Plant Cell Rep (2008) 27:1541–1550 DOI 10.



Identification of grapevine aquaporins and expression analysis in developing berries
´ line Le ´ on Æ Nathalie Ollat Æ Romain Fouquet Æ Ce Franc ¸ ois Barrieu

Received: 14 February 2008 / Revised: 24 April 2008 / Accepted: 26 May 2008 / Published online: 17 June 2008 Ó 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 identified 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). Specific probes designed on the 30 untranslated regions of each cDNA were used for the preparation of cDNA macroarray filters 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 Saı 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 Á Grape berry Á Gene expression Á In situ hybridization Á 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 first 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 first 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 final size of the berries. Indeed, water deficits from flowering to veraison can strongly affect the final 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 Á N. Ollat Á F. Barrieu (&) R. Fouquet Á 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 d’Ornon, France e-mail:


1 and the plasma membrane aquaporin VvPIP2. 2006). veraison. Sequence analysis Genes encoding putative aquaporins were identified in the grapevine genome by Blast searches in the Grape Genome Browser (http://www. Currently. Meylan). France). and harvest stages) (Deluc et al.nih.1 were highly expressed in welldefined cell types or berry tissues. but the accurate physiological functions and regulatory mechanisms of these proteins are still difficult to appreciate (Hachez et al. they are divided into four distinct families: the plasma membrane intrinsic proteins (PIPs). 2005). but recent studies have shown that SIPs are localized in the ER fraction of Arabidopsis cells (Ishikawa et al. According to sequence Browser/Vitis/) (Jaillon et al. ncbi. soft berries showing no signs of color change were selected for the veraison stage sample. 1993). the nodulin 26-like intrinsic proteins (NIPs) and the small and basic intrinsic proteins (SIPs) (Kaldenhoff and Fischer 2006). 35 members of the MIP superfamily have been identified in Arabidopsis thaliana (Johanson et al. Cabernet Sauvignon) were harvested during the growing seasons 2002 and 2003 in the Domaine du Grand Parc (INRA. (2005).gov). In situ hybridization experiments showed that genes encoding the tonoplast aquaporin VvTIP2. AtTIP1 reverse (50 -CCCAGTAGA CCCAGTGGTTG-30 ).nlm. we have identified 28 genes encoding putative aquaporins in the grapevine genome. 111 DAF] were either frozen in liquid nitrogen and stored at -80°C until RNA extraction or fixed in paraformaldehyde for in situ hybridization as previously described by Vignault et al. the berries were selected according to their softness and color: when the first signs of color change appeared on berry clusters. 2006). Berries corresponding to four developmental stages [green. veraison. the tonoplast intrinsic proteins (TIPs). 20 days after flowering (DAF). AtTIP2 reverse (50 -ATCACGA TCTCGAAGAC-30 ) were used in combination with the forward 50 primer of the pTriplex vector (Clontech). 2007) and at the National Center for Biotechnological Information (http://www. 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 modifications (Luu and Maurel 2005). (1993). In plants. as estimated by density measurement on NaCl solutions. These water channel proteins belong to the major intrinsic protein (MIP) superfamily. The Tree View software (Page 1996) was used to optimize the presentation of the phylogenic trees. Amplification products were cloned into the pGEM-T Easy vector (Promega) according to manufacturer’s instructions and sequenced on both strands (Genome express. Nine cDNAs encoding putative PIP and TIP aquaporins have been isolated from grape berry cDNA libraries at various developmental stages.cns. 1997) and the Vector NTI 7 software (Invitrogen) were used for sequence alignment. PCR cloning was conducted using primers designed in conserved regions of Arabidopsis thaliana PIPs and TIPs subgroups of plant aquaporins. AtPIP2 reverse (50 -CACTGA GCCACCATGTA-30 ). mid-ripening. 2001) and 31 in Zea mays (Chaumont et al. During ripening. 70 DAF. All RNA Materials and methods Plant material and growth conditions Grape berries (Vitis vinifera L. aquaporins are present in the tonoplast and in the plasma membrane in almost all types of tissues. 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. 2001). The subcellular location of members from the NIP and SIP families is still uncertain. Aquaporins are likely to be important for water transport both at the cellular and at the whole plant level. 123 . 90 DAF. The ClustalX program (Thompson et al. Expression analyses using cDNA macroarray indicated that aquaporin gene expression is strongly regulated along berry development and globally decreases during ripening. In this work. 1997). an ancient family of membrane proteins (Reizer et al. 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.1542 Plant Cell Rep (2008) 27:1541–1550 Water transport across biological membranes is facilitated by water channel proteins called aquaporins (Chrispeels and Maurel 1994). These primers called: AtPIP1 reverse (50 -CTTGAAT GGAATGGCTCTGAT-30 ). At veraison. berries were selected according to their size and soluble sugar content. Full-length cDNAs were obtained using specific primers designed within the 50 non-coding region of each cDNAs in combination with the T7 primer of the pTriplex vector (Clontech). Latresne. Whole berries were sorted individually according to diameter and average weight up to the veraison stage. harvest. Analysis of DNA sequences and comparison with known sequences were carried out using NCBI Blast server (Altschul et al. cv.

