
PP2C regulates cold acclimation 465
ã Blackwell Science Ltd, The Plant Journal, (2001), 26, 461±470
recently stated that ABI3 regulates differentiation and
dormancy both in seeds and vegetative meristems, sug-
gesting that ABI3 might regulate expression of some
genes also in vegetative tissues. Taken together, our
results demonstrate clear differences in expression of the
two related protein phosphatase genes, AtPP2CA and
ABI1, suggesting separate functions for the corresponding
protein phosphatases in abiotic stress responses.
Contribution of AtPP2CA to freezing tolerance and ABA
sensitivity
Our results indicate that AtPP2CA is an integral component
of low temperature signal transduction and controls ABA
responsiveness in plant cold acclimation. The inhibition of
AtPP2CA expression in transgenic antisense plants accel-
erated cold acclimation and led to higher freezing toler-
ance already after 16 h exposure to low temperature. This
increase in tolerance correlates well with the transient
increase of endogenous ABA levels after 15 h exposure to
low temperature in Arabidopsis (LaÊng et al., 1994). The
central role of ABA in the enhancement of freezing
tolerance was further supported by the notion that the
development of tolerance of antisense plants was acceler-
ated also by exogenous ABA. Dose±response studies
demonstrated that antisense plants were more sensitive
to ABA as compared to wild-type plants. Consequently, the
observed acceleration of cold acclimation is readily
explained by enhanced responsiveness to ABA in the
AtPP2CA antisense plants and suggests that AtPP2CA is a
negative regulator of cold acclimation. This is supported
by the studies of Sheen (1998) suggesting that both ABI1
and AtPP2CA act as negative regulators of ABA signalling.
Interestingly, the transgenic AtPP2CA antisense plants
were also more sensitive than wild type to the ABA
inhibition of seed germination. However, we could not
detect any alteration in another ABA-mediated response,
development of drought tolerance. This is in contrast to
results of Gosti et al. (1999) who recently demonstrated
that a loss of ABI1 PP2C activity leads, in addition to an
enhanced sensitivity to ABA in seed dormancy, also to
enhanced drought tolerance. Similar to abi1±1 revertants
also era1 mutants show hypersensitivity to ABA inhibition
of seed germination and enhanced drought tolerance
(Cutler et al., 1996; Pei et al., 1998). Indeed, all these
regulators of ABA signalling have an effect on seed
germination. However, so far only AtPP2CA seems to
have more speci®c control of ABA-dependent processes
during cold acclimation.
AtPP2CA±a negative regulator of cold-and ABA-inducible
gene expression
Our results indicate that AtPP2CA is a negative regulator of
cold and ABA-inducible gene expression. AtPP2CA was
shown to modulate the expression of several cold-
inducible genes. These genes are also responsive to
exogenous ABA but belong to different classes based on
how ABA is involved in the regulation of their expression.
The cold-induced expression of LTI78 can be ABA-inde-
pendent (Nordin et al., 1991) whereas the cold-induced
expression of RAB18 and RCI2A/LTI6 is ABA-dependent
(LaÊng and Palva, 1992; Nylander and Palva, unpublished
results). However, RAB18 and RCI2A/LTI6 belong to differ-
ent signalling pathways, since the expression of RAB18 is
also ABI1-dependent, whereas the expression of RCI2A/
LTI6 is ABI1-independent. Enhanced low temperature-
induced expression of these genes as well as enhanced
ABA-induced expression of RAB18 suggests that at the
onset of cold acclimation AtPP2CA modulates ABA signal-
ling thus regulating the expression of ABA-responsive
genes. However, there are differences in this regulation
due to the differences in ABA-dependency. Although all of
the above genes have putative ABA responsive elements
(ABRE) in their promoters and are induced by ABA, some
of them can also be triggered through an ABA-independ-
ent pathway (LaÊng and Palva, 1992; Nordin et al., 1993;
Nylander et al. 2001). Consequently, the expression of
RAB18, which is dependent on both ABA and ABI1,
exhibits the strongest response to silencing of AtPP2CA.
