17
The Plant Cell, Vol. 15, 2165–2180, September 2003, www.plantcell.org © 2003 American Society of Plant Biologists A Grape ASR Protein Involved in Sugar and Abscisic Acid Signaling Birsen Çakir, Alice Agasse, Cécile Gaillard, Amélie Saumonneau, Serge Delrot, and Rossitza Atanassova 1 Unité Mixte de Recherche Centre National de la Recherche Scientifique 6161, Transport des Assimilats, Laboratoire de Physiologie, Biochimie et Biologie Moléculaire Végétales, Bâtiment Botanique, Unité de Formation et de Recherches Sciences, 86022 Poitiers Cédex, France The function of ASR (ABA [abscisic acid]-, stress-, and ripening-induced) proteins remains unknown. A grape ASR, VvMSA, was isolated by means of a yeast one-hybrid approach using as a target the proximal promoter of a grape putative monosaccharide transporter (VvHT1). This promoter contains two sugar boxes, and its activity is induced by sucrose and glucose. VvMSA and VvHT1 share similar patterns of expression during the ripening of grape. Both genes are inducible by sucrose in grape berry cell culture, and sugar induction of VvMSA is enhanced strongly by ABA. These data suggest that VvMSA is involved in a common transduction pathway of sugar and ABA signaling. Gel-shift assays demonstrate a specific binding of VvMSA to the 160-bp fragment of the VvHT1 promoter and more precisely to two sugar-responsive elements present in this target. The positive regulation of VvHT1 promoter activity by VvMSA also is shown in planta by coexpression experiments. The nuclear localization of the yellow fluorescent protein–VvMSA fusion protein and the functionality of the VvMSA nuclear localization signal are demonstrated. Thus, a biological function is ascribed to an ASR protein. VvMSA acts as part of a transcription-regulating complex involved in sugar and ABA signaling. INTRODUCTION The ASR proteins, which are induced by abscisic acid (ABA), stress, and ripening, were first described in tomato (Iusem et al., 1993; Amitai-Zeigerson et al., 1994; Rossi and Iusem, 1994). They are characterized as small, basic proteins with strong hydrophilicity as a result of high levels of His, Glu, and Lys. Subcellular fractionation experiments and immunodetec- tion in tomato fruit chromatin fractions suggested that tomato ASR1 is localized to the nucleus (Iusem et al., 1993). This con- clusion agrees with the fact that loblolly pine and melon ASR possess a putative nuclear targeting signal at the C terminus (Padmanabhan et al., 1997; Hong et al., 2002). Moreover, these nuclear basic proteins can bind DNA, as demonstrated by DNA gel blot and filter binding experiments for tomato ASR1 (Gilad et al., 1997). Together, these data support the idea that ASRs resemble eukaryotic nonhistone chromosomal proteins (Iusem et al., 1993). After the cloning of the first ASR gene in tomato, several orthologs were isolated from many different species: dicotyle- donous and monocotyledonous plants, grasses, and trees (Maskin et al., 2001). However, no ASR-like gene has been identified in Arabidopsis. All available data suggest that ASR proteins are encoded by small multigene families: five genes in tomato (Rossi et al., 1996; Gilad et al., 1997), four genes in loblolly pine (Chang et al., 1996), and at least three genes in maize (Riccardi et al., 1998). Almost all known ASR genes contain two strongly conserved regions. The first region is a short N-terminal stretch containing six to seven His residues that might constitute a Zn binding site. The second region is a large part of the C-terminal region, corresponding to 70 amino acids. In different species, ASR genes are expressed in various or- gans, such as the fruit of tomato, pomelo, and apricot (Iusem et al., 1993; Canel et al., 1995; Mbeguie-A-Mbeguie et al., 1997), the roots and leaves of tomato, rice, pine, and maize (Amitai- Zeigerson et al., 1994; Chang et al., 1996; Riccardi et al., 1998; Vaidyanathan et al., 1999), the tubers of potato (Silhavy et al., 1995), and the pollen of lily (Wang et al., 1998). Thus, distinct members of one ASR family may be expressed in different or- gans, under different conditions, and with different expression patterns (Canel et al., 1995; Maskin et al., 2001). The sequence homology among the family members, including even the 3 noncoding regions, and their similar sizes hindered studies of the expression of individual members. However, their expres- sion patterns may differ considerably between transcripts and proteins, as described previously for cold-regulated genes of potato (Schneider et al., 1997). Finally, ASR genes seem to be involved in processes of plant development, such as senes- cence and fruit development, and in responses to abiotic stresses, such as water deficit, salt, cold, and limited light (Schneider et al., 1997; de Vienne et al., 1999; Maskin et al., 2001; Jeanneau et al., 2002). Variations in abiotic environmental factors (light, water, and temperature) may lead to a significant decrease of photosyn- thetic efficiency in source tissues and thus to a reduced carbo- hydrate supply to sink organs. According to a recent report, the response to sugar starvation is one of the adaptive mecha- nisms of plants to cold and water deficit (Yu, 1999). Sugar star- 1 To whom correspondence should be addressed. E-mail rossitza. [email protected]; fax 33-[0]5-49-45-41-86. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.013854.

A Grape ASR Protein Involved in Sugar and Abscisic … Grape ASR Protein Involved in Sugar and Abscisic Acid Signaling ... and the pollen of lily (Wang ... Variations in abiotic environmental

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The Plant Cell, Vol. 15, 2165–2180, September 2003, www.plantcell.org © 2003 American Society of Plant Biologists

A Grape ASR Protein Involved in Sugar and Abscisic Acid Signaling

Birsen Çakir, Alice Agasse, Cécile Gaillard, Amélie Saumonneau, Serge Delrot, and Rossitza Atanassova

1

Unité Mixte de Recherche Centre National de la Recherche Scientifique 6161, Transport des Assimilats, Laboratoire de Physiologie, Biochimie et Biologie Moléculaire Végétales, Bâtiment Botanique, Unité de Formation et de Recherches Sciences, 86022 Poitiers Cédex, France

The function of ASR (ABA [abscisic acid]-, stress-, and ripening-induced) proteins remains unknown. A grape

ASR

,

VvMSA

,was isolated by means of a yeast one-hybrid approach using as a target the proximal promoter of a grape putativemonosaccharide transporter (

VvHT1

). This promoter contains two sugar boxes, and its activity is induced by sucrose andglucose.

VvMSA

and

VvHT1

share similar patterns of expression during the ripening of grape. Both genes are inducible bysucrose in grape berry cell culture, and sugar induction of

VvMSA

is enhanced strongly by ABA. These data suggest thatVvMSA is involved in a common transduction pathway of sugar and ABA signaling. Gel-shift assays demonstrate a specificbinding of VvMSA to the 160-bp fragment of the

VvHT1

promoter and more precisely to two sugar-responsive elementspresent in this target. The positive regulation of

VvHT1

promoter activity by

VvMSA

also is shown in planta by coexpressionexperiments. The nuclear localization of the yellow fluorescent protein–VvMSA fusion protein and the functionality of theVvMSA nuclear localization signal are demonstrated. Thus, a biological function is ascribed to an ASR protein. VvMSA actsas part of a transcription-regulating complex involved in sugar and ABA signaling.

INTRODUCTION

The ASR proteins, which are induced by abscisic acid (ABA),stress, and ripening, were first described in tomato (Iusem etal., 1993; Amitai-Zeigerson et al., 1994; Rossi and Iusem,1994). They are characterized as small, basic proteins withstrong hydrophilicity as a result of high levels of His, Glu, andLys. Subcellular fractionation experiments and immunodetec-tion in tomato fruit chromatin fractions suggested that tomatoASR1 is localized to the nucleus (Iusem et al., 1993). This con-clusion agrees with the fact that loblolly pine and melon ASRpossess a putative nuclear targeting signal at the C terminus(Padmanabhan et al., 1997; Hong et al., 2002). Moreover, thesenuclear basic proteins can bind DNA, as demonstrated by DNAgel blot and filter binding experiments for tomato ASR1 (Giladet al., 1997). Together, these data support the idea that ASRsresemble eukaryotic nonhistone chromosomal proteins (Iusemet al., 1993).

