10
Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana Sari Ta ¨ htiharju 1 and Tapio Palva 1,2 * 1 Department of Biosciences, Division of Genetics, 2 Institute 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.fi). 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-specific 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 identified 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-deficient (aba1) and ABA-insensi- tive (dominant negative abi1–1 allele) mutants of Arabidopsis are impaired in development of freezing tolerance (Heino et al., 1990; La ˚ ng et al., 1994; Ma ¨ ntyla ¨ et al., 1995). Mutants in ABA responsiveness have defined 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 ª 2001 Blackwell Science Ltd 461

Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana

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Page 1: Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana

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

Page 2: Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana

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

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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

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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

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Page 5: Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana

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

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Page 6: Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana

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

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Page 7: Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana

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.

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