cns.1 VvPIP2.1 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) 107 164 211 260 177 179 149 196 234 258 123 .2 56 56 38 24 48 50 34 100 VvTIP2. and after a denaturation step of 5 min at 95°C. Incorporation of [32P]dCTP was determined by scintillation counting. genoscope.2 VvTIP2.2 51 100 VvPIP1.2 VvPIP1. Table 1 summarizes the homologies observed between the 30 UTR of the different cDNAs encoding putative Cabernet Sauvignon aquaporins.1 VvPIP1. total RNA (40 lg) was reverse transcribed using the LabelStar Array kit (Qiagen) according to the manufacturer’s instructions.1 100 VvPIP1.2 49 45 47 26 100 VvPIP2. the untranslated regions (UTRs) appear significantly divergent.2 VvTIP2.3 33 30 100 VvPIP2.1 VvPIP1. 2000).1 20 20 33 100 VvPIP2.1 26 32 38 34 33 35 100 VvTIP1.5 lM of each gene-specific primer (Table 2) and 50 ng of aquaporin cDNA sample.3 VvPIP2.1 39 43 38 29 41 36 40 48 100 Table 2 Primers used for PCR amplification of the 30 UTRs of aquaporin and elongation factor 1c cDNAs cDNAs VvPIP1. Previously published data related to microarrays experiments on Arabidopsis thaliana seeds indicate that cross hybridizations of immobilized probes with non-specific transcripts are unlikely to happen between fragments having less than 80% identity (Girke et al. Finally. 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.1 VvPIP1.2 VvPIP2. Spotted membranes were then washed with 29 SSC for 5 min and treated with UV radiation for stabilization of DNA on the membranes.Plant Cell Rep (2008) 27:1541–1550 1543 samples were treated with RNase-free DNase I (Promega) followed by phenol/chloroform extraction and ethanol precipitation.2 VvPIP1. All PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and finally quantified using GeneQuant pro (Amersham) spectrophotometer.3 VvPIP2. The 30 UTR of each cDNA was amplified by PCR using the primers listed in Table 2 in a total volume of 100 lL using 0.1 VvPIP2.2 VvPIP2. For probes synthesis.3 VvTIP1. 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 specific probes in a macroarray experiment. No DNA contamination was detected based on PCR amplification.3 VvTIP1.1 VvTIP1. According to Table did not indicate possible cross-hybridizations with other transcripts identified to date. BLAST searches against the grape genome (http://www.3 46 48 32 26 35 100 VvTIP1. the labelled first strand cDNA was used for hybridization at the final Table 1 Primary nucleotide sequences homologies observed between the 30 UTRs of aquaporin encoding cDNAs from Cabernet Sauvignon berries VvPIP1.1 VvTIP1. Probe labelling and macroarray hybridization Despite the high degree of homology found within the coding regions of the cDNAs cloned in this study.