After the cloning of the first

ASR

gene in tomato, severalorthologs were isolated from many different species: dicotyle-donous and monocotyledonous plants, grasses, and trees(Maskin et al., 2001). However, no

ASR-

like gene has beenidentified in Arabidopsis. All available data suggest that ASRproteins are encoded by small multigene families: five genesin tomato (Rossi et al., 1996; Gilad et al., 1997), four genes inloblolly pine (Chang et al., 1996), and at least three genes inmaize (Riccardi et al., 1998). Almost all known

ASR

genes

contain two strongly conserved regions. The first region is ashort N-terminal stretch containing six to seven His residuesthat might constitute a Zn binding site. The second region is alarge part of the C-terminal region, corresponding to

70amino acids.

In different species,

ASR

genes are expressed in various or-gans, such as the fruit of tomato, pomelo, and apricot (Iusem etal., 1993; Canel et al., 1995; Mbeguie-A-Mbeguie et al., 1997),the roots and leaves of tomato, rice, pine, and maize (Amitai-Zeigerson et al., 1994; Chang et al., 1996; Riccardi et al., 1998;Vaidyanathan et al., 1999), the tubers of potato (Silhavy et al.,1995), and the pollen of lily (Wang et al., 1998). Thus, distinctmembers of one

ASR

family may be expressed in different or-gans, under different conditions, and with different expressionpatterns (Canel et al., 1995; Maskin et al., 2001). The sequencehomology among the family members, including even the 3

noncoding regions, and their similar sizes hindered studies ofthe expression of individual members. However, their expres-sion patterns may differ considerably between transcripts andproteins, as described previously for cold-regulated genes ofpotato (Schneider et al., 1997). Finally, ASR genes seem to beinvolved in processes of plant development, such as senes-cence and fruit development, and in responses to abioticstresses, such as water deficit, salt, cold, and limited light(Schneider et al., 1997; de Vienne et al., 1999; Maskin et al.,2001; Jeanneau et al., 2002).

Variations in abiotic environmental factors (light, water, andtemperature) may lead to a significant decrease of photosyn-thetic efficiency in source tissues and thus to a reduced carbo-hydrate supply to sink organs. According to a recent report, theresponse to sugar starvation is one of the adaptive mecha-nisms of plants to cold and water deficit (Yu, 1999). Sugar star-

1

To whom correspondence should be addressed. E-mail [email protected]; fax 33-[0]5-49-45-41-86.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.013854.

2166 The Plant Cell

vation also has been described as a component of senescence(Dieuaide et al., 1992). In addition to their roles as major struc-tural components and cell nutrients, sugars may act as poten-tial signals in plant growth and development (Smeekens andRook, 1997; Gibson, 2000; Smeekens, 2000). Interactions be-tween sugar signaling and ethylene (Zhou et al., 1998), ABA(Arenas-Huertero et al., 2000; Finkelstein and Gibson, 2001),cytokinin (Riou-Khamlichi et al., 1999), and light (Mita et al.,1995) signaling have been established. The crosstalk betweensugar signal transduction and some plant hormones has beenstudied further in Arabidopsis mutants (i.e., sugar-insensitivemutants affected in ABA or ethylene response) (Zhou et al.,1998; Arenas-Huertero et al., 2000; Finkelstein and Gibson,2001; Gazzarrini and McCourt, 2001; Rook et al., 2001).

As suggested initially by Maskin et al. (2001), ASR proteinsmight act as downstream components of a common signaltransduction pathway involved in the responses of plant cells toenvironmental factors. However, to our knowledge, there is noprecise information available concerning the biological func-tions of these proteins.

Here, we describe the isolation of a grape

ASR

gene (

VvMSA

)by means of the one-hybrid approach, using as a target theproximal promoter of a putative grape monosaccharide trans-porter (

VvHT1

), which contains two sugar boxes and is regu-lated by sugars (Atanassova et al., 2003). We show that

VvMSA

expression, which is upregulated at early stages of fruit devel-opment and at late grape ripening, is inducible by sucrose andthat this sugar induction is enhanced strongly by ABA. Further-more, using two different expression systems, we demonstratea specific in vitro binding activity of VvMSA to the target

VvHT1

promoter and the requirement of two sugar boxes for this inter-action. The positive regulation of

VvHT1

promoter activity byVvMSA is further confirmed by their coexpression in planta.The study of a yellow fluorescent protein (YFP)–VvMSA fusionprotein demonstrates its preferential nuclear localization andthe functional role of its intrinsic nuclear localization signal(NLS). Therefore, VvMSA appears to act as part of a complexthat regulates the expression of a monosaccharide transporter.

RESULTS

The One-Hybrid Approach

To clone transcription factors involved in the regulation of

VvHT1

expression, the yeast one-hybrid approach was devel-oped according to Kim et al. (1997). VvHT1 is highly similar toseveral monosaccharide transporters (Fillion et al., 1999; Leterrieret al., 2003). During the ripening of grape berries, it exhibits abiphasic expression pattern with a first peak soon after fruitset and a second peak after véraison (Fillion et al., 1999). Itspromoter contains several sugar boxes, and its activity is in-duced by sucrose and glucose treatment (Atanassova et al.,2003). To avoid the isolation of general transcription factors,the proximal 160-bp region of the

VvHT1

promoter upstream ofthe TATA box was chosen as a target. The 160-bp part of the

VvHT1

promoter contains two positive sugar-responsive mo-tifs, a perfect “sucrose box 3” encompassing an imperfectSURE1 sequence (Tsukaya et al., 1991; Grierson et al., 1994).

This target

VvHT1

promoter was fused in front of two reportergenes,

HIS3

and

LacZ

, for expression in yeast. A cDNA expres-sion library from grape berries at the véraison stage was fusedto the sequence coding the activation domain of the GAL4transcription factor of yeast. The use of two reporter genes de-creases the number of false-positive clones (Kim et al., 1997).

After transformation of the host strain bearing both reporterconstructs with the activation domain/cDNA fusion library anddouble selection of transformants, 10 positive clones were ob-tained. Among these positive clones, four displayed similaritieswith unknown proteins, three were expressed in Arabidopsis,and one was expressed in rice. Five clones shared identity withproteins involved in gene transcription regulation: an AUX/IAAprotein, a MADS-box protein, a Ser/Thr protein kinase, a Gly-rich protein, and a histone variant H3.3. The complete cDNAsof all of these clones were obtained, analyzed by sequencing,and submitted to GenBank as new grape sequences, except

VvMADS1

, which already was known. A 10th clone showedstrong identity with a family of proteins induced by ABA, stress,and ripening (ASR) that is known to be expressed in fruits. Thisclone was selected for further analysis.

Cloning of an

ASR

Gene from Grape

The

ASR

homolog was named

VvMSA

(for

Vitis vinifera

matura-tion-, stress-, ABA-induced protein).

VvMSA

cDNA is 664 bplong and contains a 5

untranslated region (UTR) of 59 bp, anopen reading frame of 450 bp, and a 214-bp 3

UTR. The pre-dicted polypeptide (pI 5.67) is 149 amino acids long, with a mo-lecular mass of 16.5 kD. This is a small protein containing fourhydrophilic domains and similar amounts of His (14.8%, on afrequency basis), Lys (10.7%), and Glu (14.1%). VvMSA sharesconsiderable identity with many ASR proteins from differentspecies (Figure 1). There are two main highly conserved re-gions: a small N-terminal consensus of

18 to 20 amino acidscontaining a typical stretch of six His residues in an 8–aminoacid sequence, and a large C-terminal region of at least 80amino acids. Checking for specific sequences in VvMSA usingthe BLOCKS method (http://blocks.fhcrc.org) revealed the pres-ence of two ABA/WDS signatures, which are described in ABAstress– and ripening-induced proteins (Canel et al., 1995) and inwater deficit stress–induced proteins (Padmanabhan et al., 1997):5

-DYRKEEHHKHLEHLGELGVA-3

and 5

-AGAYALHKKHKS-EKDPEHAHKHKIEEEIAAAAA-3

. In addition, the 3

end of theC-terminal part of VvMSA contains a putative signal for nucleartargeting (5

-KKEAKEEDEEAHGKKHHHLF-3

). PROSCAN (http://npsa-pbil.ibcp.fr) analysis through PROSITE.BASE indicates thatthe VvMSA sequence contains three potential sites for three differ-ent types of phosphorylation and one site for

N

-myristoylation.The comparison of ASR proteins obtained using the CLUSTAL

method (DNAstar, Madison, WI) indicated that the closest ho-mologs to VvMSA are pomelo, apricot, peach, and pear ASRclones (Figure 1). The next group of orthologs sharing importantidentity with VvMSA corresponds to potato and tomato ASRclones. It is followed by a third group, formed mainly by mono-cotyledonous species (rice, maize, and sugarcane). The fourthand last cluster consists exclusively of the four pine clones.