Interestingly, neither expression of CBF/DREB1 (Gilmour
et al., 1998; Liu et al., 1998; Stockinger et al., 1997) nor
DREB2A genes (Liu et al., 1998), transcriptional activators
involved in cold-and dehydration-induced gene expres-
sion, was affected by antisense inhibition of AtPP2CA. Our
results indicate that AtPP2CA is a component of the CBF/
DREB-independent signal pathway controlling expression
of cold-and ABA-inducible genes and suggest a possible
role for ABRE binding factors (Choi et al. 2000; Uno et al.
2000) in this process. Furthermore, the results suggest that
enhanced expression of the above stress-inducible genes
in transgenic antisense plants appeared to contribute, at
least in part, to the improved freezing tolerance.
AtPP2CA±a negative regulator controlling cold
acclimation
The present study demonstrates that AtPP2CA is an
important modulator of the ABA action during cold stress.
Hypothetical model for the role of AtPP2CA in ABA
signalling during cold acclimation and dehydration stress
is presented in Figure 6. When plants are exposed to cold
or dehydration stress the endogenous ABA levels increase
(LaÊng et al., 1994). The dissimilarities in the expression of
ABI1 and AtPP2CA in different ABA signalling mutants and
the different stress tolerance phenotypes of transgenic
AtPP2CA antisense plants and intragenic abi1±1 revertants
(Gosti et al., 1999) suggest the existence of distinct
upstream regulatory mechanisms in ABA signalling during
466 Sari TaÈhtiharju and Tapio Palva
ã Blackwell Science Ltd, The Plant Journal, (2001), 26, 461±470
cold acclimation and during dehydration stress. We thus
propose that at the onset of cold acclimation the ABA
signalling goes through an AtPP2CA-modulated pathway,
which regulates the expression of both AtPP2CA and
target genes downstream of AtPP2CA. Induction of
AtPP2CA would allow feedback regulation of ABA signal-
ling and hence controlled induction of the target genes and
development of freezing tolerance. In this way AtPP2CA
continuously modulates ABA signalling during the cold
acclimation process. Interestingly ERA1, another negative
regulator of ABA signalling, appears to repress the
expression of AtPP2CA during cold acclimation. Indeed,
recent studies suggest the presence of several negative
regulators of ABA signalling in plants (Cutler et al., 1996;
Foster and Chua, 1999; Gosti et al., 1999; Sheen, 1998),
which could re¯ect the complexity of ABA-controlled
responses in plants. It is likely that these regulators of
ABA signalling, including AtPP2CA, monitor and reset the
ABA-signalling network allowing the cell to continuously
monitor the presence or absence of ABA as suggested for
ABI1 by Gosti et al. (1999).
In contrast to low temperature-induced acclimation, the
action of AtPP2CA during drought stress appears to be
regulated by ABI1, whose expression in turn is regulated
by ERA1 (Figure 6). Indeed, ERA1 seems to enhance one
pathway while repressing another, thus allowing the cross
talk between dehydration-and cold stress-induced ABA
signalling. We propose that, in addition to ERA1, AtPP2CA
also could mediate the interaction between dehydration
and cold signal pathways during acclimation and provide
one path to integrate stress signals leading to enhanced
freezing tolerance. Since AtPP2CA antisense plants did not
show alterations in their drought tolerance but exhibited
enhanced freezing tolerance and expression of cold
induced genes, we propose AtPP2CA to be involved in
development of tolerance to cellular dehydration during
cold acclimation.
Our study demonstrates that AtPP2CA is an important
negative regulator of ABA signalling during cold stress
although the precise role of AtPP2CA in this process
remains to be elucidated. However, our study does not rule
out the role of other PP2Cs and protein phosphatases in
this process. Possible roles of protein phosphatases 2 A
and 1 as well as cADPR in ABA signalling, as proposed by
Mùller and Chua (1999), remain to be investigated. It
would be also informative to test freezing tolerance of the
intragenic abi1±1 revertant (Gosti et al., 1999).
