Plant Cell Rep (2008) 27:15411550 DOI 10.

1007/s00299-008-0566-1

PHYSIOLOGY AND BIOCHEMISTRY

Identication of grapevine aquaporins and expression analysis in developing berries

line Le on Nathalie Ollat Romain Fouquet Ce Franc ois Barrieu

Received: 14 February 2008 / Revised: 24 April 2008 / Accepted: 26 May 2008 / Published online: 17 June 2008 Springer-Verlag 2008

Abstract Aquaporins are membrane water channels that play critical roles in controlling the water content of cells and tissues. In this work, nine full-length cDNAs encoding putative aquaporins were isolated from grape berry cDNA libraries. A phylogenetic analysis conducted with 28 aquaporin genes identied in the grapevine genome and previously characterized aquaporins from Arabidopsis indicates that three cDNAs encode putative tonoplast aquaporins (TIPs) whereas six cDNAs belong to the plasma membrane aquaporin subfamily (PIPs). Specic probes designed on the 30 untranslated regions of each cDNA were used for the preparation of cDNA macroarray lters and in situ hybridization experiments. Macroarray data indicate that expression levels of most TIP and PIP genes depend on grape berry developmental stages and point out to a global decrease of aquaporin gene expression during berry ripening. In young berries, high expression of aquaporin genes was preferentially observed in dividing and elongating cells and in cells involved
This paper is dedicated to the memory of our friend and colleague Pr. d Hamdi. This work was supported by grants from the Conseil Sa Interprofessionnel du Vin de Bordeaux (CIVB). Communicated by L. Jouanin.

in water and solutes transport. Taken together, the data provided in this paper indicate that aquaporins are implicated in various physiological aspects of grape berry development. Keywords Aquaporins Grape berry Gene expression In situ hybridization Fruit development

Introduction Berries from wine and table grape are the most widely cultivated and economically important fruit crop worldwide. Grape berry is a non-climacteric fruit following a biphasic growth (Coombe 1992). The rst growth period involves cell divisions and expansion and is characterized by the development of seed embryos and the accumulation of organic acids. Cell division occurs only during the rst 2 weeks after anthesis in the pericarp (Ojeda et al. 1999) except for the skin cells where divisions are observed up to 30 days after anthesis (Considine and Knox 1981). When cell divisions are completed, berry growth is mainly related to cell enlargement. The onset of ripening, called veraison, occurs with the initial stages of color development and softening, and indicates the beginning of the second growth phase involving only cell expansion driven by sugars accumulation in the berries. Before veraison, berry water is mainly provided by xylem tracheids, whereas phloem constitutes the preferential pathway in post-veraison berries (Greenspan et al. 1994) even if some recent evidences indicate that the xylem may remain functional in postveraison berries (Bondada et al. 2005; Keller et al. 2006). In any case, the grapevine water status strongly affects the nal size of the berries. Indeed, water decits from owering to veraison can strongly affect the nal cell volumes (Ojeda et al. 2001).

Electronic supplementary material The online version of this article (doi:10.1007/s00299-008-0566-1) contains supplementary material, which is available to authorized users.
on N. Ollat F. Barrieu (&) R. Fouquet C. Le Institut des Sciences de la Vigne et du Vin (ISVV), Mixte de Recherche Ecophysiologie et Ge nomique Unite Fonctionnelle de la Vigne, Institut National de la Recherche Agronomique, Domaine de la Grande Ferrade, de Bordeaux 1, Universite Victor Universite galen Bordeaux 2, BP 81, Se 33883 Villenave dOrnon, France e-mail: francois.barrieu@bordeaux.inra.fr