A Function for an ASR Protein 2167

Figure 1. Amino Acid Alignment of VvMSA and 22 Known ASR Proteins from Different Species Performed with the CLUSTAL Program.

2168 The Plant Cell

Determination of

VvMSA

Gene Number in the Grape Genome

To determine whether

VvMSA

belongs to a small multigenefamily, like the majority of known

ASR

genes in other species,DNA gel blot analysis of grape genomic DNA was performed.After digestion with each of four different enzymes (BglII,EcoRI, HindIII, and KpnI), genomic DNA was hybridized with aprobe corresponding to

VvMSA

cDNA. The presence of a sin-gle hybridizing band for DNA digested with each of the testedenzymes strongly suggested that there is only one copy of this

ASR

gene in the grape genome (Figure 2).

VvMSA

and

VvHT1

Expression Are Regulated Developmentally in Grape

RNA gel blot analysis was used to study the expression of

VvHT1

and

VvMSA

at five different stages of grape develop-ment: fruit set, before véraison, véraison, ripening, and harvest(Figure 3). The expression of both genes exhibited nearly simi-lar patterns. The highest amounts of

VvHT1

and

VvMSA

tran-scripts were found at fruit set and before véraison. Both

VvHT1

and

VvMSA

decreased approximately at véraison and in-creased slightly during the last stages of ripening.

Regulation of

VvMSA

Expression by ABA

To determine whether VvMSA is regulated by ABA, like otherASRs,

VvMSA

expression in a grape berry cell culture wasstudied by RNA gel blot analysis. The effects were tested in amedium containing 58 mM sucrose or no sucrose after ABAtreatment.

VvMSA

expression was induced by sucrose, andthis induction was enhanced strongly by ABA at 48 and 72 h

(Figure 4A). Under our experimental conditions, the ABA effectwas consistent only in the presence of sucrose (Figure 4A). Inthe absence of sucrose, ABA did not affect the amount of

VvMSA

transcripts.Like

VvMSA

,

VvHT1

expression was induced strongly byABA at 24 h after treatment in the presence of sucrose (Figure4B). ABA enhanced a strong but transient increase of

VvHT1

messengers at 24 h, whereas it induced the

VvMSA

transcriptfor at least 72 h after treatment. These results were confirmedwhen sucrose in culture medium was substituted by glucose(data not shown).

Sucrose Effect on

VvMSA

and

VvHT1

Expression in Grape Berry Cell Suspension

To study the effect of sucrose, 3 days after subculture of thegrape berry cell suspension, the cells were washed gently andresuspended without dilution in fresh culture medium contain-ing 58 mM sucrose. This addition of sucrose was followed by atransient increase in the level of

VvMSA

transcripts after 1 and4 h of treatment, but this effect disappeared for incubationtimes up to 24 h. By contrast, the amount of

VvHT1

transcriptsaccumulated gradually and reached a maximal level at 24 h af-ter sucrose addition (Figure 5).

VvMSA 6xHis-Tagged Protein May Interact with Target VvHT1 Promoter in Vitro

The cloning of

VvMSA

and the results of the regulation of

VvMSA

and

VvHT1

expression suggested a possible interac-tion between this protein and the target promoter. To checkthis presumption,

VvMSA

cDNA was cloned in the pQE30 ex-

Figure 2. Gel Blot Analysis of Grape Genomic DNA.

Ten micrograms of genomic DNA was digested with different enzymes:BglII, EcoRI, HindII, or KpnI. The DNA gel blot was hybridized withVvMSA cDNA as an �-32P labeled probe. M, Smart ladder marker DNA.

Figure 3. VvMSA and VvHT1 Gene Expression during Grape Develop-ment.

Gel blot hybridization of RNA from berries at different stages of ripeningwith VvMSA and VvHT1 probes. Twenty micrograms of total RNA wasloaded in each well. Equal loading was checked by staining of 25SrRNA with methylene blue.

A Function for an ASR Protein 2169

pression vector, translated in bacteria as a 6xHis-tagged pro-tein, and purified on nickel–nitrilotriacetic acid agarose (Ni-NTA) . The purified VvMSA protein was revealed by protein gelblot analysis with a RGS-6xHis–specific antibody (Qiagen,Hilden, Germany). The tagged VvMSA protein had an apparentmolecular mass of

25 kD by 12% SDS-PAGE (Figure 6A), some-what higher than the predicted mass (16.5 kD). This antibodydid not detect any protein in the control M15 bacterial extract.

Purified 6xHis-tagged protein was checked in gel mobilityexperiments for interaction with the target 160-bp fragment of

VvHT1

promoter (Figure 6B). Two complexes were observed inall assays performed with the 6xHis-tagged protein. The same160-bp fragment, used as unlabeled probe in 100- and 200-fold molar excesses, competed successfully with the labeledfragment, suggesting a specific interaction between the VvMSAprotein and the

VvHT1

proximal promoter in vitro (Figure 6B,lanes 2 to 4). To confirm the specificity of this interaction, aDNA fragment of the same length with no identity to the targetsequence was chosen on the

VvHT1

promoter and used as acompetitor. Even in 100- and 200-fold molar excesses, thisDNA fragment unrelated to the target DNA did not competewith the labeled target sequence (Figure 6B, lanes 5 and 6).

However, both complexes displayed different stability levels inthe presence of unrelated competitor. Thus, the upper complexpartially disappeared, whereas the lower complex was stableeven after the addition of 100- and 200-fold molar excess ofunrelated unlabeled fragment.

Coupled Transcription and Translation–Produced VvMSA Protein Binds to the 160-bp

VvHT1

Promoter

To check for possible interference in DNA binding activity be-tween the N-terminally located 6xHis tag and the N-terminalstretch of six His residues intrinsically present in the

VvMSA

se-quence (at positions

6 to

13 aa; Figure 1), the protein wasproduced by coupled transcription and translation (TnT) in rab-bit reticulocytes. Biotinylated VvMSA protein was revealed byanti-streptavidin antibody in the reaction system supplementedwith the

VvMSA

cDNA, but it was not detected in the free lysate(Figure 7A). The VvMSA protein produced by the TnT systemhad the same apparent molecular mass as the 6xHis-taggedprotein described above

(i.e.,

25 kD).Only one complex caused by the interaction between the tar-

get 160-bp fragment and TnT-produced VvMSA was apparentin gel mobility-shift assays (Figure 7B). This complex had thesame mobility as the lower complex obtained with the 6xHis-tagged protein. Moreover, the complex was competed forcompletely by the unlabeled 160-bp

VvHT1

promoter fragmentand was not competed for by the unrelated promoter fragment,both of which were provided at the same molar excess (100-and 200-fold).

Interaction of the VvMSA Protein with Known Consensus Sequences of the

VvHT1

Promoter

Double-stranded oligonucleotides containing

cis

elements ofthe

VvHT1

promoter were used for a more detailed study of

Figure 4. ABA Induction of VvMSA and VvHT1 Gene Expression inGrape Berry Cell Suspension.

(A) RNA gel blot analysis of VvMSA messenger accumulation at 48 and72 h after ABA treatment (10 �M) in either the presence (�S) or absence(�S) of sucrose.(B) RNA gel blot analysis of VvMSA and VvHT1 transcript amounts at24, 48, and 72 h after ABA treatment in sucrose-supplemented medium.Twenty micrograms of total RNA was loaded in each well. VvMSA- andVvHT1-specific labeled probes were used for hybridization. Equal load-ing was checked by 25S rRNA staining with methylene blue.