19 SSC.1 VvTIP1.5% acetic anhydride for 5 min) and sections were finally dehydrated through an alcohol series to 100% ethanol and dried under vacuum.2 VvTIP2.285 1.29 SSC.1 VvPIP2.0 software (Imaging Research Inc. 0. Washes were performed at 65°C in 29 SSC.5% SDS for 5 min. Digestion was stopped with 0. 0.1544 Plant Cell Rep (2008) 27:1541–1550 concentration of 3 9 105 cpm mL-1.2 VvPIP2. an acetylation step was added to decrease hybridization background (0. The membranes were finally wrapped with plastic film and exposed to an IP image plate (Kodak Photo Film) for 2 days. 0. In situ hybridization was conducted overnight at 55°C as described by Bostwick et al. Slides were first incubated in a proteinase K solution (1 lg mL-1) during 30 min at 37°C to improve in situ staining by proteolytic digestion of the fixed tissues.).1% SDS for 20 min. After several PBS washes.085 970 1. (1992). Sense and antisense digoxigenin-labelled riboprobes were generated using the DIG RNA labelling kit (Roche) according to the manufacturer’s instructions with either SP6 or T7 RNA polymerase. Hybridization was performed at 65°C for more than 12 h in a hybridization buffer containing 59 SSC.1 and VvTIP2.1% SDS for 15 min. This is especially true for berries collected during the ripening period that contain high amounts of water and sugars. 59 Denhardt reagent. Vignault et al. Tissues were sectioned transversely into 8 lm slices and mounted on poly-L-lysine coating slides.1 riboprobes were synthesized by in vitro transcription as follows. 0.2 M glycine in PBS buffer. Experiments were performed on tissue samples from Cabernet Sauvignon berries fixed by 4% paraformaldehyde in PBS buffer (50 mM sodium phosphate. The data were treated using analysis of variance procedures and means were separated by Tukey test (P \ 0.1 123 . pH 7. 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).075 1.261 1. For each developmental stage studied.2 VvPIP1. 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. which is repeated as a shorter NPA motif in the second half of the protein (Park and Saier 1996). All the deduced sequences contain the MIP family signature sequence SGxHxNPAVT in the first half of the protein.3 VvPIP2. 1. The 30 UTR of each cDNA was amplified by PCR with the corresponding specific primers (Table 2) and subcloned into the pGEM-T Easy vector (Promega). Digoxigenin-labelled VvPIP2. 29 SSC. 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.05).099 1.5% SDS and 100 lg mL-1 denatured salmon sperm DNA.2) and embedded in paraffin. RNA in situ hybridization As already reported (Diakou and Carde 2001.3 VvTIP1. Pre-hybridization treatments were performed to eliminate non-specific binding of the probes and increase their access to the target mRNAs. the use of young berries collected 20 DAF (berry diameter from three to five mm) allowed us to avoid the fixation problems and to obtain reproducible in situ hybridization results. Macroarray data analysis Signals of the IP image plates were scanned with a FX PhosphorImager (Bio-Rad) and quantified using Array Vision 8.170 1. Alignment of the deduced amino acid sequences is shown in Fig. 2007). 2005).050 1. Sections were then dewaxed with Histoclear (Shandon) and hydrated by passing through an alcohol series to water.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. For each membrane.1 VvPIP1.1 M triethanolamin. Table 3 Characteristics of the aquaporin encoding cDNAs isolated in this study cDNA name cDNA length (bp) 1. 0.1% SDS for 15 min and 0. However. mainly because of tissues fixation problems. 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. three independent RNA extractions were performed and used to generate three sets of macroarray data. the use of developing grape berries for in situ hybridization is difficult. 0. This led to the isolation of nine full-length cDNAs encoding putative aquaporins (Table 3).

VvPIP2.1: DQ834698. Analysis of the remaining expression profiles indicates an heterogeneity of aquaporin gene expression during grape berry development..1) after veraison (Table 4). these data indicate a global decrease of aquaporin gene expression during grape berry development. and SIPs.1 gene in all the samples studied.. with a decrease in expression beginning at the veraison stage. TIPs. Aquaporin gene expression during grape berry development Aquaporin gene expression was investigated by hybridizing macroarrays filters with 32P-labelled cDNAs probes retrotranscribed from RNAs samples representing four grape berry developmental stages. Taken together. 2007. or skin. Fig. 2001). VvPIP2.1 gene that showed no variation in expression during berry development. 4a. 2007) allowed us to search for putative aquaporin genes. The first group is constituted by the VvPIP1.1: DQ834701. 1 Alignment of the deduced amino acid sequences encoded by the cDNAs isolated in this study. VvPIP1. Eight out of the nine genes studied showed hybridization signals significantly above the background signal (Fig. VvPIP2.e.1 and VvPIP2. Moreover. a comprehensive phylogenetic analysis of the deduced protein sequences indicate that grape aquaporins can be divided into four distinct subfamilies (PIPs. the overall highest level of expression was observed for the first stage of development studied (i. Genbank accession numbers. indicating that this gene was probably expressed at very low levels during grape berry development. consists of a single layer of epidermal cells and about 11–12 layers of hypodermal cells (Fig. According to Fig. 4a). The second group is constituted by VvPIP1. 