FEBS Letters 580 (2006) 4160–4165

Arabidopsis RNA-binding protein UBA2a relocalizes into nuclearspeckles in response to abscisic acid

Marta Riera*, Yulia Redko, Jeffrey Leung

Institut des Sciences du Vegetal, Centre National de la Recherche Scientifique UPR2355, Av de la Terrasse 91198 Gif-sur-Yvette, France

Received 11 April 2006; revised 6 June 2006; accepted 20 June 2006

Available online 30 June 2006

Edited by Ulf-Ingo Flugge

Abstract The Arabidopsis thaliana RNA binding proteinUBA2a is the closest homologue of the Vicia faba AKIP1(56% identity). Like AKIP1, UBA2a is a constitutively-ex-pressed nuclear protein and in response to ABA it is also reorga-nized within the nucleus in ‘‘speckles’’ suggesting a possible roleof this protein in the regulation of mRNA metabolism duringABA signaling. AKIP1 interacts with, and is phosphorylatedby, the upstream ABA-activated protein kinase AAPK. We haveinvestigated if a pathway similar to that described in Vicia fabaalso exists in Arabidopsis. Our results showed that despite theresemblance between the corresponding Vicia and Arabidopsisproteins, it appears that the function of UBA2a is independentof OST1 phosphorylation.� 2006 Federation of European Biochemical Societies. Publishedby Elsevier B.V. All rights reserved.

Keywords: Abscisic acid; RNA binding protein; OST1 kinase;Arabidopsis thaliana

1. Introduction

Abscisic acid (ABA) is a plant hormone that regulates essen-

tial processes in growth and development, including seed mat-

uration or adaptive responses to drought stress [1,2]. With

increasing frequency, novel data indicate that RNA metabo-

lism underpins many ABA-responsive pathways [3]. In partic-

ular, several genes with homology to RNA-binding proteins

have been identified in Arabidopsis mutants altered in ABA

sensitivity. These include, for example, the mRNA cap-binding

protein ABH1 [4], the Sm-like small nuclear ribonucleoprotein

SAD1 [5], or HYL1, which binds specifically double-stranded

RNA in vitro [6]. Significantly, the RNA-binding protein

FCA which controls flowering time has also been described re-

cently as an ABA receptor [7], reinforcing the importance of

RNA processing to ABA signalling. Characterization of

RNA-binding proteins involved in ABA response might pro-

vide insights on how they coordinately regulate sets of mRNA

Abbreviations: ABA, abscisic acid; UBA2a, UBP1 interacting protein2a; OST1, open stomata 1; AAPK, ABA activated kinase; AKIP1,AAPK interacting protein

*Corresponding author. Present address: Unite de Mycologie Mole-culaire, Institut Pasteur, 25 rue du Dr-Roux, 75724 Paris Cedex 15,France.E-mail address: mriera@pasteur.fr (M. Riera).

0014-5793/$32.00 � 2006 Federation of European Biochemical Societies. Pu

doi:10.1016/j.febslet.2006.06.064

during stress adaptation, a mechanism that is conceptually

similar to that of operons in bacteria.

The physiological context in which ABA signals modify

post-transcriptional events has been established in the greatest

detail in Vicia faba through the characterization of AAPK

interacting protein (AKIP1), an RNA-binding protein with

homology to heterogeneous nuclear RNA-binding proteins

A/B and D. AKIP is constitutively nuclear, but in response

to ABA it is reorganized within the nucleus in ‘‘speckles’’