Figure 5. Time Course of VvMSA and VvHT1 Induction by Sucrose inGrape Berry Cell Suspension.

RNA gel blot analysis of RNA from grape berry cells with VvMSA andVvHT1 probes. The cells were harvested at the times indicated aftertransfer in fresh culture medium containing 58 mM sucrose. Twenty mi-crograms of total RNA was loaded in each well. Equal gel loading wasconfirmed by staining of 25S rRNA.

2170 The Plant Cell

motifs recognized specifically by VvMSA. Sequences of onestrand of these oligonucleotides are presented in Figure 8A.The first probe, S3S1, corresponds to a 29-bp sequence of the160-bp target combining two overlapping elements, a com-plete sucrose box 3 and an imperfect SURE1. The second oli-gonucleotide, S3, contains the sucrose box 3 motif alone. Thethird probe, S1, is the perfect consensus sequence of the

SURE1 box. The last probe corresponds to a GT-1 element (5�-AGTTTTCCTTGAAAGAAGATTTAATTCA-3�), which is presentdownstream of the S3S1 combination within the 160-bp targetpromoter (Villain et al., 1996). All three oligonucleotides carryingonly one motif correspond to consensus sequences found inthe context of the distal part of the VvHT1 promoter.

To test the DNA sequence specificity of protein binding, theinteraction of TnT-produced VvMSA protein with labeled S3S1probe was determined by gel-shift assays in the presence ofcompetitors (Figure 8B). The competitors used were the fourdistinct unlabeled probes in increasing amounts (i.e., 50- and100-fold molar excess). The free DNA probe (Figure 8B, lane 1)and its interaction with TnT-expressed proteins in the absenceof VvMSA cDNA (Figure 8B, lane 2) were used as controls. In allexperiments, only one specific VvMSA/S3S1 complex was ob-served (Figure 8B, lane 3, arrow). This specific complex is lo-cated between two nonspecific complexes and just a littlehigher than the lower complex in the TnT control, as demon-strated for the TnT-produced VvMSA protein interaction withthe 160-bp fragment of the VvHT1 promoter (Figure 7B). In ad-dition, this complex is very strong and appears with the sameintensity in the three repetitions involving the positive VvMSA–S3S1 interaction (Figure 8B, lanes 3, 6, and 9), correspondingto competition with the oligonucleotides S3S1, S3, and S1, re-spectively. This complex was competed for specifically only bythe same unlabeled S3S1 probe (Figure 8B, lanes 4 and 5),which corresponds to the target VvHT1 promoter sequenceand combines both elements mentioned above. The S3 and S1oligonucleotides applied individually as unlabeled competitorsdid not compete efficiently (Figure 8B, lanes 7 and 8 for S3,lanes 10 and 11 for S1). Such a lack of effective competitionalso was observed with the GT-1 unlabeled sequence (data notshown).

Nuclear Localization of VvMSA

The C-terminal region of VvMSA carries a putative NLS, sug-gesting the possible trafficking of the protein in the nucleus. Tostudy VvMSA subcellular compartmentalization and the func-tionality of its NLS, the protein was fused downstream of YFP.The constructs shown in Figures 9A to 9C were used for tran-sient expression experiments in protoplasts of tobacco BY2cells. The efficiency of the transformation procedure waschecked by immunodetection of VvMSA expression in proto-plasts (data not shown). The cellular sections presented werechosen after three-dimensional reconstitution of whole BY2cells because of particularly obvious nuclei. The positive con-trol for nuclear localization was YFP alone (Figure 9A), becauseof its spontaneous import within the nuclear compartment(Chiu et al., 1996). To create a negative control with a preferen-tial cytoplasmic location, the VvMSA NLS was deleted com-pletely in the YFP-VvMSA NLS fusion protein (Figure 9C). Forthe YFP protein alone, confocal microscopy revealed greaterfluorescence in the nucleus than in the cytoplasm (Figure 9A).The fluorescence caused by the YFP-VvMSA fusion proteinalso was localized preferentially in the nucleus, and its levelclearly was stronger than the residual fluorescence detected inthe cytoplasm (Figure 9B).

Figure 6. Production and DNA Binding Activity of 6xHis-TaggedVvMSA.

(A) SDS-PAGE of 6xHis-tagged VvMSA visualized by silver staining(lanes 1, 2, and 4) and protein gel blot analysis (lanes 3 and 5) with anti-body against RGS 6xHis tag. Lane 1, total protein extract of M15 bacte-rial cells; lane 2, production of VvMSA protein in transformed M15 cellsharvested 3 h after isopropylthio-�-galactoside induction; lane M, mo-lecular mass markers; lane 3, 6xHis-tagged VvMSA protein revealedwith the anti-RGS 6xHis antibody in a total protein extract of trans-formed M15 cells harvested 3 h after isopropylthio-�-galactoside induc-tion; lanes 4 and 5, SDS-PAGE (lane 4) and immunostaining (lane 5) of6xHis-tagged VvMSA after purification on an Ni-NTA affinity column.(B) In vitro binding activity of purified VvMSA to the target VvHT1 pro-moter fragment. Lane 1, DNA-labeled free probe corresponding to the160-bp fragment of the VvHT1 promoter; lane 2, DNA/protein com-plexes formed by the interaction of VvMSA and the labeled 160-bp frag-ment of the VvHT1 promoter; lanes 3 and 4, competition assays with theunlabeled 160-bp fragment of the VvHT1 promoter at 100- and 200-foldmolar excess, respectively; lanes 5 and 6, competition assays with un-related unlabeled probe (150 bp) at 100- and 200-fold molar excess, re-spectively.

A Function for an ASR Protein 2171

Given that these images were collected using the same val-ues for laser power, photomultiplier gain, iris, and black level,the fluorescence signal in nuclei of YFP-VvMSA–transfectedcells appears at least as strong or even stronger than that innuclei of YFP-expressing cells. By contrast, the YFP-VvMSANLS fusion protein, which is truncated for the NLS sequence,conferred a clear fluorescence in the cytoplasm and only aslight signal in the nucleus (Figure 9C). In all of the cells ob-served, no fluorescence signal was detected in nucleoli andvacuoles. The observed differences were confirmed by the pro-files of fluorescence intensity in subcellular compartments (Fig-ure 9, right).

Coexpression of the VvMSA Protein and the VvHT1 Promoter in Planta

The interaction between the VvMSA protein and the VvHT1promoter demonstrated above in vitro was checked further inplanta. For this purpose, a coexpression system was devel-oped. The construct of VvMSA cDNA under the control of the35S RNA promoter of Cauliflower mosaic virus (Figure 10A) wasprepared in the binary vector pBI121 (Jefferson et al., 1987)and introduced by electroporation into Agrobacterium tumefa-ciens strain LBA 4404. The transient expression assays wereperformed via Agrobacterium-mediated transformation of to-bacco leaves in planta. A suspension of Agrobacterium was in-troduced by infiltration in young leaves of transgenic tobaccoplants and transformed in a stable manner with either the pro-moter VvHT1/GUS or the p35S/GUS reporter gene construct(Atanassova et al., 2003) (Figure 10A). In each of four indepen-dent experiments, two different clones per construct were usedand two leaves per plant were agroinfiltrated. Three differentcontrols were included: the first corresponded to the Agrobac-terium strain free of binary vector carrying VvMSA cDNA; thesecond consisted of the infiltration buffer free of bacteria; andthe last was the blank (i.e., untreated leaf). All of these treat-ments were applied in parallel to the same leaf, and the sam-pling corresponded to different areas treated. The expressionof VvMSA cDNA in zones of infiltration was revealed by RNAgel blot analysis (Figure 10B).

The GUS fluorimetric assay (Figure 10C) demonstrated thatVvMSA expression induced a 2.5- to 3-fold increase of reportergene activity conferred by the full-length VvHT1 promoter (2.4kb) in two independent clones studied (pVvHT1-4 and pVvHT1-14). Furthermore, in the same experimental conditions, the ac-tivity of the proximal VvHT1 promoter (0.3 kb), corresponding tothe target 160-bp fragment plus the minimal promoter, was up-regulated by �10-fold by VvMSA (data not shown). This inplanta analysis of the promoter and the trans-acting factor re-

Figure 7. Production and DNA Binding Activity of the Native VvMSAProtein.