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Water transport across biological membranes is facilitated by water channel proteins called aquaporins (Chrispeels and Maurel 1994). These water channel proteins belong to the major intrinsic protein (MIP) superfamily, an ancient family of membrane proteins (Reizer et al. 1993). According to sequence homologies, they are divided into four distinct families: the plasma membrane intrinsic proteins (PIPs), the tonoplast intrinsic proteins (TIPs), the nodulin 26-like intrinsic proteins (NIPs) and the small and basic intrinsic proteins (SIPs) (Kaldenhoff and Fischer 2006). In plants, aquaporins are present in the tonoplast and in the plasma membrane in almost all types of tissues. The subcellular location of members from the NIP and SIP families is still uncertain, but recent studies have shown that SIPs are localized in the ER fraction of Arabidopsis cells (Ishikawa et al. 2005). Aquaporins are likely to be important for water transport both at the cellular and at the whole plant level, but the accurate physiological functions and regulatory mechanisms of these proteins are still difcult to appreciate (Hachez et al. 2006). Aquaporin genes can be regulated at the transcriptional level in response to stress and the protein channel activities can be modulated in response to post-translational modications (Luu and Maurel 2005). Currently, 35 members of the MIP superfamily have been identied in Arabidopsis thaliana (Johanson et al. 2001) and 31 in Zea mays (Chaumont et al. 2001). In this work, we have identied 28 genes encoding putative aquaporins in the grapevine genome. Nine cDNAs encoding putative PIP and TIP aquaporins have been isolated from grape berry cDNA libraries at various developmental stages. Expression analyses using cDNA macroarray indicated that aquaporin gene expression is strongly regulated along berry development and globally decreases during ripening. In situ hybridization experiments showed that genes encoding the tonoplast aquaporin VvTIP2;1 and the plasma membrane aquaporin VvPIP2;1 were highly expressed in welldened cell types or berry tissues.

During ripening, berries were selected according to their size and soluble sugar content, as estimated by density measurement on NaCl solutions. Berries corresponding to four developmental stages [green, 20 days after owering (DAF); veraison, 70 DAF; mid-ripening, 90 DAF; harvest, 111 DAF] were either frozen in liquid nitrogen and stored at -80C until RNA extraction or xed in paraformaldehyde for in situ hybridization as previously described by Vignault et al. (2005). Cloning of cDNAs encoding putative aquaporins Nine full-length cDNAs encoding putative aquaporins were isolated using as templates three cDNA libraries prepared from berries of Cabernet Sauvignon collected at three developmental stages (green, veraison, and harvest stages) (Deluc et al. 2006). PCR cloning was conducted using primers designed in conserved regions of Arabidopsis thaliana PIPs and TIPs subgroups of plant aquaporins. These primers called: AtPIP1 reverse (50 -CTTGAAT GGAATGGCTCTGAT-30 ); AtPIP2 reverse (50 -CACTGA GCCACCATGTA-30 ); AtTIP1 reverse (50 -CCCAGTAGA CCCAGTGGTTG-30 ); AtTIP2 reverse (50 -ATCACGA TCTCGAAGAC-30 ) were used in combination with the forward 50 primer of the pTriplex vector (Clontech). Amplication products were cloned into the pGEM-T Easy vector (Promega) according to manufacturers instructions and sequenced on both strands (Genome express, Meylan). Full-length cDNAs were obtained using specic primers designed within the 50 non-coding region of each cDNAs in combination with the T7 primer of the pTriplex vector (Clontech). Sequence analysis Genes encoding putative aquaporins were identied in the grapevine genome by Blast searches in the Grape Genome Browser (http://www.genoscope.cns.fr/externe/Genome Browser/Vitis/) (Jaillon et al. 2007) and at the National Center for Biotechnological Information (http://www. ncbi.nlm.nih.gov). Analysis of DNA sequences and comparison with known sequences were carried out using NCBI Blast server (Altschul et al. 1997). The ClustalX program (Thompson et al. 1997) and the Vector NTI 7 software (Invitrogen) were used for sequence alignment. The Tree View software (Page 1996) was used to optimize the presentation of the phylogenic trees. Isolation of total RNA Frozen seeded berries were ground to powder in liquid nitrogen and RNA extraction was carried out according to the method described by Chang et al. (1993). All RNA

Materials and methods Plant material and growth conditions Grape berries (Vitis vinifera L. cv. Cabernet Sauvignon) were harvested during the growing seasons 2002 and 2003 in the Domaine du Grand Parc (INRA, Latresne, France). Whole berries were sorted individually according to diameter and average weight up to the veraison stage. At veraison, the berries were selected according to their softness and color: when the rst signs of color change appeared on berry clusters, soft berries showing no signs of color change were selected for the veraison stage sample.