3). Velasco et al. No clear signal was detected for the VvPIP1. 2). 3. 1997). Localization of aquaporin transcripts by in situ hybridization in young berries To examine the preferential sites of aquaporin gene expression in grape berries.1: DQ834694. Like in other plant species. VvPIP1. Twenty-eight genes encoding complete aquaporin proteins were identified.2: DQ834695.2: DQ834702. and VvTIP2.3: DQ834700. VvTIP1. in preparation) using the same macroarray filters indicate that the VvPIP1. b).3: DQ834696. Deduced amino acid sequences were compared using the ClustalX multiple alignment program (Thompson et al. S1. the nine genes corresponding to the cDNAs cloned in this study have been clearly identified in the genome and encode six proteins belonging to the PIP subfamily and three related to the TIP subfamily (Fig.1.2.3 genes that showed an increase of expression at the veraison stage followed by a stabilization (VvPIP2. 123 . further studies from our group (Fouquet et al.2: DQ834699. 20 DAF). NIPs. ruling out the possibility of a hybridization mismatch.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. 2).3 and VvTIP1. the mesocarp and the endocarp constituted by a thin layer of cells in contact with the developing seeds (Fig. Further analysis showed that aquaporin genes could be clustered into four distinct groups according to their expression profiles during grape berry development (Table 4). VvTIP1. Identical amino acid residues are shaded in black and residues shaded in grey indicate a conservative amino acid substitution. whereas the fourth group contains the VvPIP2. in situ hybridization experiments were carried out using berries collected 20 DAF.Plant Cell Rep (2008) 27:1541–1550 1545 Fig.2 and VvTIP2.1: DQ834703 The recent releases of the grapevine genome (Jaillon et al. At this stage. As expected.3) or a decrease of expression (VvPIP2.1 belong to a third group characterized by a decrease in the amount of transcripts occurring after veraison.1 gene is highly expressed in Cabernet Sauvignon roots. the pericarp is composed of three distinct tissues: the exocarp. VvPIP2. on-line supplementary data) and in full respect of the current nomenclature (Johanson et al. VvPIP1. The exocarp. VvPIP1. VvTIP1.

A global transcriptome analysis approach has recently identified 16 transcripts encoding putative PIP and TIP proteins differentially expressed during berry development (Deluc et al. Among the 28 grape aquaporin genes. The VvPIP2.1 (GSVIVP00034350001).1 (GSVIVP00029248001). these parenchymatous cells belong to the developing outer integument.1 (GSVIVP00023346001). VvTIP1. our results indicate that at least nine genes encoding PIP and TIP aquaporins are expressed in developing berries. 123 .5 (GSVIVP00026882 001).1 labelled RNA probes. The proposed nomenclature for the grapevine aquaporins has been established according to the results of multiple alignments with MIP genes from Arabidopsis (Fig. a strong signal was observed in developing seeds in cells surrounding the apical side of the embryo (Fig. VvNIP6.1 (GSVIVP00019910001). (2007) for the Experiments carried out with VvPIP2. additional file 2). when considering the recent genome data.2 (GSVIVP00003903001). VvPIP2.1 sense probes did not reveal any specific signal in the epidermis and the hypodermal cells (Fig. A high level of signal was also detected in cells located under the epidermis and belonging to the first hypodermal cell layer (Fig.2 (GSVIVP00012703001). S1. However.1 protein (Genbank accession: DQ086835) appears in the grape genome draft with accessions GSVIVP00025504001 and GSVIVP00025505001.1 sense probe on the same tissues (Fig. 2005). 1997) and Tree View (Page 1996) programs. 4d).2 (GSVIVP00026881001). 2001). 2). 2 Phylogenetic analysis of putative grapevine aquaporins. Accessions of sequences retrieved from the Grape Genome sequencing draft are as follow: VvNIP1.1 (GSVIVP 00018548001). According to the histological study of Cadot et al. Sequences identified in the present study are framed. VvTIP4.1 (GS VIVP00033750001). VvPIP2. Pilati et al. 4g).2 (GSVIVP0001917 0001).1 genes were highly expressed in well-defined tissues or cell types within young developing grape berries. whereas maize appears to have a reduced number of NIPs genes (Chaumont et al.1 (GSVIVP00007127001). 2001). Sakurai et al. The VvTIP2. 2007. VvPI P1. VvTIP5. 2001) and rice (Sakurai et al. VvPIP1. The use of VvTIP2. whereas no specific signal was detected using a control VvPIP2. in situ hybridization experiments indicated that the VvPIP2.3 (GSVI VP00022146001). VvPIP1. VvTIP2. (2007) In situ hybridization experiments were also carried out using VvTIP2. The VvSIP1. VvNIP8. VvTIP1. The distribution between these four subgroups appears similar to the ones observed in Arabidopsis (Johanson et al. (2006).1 sense probes confirmed the absence of a specific hybridization signal around the vascular bundles (Fig. VvNIP4.1 (GSVIVP00029946001).2 (GSVIVP00036133001). Velasco et al. In conclusion. (2007) detected the differential expression of 8 PIP and 4 TIP genes during berry development. online supplementary data) and in full respect of the current nomenclature (Johanson et al.1 transcripts appeared abundant in some cells associated with the peripheral vascular bundles located at the outer part of the mesocarp. 