The grape aquaporin VvSIP1 transports water across the ER membrane

Abstract

Water diffusion through biological membranes is facilitated by aquaporins, members of the widespread major intrinsic proteins (MIPs). In the present study, the localization, expression, and functional characterization of a small basic intrinsic protein (SIP) from the grapevine were assessed. VvSIP1 was expressed in leaves and berries from field-grown vines, and in leaves and stems from in vitro plantlets, but not in roots. When expressed in tobacco mesophyll cells and in Saccharomyces cerevisiae, fluorescent-tagged VvSIP1 was localized at the endoplasmic reticulum (ER). Stopped-flow spectroscopy showed that VvSIP1-enriched ER membrane vesicles from yeast exhibited higher water permeability and lower activation energy for water transport than control vesicles, indicating the involvement of protein-mediated water diffusion. This aquaporin was able to transport water but not glycerol, urea, sorbitol, glucose, or inositol. VvSIP1 expression in Xenopus oocytes failed to increase the water permeability of the plasma membrane. VvSIP1-His-tag was solubilized and purified to homogeneity from yeast ER membranes and the reconstitution of the purified protein in phosphatidylethanolamine liposomes confirmed its water channel activity. To provide further insights into gene function, the expression of VvSIP1 in mature grapes was studied when vines were cultivated in different field conditions, but its transcript levels did not increase significantly in water-stressed plants and western-exposed berries. However, the expression of the aquaporin genes VvSIP1, VvPIP2;2, and VvTIP1;1 was up-regulated by heat in cultured cells.

Functional characterization of FaNIP1;1 gene, a ripening-related and receptacle-specific aquaporin in strawberry fruit

“…A similar function has been also proposed for FaPIP1;1 and FaPIP2;1 in ripening stages of the strawberry cultivar Selva [20,21] and for VvSIP1 from Vitis [91]. In this sense, our results suggest that FaNIP1;1 protein together with FaPIP1;1 and FaPIP2;1 could play a decisive role in the fine tuning of the fruit receptacle water content and hence influence the receptacle the high moisturizing of the fruit receptacle during the last steps of ripening.…”

Exploring Three PIPs and Three TIPs of Grapevine for Transport of Water and Atypical Substrates through Heterologous Expression in aqy-null Yeast

“…Pinot Noir) aquaporins can be distributed in the four main subfamilies: PIPs (8 genes), TIPs (10 genes), NIPs (8 genes) and SIPs (2 genes) [12]. Despite being a very important economical plant, only few reports are available on Vitis aquaporins, explaining their quantitative expression in various rootstocks [13], during water stress [14], [15] and their cloning and expression in planta [8], [16] or in heterologous systems, such as Xenopus oocytes [1], [12] and S. cerevisiae [17], [18]. Although from a molecular point of view the presence of aquaporins can be easily recognized in a genome, their physiological role in planta is still difficult to understand.…”

Identification and functional characterization of grapevine transporters that mediate glucose-6-phosphate uptake into plastids

“…Total RNA was isolated with RNeasy Plant Mini Kit (Qiagen) following manufacturer's instructions, except that the extraction buffer was changed to 2 % cetrimonium bromide (CTAB), 2 % soluble polyvinylpyrrolidone K-30, 300 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2.0 M NaCl and 2 % (v/v) b-mercaptoethanol (Noronha et al 2014). After DNAse treatment, RNA integrity was checked in a 1 % agarose gel, and first strand cDNA synthesis was performed with the Omniscript RT kit (Qiagen), following the manufacturer's instructions.…”

“…The 35S-VvGPT1-GFP, 35S-VvGPT2b-GFP and 35S-VvGPT2X-GFP constructs were prepared using GATE-WAY as described previously using the primers given in Table S1 (Noronha et al 2014). Constructs were introduced in Agrobacterium tumefaciens (GV3101) and transient transformation Nicotiana benthamiana leaf epidermal cells was performed according to Sparkes et al (2006).…”