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samples were treated with RNase-free DNase I (Promega) followed by phenol/chloroform extraction and ethanol precipitation. No DNA contamination was detected based on PCR amplication. Probe labelling and macroarray hybridization Despite the high degree of homology found within the coding regions of the cDNAs cloned in this study, the untranslated regions (UTRs) appear signicantly divergent. Table 1 summarizes the homologies observed between the 30 UTR of the different cDNAs encoding putative Cabernet Sauvignon aquaporins. Previously published data related to microarrays experiments on Arabidopsis thaliana seeds indicate that cross hybridizations of immobilized probes with non-specic transcripts are unlikely to happen between fragments having less than 80% identity (Girke et al. 2000). According to Table 1, this 80% threshold is not reached between the 30 UTRs of the cDNAs of interest indicating that these regions are suitable to be used as specic probes in a macroarray experiment. Finally, BLAST searches against the grape genome (http://www.

genoscope.cns.fr/externe/GenomeBrowser/Vitis/) did not indicate possible cross-hybridizations with other transcripts identied to date. The 30 UTR of each cDNA was amplied by PCR using the primers listed in Table 2 in a total volume of 100 lL using 0.5 lM of each gene-specic primer (Table 2) and 50 ng of aquaporin cDNA sample. All PCR products were puried using the QIAquick PCR Purication Kit (Qiagen) and nally quantied using GeneQuant pro (Amersham) spectrophotometer. Nylon membranes (Hybond XL, Amersham) were wetted with 29 SSC before DNA spotting and 25 ng of each PCR product were arrayed on the membranes using a Hybri-dot Manifold 1050 MM (BRL) under vacuum. Spotted membranes were then washed with 29 SSC for 5 min and treated with UV radiation for stabilization of DNA on the membranes. For probes synthesis, total RNA (40 lg) was reverse transcribed using the LabelStar Array kit (Qiagen) according to the manufacturers instructions. Incorporation of [32P]dCTP was determined by scintillation counting, and after a denaturation step of 5 min at 95C, the labelled rst strand cDNA was used for hybridization at the nal

Table 1 Primary nucleotide sequences homologies observed between the 30 UTRs of aquaporin encoding cDNAs from Cabernet Sauvignon berries VvPIP1;1 VvPIP1;1 VvPIP1;2 VvPIP1;3 VvPIP2;1 VvPIP2;2 VvPIP2;3 VvTIP1;1 VvTIP1;2 VvTIP2;1 100 VvPIP1;2 51 100 VvPIP1;3 33 30 100 VvPIP2;1 20 20 33 100 VvPIP2;2 49 45 47 26 100 VvPIP2;3 46 48 32 26 35 100 VvTIP1;1 26 32 38 34 33 35 100 VvTIP1;2 56 56 38 24 48 50 34 100 VvTIP2;1 39 43 38 29 41 36 40 48 100

Table 2 Primers used for PCR amplication of the 30 UTRs of aquaporin and elongation factor 1c cDNAs cDNAs VvPIP1;1 VvPIP1;2 VvPIP1;3 VvPIP2;1 VvPIP2;2 VvPIP2;3 VvTIP1;2 VvTIP2;1 VvTIP1;1 VvEF1c 30 UTR-forward primer 50 -AGTGGTGCTGGGCGTTGA 5 -TTCCTCCATTTTCTGTTTC 50 -ACTTCCATCGCCTTTCTC 50 -CCTTCTTCCACTGTTATTGGG 50 -AAACCCACAACACCCTCC 50 -TAGTGGTGAGGATGTGTGA 5 -CAGTTCCGAGGCTTTTGT 50 -TTGTTTGTTGTTGTCTCA 50 -CTTGCTATGAATTTCAGGG 50 -GCGGGCAAGAGATACCTCAA
0 0

30 UTR-reverse primer 50 -AGTGGAATGCTACAGACA 50 -CATTCAAAAGCTGCCCAT 50 -GCCACAAAAATAGATACTC 50 -AGACAAGCCACAACACAG 50 -GAAGGATTAAATTATGGA 50 -AGGAGCTTATTAGAGCAGAG 50 -AACTAAATGGGAGCCGAT 50 -CATCACCAACCTCATTCA 50 -TTTAAGTTCCAAGGACAT 50 -TCAATCTGTCTAGGAAAGGAAG