Furthermore, the existence of homologous and possibly
functionally redundant PP2Cs also make evident that
further studies using double mutants are needed to
examine whether simultaneous disruption of PP2C activ-
ities leads to a further increase in freezing tolerance. In
addition, our study clearly shows that genetic engineering
of signalling components other than transcriptional acti-
vators (Jaglo-Ottosen et al., 1998; Liu et al., 1998) can be
employed to increase stress tolerance in plants, in this
case speci®cally freezing tolerance.
Experimental procedures
Plant material
Arabidopsis thaliana (L.) Heynh., ecotype Landsberg erecta andmutants affecting ABA metabolism, aba1±1, or ABA responsive-ness, abi1±1, abi2±1 and abi3±1, were kindly provided byArabidopsis Biological Resource Center (The Ohio StateUniversity, Columbus, Ohio, USA). era1±1 seeds were a gift ofDr Peter McCourt (University of Toronto, Toronto, Canada). Thephenotypes of abi mutants veri®ed by germinating seeds on themedia containing 10 mM ABA.
Axenic cultures of Arabidopsis were prepared as described byTaÈhtiharju et al. (1997). For scoring kanamycin resistance themedium included 50 mg ml±1 kanamycin. The plants were incu-bated for 3 days at 4°C to break any residual dormancy, and thentransferred to a controlled-environment chamber with an airtemperature of 22°C using a 14-h photoperiod (120 mmol m±2 s±1
photosynthetically active radiation). One week after sowing the
Figure 6. A hypothetical model for AtPP2CA-mediated cold anddehydration stress signal transduction.At the onset of cold acclimation the ABA signalling goes through theAtPP2CA controlled pathway. Perception of cold (solid line) ordehydration (dotted line) signal triggers an increase in endogenous ABA,which is blocked in ABA-de®cient, aba1 mutants (indicated by doubleline). These increases in ABA trigger signal cascades leading to aexpression of ABI1 and AtPP2CA as well as cold-inducible target genesdownstream of AtPP2CA. These signal pathways are regulated by ERA1either positively (arrowhead line) or negatively (end line) depending onstress. AtPP2CA mediates the interaction between dehydration and coldsignal pathways during acclimation and integrates stress signals leadingto enhanced freezing tolerance. Induction of AtPP2CA allows feedbackregulation of ABA signalling and hence controlled induction of the cold-inducible target genes.
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seeds plantlets were transferred onto 24-well plates containing1 ml of MS medium described in TaÈhtiharju et al. (1997), oneplantlet per well. ABA treatment was performed as describedpreviously (LaÊng et al., 1989). Control plants were grown as above(without ABA addition). Low temperature treatment was done byexposing plants to 4°C under 14-h photoperiod for times indicatedin ®gure legends. Salt treatment was performed by exposingplants to 300 mM NaCl for 24 h. Dehydration of excised shoots ofArabidopsis was performed by subjecting them to desiccation for4 h on chromatography paper (3 MM; Whatman, Maidstone,England) moisturized with liquid 0.5 3 MS media.