[8,9], discrete cytological entities that are thought to function

as mRNA storage/processing factories. AKIP1 interacts with

and is phosphorylated by AAPK (ABA activated protein ki-

nase) and once activated it is able to bind a dehydrin mRNA

[8]. The protein kinase OPEN STOMATA1 (OST1, also

known as SNRK2E and SnRK2.6) is the Arabidopsis ortho-

logue of AAPK. Like AAPK, OST1 is activated by ABA

and is a key regulator of stomatal closure in response to osmo-

tic stress [10–13]. However, it was not known if OST1 could be

involved in post-transcriptional regulation. The closest Arabid-

opsis homologue of the Vicia faba AKIP1 is UBP1 interacting

protein 2a (UBA2a) (56% identity). UBA2a has been described

as the interacting partner of UBP1, also an RNA-binding pro-

tein [14]. Whereas UBP1 is able to stimulate pre-mRNA splic-

ing in transfected protoplasts using reporter constructs, the

role of the UBA2a protein in RNA metabolism remains un-

known. UBA2a belongs to a family of three related genes,

UBA2a, UBA2b, and UBA2c. However, analyses of the

UBA2 proteins have not been systematic and none has been

studied in the context of physiological functions. Here we

tested the possibility that UBA2a might be a downstream tar-

get of OST1, and that a pathway similar to that described in

Vicia faba might also exist in Arabidopsis.

2. Materials and methods

Arabidopsis of the Col-0 accession was used throughout for the prep-aration of protoplasts and transgenic lines. All of the primers used inthe present study are described in supplementary data (Table 1). ThecDNAs of UBA2a (AGI Accession Number AT3G56860) cloned inpGEX2T, pDEDH and pGBT9 were provided by Z. Lorkovic (MaxF Perutz Laboratories, Vienna, Austria).

2.1. Expression of the UBA2 genesThe expression of the UBA2 genes was semi-quantified by RT-PCR

in leaves from 10-days old plantlets, grown in soil under controlledconditions in growth chambers (200 lE/m2/s; 24 �C, 16 h light, 50%humidity) either well-watered or withheld from water for 6 days. To-tal RNA was extracted using QIAGEN purification kits. For cDNAsynthesis, 5 lg of total RNA were reverse transcribed using Power-

blished by Elsevier B.V. All rights reserved.

OST1 KIN1 EF1α

UBA2cUBA2bUBA2aC S C S C S

C S C S C S

Fig. 1. Gene regulation of UBA2 and OST1 genes under progressivestress conditions. Total RNA was extracted from A. thaliana ecotypeColumbia plants grown in soil under standard conditions (C) orstressed plants not watered during 6 days (S) and semi-quantitativeRT-PCR was done using specific primers. Constitutive EF1a and stressinduced KIN1 genes were used as controls.

M. Riera et al. / FEBS Letters 580 (2006) 4160–4165 4161

Script RT (Clontech) according the manufacturer’s protocol. As neg-ative controls, each PCR reaction was performed in parallel withoutRT. The following conditions were used for PCR reactions: UBA2a:26 cycles, T annealing at 58 �C; UBA2b and OST1: 26 cycles, Tannealing at 56.4 �C; UBA2c: 32 cycles, T annealing at 51 �C;EF1a 22 cycles, T annealing at 58 �C; KIN1: 32 cycles, T annealingat 48 �C.

2.2. Protoplast transformation, screening of T-DNA mutants, production

and analysis of stable transgenic ArabidopsisThe protoplasts were prepared and transformed with constructs

pDEDH-35S::UBA2aGFP [14] using the PEG-mediated method [15].To produce stable transgenic Arabidopsis plants, the 35S::UBA2afused either to GFP or HA were released from the correspondingpDEDH constructs [14] by digestion with EcoRI and recloned in thebinary vector pGREEN0229 containing the plant selection markerBASTA [16]. The 3 0-UTR of UBA2a was retained as terminator topreserved potential post-transcriptional regulatory signals. These con-structs were introduced into Arabidopsis by Agrobacterium-mediatedtransformation [17]. Soil-grown transgenic plants were selected byspraying with a gardening herbicide (Contour� from a local gardeningcenter) containing the active selection ingredient Glufosinate (used at afinal concentration of 60 mg/L), or in case of in vitro selection, germi-nation on MS-containing agar with glufosinate ammonium (10–15 mg/L; Sigma). The fusion protein UBA2a-GFP was visualized in protop-lasts and in transgenic plants by using a Leica TCS SP confocal laser-scanning microscope (Leica, Heidelberg). For ABA treatment, abscisicacid (mixed isomers, Sigma) was diluted from 10 mM stock solutionprepared in methanol; equivalent volumes of methanol were includedin the ABA free controls. Five-day old seedlings grown in MS mediumwere transferred to similar plates containing 10 lM ABA for 30 min.For Western analysis, protein extraction and blotting were done as de-scribed [18], using the rat monoclonal anti-HA antibody 3F10 (Amer-sham).