Product size (bp) 107 164 211 260 177 179 149 196 234 258

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concentration of 3 9 105 cpm mL-1. Hybridization was performed at 65C for more than 12 h in a hybridization buffer containing 59 SSC, 59 Denhardt reagent, 0.5% SDS and 100 lg mL-1 denatured salmon sperm DNA. Washes were performed at 65C in 29 SSC, 0.5% SDS for 5 min, 29 SSC, 0.1% SDS for 15 min, 19 SSC, 0.1% SDS for 15 min and 0.29 SSC, 0.1% SDS for 20 min. The membranes were nally wrapped with plastic lm and exposed to an IP image plate (Kodak Photo Film) for 2 days. Macroarray data analysis Signals of the IP image plates were scanned with a FX PhosphorImager (Bio-Rad) and quantied using Array Vision 8.0 software (Imaging Research Inc.). For each membrane, the relative expression levels were estimated by calculating the ratio of the intensity of each individual aquaporin signal over the signal intensity of the Elongation factor 1 c (EF1c) gene (Hanana et al. 2007). For each developmental stage studied, three independent RNA extractions were performed and used to generate three sets of macroarray data. The mean value of the relative expression levels was taken as an indicator of the relative abundance of each aquaporin transcript compared with the abundance of EF1c. The data were treated using analysis of variance procedures and means were separated by Tukey test (P \ 0.05). RNA in situ hybridization As already reported (Diakou and Carde 2001; Vignault et al. 2005), the use of developing grape berries for in situ hybridization is difcult, mainly because of tissues xation problems. This is especially true for berries collected during the ripening period that contain high amounts of water and sugars. However, the use of young berries collected 20 DAF (berry diameter from three to ve mm) allowed us to avoid the xation problems and to obtain reproducible in situ hybridization results. Experiments were performed on tissue samples from Cabernet Sauvignon berries xed by 4% paraformaldehyde in PBS buffer (50 mM sodium phosphate, pH 7.2) and embedded in parafn. Tissues were sectioned transversely into 8 lm slices and mounted on poly-L-lysine coating slides. Sections were then dewaxed with Histoclear (Shandon) and hydrated by passing through an alcohol series to water. Pre-hybridization treatments were performed to eliminate non-specic binding of the probes and increase their access to the target mRNAs. Slides were rst incubated in a proteinase K solution (1 lg mL-1) during 30 min at 37C to improve in situ staining by proteolytic digestion of the xed tissues. Digestion was stopped with

0.2 M glycine in PBS buffer. After several PBS washes, an acetylation step was added to decrease hybridization background (0.1 M triethanolamin, 0.5% acetic anhydride for 5 min) and sections were nally dehydrated through an alcohol series to 100% ethanol and dried under vacuum. Digoxigenin-labelled VvPIP2;1 and VvTIP2;1 riboprobes were synthesized by in vitro transcription as follows. The 30 UTR of each cDNA was amplied by PCR with the corresponding specic primers (Table 2) and subcloned into the pGEM-T Easy vector (Promega). Sense and antisense digoxigenin-labelled riboprobes were generated using the DIG RNA labelling kit (Roche) according to the manufacturers instructions with either SP6 or T7 RNA polymerase. In situ hybridization was conducted overnight at 55C as described by Bostwick et al. (1992). Detection of hybridized probes was performed using anti-digoxygenin antibodies conjugated to alkaline phosphatase (Roche) and signals were visualized by colour development with 5-bromo-4-chloro-3-indolyl-phosphate and tetrazolium nitroblue (Roche).

Results Cloning and analysis of grapevine aquaporin cDNAs Degenerate oligonucleotide primers designed from highly conserved regions of plant aquaporins (HI/VNPAVT) were used to screen grape berry cDNA libraries. This led to the isolation of nine full-length cDNAs encoding putative aquaporins (Table 3). Alignment of the deduced amino acid sequences is shown in Fig. 1. All the deduced sequences contain the MIP family signature sequence SGxHxNPAVT in the rst half of the protein, which is repeated as a shorter NPA motif in the second half of the protein (Park and Saier 1996).
Table 3 Characteristics of the aquaporin encoding cDNAs isolated in this study cDNA name cDNA length (bp) 1,050 1,099 1,170 1,285 1,075 1,261 1,085 970 1,047 ORF (bp) 861 861 864 852 840 864 756 753 750 Protein length (aa) 286 286 287 284 279 287 251 250 251 Accession number DQ834694 DQ834695 DQ834696 DQ834698 DQ834699 DQ834700 DQ834701 DQ834702 DQ834703