2007.1 (GSVIVP00000446001). Amino acid sequence alignment and subsequent neighbor-joining tree were generated using the ClustalX (Thompson et al. VvTIP1. VvNIP7.1 transcripts were not detected in epidermal cells and only a very weak signal was occasionally observed in cells from the second hypodermal layer.1546 Plant Cell Rep (2008) 27:1541–1550 Fig. Discussion Analysis of grape aquaporins and identification of genes expressed in grape berry Like in Arabidopsis and rice. 4f).1 (GSVIVP00032441001).1 RNA probes did not show any clear hybridization signals in the skin and pulp tissues of young berries (data not shown). VvTI P3.1 and VvTIP2. VvTIP2. VvTIP5.3 (GSVIVP000 23192001). A search in the first releases of the grape genome (Jaillon et al. Transcripts accumulations were observed in two distinct cell types of the pericarp. VvSIP2.3 (GSVIVP00000433001). VvPIP1. VvTIP1.1 (GSVIVP00022377001).4 (GSVIVP00024394001). aquaporins appear to form an important multigenic family in grapevine. Using the same technology with berries sampled in three growing years to limit the influence of climatic conditions.1 (GSVIVP00013854001). However.1 (GSVI VP00011149001).1 (GSVIVP 00035815001) VvNIP3. 2005). 4c). VvNIP8. 2001. 2007) allowed us to identify 28 genes encoding putative aquaporins that can be divided into four subgroups on the basis of sequence homology (Fig. VvTIP2.2 (GSVIVP00000605001). where 35 and 33 isoforms have been respectively identified (Johanson et al. 4h). Experiments carried out with VvTIP2. and as stated by Pilati et al. VvNIP5. 4e).1 protein (Genbank accession: CAN75442) has been identified by Velasco et al. 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.

other appeared preferentially expressed during a particular growth period. while some aquaporin genes did not show any significant variation in expression during berry development. a and b indicate that means are significantly different as determined by ANOVA followed by Tukey test (P \ 0. If we consider only this water channel function. encoding. VvTIP1. 3 Analysis of aquaporin gene expression during berry development.2.1 VvPIP1.3 gene. has also been detected in grape berries (Deluc et al. Although some aquaporins may be involved in the transport of small neutral solutes and/or gazes (Kaldenhoff and Fischer 2006). 90 or 111 days after flowering (DAF). Taking account this redundancy. It is tempting to hypothesize that spatiotemporal regulation along fruit development may be linked to particular physiological events and reflects a possible specialization of some aquaporins in these events. According to our results.Plant Cell Rep (2008) 27:1541–1550 1547 Fig. Similar results were obtained by Deluc et al. For each gene. 2007). these particular cells are dividing up to three weeks after anthesis. it appears that our screen led to the identification of a representative set of the various TIP and PIP genes expressed in berries. the central function of all characterized proteins belonging to the aquaporin family is water transport. n = 3). (2007) and Pilati et al. Expression levels were normalized relative to EF1c transcripts (see ‘‘Materials and methods’’).1 aquaporin.1. 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. VvTIP2. one SIP and one NIP aquaporin.1 VvPIP2. Pilati et al. the expression of two other genes. a process that requires. who studied cell division in the developing dermal system of grape berry. b Relative expression levels of aquaporin gene during berry development. except for the VvPIP2. The localization of the VvTIP2.2. (2007) who observed a preferential expression of aquaporin genes during the preveraison phase. VvPIP2. 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 first berry growth stage. Expression of aquaporin genes during berry development The data presented in this paper indicate that. As reported by Considine and Knox (1981). 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. like cell 123 . respectively. (2008). To date. 2007.1 transcripts in cells of the first hypodermal layer also supports this hypothesis. After veraison. VvTIP1.05. most of the genes showing significant variation in expression appeared preferentially expressed before or up to the veraison stage. 70. berry ripening can be characterized by a global decrease of aquaporin gene expression.3. a Representative macroarray hybridization experiment using complex probes prepared from Cabernet Sauvignon berries harvested at 20. However.2.3 PIP2. some probe sets of the microarrays used in these studies target in fact an unique transcript. as recently demonstrated by Schlosser et al. VvPIP2. Dividing cells must generate equal amounts of tonoplast and plasma membrane between rounds of cell division. it is likely that the outcomes from the grape genome sequencing will allow the identification of additional aquaporin genes expressed in developing berries.