Mutants homozygous for the T-DNA insertion into the UBA2a gene(SALK 53281) were selected and confirmed by PCR using specific oli-gonucleotide primers.

2.3. Expression of recombinant proteins in E. coli and in vitro

phosphorylation assaysSince the UBA2 genes contain no introns, UBA2b (AT2G41060)

and UBA2c genes (AT3G15010) were amplified from genomicDNA, cloned into the corresponding EcoRI/XhoI sites in pTOPO(Invitrogen) and confirmed by sequencing. For protein expression,UBA2b and UBA2c were cloned in-frame with the glutathione-S-transferase (GST) in pGEX-2T. The fusion proteins were expressedin E. coli and purified according the manufacturer’s protocol (Amer-sham). The purified histidine tagged OST1 kinase produced in E coliwas a gift from C. Belin and S. Thomine (ISV, Gif-surYvette). Forin vitro kinase assay, 100 ng of purified OST1 kinase and 500 ng ofeach of the UBA2 proteins were used. The reaction was carried outin 20 mM Hepes pH 8, 0.5% Triton X-100, 2 mM MnCl2, 10 mMNaF, 5 mM b-glycerophosphate, 10 mM orthovanadate, completeprotease inhibitors cocktail tablets (Roche) and 3 lCi of c-[32P]ATP in a final volume of 25 ll. The reactions were stopped by add-ing the Laemmli buffer after 30 min of incubation at 30 �C and thesamples were separated by SDS–PAGE and visualized by autoradi-ography. Western blot, using anti-GST antibody was done asdescribed [18].

2.4. Directed two-hybrid interactionThe cDNAs encoding proteins to be tested for interaction by the

yeast two-hybrid were cloned in-frame with the respective GAL4 do-mains in both the vectors pGBKT7 and pGADT7. UBA2a was re-leased from pGBT9 [14], and UBA2b from pTOPO and cloned asEcoRI/XhoI fragments. The UBA2c gene was amplified by PCR fromgenomic DNA and cloned as an NdeI/SalI fragment, while the cDNAof OST1 was PCR-amplified from leaves and cloned as EcoRI and SalIfragments. The plasmids were transformed into the yeast strain AH109according to the supplier’s instructions (Clontech) and transformantswere selected for prototrophy on Drop Out medium without leucine,thiamine, (-L-T) and for protein interaction based on prototrophyon the same selective medium without histidine and adenine (-L-T-H-A).

3. Results

3.1. Expression of the UBA2 and OST1 genes under progressive

stress conditions

Since the AAPK-AKIP pathway has been described in Vicia

guard cells and the Arabidopsis AAPK homolog OST1 is in-

volved in regulation of stomatal aperture [8,11], we checked

by semi-quantitative RT-PCR the expression in leaves of the

three AKIP-homologous genes encoding UBA2a, UBA2b

and UBA2c and that for the OST1 kinase (Fig. 1). The tran-

script levels of the UBA2 genes remained constant when plants

were submitted to a progressive drought regime over a 6-day-

period. However, the expression of the OST1 gene is enhanced

under these same long-term stress conditions.