RNA isolation and RNA gel blot analysis
Isolation of total RNA from shoots and the northern blotting wereperformed as described previously (TaÈhtiharju et al., 1997). TheRNA was transferred to a nylon membrane (BoehringerMannheim, Mannheim, Germany) and hybridised with digox-igenin (DIG)-labelled DNA or RNA probes. DNA Probes werelabelled by incorporating of digoxigenin-11-dUTP during PCR.Primer pairs 5¢-GGAATTCCATATGGCTGGGATTTGTTGCGGTG-TTG-3¢ and 5¢-GTGGATCCGACGGCGTCACA-3¢ for AtPP2CA(Kumori and Yamamoto, 1994) and 5¢-ATCAAATCTGCACCG-CATATGGAG-3¢ and 5¢-GAAATGAGCGGCGGATTGAGGATC-3¢for ABI1 (Leung et al., 1994; Meyer et al., 1994) were designed toamplify N-terminal part of the coding sequence of the genes fromcDNA templates, to give a 668-bp and a 534-bp PCR products,respectively. Primer was also designed to amplify part of thecoding sequence of the Arabidopsis transcript LTI78 (Nordin et al.,1991; Nordin et al., 1993) to give PCR product of 1075 from cDNAtemplate. Primer pair used was 5¢-CGATGGGCTTTGGTAGTGA-3¢and 5¢-CTTAAAGCTCCTTCTGCACC-3¢ for LTI78. Primers 5¢-GGG-GTCTAGAACCGTCCAGGAGGTC-3¢ and 5¢-CCATGCCATGGCCA-CCACCGGGAAGCTTTTCC-3¢ for RAB18 (LaÊng et al. 1992) and5¢-ATGAGTACAGCTACTTTCGT-3¢ and 5¢-ATGATAGATGGTAA-ATCATT-3¢ for LTI6 (Nylander, M., Helenius, E., Lindgren, O.,Baudo, M.M., Heino, P. Genbank accession AF104221) weredesigned to amplify the full length coding sequences of thesegenes from cDNA templates and to give PCR products of 560 bpand 476 bp, respectively. CBF1 gene speci®c probe was asdescribed by Gilmour et al. (1998). Antisense RNA probe corres-ponding to AtPP2CA was labelled using DIG-RNA-Labelling Kit(Boehringer Mannheim, Mannheim, Germany).
The hybridizations were performed according to manufacturersinstructions (Boehringer Mannheim, Mannheim, Germany). Equalloading was con®rmed by hybridization of the ®lters to a DIG-labelled rRNA probe. DIG-labelled partial 18S rRNA was obtainedby PCR with universal T7 and SP6 primers using Betula pendulapartial 18S rRNA gene (GenBank accession AJ279693) as atemplate. The DIG-labelled nucleic acids were immuno-detectedwith an alkaline phosphatase-conjugated antidigoxigenin anti-body and then visualized with the chemiluminescent substrateCSPDâ (Boehringer Mannheim, Mannheim, Germany).Experiments were repeated three times with similar results eachtime.
Cloning of AtPP2CA, vector construction and plant
transformation
CDNA for AtPP2CA (Kumori and Yamamoto, 1994) was clonedfrom cDNA of Arabidopsis plants by polymerase chain reaction byusing the following primer pairs: 5¢-ACGCGTCGACATGGCTGGG-ATTTGTTGCGGTGTTG-3¢ and 5¢-ACGCGTCGACTTAAGACGACG-
CTTGATTATTCCTC-3¢. The PCR fragment was digested with Sal1,subcloned into pGEM-3Z vector (Promega, Madison, WI, USA)resulting in the plasmid called pSTM6 and sequenced.
Transgenic Arabidopsis plants that underexpress AtPP2CAwere created by placing cDNA encoding this phosphatase inantisense orientation under the control of the strong CaMV 35SRNA promoter and transforming the chimeric gene intoArabidopsis plants. AtPP2CA was cloned by PCR from plasmidpSTM6 (see above) containing full length AtPP2CA cDNA usingtwo primers: (i) 5¢-CCGTGATCAATGGCTGGGATTTGTTGCGG-TGTTG, for creating an Bcl1 site at the start codon of AtPP2CAgene, and (ii) 5¢-CCGTGATCATTAAGACGACGCTTGATTATTCCTC,for creating an Bcl1 site at the stop codon of AtPP2CA gene. ThePCR fragment was digested with Bcl1 and the resulting fragmentwas cloned to BamH1 site of pHTT202 (a kind gift of Dr TeemuTeeri, Institute of Biotechnology, Helsinki, Finland) a vectorsimilar to pHTT370 in Elomaa et al. (1993), which contains theCaMV 35S RNA promoter. Antisense construct was identi®ed byrestriction analysis and subsequently was sequenced.
The chimeric plasmid was transferred to Agrobacteriumtumefaciens as described in Elomaa et al. (1993). Arabidopsisplants were transformed by the vacuum in®ltration procedure(Bechtold et al., 1993). Seeds were harvested from individual potsand plated on selection medium to identify transgenic progeny.For each T1, T2 and T3 progeny seeds were plated on selectionmedium to assay the segregation ratio of kanamycin resistanceand the copy number of the transgene. Transgenic lines carryinga single copy were selected, and T3 progenies homozygous forkanamycin resistance were used for subsequent studies.