expression of VvPIP2. However. the synthesis of new membrane components. these findings support the implication of the PIP2. OI outer integument. 4 In situ localization of VvPIP2. Moreover.1 and VvTIP2. MI medium integument.1 aquaporin in seed development.1. Ep epidermis. found that the tomato VvPIP2. 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. expression of VvTIP2.1 mRNAs in berry tissues. expression of the VvPIP2.1 gene occurs in cells of the outer integument of developing seeds. h) probes. before veraison. (2006).1548 Plant Cell Rep (2008) 27:1541–1550 Fig. Because of the lack of in situ hybridization data from the veraison to the harvest stage (see materials and methods). EL epidermal cell layer. Nu nucellus. 2006). En endosperm. Thus.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. According to the multiple functions proposed for aquaporins in plants (Kaldenhoff and Fischer 2006). 2005). Our data indicate that. called LePIP2.1 in xylem parenchyma cells before veraison indicates that aquaporins may also participate to the xylem unloading process. Transverse sections of Cabernet Sauvignon berries harvested 20 days after flowering were hybridized either with digoxigenin-labelled VvPIP2.1 antisense (e. g) or sense (f. some other implications may be proposed. A grape berry half-section stained with toluidine blue (a) and a magnified view of the epicarp (b) are showed to facilitate the localization of the hybridization signals. Cell division activity in the outer integuments peaks at 20–25 days after anthesis (Ristic and Iland 2005) and cell elongation occurs until veraison (Cadot et al.1 homolog. it is difficult to assign putative functions for aquaporins in ripening berries. II inner integument. 123 . Aquaporins are also required in the phloem unloading mechanisms to allow water exchange between phloem conducting cells and surrounding tissues (Fraysse et al. Boxed areas represent the berry tissues where hybridization signals were observed and are linked by arrows to the corresponding in situ hybridization data. was also expressed in seeds throughout fruit development with a peak of expression 20 DAF. HLs hypodermal cell layers. The transcript signal appears dark-blue and is observed only on sections hybridized with antisense probes. Shiota et al. Together with our data. as discussed below.1 antisense (c) or sense (d) probes or with VvTIP2. XT xylem tracheids elongation.

aquaporins might play a role in regulating the berry hydraulic conductance. Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Additional work is now needed to elucidate the precise role of the different aquaporins throughout berry development. 2006) and may represent the next step towards a better understanding of aquaporin function in developing fleshy fruits. ˜ ana-Castello Cadot Y. As a consequence. Chaumont F. Carde JP (2001) In situ fixation of grape berries. Keller et al. a weak expression of aquaporin genes in xylem parenchyma cells may account for the decrease of berry hydraulic conductance observed during ripening. Cabernet franc during fruit development. 2006). Chevalier M (2006) Anatomical. Larkins BA. Ruuska S. Thus. (2004). 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. J Exp Bot 56:2949–2957 Bostwick DE. Min histological. These authors suggested that in addition to possible changes in xylem anatomy (Findlay et al. Schooley DA. J Exp Bot 38:668–679 Fraysse LC. when water is provided mainly by the xylem tracheids (Greenspan et al. Dannenhoffer JM. and as suggested by Tyerman et al. Modulation of aquaporin expression in planta by genetic engineering is a strategy of choice for investigating the role of these proteins (Hachez et al. Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search Another interesting possibility relies on the implication of tonoplast aquaporins in the regulation of berry water status. Cramer GR (2007) Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development. Merillon JM. 1987). Protoplasma 218:225–235 Findlay N. springerlink. and despites the presence of functional xylem (Bondada et al. water and solute exchanges between the xylem and the berry tissues will be facilitated. 