3.2. The UBA2a protein relocalizes into ‘‘nuclear speckles’’ in

response to ABA

To study the regulation of the protein UBA2a in plants,

transient expression of the protein fused to GFP in Arabidopsis

protoplasts was carried out. Our results indicated that the fu-

sion protein is largely or exclusively nuclear and it is noticeable

that the protein is excluded from the nucleolar structures

(Fig. 2A). This subcellular localization is confirmed in stable

transgenic plants (Fig. 2C, left). Observation by confocal

microscopy revealed that UBA2a-GFP expressed under the

35S RNA promoter of CaMV is highly abundant in the nuclei

of root cells (Fig. 2B). Although the overall expression is weak-

er in leaves, UBA2a-GFP seemed nonetheless enriched in

guard cells (data not shown). The much higher accumulation

of the UBA2a protein in the root relative to the arial parts

was confirmed by immunoblotting analyses using an anti-HA

specific antibody of two independent transgenic Arabidopsis

lines also expressing UBA2a-HA under the 35S promoter

(Fig. 2D). To check if UBA2a is responsive to ABA, transgenic

plants were treated with ABA (10 lM for 30 min). The

UBA2a-GFP signal was observed to coalesce into structures

resembling ‘speckles’ (Fig. 2C, right) similar to those described

for AKIP1 in Vicia faba guard cells [8,9].

-L-T -L-T-H-A

2a/2a 2a/

2b/2b 2b/

2c/2c 2c/

2c/2b 2b/

2a/

2a/AD

2c/AD

2b/

Fig. 3. Interaction between UBA2 proteins and OST1 kinase using twotransformed into the yeast strain AH109 and transformants were selected forinteraction based on prototrophy on the same selective medium without histfilter assays using b-galactosidase as an independent marker (data not show

Fig. 2. Subcellular localization of UBA2a protein. Confocal images of(A) Arabidopsis protoplast transformed with 35S::UBA2aGFP. Con-focal and bright field were captured from the same cell. (B) Confocal anfbright field of root tip of transgenic Arabidopsis line 1 35S::UBA2aGFP.(C) Root cells nuclei of transgenic Arabidopsis line 1 35S::UBA2aGFP.Single nucleus not treated (left) and treated for 30 min with 10 lM ABA(right) (D) Western blot analysis of transgenic Arabidopsis lines35S::UBA2aHA using anti HA antibody. Lane 1: Leaves line 1, lane2: Leaves line 2, lane 3: Roots line 1, lane 4: Roots line 2.

4162 M. Riera et al. / FEBS Letters 580 (2006) 4160–4165

3.3. ABA responsiveness of transgenic lines overexpressing

UBA2a and uba2a mutant

A uba2a mutant that presents a T-DNA insertion in position

1981 of UBA2a coding sequence was obtained from the SALK

Institute collection [19] and homozygous seeds were selected

and confirmed by PCR. The germination behaviour of seeds

from uba2a mutant and transgenic lines overexpressing UBA2a

fused with either HA or GFP were compared to the wild type

(Col). In three independent assays, no statistically significant

differences were observed neither in the T-DNA mutants nor

in the overexpressing lines (Fig. 1, supplementary data). Simi-

larly, other assays such as transpiration/water loss measures,

root elongation in different ABA concentrations, or visualiza-

tion of stomatal aperture/closure in response to drought by

infrared thermography of these different transgenic lines did

not show any ABA-related phenotype (data not shown).

3.4. Interaction between the UBA2 proteins and the OST1

kinase

To investigate whether OST1 can interact with the UBA2

proteins, in particular UBA2a, as well as the possibility that

some of the UBA2 proteins could interact among themselves,

we carried out a two-hybrid test in all possible pair-wise inter-

actions (Fig. 3). The results showed that both UBA2a and

UBA2b can homo- as well as heterodimerize. In contrast,

UBA2c did not interact with itself nor with the other two

UBA2 proteins. OST1 kinase can interact with UBA2a only

when OST1 was fused to the DNA-binding domain in the vec-

tor pGBKT7, however it does not interact with UBA2b or

UBA2c proteins (Fig. 3).

3.5. In vitro phosphorylation assays using OST1 kinase

The possibility that OST1 can phosphorylate the UBA2 pro-

teins was tested using recombinant proteins produced in E. coli.

All three GST-UBA2 proteins were slightly phosphorylated by

OST1-HIS6· in vitro (Fig. 4A). However, the level of phosphor-

ylation of the three UBA2 proteins was very weak compared to

that obtained using the myelin basic protein (MBP) as the

-L-T -L-T-H-A

2b

2a

2a

2c

2c

AD

OST1/2a

2a/OST1

OST1/2b

2b/OST1

OST1/2c

2c/OST1

-hybrid system. Plasmids containing the three UBA2 proteins wereprototrophy on plates without leucine, thiamine, (-L-T) and for proteinidine and adenine (-L-T-A-H). Positive interactions were confirmed onn).

