Expression of Water Channel Proteins in <em>Mesembryanthemum crystallinum</em>^{<a href="#FN1" rid="FN1" class=" fn">1</a>}

Abstract

The physiology of plant water relations is receiving renewed attention following the detection of a superfamily of “major intrinsic proteins” (MIPs) (Reizer et al., 1993), which function as channels facilitating the movement of water and/or uncharged, low-M_r solutes (Maurel, 1997; Agre et al., 1998; Biela et al., 1999; Gerbeau et al., 1999). MIPs are called water channels or aquaporins once their ability to facilitate water flux has been demonstrated. Although their existence is irrefutable, conceptual reservations exist as to whether they are important conduits for water flux or minor players in plant water relations (Steudle, 1997; Tyerman et al., 1999). Information about the size of the Mip gene family, transcript expression, and the regulation of expression is available, but less is known about proteins and their dynamic behavior under normal conditions or conditions that force changes in water status.

Plant genomes include a large number of Mip genes (Chrispeels et al., 1999). In Arabidopsis, for example, at least 23 different transcripts are found, and an analysis of corn expressed sequence tags indicates that more than 30 Mip genes should be present (Weig et al., 1997; Barrieu et al., 1998; Tyerman et al., 1999). Plant Mip genes can be grouped into three subfamilies based on phylogenetic analysis (Yamada et al., 1995; Weig et al., 1997). The two major groups divide MIPs according to location in either the plasma membrane (PM) or tonoplast and, possibly, according to functions (Daniels et al., 1994; Kammerloher et al., 1994; Yamada et al., 1995; Maurel, 1997; Tyerman et al., 1999).

Mip transcripts have been detected in every tissue analyzed. They may be abundant or rare, and some are under developmental control. Mip genes are seed, root, and leaf specific, and are associated with leaf expansion, root tip elongation, or seedling development (Guerrero et al., 1990; Yamamoto et al., 1991; Höfte et al., 1992; Jones and Mullet, 1995; Fukuhara et al., 1999). Cell specificity has been reported for some Mip genes based on in situ hybridizations or by monitoring Mip-promoter-controlled uidA (β-glucuronidase [GUS]) activity (Yamamoto et al., 1991; Jones and Mullet, 1995; Kaldenhoff et al., 1995; Yamada et al., 1995, 1997; Barrieu et al., 1998; Chaumont et al., 1998). Studies on Mip expression have indicated regulation by environmental factors such as drought, salinity, or temperature (Yamaguchi-Shinozaki et al., 1992; Jones and Mullet, 1995; Yamada et al., 1995; Maurel, 1997; Johansson et al., 1998).

The only large-scale analysis of Mip transcripts has been conducted with Arabidopsis (Weig et al., 1997). Based on PCR amplification, this study provided evidence for dramatic differences in transcript abundance between Mip expressed in various organs. Studies on water channel activity are based on the expression of Mip RNA in Xenopus oocytes. Measurements of volume changes and water permeability in oocytes expressing plant Mip show water channel activity upon changes in the external osmoticum (Maurel et al., 1993; Daniels et al., 1994; Kammerloher et al., 1994; Yamada et al., 1995). However, even for Arabidopsis, activity has been tested only for a minority of the presumptive aquaporins. A few studies indicate that MIPs are also active in water flux in planta (Kaldenhoff et al., 1995, 1998). In transgenic plants, antisense expression of a MIP-coding region reduced the number of water channels. When protoplasts were isolated from these plants and transferred to medium with lower osmolarity, they resisted water influx longer and burst later than protoplasts from non-transformed plants.

Missing in the analysis of plant MIPs are comparative studies of proteins and their distribution in individual cells and tissues. Following initial studies on Mip transcript expression in the ice plant Mesembryanthemum crystallinum (Yamada et al., 1995), we focused on the proteins to explore cell specificity, because location might provide further clues about function. With antibodies directed against peptides selected to distinguish different MIPs, we were able to identify several proteins. Most antibodies identify proteins in more than one organ, but show remarkable diversity in the amount present in different cells of a tissue. MIP-A, MIP-B, and MIP-C can be identified by signature cells in tissues in which they are highly expressed, while the tonoplast-located MIP-F seems to be ubiquitously present albeit with different amounts in different cell types. In addition, we present evidence for differential regulation of MIP under salt stress and the localization of PM MIP in internal membranes, suggesting endosomal trafficking of these proteins.

ACKNOWLEDGMENTS

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