Shelden Megan C, Howitt Susan M, Kaiser Brent N, Tyerman Stephen D
School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia.
Biochemistry and Molecular Biology, Research School of Biology, Australian National University, Canberra, ACT 0200, Australia.
Funct Plant Biol. 2010 Jan;36(12):1065-1078. doi: 10.1071/FP09117.
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.
植物水通道蛋白属于一个被称为主要内在蛋白（MIPs）的保守蛋白大家族。关于葡萄中MIPs的多样性及其水分运输能力的信息有限。本研究的目的是从葡萄中鉴定MIPs，并对一部分MIPs的水分运输功能进行表征。通过用基因特异性探针筛选赤霞珠葡萄（Vitis vinifera L. cv. Cabernet Sauvignon）的cDNA文库，鉴定出编码质膜内在蛋白（PIP）和液泡膜内在蛋白（TIP）亚家族成员的水通道蛋白cDNA的候选基因。筛选结果鉴定出11个全长和2个部分长度的水通道蛋白cDNA。VvTIP2;1异构体在多聚腺苷酸尾巴上游紧邻处有不同的3'非翻译区（UTR），这表明存在多个聚腺苷酸化切割位点。利用已公布的葡萄基因组序列，我们对MIPs与拟南芥中先前表征的MIPs进行了系统发育分析。我们从近纯合系（PN40024）的葡萄（V. vinifera）基因组序列中鉴定出23个全长MIP基因，它们聚类到在其他物种中鉴定出的四个主要亚家族（以及其中的亚组）中。然而，基于在赤霞珠中鉴定出的PN40024基因组中不存在的PIP2基因，其他杂合葡萄品种中可能存在超过23个MIP基因。通过在非洲爪蟾卵母细胞中表达，测定了几种PIPs和TIPs的水分运输能力。除了VvPIP2;5外，只有VvPIP2和VvTIP蛋白作为水通道发挥功能。VvPIP2;5与具有水分传导功能的VvPIP2;1的不同之处在于，其环B中有两个高度保守的氨基酸发生了替换（G97S，G100W），同源建模显示这可能形成水孔的疏水屏障。