50

75

100 **

OS

T1

OS

T1+

UB

A2a

UB

A2a

50

75

100

** *

OS

T1

OS

T1+

UB

A2b

OS

T1+

UB

A2c

UB

A2b

UB

A2c

75

100

UB

A2a

UB

A2b

UB

A2c

100

37

50

75

OS

T1

OS

T1+

AB

A

OS

T1

OS

T1+

AB

A

GST MBP

OS

T1

OS

T1+

AB

A

OS

T1

OS

T1+

AB

A

GST MBP

A

B

Fig. 4. In vitro phosphorylation assays using the recombinant proteins UBA2-GST and OST1-His kinase. (A) In vitro phosphorylation of UBA2proteins by OST1 kinase is shown and phosphorylated proteins are detected by autoradiography (right and central panels). Asterisks show positionsof the UBA2 proteins. Highly-phosphorylated band about 50 kDa corresponds to autophosphorylation of the OST1 kinase. The phosphorylatedproducts in these experiments were analyzed in the same gel and for the same duration of exposure. Only the relevant lanes are shown. Left panelshows Western blot of the UBA2 proteins loaded in the assay using anti-GST antibody. The two bands represent the intact proteins and theproteolytic products. (B) Control in vitro phosphorylation assays using MBP or GST as substrates. ABA (50 lM) was added to the in vitro proteinphosphorylation assays (lanes 2, 4) without modifying the OST1 kinase activity. Left panel corresponds to the autoradiography of the gel and rightpanel is the Western blot using anti-GST antibody.

M. Riera et al. / FEBS Letters 580 (2006) 4160–4165 4163

in vitro substrate (Fig. 4B). Furthermore, we did not observe an

improvement in the phosphorylation signal by varying the pH

or the ion (Mg2+ and or Mn2+) concentrations. The catalytic

activities of the Vicia AAPK and the OST1 isolated from plant

cells are stimulated by exogenous ABA [8,11–13]. In our case,

the efficiency of in vitro phosphorylation towards either MBP

or UBA2 proteins was not significantly enhanced by using

OST1-HA recovered from transfected Arabidopsis cells pre-

treated with exogenous ABA (data not shown). Similar nega-

tive results were also obtained by direct ABA treatment of

OST1-His6· produced in E. coli (Fig. 4B). Thus, under our

experimental conditions, OST1 has only weak and most likely

non-physiological kinase activity towards the three UBA2

proteins.

4. Discussion

Based on its close homology to Vicia faba AKIP1, we decided

to investigate the possibility that UBA2 proteins may have a

role in ABA signal transduction. Few experimental data are

available for the Arabidopsis UBA2 family. A systematic classi-

fication of plant RNA binding proteins includes the UBA2

family in the oligouridylate-specific group of RRM proteins

[20]. A common feature of this group of nuclear RRM proteins

(that also includes UBP1, RBP45, RBP47, UBA1 families) is

their in vitro specificity for oligouridylates. Neither UBA1

nor UBA2 proteins have obvious orthologues in metazoan gen-

omes and no physiological role for the UBA2 proteins has been

demonstrated so far. We first tested the expression of the UBA

genes in leaves and we found that under stress conditions, the

expression of the UBA2 genes does not change whereas that

of the OST1 gene is upregulated. It was known previously that

the level of the OST1 transcript is refractory to short treatments

of ABA [11] but here we showed that the transcript accumu-

lated in response to long progressive drought.