(A) SDS-PAGE of TnT proteins produced in reticulocyte lysate and visu-alized after protein gel blot transfer by Ponceau S staining (top gel) andafter immunochemical reaction developed using streptavidin-peroxi-dase and enhanced chemiluminescence (bottom gel). Lane 1, molecularmass marker; lane 2, TnT-positive control based on luciferase detec-tion; lane 3, TnT-negative control (proteins produced in the absence ofthe VvMSA cDNA); lane 4, molecular mass marker; lanes 5 and 6, TnTproteins produced in the presence of the VvMSA cDNA. Two microlitersof TnT reaction mixture was loaded in lanes 2 and 3, 4 �L was loaded inlane 5, and 8 �L was loaded in lane 6.(B) In vitro binding activity of TnT-produced VvMSA protein to the targetfragment of the VvHT1 promoter. Lanes 1 and 6, free DNA-labeledprobe corresponding to the 160-bp fragment of the VvHT1 promoter;lane 2, unspecific DNA/protein complex obtained in the control bindingassay with proteins produced in the VvMSA cDNA–free TnT system;

lanes 3 and 7, complex corresponding to the interaction of the VvMSAprotein and the labeled 160-bp fragment of the VvHT1 promoter; lanes 4and 5, competition assays with the unlabeled 160-bp fragment of theVvHT1 promoter at 100- and 200-fold molar excess, respectively; lanes8 and 9, competition assays with unrelated unlabeled probe (150 bp) at100- and 200-fold molar excess, respectively.

2172 The Plant Cell

vealed a positive interaction between the studied protein andthe target promoter that did not appear in the different controlsamples. This finding indicates that the observed effect was at-tributable neither to the presence of Agrobacterium as a bioticfactor nor to the infiltration procedure as an abiotic stress. Theparallel agroinfiltration of transgenic tobacco plants carryingthe GUS reporter gene under the control of the 35S promoter(p35S-2 and p35S-4) did not affect viral promoter-conferred GUSactivity (Figure 10D). The latter result confirmed that the regula-tion of pVvHT1 activity by VvMSA is specific for this promoter.

DISCUSSION

Cloning and Characterization of VvMSA, a Member of the ASR Family

VvHT1 expression is induced by various sugars, including glu-cose, sucrose, and palatinose, and the promoter of this genecontains several sugar boxes (Atanassova et al., 2003). Toidentify transcription factors binding to this promoter, we devel-oped a one-hybrid approach and succeeded in the cloning

Figure 8. Interaction of VvMSA with Some Consensus Motifs of the VvHT1 Promoter.

(A) Sequences of the positive strain of double-stranded oligonucleotides used in the DNA binding assays. DNA sequences are presented from the 5�

to the 3� end. cis element sequences are shown in boldface, and when they overlap, the second one is underlined.(B) S3S1 sequence was used as a labeled probe in all experiments, and competition assays were performed with each of three unlabeled probes at50- and 100-fold molar excess, as indicated. Lane 1, free S3S1 labeled probe; lane 2, unspecific DNA/protein complexes obtained in a control bindingassay with proteins produced in the VvMSA cDNA–free TnT system; lanes 3, 6, and 9, complex corresponding to the interaction of VvMSA and S3S1labeled probe; lanes 4 and 5, competition assay with S3S1 unlabeled oligonucleotide in 50- and 100-fold molar excess, respectively; lanes 7 and 8,competition assay with S3 unlabeled oligonucleotide in 50- and 100-fold molar excess, respectively; lanes 10 and 11, competition assay with S1 un-labeled oligonucleotide in 50- and 100-fold molar excess, respectively. These gel-shift assay results are representative of three to five independentexperiments with similar results.

A Function for an ASR Protein 2173

of several cDNAs that encode regulatory proteins, includingVvMSA. Although a partial mRNA was present in GenBank(GASR), our work yields a complete cDNA that encodes anASR protein in grape. The 93% identity between the 3� UTRs ofVvMSA and the GASR partial sequence available in the data-base also indicates that they correspond to the same gene. Noidentity was found with the 3� UTRs of ASR genes from otherplant species.

Genomic DNA analysis using the VvMSA cDNA as a probestrongly suggests the presence of a single copy of the ASRgene in grape (Figure 2). The presence of a single copy in grapeis in contrast to what has been described in most other spe-cies, such as tomato (Iusem et al., 1993), pomelo (Canel et al.,1995), apricot (Mbeguie-A-Mbeguie et al., 1997), and maize(Riccardi et al., 1998).

Several lines of evidence allow us to conclude that VvMSAbelongs to the ASR family. Many ASR genes have been iso-lated in ripening fruit of tomato, pomelo, and apricot (Iusem etal., 1993; Canel et al., 1995; Mbeguie-A-Mbeguie et al., 1997).VvMSA was cloned from a cDNA library corresponding to theonset of grape ripening and is expressed until the time of har-vest (Figure 3). As described for other ASRs (Rossi et al., 1998),VvMSA expression is upregulated by ABA. The VvMSA se-quence displays identity to all previously characterized ASR se-quences (Figure 1). VvMSA shares the main characteristics ofASR (i.e., low molecular mass and high level of hydrophilicity),which indicates that the protein is soluble. Furthermore, theprotein sequence contains two ABA/WDS (ABA and water defi-cit stress) signatures typical of ASR proteins (see Results).

Sugar and ABA in the Regulation of VvMSA and VvHT1 Gene Expression

During grape ripening, VvMSA messengers are accumulatedstrongly at the stages before véraison (Figure 3), and the sig-nals are present until harvest. Both VvMSA and its targetVvHT1 display similar patterns of expression. In grape, which isa nonclimacteric fruit, ABA is the only endogenous hormonewhose content increases from véraison to late ripening, in par-allel with sugar accumulation (Blouin and Guimberteau, 2000).

This study offers new insight into the role of ABA in the sugarregulation of ASR gene expression. In grape cell suspension,sugar-induced VvMSA messenger accumulation was enhancedstrongly by ABA (Figure 4A). Furthermore, the induction ofVvMSA mRNA steady state levels by ABA occurs in the pres-ence but not in the absence of sucrose, which means that su-crose is required for the ABA upregulation of this gene. Simi-larly, recent studies of two sugar-induced genes involved instarch biosynthesis in Arabidopsis (the ApL3 subunit of ADP-glucose pyrophosphorylase and starch-branching enzyme) haveshown that ABA strongly enhances their sucrose-induced ex-pression but has no effect in the absence of sucrose (Rook etal., 2001). The existence of Arabidopsis mutants affected inboth ABA signaling and sugar sensing also is a strong indicationfor interactions between these signaling pathways (Gazzarriniand McCourt, 2001).

This ABA enhancement of VvMSA sucrose-induced expres-sion is in good correlation with the ABA upregulation of VvHT1

gene expression, as demonstrated at the transcriptional level(Leterrier, 2002) and at the RNA steady state level in the currentstudy (Figure 4B). All of these results suggest that VvMSA andVvHT1 gene expression may be regulated in a common sugar-and ABA-dependent pathway.

The possible interference of VvMSA with two signal trans-duction pathways, that of sugar and that of ABA, led us to in-vestigate in more detail the control exerted by sucrose. Ingrape cell suspension culture, both VvHT1 and VvMSA tran-scripts are regulated positively by sugar, but with different ki-netics. The sudden and strong increase of VvMSA expressionpreceded the accumulation of VvHT1 transcripts, which dem-onstrates that the expression of both genes is correlated posi-tively and furthermore that VvMSA may act upstream of VvHT1.

Interaction between VvMSA and the VvHT1 Promoter

Our initial attempts to study the interaction between VvMSAand the VvHT1 promoter fragment of 160 bp in gel mobility as-says used the purified 6xHis-tagged protein and suggested thepresence of two DNA/protein complexes. These two com-plexes may correspond to the binding of monomer and dimerforms. The band-shift effect corresponding to the lower com-plex is nearly doubled for the upper complex (Figure 6B). TheHis block located at the N-terminal end of VvMSA may mimic azinc binding structure and may be involved in the formation ofdimers, homodimers, or heterodimers. Examples of hetero-dimerization of plant transcription factors have been described,and among them are many basic domain/Leu zipper proteins,including some that are involved in ABA-inducible gene expres-sion (Riechmann and Ratcliffe, 2000).