VvPIP1;1 VvPIP1;2 VvPIP1;3 VvPIP2;1 VvPIP2;2 VvPIP2;3 VvTIP1;1 VvTIP1;2 VvTIP2;1

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Fig. 1 Alignment of the deduced amino acid sequences encoded by the cDNAs isolated in this study. Deduced amino acid sequences were compared using the ClustalX multiple alignment program (Thompson et al. 1997). Identical amino acid residues are shaded in black and residues shaded in grey indicate a conservative amino acid

substitution. Genbank accession numbers; VvPIP1;1: DQ834694, VvPIP1;2: DQ834695, VvPIP1;3: DQ834696, VvPIP2;1: DQ834698, VvPIP2;2: DQ834699, VvPIP2;3: DQ834700, VvTIP1;1: DQ834701, VvTIP1;2: DQ834702, and VvTIP2;1: DQ834703

The recent releases of the grapevine genome (Jaillon et al. 2007; Velasco et al. 2007) allowed us to search for putative aquaporin genes. Twenty-eight genes encoding complete aquaporin proteins were identied. Like in other plant species, a comprehensive phylogenetic analysis of the deduced protein sequences indicate that grape aquaporins can be divided into four distinct subfamilies (PIPs, TIPs, NIPs, and SIPs; Fig. 2). The proposed nomenclature for these proteins and their corresponding cDNAs has been established with special care according to the results of multiple alignments with MIP genes from Arabidopsis (Fig. S1, on-line supplementary data) and in full respect of the current nomenclature (Johanson et al. 2001). As expected, the nine genes corresponding to the cDNAs cloned in this study have been clearly identied in the genome and encode six proteins belonging to the PIP subfamily and three related to the TIP subfamily (Fig. 2). Aquaporin gene expression during grape berry development Aquaporin gene expression was investigated by hybridizing macroarrays lters with 32P-labelled cDNAs probes retrotranscribed from RNAs samples representing four grape berry developmental stages. Eight out of the nine genes studied showed hybridization signals signicantly above the background signal (Fig. 3). No clear signal was detected for the VvPIP1;1 gene in all the samples studied, indicating that this gene was probably expressed at very low levels during grape berry development. Moreover, further studies from our group (Fouquet et al., in preparation) using the same macroarray lters indicate that the VvPIP1;1 gene is highly expressed in Cabernet Sauvignon roots, ruling out the possibility of a hybridization

mismatch. Analysis of the remaining expression proles indicates an heterogeneity of aquaporin gene expression during grape berry development. According to Fig. 3, the overall highest level of expression was observed for the rst stage of development studied (i.e., 20 DAF). Further analysis showed that aquaporin genes could be clustered into four distinct groups according to their expression proles during grape berry development (Table 4). The rst group is constituted by the VvPIP1;1 gene that showed no variation in expression during berry development. The second group is constituted by VvPIP1;2, VvPIP2;2, VvTIP1;2 and VvTIP2;1, with a decrease in expression beginning at the veraison stage. VvPIP1;3 and VvTIP1;1 belong to a third group characterized by a decrease in the amount of transcripts occurring after veraison, whereas the fourth group contains the VvPIP2;1 and VvPIP2;3 genes that showed an increase of expression at the veraison stage followed by a stabilization (VvPIP2;3) or a decrease of expression (VvPIP2;1) after veraison (Table 4). Taken together, these data indicate a global decrease of aquaporin gene expression during grape berry development. Localization of aquaporin transcripts by in situ hybridization in young berries To examine the preferential sites of aquaporin gene expression in grape berries, in situ hybridization experiments were carried out using berries collected 20 DAF. At this stage, the pericarp is composed of three distinct tissues: the exocarp, the mesocarp and the endocarp constituted by a thin layer of cells in contact with the developing seeds (Fig. 4a). The exocarp, or skin, consists of a single layer of epidermal cells and about 1112 layers of hypodermal cells (Fig. 4a, b).

