Journal of Experimental Botany, Vol. 52, No. 362, pp. 1949-1951,
September 1, 2001
© 2001 Oxford University Press
Gene Note |
Eight cDNA encoding putative aquaporins in Vitis hybrid Richter-110 and their differential expression
1 Unitat d'Enologia-CeRTA, Departament de Bioquímica i Biotecnologia, Facultat d'Enologia de Tarragona, Universitat Rovira i Virgili, Ramon y Cajal, 70, 43005 Tarragona, Spain
2 Institute of Biochemical Plant Pathology, GSF Research Center for Environment and Health, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany
Received 10 May 2001; Accepted 15 June 2001
Abstract
The nucleotide sequences of eight cDNAs encoding putative aquaporins obtained from a leaf Vitis hybrid Richter-110 cDNA library are reported. They encode proteins ranging from 249 to 287 amino acids with characteristic sequences that clearly include them within the MIP family. According to available database sequence homologies, they can be classified into four groups belonging to two subfamilies: PIP (PIP1 and PIP2) and TIP (
-TIP and 
-TIP). In order to elucidate the expression patterns of these putative aquaporins in the plant, specific probes were developed and tissue specific differential expression was tested by reverse Northern and compared with two reference genes (malic enzyme and glutamate dehydrogenase). Clearly, most of the putative aquaporins had higher expression in roots, whereas expression in shoot and leaves was generally weaker than the reference genes.
Key words: Vitis, aquaporin, water channel, sequence, differential expression.
Introduction
Water channels or aquaporins have been identified as part of the major intrinsic proteins (MIP), an integral membrane protein family, in numerous organisms since the presence and function of such selective water channels in cellular membranes was discovered (Preston et al., 1992
). Plant MIP members contain highly conserved amino acid motifs (Schäffner, 1998) that allow them to be classified into one of the following four groups: PIP1, PIP2, TIP, and NOD26-like intrinsic proteins. Recently, an additional small subfamily of SIP (small intrinsic proteins) has been constituted based on the Arabidopsis genome sequence (Johanson et al., 2001). These groups are conserved through plant evolution, which suggests a specific role, location or regulation of the members belonging to each group.
Compared with ion channels and other transporters, aquaporins are very abundant in the plasma membrane and tonoplast. Water channels may play a role in the plant response to drought stress since osmotic adjustment is a mechanism of water stress adaptation involving both plasma membrane and tonoplast. Aquaporins accelerate the transport of water through these membranes by considerably increasing the membrane permeability. A high number of aquaporins are expressed in the same plant at the same time. This diversity allows organ-, tissue- or even compartment-specific expression as well as response to different stimuli.
Grapevines are known to be extremely stress-tolerant plants, adapting well to dry environments. Indeed, for adequate ripening of some varieties, periods of drought are a requirement. Vitis Richter R-110 rootstock is broadly used in mediterranean countries because of its high vigour and resistance to drought stress (Galet, 1988). This rootstock is a hybrid of two American Vitis species: Vitis berlandieri and Vitis rupestris.
The cDNA and the amino acid sequences of eight members of the MIP family, identified in Vitis rootstock Richter-110 by screening a cDNA library with specific homologous probes, is reported here. In order to ascertain the expression of these aquaporins, highly specific probes have been developed and differential expression has been analysed in hydroponically cultured Vitis Richter R-110 plants.
Materials and methods
Plant material and propagation
Plant material used to analyse tissue differential expression was obtained from hydroponically cultured Vitis R-110. Vines were first obtained from field-cultured vine rootstock and cultured in solid, modified Murashige and Skoog (Murashige and Skoog, 1962) media for 2 months and then transferred to continuously aerated hydroponic medium (Hewitt, 1956). Plants were grown under 16 h light at a photosynthetically active radiation of c. 300 µmol m-2 s-1. Temperature was controlled between 22 and 25 °C and relative humidity ranged from 45 to 60%.
Upon harvest the main root was separated from the secondary roots and was further dissected by cutting the first 3–5 cm (root tip). The cortex was separated from the stele in the rest. In addition, whole roots were analysed. Whole leaves, leaf ribs, leaves without ribs, and petioles were separately analysed. In the shoot, total RNA from buds, expanding leaves (developing from buds) and stems was isolated.
Vitis RNA isolation
RNA to construct a cDNA library was obtained from field-cultured vine rootstock Vitis R-110 leaves. The method for Vitis DNA extraction (Steenkamp et al., 1994) was adapted for RNA isolation. After co-extraction of DNA and RNA, DNA was eliminated by precipitation with LiCl or DNase treatment.
Method of identification
The initial identification of PIP and TIP gene expression in Vitis Richter-110 was performed by reverse transcription-coupled PCR using two batches of generic primers able to amplify PIP or TIP-like cDNA, respectively. Four different partial sequences were identified and named Vitis-PIP1, Vitis-PIP2, Vitis-TIP1, and Vitis-TIP2. The annotation of partially sequenced products to Vitis-PIP1 and Vitis-PIP2 groups was done due to the presence in their translation products of conserved amino acids through PIP1 and PIP2 subfamilies in plants (Schäffner, 1998).