Whether the 6-His tag may interfere with VvMSA interactions,either at the level of dimer formation or at the level of DNA bind-ing activity, was studied with further experiments involving TnT-produced VvMSA protein. The protein obtained in vitro boundthe proximal VvHT1 promoter fragment and displayed only onecomplex (Figure 7B). The gel mobility of this complex was com-parable to the band shift of the lower 6xHis-tagged VvMSA/pVvHT1 DNA complex. In all competition assays with the sameunlabeled fragment or with the unrelated sequence, the speci-ficity of interaction between VvMSA and the VvHT1 promoterwas confirmed.

In addition, gel-shift assays performed with oligonucleotidescorresponding to consensus sequences found in the targetpromoter clearly demonstrated that the combination present inthe context of the VvHT1 promoter of both elements involved inthe positive sugar response (i.e., sucrose box 3 and SURE1) isnecessary for specific VvMSA–pVvHT1 interaction in vitro (Fig-ure 8B). Furthermore, in planta coexpression of both of thesepartners unambiguously showed that VvMSA is involved in theregulation of VvHT1 promoter activity (Figure 10) in a positiveand specific way. Although many sugar transporters have nowbeen cloned, their regulation remains poorly understood (Delrotet al., 2000).

Here, the nuclear localization of an ASR protein is shown invivo by transient expression of different YFP-VvMSA fusionproteins in tobacco BY2 protoplasts (Figures 9A to 9C), therebyconfirming the previous assumption that tomato ASR1 may be

2174 The Plant Cell

Figure 9. Subcellular Localization of the VvMSA Protein.

(A) Subcellular compartmentalization of YFP alone, considered as a positive control for free nuclear targeting.(B) Preferential nuclear expression of the YFP-VvMSA fusion protein(C) Strong cytoplasmic localization of the YFP-VvMSA fusion protein, deleted for the VvMSA NLS.Gene structures of the constructs used are detailed above the corresponding micrographs. From left to right are transmission micrographs, confocalfluorescence images, and profiles of fluorescence intensity obtained for different subcellular compartments as indicated by the arrows. c, cytoplasm;n, nucleus; nu, nucleolus; TL, translational enhancer.

A Function for an ASR Protein 2175

Figure 10. In Planta Coexpression and Interaction of VvMSA and the VvHT1 Promoter.

(A) Effector and reporter constructs obtained in pBI121 and pBI101.1 binary vectors, respectively, and introduced in Agrobacterium tumefaciens.(B) RNA gel blot analysis of VvMSA expression 48 h after infiltration with Agrobacteria carrying VvMSA cDNA of tobacco leaves expressing thepVvHT1-GUS chimerical gene. Two independent tobacco lines were analyzed, and agroinfiltrated areas were compared with buffer-infiltrated areason the same leaves.(C) Induction of VvHT1 promoter–conferred GUS activity by VvMSA in two independent tobacco transformants. VvMSA’s effect on the VvHT1 pro-moter (Agro�VvMSA) was compared with that of three different controls: untreated areas of the same leaf (Control), buffer-infiltrated areas of thesame leaf (Buffer), and areas of the same leaf infiltrated with the Agrobacterium cell suspension without VvMSA cDNA (Agro). MU, 4-methylumbellif-erone.(D) Absence of VvMSA’s effect on 35S promoter–conferred GUS activity in two independent tobacco transformants carrying the p35S/GUS construct.In both (C) and (D), agroinfiltration results were confirmed in at least four independent experiments for each promoter. Error bars correspond to the SE.

2176 The Plant Cell

a nuclear protein (Gilad et al., 1997). Profiles of fluorescence in-tensity obtained by confocal microscopy clearly lend support tothis conclusion. In our conditions of tobacco BY2 cell culture,VvMSA was compartmentalized preferentially in the nucleus,and the residual fluorescent signal in the cytoplasm may reflectthe requirement of certain stimuli for this change of compart-ment, such as alterations in protein phosphorylation state andcooperation with other proteins. For example, VvMSA presentsspecific sites for phosphorylation by three different kinases,among them casein kinase II, which has been discussed in theliterature as a complement of NLS functionality (Kircher et al.,1999). Furthermore, the results showed the functionality of thestudied NLS sequence and proved that this signal was requiredfor VvMSA nuclear targeting.

Although VvMSA affects the transcription of VvHT1, it doesnot show any important similarity to transcription factors al-ready known. Because information is limited regarding the tran-scription factors involved in fruit development (Giovannoni,2001), this finding does not exclude the possibility of VvMSAbeing a transcription factor. The demonstrated nuclear localiza-tion of VvMSA and the functionality of its NLS, the DNA bindingactivity shown in band-shift assays, and the coexpression dataobtained in planta allow us to assign VvMSA a role as a tran-scription-regulating protein. However, VvMSA is expressed at arelatively high level throughout grape ripening. These consider-ations and the different time courses of VvHT1 and VvMSA in-duction by ABA (Figure 4B) suggest that VvMSA acts as part ofa transcriptional complex with a combinatory effect rather thanas a transcription factor acting alone.

Physiological Role of VvMSA

To date, despite the cloning of many ASR genes from differentplant species and the molecular characterization of the en-coded products, no physiological function has been ascribedto these proteins. A first hypothesis predicted that the small nu-clear and basic ASR proteins of tomato were nonhistone chro-mosomal proteins (Rossi and Iusem, 1994). Nonhistone chro-mosomal proteins are involved in DNA topology changes, inestablishing and maintaining of chromatin higher order struc-tures, and in modulating gene expression (Zlatanova, 1990).Since then, a number of ASR proteins have been characterized,and it has now been reported that they may differ in molecularmass (from 70 to 230 amino acids), in pI (from basic to acidic),in organ and tissue specificity, and in expression regulation.The nuclear localization targeting sequence is not alwayspresent in orthologs and even in paralogs of a single multigenefamily, and its physiological role remains unclear. Therefore, theputative ASR function as nonhistone chromosomal proteinsneeds to be confirmed experimentally.

Another hypothesis, based on numerous reports indicatingthe accumulation of ASR proteins in response to various abioticstresses (Maskin et al., 2001), ascribed them a potential protec-tive role against these stresses (Silhavy et al., 1995). This hy-pothesis takes into account the high proportion of chargedresidues in ASR, their high average hydrophilicity, and thepredicted helical secondary structure with enhanced accessi-bility to water molecules. In agreement with this hypothesis,

ASR proteins have been reported to be members of the wide-spread class of hydrophilins, including the seed-specific LEAproteins (Dure et al., 1989; Garay-Arroyo et al., 2000). The simi-larity between ASR and LEA proteins concerns only the N-ter-minal conserved repeat, which has been found in some ASRsof wild potato but not in tomato ASR (Silhavy et al., 1995;Maskin et al., 2001). In addition, there is no ASR homolog in Ar-abidopsis, although this species contains many LEA proteinsand the related RAB (responsive to ABA) and DHN (dehydrin)proteins. Therefore, the hypothesis of ASR involvement in seeddevelopment requires further investigation.

Together, the data in the literature and the present results al-low us to propose the tantalizing hypothesis that some ASRproteins may be involved in the crosstalk between sugar andABA. Both ABA and sugar are key players in the ripening pro-cess in grape (Coombe, 1992), which is triggered at the vérai-son stage. Our data demonstrate that VvMSA, an ASR mem-ber, acts as part of a transcription regulation complex thatcontrols the expression of a monosaccharide transporter ho-molog and that VvMSA expression itself is under the control ofboth sugar and ABA. This hypothesis gives further support tothe current concept that ascribes a primary role of sugar sens-ing and signaling in plant responses to abiotic and bioticstresses (Smeekens, 2000).

In conclusion, our study presents experimental evidence fora function of an ASR protein acting as a downstream compo-nent of a common transduction pathway for sugar and ABAsignals. Further investigations are necessary to identify VvMSAmolecular partners in this cascade of signaling.