Measurements of freezing tolerance
Freezing tolerance was determined in shoots after cold acclima-tion by measuring the extent of freezing injury by the ion-leakagemethod as described by TaÈhtiharju et al. (1997).
Acknowledgements
We thank Prof. Teemu Teeri (University of Helsinki, Finland) forkindly providing pHTT202 and Agrobacterium strain and Prof.Dirk Inze's laboratory (University of Gent, Belgium) for pABI1plasmid. We also thank Dr Peter McCourt (University of Toronto,Canada) for era1±1 seeds and Arabidopsis Biological ResourceCenter for Arabidopsis aba and abi mutant seeds. Pekka Heinoand Ilkka Tamminen are thanked for useful scienti®c discussionsand for critical reading of the manuscript. Arja IkaÈvalko is thankedfor preparing the plant culture media. This work was supported byAcademy of Finland (projects no. 38034, 44252 and 44883; FinnishCentre of Excellence Programme 2000±2005) and BiocentrumHelsinki.
References
Bechtold, N., Ellis, J. and Pelletier, G. (1993) In plantaArgobacterium mediated gene transfer by in®ltration of adultArabidopsis thaliana plants. C.R. Acad. Sci. Paris, 316,1994±1999.
Boyer, J.S. (1982) Plant productivity and environment. Science,218, 443±448.
Capel, J., Jarillo, J.A., Salinas, J. and MartõÂnez-Zapater, J.M.
(1997) Two homologous low-temperature-inducible genes from
468 Sari TaÈhtiharju and Tapio Palva
ã Blackwell Science Ltd, The Plant Journal, (2001), 26, 461±470
Arabidopsis encode highly hydrophilic proteins. Plant Physiol.115, 569±576.
Choi, H.-I., Hong, J.-H., Ha, J.-O., Kang, J.-Y. and Kim, S.Y. (2000)ABFs, a family of ABA-responsive element binding factors. J.Biol. Chem. 21, 1723±1730.
Cutler, S., Ghassemian, M., Bonetta, D., Cooney, S. and McCourt,P. (1996) A protein farnesyl transferase involved in abscisic acidsignal transduction in Arabidopsis. Science, 273, 1239±1240.
Elomaa, P., Honkanen, J., Puska, R., SeppaÈnen, P., Helariutta, Y.,Mehto, M., Kotilainen, M., Nevalainen, L. and Teeri, T.H. (1993)Agrobacterium mediated transfer of antisense chalconesynthase cDNA to Gerbera hybrida inhibits ¯owerpigmentation. Bio/Technology, 11, 508±511.
Finkelstein, R.R. and Lynch, T.J. (2000) The Arabidopsis Abscisicacid response gene ABI5 encodes a basic leucine zippertranscription factor. Plant Physiol. 12, 599±609.
Finkelstein, R.R., Wang, M.L., Lynch, T.J., Rao, S. and Goodman,H.M. (1998) The Arabidopsis abscisic acid response locus ABI4encodes an APETALA2 domain protein. Plant Cell, 10,1043±1054.
Foster, R. and Chua, N.-H. (1999) An Arabidopsis mutant withderegulated ABA gene expression: implications for negativeregulator function. Plant J. 17, 363±372.
Gilmour, S.J., Zarka, D.G., Stockinger, E.J., Salazar, M.P.,Houghton, J.M. and Thomashow, M.F. (1998) Lowtemperature regulation of the Arabidopsis CBF family of AP2transcriptional activators as an early step in cold-induced CORgene expression. Plant J. 16, 433±442.
Giraudat, J., Hauge, B.M., Valon, C., Smalle, J., Parcy, F. andGoodman, H.M. (1992) Isolation of the Arabidopsis ABI3 geneby positional cloning. Plant Cell, 4, 1251±1261.
