Molecular and Functional Characterization of Grapevine NIPs through Heterologous Expression in aqy-Null Saccharomyces cerevisiae.

Farzana Sabir,Graça Soveral,Catarina Prista,Maria C Loureiro-Dias,Sara Gomes

doi:10.3390/ijms21020663

Abstract

Plant Nodulin 26-like Intrinsic Proteins (NIPs) are multifunctional membrane channels of the Major Intrinsic Protein (MIP) family. Unlike other homologs, they have low intrinsic water permeability. NIPs possess diverse substrate selectivity, ranging from water to glycerol and to other small solutes, depending on the group-specific amino acid composition at aromatic/Arg (ar/R) constriction. We cloned three NIPs (NIP1;1, NIP5;1, and NIP6;1) from grapevine (cv. Touriga Nacional). Their expression in the membrane of aqy-null Saccharomyces cerevisiae enabled their functional characterization for water and glycerol transport through stopped-flow spectroscopy. VvTnNIP1;1 demonstrated high water as well as glycerol permeability, whereas VvTnNIP6;1 was impermeable to water but presented high glycerol permeability. Their transport activities were declined by cytosolic acidification, implying that internal-pH can regulate NIPs gating. Furthermore, an extension of C-terminal in VvTnNIP6;1M homolog, led to improved channel activity, suggesting that NIPs gating is putatively regulated by C-terminal. Yeast growth assays in the presence of diverse substrates suggest that the transmembrane flux of metalloids (As, B, and Se) and the heavy metal (Cd) are facilitated through grapevine NIPs. This is the first molecular and functional characterization of grapevine NIPs, providing crucial insights into understanding their role for uptake and translocation of small solutes, and extrusion of toxic compounds in grapevine.

Highlights

The sessile nature of plants restricts their access to water and essential minerals in their vicinity
The combined sequence and phylogenetic analysis, transport assays, and phenotypic growth data presented above demonstrate the ability of Nodulin 26-like Intrinsic Proteins (NIPs) to transport water and glycerol, and support evidence that they are involved in transmembrane flux of As, B, Se, and Cd
The finding suggests that substrate selection of the specific NIP does not merely depend on their group

Summary

Introduction

The sessile nature of plants restricts their access to water and essential minerals in their vicinity. To overcome this limitation, membrane channel diversity has been suggested as a sophisticated evolvement in plants [1,2]. Plant genomes have several-fold more Major Intrinsic Protein (MIP) genes than other organisms, implying their multiple functions for plant growth and adaptation, especially under stress [1]. Plant aquaporins are divided into four major groups: (1) Plasma membrane Intrinsic Proteins (PIPs), (2) Tonoplast Intrinsic Proteins (TIPs), (3) Small basic Intrinsic Proteins (SIPs), and (4) Nodulin 26-like Intrinsic Proteins (NIPs) [1]

Methods

Results

Conclusion