To study the physiological role of UBA2a in Arabidopsis, we

constructed transgenic lines expressing the UBA2a protein

fused to either the GFP or the HA epitope tags. We found that

the protein is constitutively nuclear and is mainly detectable in

root cells rather than in leaves of Arabidopsis, even though the

gene was expressed from the constitutive 35S promoter. This

suggests some form of post-transcriptional regulation affecting

the differential translatability of the mRNA and/or the stability

of the protein. When the Arabidopsis transgenic plants express-

ing UBA2a-GFP were treated with ABA UBA2a-GFP re-

sponded by reorganization into multiple foci resembling

nuclear speckles. However, immunoblotting experiments

showed that ABA has no significant influence on the stability

of the protein (1–10 lM ABA for 1–24 h; data not shown).

4164 M. Riera et al. / FEBS Letters 580 (2006) 4160–4165

In other organisms, these interchromatic structures have also

been referred to as stress granules and are thought to be in-

volved in RNA storage and processing [21] thus suggesting a

possible role of the UBA2a protein in the regulation of mRNA

metabolism in response to stress or ABA. Work is in progress

to define more precisely the role of UBA2a in mRNA metab-

olism through the isolation of its mRNA targets to determine

whether some of them encode known elements in stress or

ABA signaling.

Neither the transgenic lines overexpressing UBA2a nor the

uba2a T-DNA mutant showed any obvious ABA-related phe-

notype. In the uba2a mutant, this absence of phenotype could

be due to a compensatory effect provided by UBA2b (highly

homologue to UBA2a).

Interactions between the different UBA2 proteins were

tested by using the two-hybrid system. Many RNA-binding

proteins form homo- and heterodimers. The different subunit

compositions in the complex may contribute to the binding

specificity of the proteins. It was previously described that

UBA2a is able to form homodimers [14]; however, nothing

was known about UBA2b or UBA2c. Results obtained here

demonstrated that only UBA2a and UBA2b but not UBA2c

could form homo and heterodimers. Thus UBA2b, like its

close homolog UBA2a, could also be found in a complex with

UBP1 [14]. In the case of UBA2c, the absence of the acidic N-

terminal domain present in UBA2a and 2b could be a reason

for this lack of interaction with the others. In Vicia faba

AKIP1 protein interacts with AAPK kinase in two-hybrid sys-

tem [8]. In this same assay we did not detect interaction be-

tween OST1 and UBA2b or UBA2c proteins, but we were

able to demonstrate a weak interaction between OST1 and

UBA2a protein only when OST1 is cloned in-frame with the

yeast GAL4 DNA-binding domain. One possibility is that

the affinity of OST1 for UBA2a is relatively weak and can

be disrupted by subtle changes in the conformations of either

one or both of the partner proteins caused by the different fu-

sion tags. This interpretation is consistent with the observation

that OST1 has only a very modest kinase activity towards

UBA2a as an in vitro substrate (see below). OST1 can also

interact with itself in this two-hybrid analysis, which might

suggest its capacity to trans autophosphorylate in vitro. More-

over, phosphorylation assays showed that at least under the

in vitro conditions tested, the other UBA2 proteins are also

barely phosphorylated by the OST1 kinase. There are 11 ser-

ines and nine threonines theoretically phosphorylable in the

UBA2a, but none is associated with the core consensus motif

(R-XX-S/T) found in the putative substrates of rice and Ara-

bidopsis SnRK2 kinases [22,23]. One possibility is that OST1

docks onto UBA2a via intermediates that were not reproduced

in our experimental systems (yeast two-hybrid, and in vitro

using OST1 produced in protoplasts or bacteria). An alterna-

tive explanation is that despite the functional similarities in

the individual components, the physiological relationship be-

tween the kinases and their respective RNA-binding protein(s)

in ABA signalling has not been conserved between Vicia faba

and Arabidopsis thaliana.

Acknowledgements: We thank Z. Lorkovic and W. Filipowicz for pro-viding plasmids, C. Belin for the purified OST1 kinase and S. Thominefor thoughtful suggestions. M.R. was supported by a Marie Curie FP5Individual Post-doctoral Fellowship (MCFI-2002-01624) and Y.R. bya CNRS post-doctoral fellowship. The project is supported in part bythe Genoplante program Phase III GEN026 (J.L.).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,

in the online version, at doi:10.1016/j.febslet.2006.06.064.

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