Gosti, F., Beaudoin, N., Serizet, C., Webb, A.A., Vartanian, N. andGiraudat, J. (1999) ABI1 protein phosphatase 2C is a negativeregulator of abscisic acid signaling. Plant Cell, 11, 1897±1909.
Heino, P., Sandman, G., LaÊng, V., Nordin, K. and Palva, E.T. (1990)Abscisic acid de®ency prevents development of freezingtolerance in Arabidopsis thaliana (L.) Heynh. Theor. Appl.Genet. 79, 801±806.
Hunter, T. (1995) Protein kinases and phosphatases: the yin andyang of protein phosphorylation and signaling. Cell, 80,225±236.
Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., Schabenberger,O. and Thomashow, M.F. (1998) Arabidopsis CBF1overexpression induces COR genes and enhances freezingtolerance. Science, 280, 104±106.
Kumori, T. and Yamamoto, M. (1994) Cloning of cDNAs fromArabidopsis thaliana that encode putative protein phosphatase2C and human Dr-1-like protein by transformation of a ®ssionyeast mutant. Nuc. Acid Res. 22, 5296±5301.
Leung, J. and Giraudat, J. (1998) Abscisic acid signaltransduction. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49,199±222.
Leung, J., Bouvier-Durand, M., Morris, P.-C., Guerrier, D., Chefdor,F. and Giraudat, J. (1994) Arabidopsis ABA response gene ABI1:Features of a calcium-modulated protein phosphatase. Science,264, 1448±1452.
Leung, J., Merlot, S. and Giraudat, J. (1997) The ArabidopsisABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encodehomologous protein phophatases 2C involved in abscisic acidsignal transduction. Plant Cell, 9, 759±771.
Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K. and Shinozaki, K. (1998) Two transcriptionfactors, DREB1 and DREB2, with an EREBP/AP2 DNA bindingdomain separate two cellular signal transduction pathways in
drought- and low temperature-responsive gene expression,respectively. In: Arabidopsis. Plant Cell, 10, 1391±1406.
LaÊng, V. and Palva, E.T. (1992) The expression of a rab-relatedgene, rab18, is induced by abscisic acid during the coldacclimation process of Arabidopsis thaliana (L.) Heynh. PlantMol. Biol. 20, 951±962.
LaÊng, V., Heino, P. and Palva, E.T. (1989) Low temperatureacclimation and treatment with exogenous abscisic acid inducecommon polypeptides in Arabidopsis thaliana (L.) Heynh.Theor. Appl. Genet. 77, 729±734.
LaÊng, V., MaÈntylaÈ , E., Welin, B., Sundberg, B. and Palva, E.T.
(1994) Alterations in water status, endogenous abscisic acidcontent, and expression of rab18 gene during the developmentof freezing tolerance in Arabidopsis thaliana. Plant Physiol. 104,1341±1349.
Meyer, K., Leube, M.P. and Grill, E. (1994) A protein phosphatase2C involved in ABA signal transduction in Arabidopsis thaliana.Science, 264, 1452±1455.
Mùller, S.G. and Chua, N.-H. (1999) Interactions and intersectionsof plant signaling pathways. J. Mol. Biol. 293, 219±234.
Monroy, A.F., Sangwan, V. and Dhindsa, R.S. (1998) Lowtemperature signal transduction during cold acclimation:protein phosphatase 2A as an early target for cold-inactivation. Plant J. 13, 653±660.
MaÈntylaÈ , E., LaÊng, V. and Palva, E.T. (1995) Role of abscisic acid indrought-induced freezing tolerance, cold acclimation, andaccumulation of LTI78 and RAB18 proteins in Arabidopsisthaliana. Plant Physiol. 107, 141±148.
Nordin, K., Heino, P. and Palva, E.T. (1991) Separate signalpathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh. Plant Mol.Biol. 16, 1061±1071.
Nordin, K., Vahala, T. and Palva, E.T. (1993) Differentialexpression of two related, low-temperature-induced genes inArabidopsis thaliana (L.) Heynh. Plant Mol. Biol. 21, 641±653.