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Fig. 2 Phylogenetic analysis of putative grapevine aquaporins. Amino acid sequence alignment and subsequent neighbor-joining tree were generated using the ClustalX (Thompson et al. 1997) and Tree View (Page 1996) programs. The proposed nomenclature for the grapevine aquaporins has been established according to the results of multiple alignments with MIP genes from Arabidopsis (Fig. S1, online supplementary data) and in full respect of the current nomenclature (Johanson et al. 2001). Sequences identied in the present study are framed. Accessions of sequences retrieved from the Grape Genome sequencing draft are as follow: VvNIP1;1 (GSVIVP 00035815001) VvNIP3;1 (GSVIVP00022377001), VvNIP4;1 (GSVI VP00011149001), VvNIP5;1 (GSVIVP00000446001), VvNIP6;1 (GS VIVP00033750001), VvNIP7;1 (GSVIVP00019910001), VvNIP8;1 (GSVIVP00007127001), VvNIP8;2 (GSVIVP00003903001), VvPI P1;1 (GSVIVP00029248001), VvPIP1;2 (GSVIVP00026881001), VvPIP1;3 (GSVIVP00000433001), VvPIP1;5 (GSVIVP00026882 001), VvPIP2;2 (GSVIVP00036133001), VvPIP2;3 (GSVIVP000 23192001), VvSIP2;1 (GSVIVP00023346001), VvTIP1;1 (GSVIVP 00018548001), VvTIP1;2 (GSVIVP00000605001), VvTIP1;3 (GSVI VP00022146001), VvTIP1;4 (GSVIVP00024394001), VvTIP2;1 (GSVIVP00034350001), VvTIP2;2 (GSVIVP00012703001), VvTI P3;1 (GSVIVP00013854001), VvTIP4;1 (GSVIVP00032441001), VvTIP5;1 (GSVIVP00029946001), VvTIP5;2 (GSVIVP0001917 0001). The VvSIP1;1 protein (Genbank accession: DQ086835) appears in the grape genome draft with accessions GSVIVP00025504001 and GSVIVP00025505001. The VvPIP2;1 protein (Genbank accession: CAN75442) has been identied by Velasco et al. (2007)

In situ hybridization experiments were also carried out using VvTIP2;1 labelled RNA probes. Transcripts accumulations were observed in two distinct cell types of the pericarp. The VvTIP2;1 transcripts appeared abundant in some cells associated with the peripheral vascular bundles located at the outer part of the mesocarp. Close observations of the labelled cells revealed that these cells were associated for their majority with the xylem tracheids and thus belong to the xylem parenchyma (Fig. 4e). The use of VvTIP2;1 sense probes conrmed the absence of a specic hybridization signal around the vascular bundles (Fig. 4f). A high level of signal was also detected in cells located under the epidermis and belonging to the rst hypodermal cell layer (Fig. 4g). VvTIP2;1 transcripts were not detected in epidermal cells and only a very weak signal was occasionally observed in cells from the second hypodermal layer. Experiments carried out with VvTIP2;1 sense probes did not reveal any specic signal in the epidermis and the hypodermal cells (Fig. 4h). In conclusion, in situ hybridization experiments indicated that the VvPIP2;1 and VvTIP2;1 genes were highly expressed in well-dened tissues or cell types within young developing grape berries.

Discussion Analysis of grape aquaporins and identication of genes expressed in grape berry Like in Arabidopsis and rice, where 35 and 33 isoforms have been respectively identied (Johanson et al. 2001; Sakurai et al. 2005), aquaporins appear to form an important multigenic family in grapevine. A search in the rst releases of the grape genome (Jaillon et al. 2007; Velasco et al. 2007) allowed us to identify 28 genes encoding putative aquaporins that can be divided into four subgroups on the basis of sequence homology (Fig. 2). The distribution between these four subgroups appears similar to the ones observed in Arabidopsis (Johanson et al. 2001) and rice (Sakurai et al. 2005), whereas maize appears to have a reduced number of NIPs genes (Chaumont et al. 2001). Among the 28 grape aquaporin genes, our results indicate that at least nine genes encoding PIP and TIP aquaporins are expressed in developing berries. A global transcriptome analysis approach has recently identied 16 transcripts encoding putative PIP and TIP proteins differentially expressed during berry development (Deluc et al. 2007; additional le 2). Using the same technology with berries sampled in three growing years to limit the inuence of climatic conditions, Pilati et al. (2007) detected the differential expression of 8 PIP and 4 TIP genes during berry development. However, when considering the recent genome data, and as stated by Pilati et al. (2007) for the

Experiments carried out with VvPIP2;1 RNA probes did not show any clear hybridization signals in the skin and pulp tissues of young berries (data not shown). However, a strong signal was observed in developing seeds in cells surrounding the apical side of the embryo (Fig. 4c), whereas no specic signal was detected using a control VvPIP2;1 sense probe on the same tissues (Fig. 4d). According to the histological study of Cadot et al. (2006), these parenchymatous cells belong to the developing outer integument.