1994). Planta 151:403–412 Coombe BG (1992) Research on development and ripening of the grape berry. Grimplet J. 1998). Centre INRA de Bordeaux) for providing the grape berry half-section pictures. Knox RB (1981) Tissue origins. we observed a high expression of the VvTIP2. Perfusion of pericarp apoplast via the pedicel and evidence for xylem malfunction in ripening berries. Plant Cell 4:1539– 1548 ` MT. Nil N. (2004) reported a tenfold reduction of the whole berry hydraulic conductance between the veraison and harvest stages. Aubourg (Plant Genomics Research Unit. Plant Physiol 125:1206–1215 Chrispeels MJ. Chrispeels MJ (1998) High expression of the tonoplast aquaporin ZmTIP1 in epidermal and conducting tissues of maize. Ohlrogge J (2000) Microarray analysis of developing Arabidopsis seeds. Wheatley MD. Richard T. This shift from xylem to phloem water uptake coincides with a strong decrease of VvTIP2. Benning C. References Altschul SF. Wojcik E. Before veraison. Schaffer AA. At the transcript level. Expression of VvTIP2. After veraison. Barrieu F. Shackel KA. Kjellbom P (2005) Specific plasma membrane aquaporins of the PIP1 subfamily are expressed in sieve elements and guard cells.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 flow (Barrieu et al. Jean-Pierre Carde (UMR 619 Biologie du Fruit. Oliver KJ. Miller W. Todd J. BMC Genomics 8:429 Diakou P.1 expression. Plant Cell Environ 17:811–820 Supplementary data The phylogenetic analysis of aquaporins from Vitis vinifera and Arabidopsis thaliana is available on-line at http://www. 2005. Maurel C (1994) Aquaporins: the molecular basis of facilitated water movement through living plant cells? Plant Physiol 105:9–13 Considine JA. phloem sap becomes the primary water source for the berries (Greenspan et al. Zhang J. Lister RM. Cushman JC. A decrease of membrane hydraulic conductance can be linked to a decrease of aquaporins activity by post-transcriptional modifications and/or to a modification of aquaporins abundance in the membrane (Hachez et al. Jung R (2001) Aquaporins constitute a large and highly divergent protein family in maize. Plant Mol Biol Rep 11:113– 116 Chaumont F. Nucleic Acids Res 25:3389–3402 Barrieu F. White J. Thompson GA (1992) Pumpkin phloem lectin genes are specifically expressed in companion cells. Tyerman et al. Puryear J. Madden TL. Barrieu F. David Lecourieux for comments and advice on the manuscript and Dr. Decendit A. Matthews MA (1994) Developmental changes in the diurnal water budget of the grape berry exposed to water deficits. Plant Physiol 117:1153–1163 Bondada BR. 1994). cell lineages and patterns of cell division in the developing dermal system of the fruit of Vitis vinifera L. Evry) for his help and valuable advices for the identification of aquaporin genes in the grapevine genome. We thank Pr. Hamdi S (2006) Characterization of a grapevine R2R3–MYB transcription factor that regulates the phenylpropanoid pathway. Wells B. cv. Coombe BG (1987) Solute Accumulation by grape pericarp cells: IV. Quilici DR. Plant Physiol 140:499–511 Deluc LG. Marchive C. Plant Physiol 124:1570–1581 Greenspan MD. Shackel KA (2005) Functional xylem in the post-veraison grape berry. 123 . Skaggs MI. 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. Serge Delrot and Dr. Lauvergeat V. J Agric Food Chem 54:9206–9215 Chang S. 2006). Implication of aquaporins in the regulation of berry water status after veraison At the berry level. Schlauch KA. Biol Cell 97:519– 534 Girke T. Zhang Z. Chrispeels MJ. Matthews MA.Plant Cell Rep (2008) 27:1541–1550 1549 Acknowledgments The authors are very grateful to Dr S. Carde JP. Tillett RL. (2004).1 gene in xylem parenchyma cells. McCann MC. Am J Enol Vitic 43:101–110 Deluc L. Osborne C. and histochemical changes in grape seeds from Vitis vinifera L.

Stella A. Vezzi A. Stormo K. Mica E. Costa F. Gutin N. Legeai F. Macalma T. Vandepoele K. Wardell B. Scarpelli C. Taillon B. Karlsson M. Coppola G. Dedaldechamp F. Reid J. Harkins T. Wincker P. Oyzerski R. Uemura M. Ageorges A. Peng F. Lund S. Morgante M. Carbonneau A (2001) Influence of water deficits on grape berry growth. FEBS Lett 579:5814–5820 Jaillon O. Zelazny E. Pilati S. cv Shiraz: developmental changes in seed morphology and phenolic composition. Bondada BR (2006) Ripening grape berries remain hydraulically connected to the shoot. Lackey A. Crit Rev Biochem Mol Biol 28:235–257 Ristic R. J Exp Bot 57:2577– 2587 Luu DT. Lecharny A. Tanaka I (2006) Expression analysis of genes encoding plasma membrane aquaporins during seed and fruit development in tomato. Plant Cell Physiol 46:1568–1577 Schlosser J. Tilbrook J. Laucou