METHODS

cDNA Library Construction

A cDNA library was produced using mRNA isolated from grape berries(Vitis vinifera) of the variety Ugni blanc at the véraison stage. Total RNAwas prepared by ultracentrifugation in a CsCl gradient (Tesnière andVayda, 1991). Polyadenylated mRNAs were column purified with thePoly (A) Quick mRNA isolation kit from Stratagene. cDNAs were synthe-sized using a Gibco BRL SuperScript plasmid system with �5 �g ofpoly(A) mRNA. Labeling with �-32P-dCTP was used to monitor the syn-thesis of first and second strands. All cDNAs longer than 600 bp were li-gated as SalI-NotI inserts with pSPORT1 vector (Gibco BRL). Transfor-mation was achieved by electroporation of Electro Max DH10B cells(Gibco BRL), and its efficiency was estimated at 1.2 108 plates/�gcDNA. The average size of the inserts was 1 kb.

After plating of Escherichia coli transformants, plasmid DNA was pre-pared, digested with SalI-NotI, and electrophoresed on a 1% agarosegel. cDNAs ranging in size between 600 bp and 2.5 kb were excised andpurified with the Qiex II kit (Qiagen). Two hundred fifty nanograms ofthese cDNAs and 500 ng of shuttle vector pPC86 were ligated and usedto obtain 1.05 106 E. coli DH10B transformants. Thus, the cDNA ex-pression library fused downstream of the activation domain of the GAL4transcription factor was ready to be introduced into yeast.

Yeast Reporter Constructs

A 160-bp promoter fragment of the VvHT1 gene (Fillion et al., 1999) wascloned in the shuttle vectors pSK1 and pYC7 carrying the HIS3 and LacZyeast reporter genes, respectively. The proximal region of the VvHT1

A Function for an ASR Protein 2177

promoter was amplified by PCR with sequence-specific primers (for-ward primer, 5�-TAGAACGGGGAGTTAGAAACAA-3�; reverse primer,5�-AGCTGTCCCCGATAATATCTAA-3�), which allowed the removal ofminimal promoter containing the TATA and CAAT boxes. The PCR prod-uct was ligated in pGEM-T Easy vector, and two clones with inserts inthe opposite orientation were selected for further cloning. The 160-bppromoter was cloned directionally (5� → 3�) in the NotI-SpeI sites ofpSK1 shuttle vector, a Leu-marked centromeric plasmid, in front of aHIS3 reporter gene under the control of the minimal inactive promoterGAL1. For the second reporter gene construct, the VvHT1 promoterfragment was first excised from pGEM-T Easy as a 5� → 3� fragment bySpeI-EcoRI and ligated in the intermediary vector pcDNAII (Invitrogen,San Diego, CA) digested by the same enzymes. The VvHT1 promoterfragment was inserted directionally as a KpnI-XhoI fragment in the pYC7vector, a URA-marked 2-�m plasmid, in front of the LacZ reporter geneunder the control of a minimal inactive CYC1 promoter.

All vectors used, the pPC86 carrying the cDNA library, the pSK1 andpYC7 carrying the reporter genes HIS3 and LacZ, respectively, and thehost strain YN954, were kindly provided by Terry Thomas (Texas A&MUniversity, College Station).

Yeast One-Hybrid Cloning System

The reporter yeast strain was constructed by cotransforming strainYM954 with both reporter constructs (5 �g each) using a lithium acetateprotocol (Geitz and Woods, 1993). To screen the library, the reporteryeast was transformed with the library DNA. Double screening was ap-plied sequentially as described by Kim et al. (1997). Putative positiveyeast clones were grown for 3 days and assayed for �-galactosidase ac-tivity colorimetrically according to Breeden and Nasmyth (1985). Yeastplasmid DNA was prepared and transferred to E. coli DH5-� cells byelectroporation. Bacterial colonies were screened by transfer to mem-branes and hybridization with GAL4-specific probe. DNA sequencingwas performed by Eurogentec (Seraing, Belgium), and database searcheswere performed using the Basic Local Alignment Search Tool (BLAST)algorithm.

Genomic DNA Gel Blot Analysis

Genomic DNA was isolated from grape berry cell suspension by phenolextraction after RNase and proteinase K treatments. Ten micrograms ofDNA was digested by each of the four enzymes (BglII, EcoRI, HindIII, andKpnI) and blotted onto Hybond N� membranes by alkaline transfer asdescribed by Amersham Pharmacia Biotech. The VvMSA cDNA wasused for hybridization as a 32P-labeled probe. All procedures, hybridiza-tions, and washings were performed at 65C, and the final washing wasperformed stringent conditions with 0.1 SSC (1 SSC is 0.15 M NaCland 0.015 M sodium citrate) and 0.1% SDS.

RNA Gel Blot Analysis

Two different procedures of RNA extraction were performed dependingon the plant material used. (1) Total RNA from grape berries of the varietyUgni blanc was extracted according to Davies and Robinson (1996), withan additional step of selective precipitation with 2 M LiCl. (2) Total RNAfrom grape berry cell suspension (10-mL samples) and from frozen to-bacco leaves was isolated by phenol extraction (Howell and Hull, 1978)followed by selective precipitation with 2 M LiCl.

Equal amounts of purified RNA samples were separated by formalde-hyde-agarose gel electrophoresis and transferred to Hybond N mem-branes (Amersham Life Science). RNA gel blots were hybridized withrandomly primed 32P probes, and mRNA was quantified using a StormBio-Imaging Analyzer (Molecular Dynamics, Sunnyvale, CA).

Grape Berry Cell Suspension, Culture, and Treatments

The grape berry cell suspension derived from Cabernet Sauvignon ber-ries was maintained at 25C on an orbital shaker (100 rpm) by weeklysubculture in a medium supplemented with 58 mM sucrose (Decendit etal., 1996). Three days after subculture, grape berry cells were allowed tosettle, washed carefully, and suspended again in fresh medium supple-mented with sucrose (58 mM) (Figure 5). For the experiments describedin Figure 4, the cell suspension on day 3 of subculture was separatedand transferred in four different batches: (1) the same culture mediumwith sucrose; (2) the same medium with sucrose plus 10 �M abscisicacid (ABA); (3) sucrose-depleted medium; and (4) sucrose-depleted me-dium supplemented with 10 �M ABA. The cells were sampled at varioustimes after treatment, as indicated in Results, and used for RNA gel blotexperiments.

6xHis-Tagged Protein Expression and Purification

To express VvMSA in bacteria, two different constructs were prepared intwo QIAexpress pQE vectors (Qiagen). The first was introduced intopQE-30 vector with a 6xHis tag at the N-terminal end, and the secondwas introduced into the pQE-50 vector without a tag. To clone VvMSAcDNA in the same reading frame as the 6xHis affinity tag, its translationinitiation codon was modified (boldface letters) by PCR using the primer5�-ACGGATCCCTGTCGGAGGAGAA-3�. In parallel, a BamHI restrictionsite (underlined sequence in the primer above) suitable for cloning wasintroduced at the 5� end. PCR-modified cDNA was first digested by NotIand, after a Klenow fill-in reaction, digested sequentially with BamHI andcloned in the BamHI-SmaI sites of pKS plasmid. After this intermediaryligation, the VvMSA cDNA was inserted as a BamHI-EcoRV fragment inBamHI-SmaI of pQE-30 and pQE-50 vectors. For both constructs, thePCR product was checked by sequencing before and after ligation.

The E. coli M15 host strain was transformed with either the pQE-30 orthe pQE-50 construct. Luria-Bertani culture medium (100 �g/mL ampi-cillin and 25 �g/mL kanamycin) was inoculated (1:500) with overnightculture and grown at 28C with vigorous shaking until an OD600 of 0.6was reached. After induction with 2 mM isopropylthio-�-galactoside, theculture was incubated for an additional 4 to 5 h, but at 21C. Cells wereharvested at different times to determine the best expression level andthen frozen in liquid nitrogen.

Total proteins were extracted under nondenaturing conditions, andVvMSA purification by nickel–nitrilotriacetic acid agarose (Ni-NTA) affin-ity chromatography and elution were achieved under native conditionsaccording to the recommended QIAexpressionist protocol (Qiagen). Af-ter dialysis against the column fixation buffer, VvMSA protein was puri-fied a second time by Ni-NTA affinity chromatography and concentratedby centrifugation in a Centricon tube (3000 D; Pharmacia).