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Fig. 3 Analysis of aquaporin gene expression during berry development. a Representative macroarray hybridization experiment using complex probes prepared from Cabernet Sauvignon berries harvested at 20, 70, 90 or 111 days after owering (DAF). b Relative expression levels of aquaporin gene during berry development. Signals from three independent membranes were averaged for each gene and each Table 4 Summary of aquaporin genes expression during grape berry development

time point of berry development. Expression levels were normalized relative to EF1c transcripts (see Materials and methods). For each gene, a and b indicate that means are signicantly different as determined by ANOVA followed by Tukey test (P \ 0.05; n = 3). Means designated ab cannot be distinguished from a or b

Group 1 2 3 4

Expression during grape berry development No extensive variation Expression decreases at veraison Expression decreases after veraison Expression increases at veraison

Gene name VvPIP1;1 VvPIP1;2, VvPIP2;2, VvTIP1;2, VvTIP2;1 VvPIP1;3, VvTIP1;1 VvPIP2;1, VvPIP2;3

PIP2;1 aquaporin, some probe sets of the microarrays used in these studies target in fact an unique transcript. Taking account this redundancy, it appears that our screen led to the identication of a representative set of the various TIP and PIP genes expressed in berries. To date, the expression of two other genes, encoding, respectively, one SIP and one NIP aquaporin, has also been detected in grape berries (Deluc et al. 2007; Pilati et al. 2007). However, it is likely that the outcomes from the grape genome sequencing will allow the identication of additional aquaporin genes expressed in developing berries. Expression of aquaporin genes during berry development The data presented in this paper indicate that, while some aquaporin genes did not show any signicant variation in expression during berry development, other appeared preferentially expressed during a particular growth period. According to our results, most of the genes showing signicant variation in expression appeared preferentially expressed before or up to the veraison stage. After veraison, berry ripening can be characterized by a global decrease of aquaporin gene expression, except for the

VvPIP2;3 gene. Similar results were obtained by Deluc et al. (2007) and Pilati et al. (2007) who observed a preferential expression of aquaporin genes during the preveraison phase. It is tempting to hypothesize that spatiotemporal regulation along fruit development may be linked to particular physiological events and reects a possible specialization of some aquaporins in these events. Although some aquaporins may be involved in the transport of small neutral solutes and/or gazes (Kaldenhoff and Fischer 2006), the central function of all characterized proteins belonging to the aquaporin family is water transport. If we consider only this water channel function, the preferential expression of aquaporin genes before veraison is likely to be associated with the mechanisms of cell division and cell expansion occurring during the rst berry growth stage, as recently demonstrated by Schlosser et al. (2008). The localization of the VvTIP2;1 transcripts in cells of the rst hypodermal layer also supports this hypothesis. As reported by Considine and Knox (1981), who studied cell division in the developing dermal system of grape berry, these particular cells are dividing up to three weeks after anthesis. Dividing cells must generate equal amounts of tonoplast and plasma membrane between rounds of cell division, a process that requires, like cell

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Fig. 4 In situ localization of VvPIP2;1 and VvTIP2;1 mRNAs in berry tissues. A grape berry half-section stained with toluidine blue (a) and a magnied view of the epicarp (b) are showed to facilitate the localization of the hybridization signals. Boxed areas represent the berry tissues where hybridization signals were observed and are linked by arrows to the corresponding in situ hybridization data. HLs hypodermal cell layers, EL epidermal cell layer. Transverse sections

of Cabernet Sauvignon berries harvested 20 days after owering were hybridized either with digoxigenin-labelled VvPIP2;1 antisense (c) or sense (d) probes or with VvTIP2;1 antisense (e, g) or sense (f, h) probes. The transcript signal appears dark-blue and is observed only on sections hybridized with antisense probes. Ep epidermis, II inner integument, MI medium integument, OI outer integument, Nu nucellus, En endosperm, XT xylem tracheids