V. Steudle E (2004) Direct measurements of hydraulic properties in developing berries of Vitis vinifera L. Glissant D. Mraz A. Iliev D. Delrot S (2005) VvHT1 encodes a monosaccharide transporter expressed in the conducting complex of the grape berry phloem. Durand E. Maeshima M (2005) Novel type aquaporin SIPs are mainly localized to the ER membrane and show cell-specific expression in Arabidopsis thaliana. CABIOS 12:357–358 Park JH. Ugarte E. Facci M. Moser C. Cestaro A. Fraysse L. Buttner M. Chatelet P. Solovyev V. C. Plant Sci 171:277–285 Thompson JD. M. Pe Valle G. Stefanini M. Johansson I. ´ tier F. Gibson TJ. Aury JM. Smith JP. Sterck L. Le Clainche I. Artiguenave F. Sudoh T. Kjellbom P (2001) The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants. Moretto M. Viola R.1550 Hachez C. Segala C. Uemura T. Caboche M. Fitzgerald LM. Davenport ` L. Vitis 40:141–145 Ojeda H. Battilana J. Lanchbury J. Pardo C. Troggio M. Hamdi S. Shiraz and Chardonnay. BMC Genomics 8:428 of an oxidative burst at ve Reizer J. Dematte Dal Ri A. Horner D. Fontana P. Merdinoglu D. Eldredge G. Felice N. Plant Cell Environ 28:85–96 Ojeda H. Mitchell JT. Dumas V. Chen Y. Moroldo M. Van de Peer Y. Saier M Jr (1996) Phylogenetic characterization of the MIP family of transmembrane channel proteins. Reid K. Pruss D. Vico V. Malacrida G. Salamini F. Carbonneau A. Sato MH. Maurel C (2005) Aquaporins in a challenging environment: molecular gears for adjusting plant water status. Jeanmougin F. Choisne N. Adam-Blondon AF.1007/s00709-0080280-9 Shiota H. Weig AR. Fawcett JA. Policriti A. Moser C (2007) Genome-wide transcriptional analysis of grapevine berry ripening reveals a set of genes similarly modulated during three seasons and the occurrence ` raison. Tao Q. Gutin A. Delledonne ` ME. Paillard S. Perbost C. Ghorbel A. Nucleic Acids Res 25:4876–4882 Tyerman SD. Nature 449:463–467 Johanson U. Plant Physiol 126:1358–1369 Kaldenhoff R. Aubourg S. Gatto P. Bogden R. Romieu C (1999) Berry development of grapevines: relations between the growth of berries and their DNA content indicate cell multiplication and enlargement. Cartwright DA. Micheletti D. cv. Bhatnagar SK. Jublot D. Reizer A. Weis M. Suga S. Maeshima M (2005) Identification of 33 rice aquaporin genes and analysis of their expression and function. Pindo M. Blumwald E (2007) A grape berry (Vitis vinifera L. Que Consortium for Grapevine Genome Characterization (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Hugueney P. Cagnac O. Cestaro A. Ishikawa F. Zharkikh A. Gouyvenoux M. Vezzulli S. Dasilva C. Clepet C. Kotula L. Poulain J. Jubin C. Plant Cell Physiol 48:804–811 Ishikawa F. Deloire A. Perazzolli M. Sullivan W. Billault A. Gustavsson S.). Alaux M. Chaumont F (2006) Modulating the expression of aquaporin genes in planta: a key to understand their physiological functions? Biochim Biophys Acta 1758:1142– 1156 Hanana M. Juman I. Malossini A. Fontana P. French-Italian Public Weissenbach J. Fleurat-Lessard P. evolutionary relationships. Biochim Biophys Acta 1758:1134–1141 Keller M. Austr J Grape Wine Res 11:43–58 Sakurai J. Casagrande A.) cation/proton antiporter is associated with berry ripening. Sjovall S. Pezzotti M. Perazzolli M. Skolnick M. Yamaguchi T. Scalabrin S. Plewniak F. Di Gaspero G. Toppo S. Vachaud M. Sgaramella V. Bowen P (2008) Cellular expansion and gene expression in the developing grape (Vitis vinifera L. Noel B. doi:10. ` re C. Deloire A. Saier MH Jr (1993) The MIP family of integral membrane channel proteins: sequence comparisons. Viola R (2007) A high quality draft consensus sequence of the genome of a heterozygous grapevine variety. Dematte Si-Ammour A. Yamaguchi T. Pesole G. reconstructed pathway of evolution. Fischer M (2006) Functional aquaporin diversity in plants. Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Aust J Grape Wine Res 10:170–181 Velasco R. Del Fabbro C. and proposed functional differentiation of the two repeated halves of the proteins. Segurens B. Lemoine R. Vitulo N. Olsson N. Bruye Cattonaro F. Vitis 38:145–150 Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Atanassova R. Protoplasma. Cakir B. Canaguier A. Malacarne G. PLoS ONE 2:e1326 Vignault C. Velasco R. Anthouard V. J Exp Bot 56:1409–1418 123 . Grando SM. J Membr Biol 153:171–180 Plant Cell Rep (2008) 27:1541–1550 ` L. Iland PG (2005) Relationships between seed and berry development of Vitis Vinifera L.