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

Abstract

Abstract. 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 0 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.

The Grapevine Root-Specific Aquaporin VvPIP2;4N Controls Root Hydraulic Conductance and Leaf Gas Exchange under Well-Watered Conditions But Not under Water Stress

“…induced a 54-fold increase in membrane P f , a value comparable to those detected when using this experimental system for plant aquaporins (Marjanović et al, 2005;Secchi et al, 2007) and to those reported for the four grapevine PIP2 aquaporins analyzed by Shelden et al (2009). Taken together, these results suggest that VvPIP2;4N is a water-transporting aquaporin that may potentially offer a remarkable contribution to water transcellular transport in grape tissues.…”

“…3A). These results are in contrast with the very low expression levels of VvPIP2;4 in roots reported by Vandeleur et al (2009); however, no correspondence was observed between the primers used by those authors and those used in this study, confirming that VvPIP2;4N is different from VvPIP2;4 described by Shelden et al (2009) and Vandeleur et al (2009). The expression pattern of VvPIP2;4N suggests that this aquaporin could be a specific root isoform, probably involved in the regulation of root hydraulic conductance.…”

“…The isolated gene (DQ358107) was nearly identical in its coding sequence (single nucleotide mismatch, not leading to an amino acid change) to the gene predicted in the grapevine 'PN40024' 123 genome draft (http:// genomes.cribi.unipd.it/) at locus VIT06s0004g02850 on chromosome 6 and was called VvPIP2;4 by Shelden et al (2009). The same authors isolated an aquaporin sequence that they called VvPIP2;4 (EF364438) from cv Cabernet Sauvignon.…”

“…The sequence of this gene (which we refer to as VvPIP2;4N) only shares 83% amino acid identity with VvPIP2;4 isolated by Shelden et al (2009) in cv Cabernet Sauvignon, thus appearing to be an uncharacterized grape PIP2 gene. Functional assays in oocytes showed that VvPIP2;4N expression B and E), and leaf ABA content (n = 4; C and F) in wild-type (wt; triangles) and transgenic (squares) plants belonging to six lines (4,16,23,24,28, and 33) upon well-watered (black symbols) and water stress (white symbols) conditions (means 6 SE).…”

“…Eighty-three sequences annotated as aquaporins, belonging to different Vitis species, are present in GenBank, and 28 putative aquaporin genes have been identified in the grapevine genome (Fouquet et al, 2008;Shelden et al, 2009). Expression of some of these genes, belonging to the PIP and Tonoplast Intrinsic Protein subfamilies, has been studied in grape berry (Picaud et al, 2003;Fouquet et al, 2008;Choat et al, 2009) and vegetative tissues (Baiges et al, 2001;Vandeleur et al, 2009).…”