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A Grape ASR Protein Involved in Sugar and Abscisic Acid Signaling

A Grape ASR Protein Involved in Sugar and Abscisic Acid Signaling
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   This article is published in The Plant Cell   Online, The Plant Cell   Preview Section, which publishes manuscripts accepted for publication afterthey have been edited and the authors have corrected proofs, but before the final, complete issue is published online. Early posting of articles reduces normal time to publication by several weeks.  The Plant Cell   Preview, © 2003 American Society of Plant Biologists1 of 16   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 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 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 the VvMSA 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 tomato ASR1 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 DNA gel 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   genescontain 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; fax 33-[0]5-49-45-41-86. Article, publication date, and citation information can be found   2 of 16The 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 by VvMSA 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.   RESULTSThe 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/IAA protein, 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 ( revealed the pres-ence of two ABA/WDS signatures, which are described in ABA stress– 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:// 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 CLUSTALmethod (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 Protein3 of 16 Figure 1.  Amino Acid Alignment of VvMSA and 22 Known ASR Proteins from Different Species Performed with the CLUSTAL Program.   4 of 16The 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 other ASRs, 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 ABA treatment. 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 by ABA 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 with VvMSA  cDNA as an  - 32 P 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 Protein5 of 16   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 VvMSA protein 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 - and VvHT1 -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  and VvHT1  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.
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