Nylander, M., Heino, P., Helenius, E., Palva, E.T., Ronne, H., Welin,
B.V. (2001) The low temperature- and salt-induced RC12A geneof Arabidopsis complements the sodium sensitivity caused by adetection of the homologous yeast gene SNA1. Plant Mol Biol.45, 341±352.
Pei, Z.-M., Ghassemian, M., Kwak, C.M., McCourt, P. and
Schroder, J.I. (1998) Role of farnesyltransferase in ABAregulation of guard cell anion channels and plant water loss.Science, 282, 287±290.
Rodriquez, P.L., Leube, M.P. and Grill, E. (1998) Molecular cloningin Arabidopsis thaliana of a new protein phosphatase 2C (PP.2C) with homology to ABI1 and ABI2. Plant Mol Biol. 38,879±883.
Rohde, A., Kurup, S. and Holdsworth, M. (2000) ABI3 emergesfrom seed. Trends Plant Sci. 5, 418±419.
Sakai, A. and Larcher, W. eds. (1987) Frost survival of plants.Responses and Adaptation to Freezing Stress. Springer Verlag.Berlin, Heidelberg, New York.
Sheen, J. (1996) Ca2+-dependent protein kinases and stress signaltransduction in plant. Science, 274, 1900±1902.
Sheen, J. (1998) Mutational analysis of protein phosphatase 2Cinvolved in abscisic acid signal transduction in higher plants.Proc. Natl Acad. Sci. 95, 975±980.
Shinozaki, K. and Yamaguchi-Shinozaki, K. (1996) Molecularresponses to drought and cold stress. Curr. Opin. Biotech. 7,161±167.
Shinozaki, K. and Yamaguchi-Shinozaki, K. (2000) Molecularresponses to dehydration and low temperature: differences
PP2C regulates cold acclimation 469
ã Blackwell Science Ltd, The Plant Journal, (2001), 26, 461±470
and cross talk between two stress signaling pathways. Curr.Opin. Plant Biol. 3, 217±223.
Stockinger, E.J., Gilmour, S.J. and Thomashow, M.F. (1997)Arabidopsis thaliana CBF1 encodes an AP2 domain-containingtranscriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription inresponse to low temperature and water de®cit. Proc. Natl Acad.Sci. USA, 94, 1035±1040.
Stone, J.M., Collinge, M.A., Smith, R.D., Horn, M.A. and Walker,J.C. (1994) Interaction of a protein phosphatase with anArabidopsis serine-threonine receptor kinase. Science, 266,793±795.
Thomashow, M.F. (1999) Plant cold acclimation: Freezingtolerance genes and regulatory mechanisms. Annu. Rev. PlantPhysiol. Plant Mol. Biol. 50, 571±599.
TaÈhtiharju, S., Sangwan, V., Monroy, A.F., Dhindsa, R.S. and
Borg, M. (1997) The induction of kin genes in cold-acclimating
Arabidopsis thaliana. Evidence of a role for calcium. Planta,
203, 442±447.Uno, Y., Furihata, T., Abe, H., Yoshida, R., Shinozaki, K. and
Yamaguchi-Shinozaki, K. (2000) Arabidopsis basic leucine
zipper transcription factors involved in an abscisic acid-
dependent signal transduction pathway under drought and
high-salinity conditions. Proc. Natl Acad. Sci. USA, 97,
11632±11637.Wang, M.L., Belmonte, S., Kim, U., Dolan, M., Morris, J.W. and
Goodman, H.M. (1999) A cluster of ABA-regulated genes on
Arabidopsis thaliana BAC T07M07. Genome Res. 9, 325±333.Wu, Y., Kuzma, J., Mare chal, E., Graeff, R., Lee, H.C., Foster, R.
and Chua, N.-H. (1997) Abscisic acid signaling through cyclic
ADP-ribose in plants. Science, 278, 2126±2130.
470 Sari TaÈhtiharju and Tapio Palva
ã Blackwell Science Ltd, The Plant Journal, (2001), 26, 461±470