Identification and functional characterisation of aquaporins in the grapevine, Vitis vinifera

Plant aquaporins belong to a large superfamily of conserved proteins called the major intrinsic proteins (MIPs). There is limited information about the diversity of MIPs in grapevine, and their water transport capacity. The aim of the present study was to identify MIPs from grapevine and functionally characterise water transport of a subset of MIPs. Candidate genes were identified, by screening a Vitis vinifera L. (cv. Cabernet Sauvignon) cDNA library with gene specific probes, for aquaporin cDNAs encoding members of the plasma membrane intrinsic protein (PIP) and tonoplast intrinsic protein (TIP) subfamilies. The screen resulted in the identification of 11 full-length and two partial length aquaporin cDNAs. VvTIP2;1 isoforms had different 3' UTRs, immediately upstream of the poly(A) tail, suggesting the presence of multiple cleavage sites for polyadenylation. Using published genome sequences of grapevine, we conducted a phylogenetic analysis of the MIPs with previously characterised MIPs from Arabidopsis. We identified 23 full-length MIP genes from the V. vinifera genome sequence of a near homozygous line (PN40024) that cluster into the four main subfamilies (and subgroups within) identified in other species. However, based on the identification of PIP2 genes in Cabernet Sauvignon that were not present in the PN40024 genome, there are likely to be more than 23 MIP genes in other heterozygous grapevine cultivars. Water transport capacity was determined for several PIPs and TIPs, by expression in Xenopus oocytes. Only VvPIP2 and VvTIP proteins function as water channels with the exception of VvPIP2;5. VvPIP2;5 differs from the water conducting VvPIP2;1 by the substitution of two highly conserved amino acids in Loop B (G97S, G100W), which was shown by homology modelling to likely form a hydrophobic block of the water pore.