In Vitro Transcription and Translation System

Extracts containing VvMSA protein were prepared by coupled in vitrotranscription/translation of 0.75 �g of DNA (pGEM-T Easy vector carry-ing VvMSA cDNA) using the TnT Coupled Reticulocyte Lysate System ata final concentration of 56% lysate. The immunodetection of coupledtranscription and translation (TnT)–produced proteins was achieved bychemiluminescence using the Transcend nonradioactive detection sys-tem (Promega).

In Vitro Protein Binding Assay

Electrophoretic mobility-shift assay was performed according to Bernardet al. (2001). Probe DNA (20,000 to 50,000 cpm) was incubated witheither 400 ng of Ni-NTA–purified protein or 4 to 7 �L of in vitro translation

2178 The Plant Cell

product in 20 �L of binding reaction mixture containing 1 �g of poly(dI-dC) for 20 min. The reaction mixture was electrophoresed on a 5%polyacrylamide gel (29:1 acrylamide:N�,N-methylene bis-acrylamide).When competition analysis was performed, unlabeled probes were usedas competitor in the binding mixture and the reaction was continued foran additional 20 min. The unrelated fragment of the VvHT1 promoter wasamplified by PCR with sequence-specific primers (forward, 5�-ACTACG-GAAAAATTCGACCC-3�; reverse, 5�-TGGCTCTGATAGGGCTGAAA-3�).

When oligonucleotides were used in band-shift assays, both strains ofeach synthetic oligonucleotide were hybridized and 5� end labeled withthe T4 polynucleotide kinase and �-32P-dATP. One and one-half microli-ter (40,000 to 50,000 cpm) of the labeled probe was added to 18 �L ofthe binding mixture containing 7 �L of TnT-produced proteins and 1 �gof poly(dI-dC)–poly(dI-dC) as a nonspecific DNA competitor. The incu-bation mixtures were loaded on 5% polyacrylamide gels (37.5:1 acryl-amide:N�,N-methylene bis-acrylamide). After migration in 1 TGE buffer(40 mM Tris, 270 mM glycine, 4 mM EDTA, pH 8.4) at 150 V, the gelswere dried and autoradiographed.

YFP-VvMSA Fusion Proteins

To study the subcellular compartmentalization of the VvMSA protein,three different constructs based on a YFP fusion were used. The first onewas the pAVA 554 vector (Von Arnim et al., 1998) designed especially asa reporter gene with enhanced yellow fluorescence. Expression of YFPgene expression is driven by a double 35S promoter, and YFP proteinsynthesis is induced by a viral translation enhancer. The second con-struct corresponded to the YFP-VvMSA fusion, in which the completecDNA of VvMSA encompassing the nuclear localization sequence (NLS)was used. To produce this construct, the VvMSA cDNA with a modifiedATG codon and a BamHI restriction site introduced at the 5� end (see theVvMSA sequence used for 6xHis-tagged protein production) was clonedas a BamHI-XbaI (VvMSA stop codon) fragment in the BglII-XbaI sites ofpAVA 554. In the third construct, the NLS sequence of VvMSA cDNAwas deleted completely by PCR performed with the forward primermentioned above and the reverse primer 5�-GCTCTAGACTCGTGA-TGCTCGT-3�, allowing the introduction of the XbaI restriction site (un-derlined sequence) just upstream of the NLS sequence. The correct se-quence of PCR-produced VvMSA, the YFP–VvMSA gene junction, andthe stop codon present in the XbaI site were checked by sequencing ofboth strands of VvMSA in each construct.

Protoplast Preparation and Transformation

Protoplasts were isolated from tobacco BY2 cells cultured as describedpreviously (Atanassova et al., 2003) on the 4th day after subculture infresh medium. Protoplast preparation and transformation using a poly-ethylene glycol–based technique were performed essentially accordingto the method of Neuhaus and Boevink (2001). Overnight digestion ofcell walls was achieved with 0.1% pectolyase and 1% cellulase (Seishin,Tokyo, Japan) at 28C in the dark with gentle shaking. Approximately 7.5 105 protoplasts were transformed with 20 �g of plasmid DNA and 20 �gof carrier DNA to a medium containing 40% PEG-6000 (Roth, Saint-Quentin Fallavier, France), 0.1 M Ca(NO3)2, 0.4 M mannitol, and 0.1%Mes, pH 8.0. Observation by confocal microscopy was made 48 h aftertransformation, and protoplasts were maintained at 26C in the darkwithout shaking.

Confocal Imaging

The samples were examined by confocal laser scanning microscopy us-ing a Bio-Rad MRC 1024 microscope equipped with a 15-mW argon-krypton gas laser. The confocal unit was attached to an inverted micro-

scope (IX70; Olympus, Tokyo, Japan). Fluorescence signal collection,image construction, and scaling were performed using the control soft-ware (Lasersharp 3.2; Bio-Rad). The YFP protein was excited with the488-nm blue line, and emission of the dye was collected via a photomul-tiplier through a 522-nm band-pass filter.

Agroinfiltration

Agrobacteria infiltration was conducted as described by Yang et al.(2000). Agrobacterium tumefaciens strain LBA 4404 containing the bi-nary plasmid pBI121 with VvMSA cDNA was grown at 28C overnight inYEB liquid medium (5 g L�1 sucrose, 1 g L�1 yeast extract, 10 g L�1 Bac-topeptone, 5 g L�1 Gibco beef extract, pH 7.4) supplemented withrifampicin (100 �g/mL) and kanamycin (50 �g/mL). Agrobacteria thenwere inoculated in 20 mL of induction medium containing AB salts(NH4Cl 18.6 mM; MgSO4, 7H2O, 1.2 mM; KCl 1.9 mM; CaCl2 0.06 mM;FeSO4; 7H2O 0.008 mM), 2 mM phosphate, 1% glucose, 20 mM Mes, pH5.5, 100 �M acetosyringone, and rifampicin and kanamycin at the sameconcentrations given above. After overnight culture at 28C, the bacteriawere collected by centrifugation (15 min at 3000g) and washed once in asolution containing 10 mM Mes, pH 5.5, 10 mM MgSO4, and 100 �M ac-etosyringone. Cells then were resuspended in the same solution, and theOD600 of the suspension was adjusted to 0.8 before infiltration.

Young, nearly expanded leaves of 6-week-old tobacco plants were in-filtrated. The infiltrated areas were collected after 48 h and frozen in liq-uid nitrogen. The GUS fluorimetric assay was performed according tothe method of Jefferson et al. (1987).

Upon request, materials integral to the findings presented in this pub-lication will be made available in a timely manner to all investigators onsimilar terms for noncommercial research purposes. To obtain materials,please contact S. Delrot, [email protected].

Accession Numbers

The accession number for VvMSA is AF281656. Other accession num-bers are AF176655 (GASR) and AJ001062 (VvHT1).

ACKNOWLEDGMENTS

We are grateful to Terry Thomas (Texas A&M University, College Station)for the gift of pPC86, pSK1, and pYC7 vectors and of yeast strainYM954, to Matthieu Régnacq (Unité Mixte de Recherche Centre Nationalde la Recherche Scientifique [UMR CNRS] 6161, University of Poitiers,France) for helpful discussions regarding one-hybrid screening, to MarianneBernard (UMR CNRS 6558, University of Poitiers) for help with the gel-shift assays, to Anne Cantereau (UMR CNRS 6558, University of Poitiers)for confocal microscopy observations, to Marie-Thérèse Bidoyen fortechnical assistance, and to Bruno Faure for greenhouse plants. Part ofthis work was supported by the Conseil Régional Poitou-Charentes.

Received May 21, 2003; accepted June 20, 2003.

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DOI 10.1105/tpc.013854; originally published online August 8, 2003; 2003;15;2165-2180Plant Cell

Birsen Çakir, Alice Agasse, Cécile Gaillard, Amélie Saumonneau, Serge Delrot and Rossitza AtanassovaA Grape ASR Protein Involved in Sugar and Abscisic Acid Signaling

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