elongation, the synthesis of new membrane components. Moreover, expression of VvTIP2;1 in xylem parenchyma cells before veraison indicates that aquaporins may also participate to the xylem unloading process, as discussed below. Because of the lack of in situ hybridization data from the veraison to the harvest stage (see materials and methods), it is difcult to assign putative functions for aquaporins in ripening berries. According to the multiple functions proposed for aquaporins in plants (Kaldenhoff and Fischer 2006), water channels may facilitate the osmotic adjustments linked to continued sugar accumulation in pericarp cells during berry ripening and the subsequent cell expansion events driven by this sugar accumulation. Aquaporins are also required in the phloem unloading mechanisms to allow water exchange between phloem

conducting cells and surrounding tissues (Fraysse et al. 2005). However, some other implications may be proposed. Our data indicate that, before veraison, expression of the VvPIP2;1 gene occurs in cells of the outer integument of developing seeds. Cell division activity in the outer integuments peaks at 2025 days after anthesis (Ristic and Iland 2005) and cell elongation occurs until veraison (Cadot et al. 2006). Thus, expression of VvPIP2;1 in these cells may be reasonably linked to a high activity of cell division and cell expansion in this particular tissue of the developing seeds. Shiota et al. (2006), found that the tomato VvPIP2;1 homolog, called LePIP2;1, was also expressed in seeds throughout fruit development with a peak of expression 20 DAF. Together with our data, these ndings support the implication of the PIP2;1 aquaporin in seed development.

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1549 Acknowledgments The authors are very grateful to Dr S. Aubourg (Plant Genomics Research Unit, Evry) for his help and valuable advices for the identication of aquaporin genes in the grapevine genome. We thank Pr. Serge Delrot and Dr. David Lecourieux for comments and advice on the manuscript and Dr. Jean-Pierre Carde (UMR 619 Biologie du Fruit, Centre INRA de Bordeaux) for providing the grape berry half-section pictures.

Implication of aquaporins in the regulation of berry water status after veraison At the berry level, Tyerman et al. (2004) reported a tenfold reduction of the whole berry hydraulic conductance between the veraison and harvest stages. These authors suggested that in addition to possible changes in xylem anatomy (Findlay et al. 1987), aquaporins might play a role in regulating the berry hydraulic conductance. A decrease of membrane hydraulic conductance can be linked to a decrease of aquaporins activity by post-transcriptional modications and/or to a modication of aquaporins abundance in the membrane (Hachez et al. 2006). At the transcript level, berry ripening is characterized by a global decrease of the amount of transcripts encoding plasma membrane aquaporins that can explain the reduction of the berry hydraulic conductance observed by Tyerman et al. (2004). Another interesting possibility relies on the implication of tonoplast aquaporins in the regulation of berry water status. Before veraison, when water is provided mainly by the xylem tracheids (Greenspan et al. 1994), we observed a high expression of the VvTIP2;1 gene in xylem parenchyma cells. Expression of VvTIP2;1 in these particular cells should lead to an increase of the tonoplast water permeability that will not only facilitate intracellular osmotic adjustments but also transcellular water ow (Barrieu et al. 1998). As a consequence, water and solute exchanges between the xylem and the berry tissues will be facilitated. After veraison, and despites the presence of functional xylem (Bondada et al. 2005; Keller et al. 2006), phloem sap becomes the primary water source for the berries (Greenspan et al. 1994). This shift from xylem to phloem water uptake coincides with a strong decrease of VvTIP2;1 expression. A reduced abundance of tonoplast aquaporins in xylem parenchyma cells should lead to a reduction of water and solute exchanges between the tracheids and the berry tissues. Thus, and as suggested by Tyerman et al. (2004), a weak expression of aquaporin genes in xylem parenchyma cells may account for the decrease of berry hydraulic conductance observed during ripening. Additional work is now needed to elucidate the precise role of the different aquaporins throughout berry development. Modulation of aquaporin expression in planta by genetic engineering is a strategy of choice for investigating the role of these proteins (Hachez et al. 2006) and may represent the next step towards a better understanding of aquaporin function in developing eshy fruits.

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Supplementary data The phylogenetic analysis of aquaporins from Vitis vinifera and Arabidopsis thaliana is available on-line at http://www. springerlink.com/content/0721-7714.

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