In order to obtain the complete coding sequence, a cDNA library from Vitis Richter-110 leaves was constructed with the ZAP-cDNA Synthesis and Gigapack III Gold Cloning Kit (Stratagene) according to the manufacturer's instructions. The four clones obtained previously were amplified by PCR, radiolabelled and used as probes for screening the cDNA library. Three full-length clones corresponding to Vitis-PIP1 (re-named Vitis-PIP1-1), Vitis-PIP2 (re-named Vitis-PIP2-1) and Vitis-TIP1 and four new full-length clones called Vitis-PIP1-2, Vitis-PIP1-3, Vitis-PIP2-2, and Vitis-TIP3 were detected. None of the full-length clones that hybridized with TIP2 probe shared exactly the same sequence. Therefore, only the partial sequence of TIP2 is known.
Differential expression
Differential expression was analysed by reverse Northern. Specific DNA fragments were obtained by PCR from the 3'-end non-coding region of the seven fully sequenced Vitis MIPs. Specific fragments were also obtained in order to detect the glutamate dehydrogenase (Acc. No. X86924) and the malic enzyme (Acc. No. L34836) gene expression. These two genes were used as internal controls since they are described in the literature as being ubiquitously and constitutively expressed (Syntichaky et al., 1996; Franke and Adams, 1995). 100 ng of each PCR product were blotted onto a nylon membrane (Ausubel et al., 1995). Radiolabelled cDNA probes were obtained by reverse transcription of 10 µg of total RNA of the different root, leaf and shoot tissues. For normalization purposes, 0.5 ng (about 8x108 copies) of At-PIP2b mRNA were added to each total RNA sample and served as the external standard. Hybridization was performed following standard methods. The signal was detected and quantified using a phosphorimaging system (Fuji screens and a Molecular Dynamics Storm 860 reader).
Results
The characteristics of these eight sequences and their deduced amino acid sequences are detailed in Table 1. Interestingly, Vitis-PIP1-2 and Vitis-PIP1-3 share a very high homology (97.9%) with other Vitis vinifera putative aquaporins as a clear sign of evolution proximity (divergence between American species (Vitis berlandieri, Vitis rupestris) and Vitis vinifera occurred after last glacial period, about 120 thousand years ago). Also a high degree of identity (98.4%) appears between Vitis-TIP1 and a putative TIP aquaporin in Vitis vinifera. These results suggest that the eight MIP sequences reported here might be conserved in the whole Vitis class. All other Vitis-MIPs share a lower maximal homology with other plant MIPs (around 90% for PIPs and 80% for TIPs).
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Differential expression analyses
The results of the expression analysis of five PIP and two TIP genes identified in Vitis Richter-110 are shown in Fig. 1
. The expression of the two reference genes was similar in all the tissues and organs studied, ranging from 10 to 20% of the amount of the external mRNA added. Only two of the putative aquaporins, Vitis-PIP1-1 and Vitis-TIP1, had significantly higher expression than the reference genes when analysed in root tissues. Both Vitis-PIP2 assayed had similar levels of expression to the reference genes, Vitis-PIP1-1, PIP1-2 and TIP 3 had very low levels of expression. The expression of all the aquaporins is generally higher in roots than in shoots, leaves being the organs where the expression is generally lower. This is remarkable since all cDNA were isolated from a leaf cDNA library. Thus, water permeability enhanced through aquaporins and its regulation may be more important in roots.
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In all the tissues analysed, the youngest ones (root tip, young leaves, terminal buds) had the highest added expression in each organ, which could relate aquaporin expression (and pressumably activity) to growth and development.
The expression of Vitis-PIP1-1 and Vitis-TIP1 in root tip and lateral roots provide the highest expression of all aquaporins studied. This could probably be related to the high water flow activity needed in these two parts of the plant. The lateral roots and root tips contain very young cells: meristematic, elongating and absorbing cells. The non-suberized epidermis is exposed to the rapid osmotic changes caused by the nutrient uptake during the absorption process. These water potential changes probably need a cytoplasmic buffering process consisting of a rapid flow of the water out/into the vacuole through a tonoplast aquaporin as proposed earlier (Barrieu et al., 1998). The root tip is also the place where root growth occurs. Expanding and meristematic cells require water uptake to get the necessary turgor that precedes and then parallels plastic wall extension or cell division. Important flows of water are involved in these kinds of cells and processes. Vitis Richter-110 is a very vigorous hybrid and their roots are able to penetrate into deep soils and this fact is directly related to its drought resistance. It is proposed here that the high gene expression of Vitis-PIP1-1 and Vitis-TIP1 in young tissues of the root could facilitate fast water transport, giving to this plant the capacity to extend its roots more effectively than other vines.
Note in press
Recently (2 June 2001) a reassignment of plant aquaporin gene families has been proposed (http://mbclserver.rutgers.edu/CPGN/AquaporinWeb/Aquaporin.Table.html). According to this proposal, the TIP genes reported here should be renamed as: TIP 1: TIP 2-1, TIP 2: TIP 1-1, TIP 3: TIP 1-2.
Acknowledgments
This work was supported by grants of CiCYT ALI96-0497, Spain and Deutsche Forschungsgemeinschaft DFG Scha 454/5-2, Germany. IB was funded by grants from MEC and Direcció General de la Recerca de la Generalitat de Catalunya, Spain.
Notes
3 To whom correspondence should be addressed. Fax: +34 977 250347. E-mail: amb{at}astor.urv.es 
Abbreviations
At, Arabidopsis thaliana; PIP, Plasma membrane Intrinsic Protein; TIP, Tonoplast Intrinsic Protein; NLM, NOD26-like MIP; MIP, Major Intrinsic Protein..
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