Illinois 61801-3838 (H.J.B.) Aquaporin protein regulation and redistribution in response to osmotic stress was investigated. Ice plant (Mesembryanthemum crystallinum) McTIP1;2 (McMIPF) mediated water flux when expressed in Xenopus leavis oocytes. Mannitol-induced water imbalance resulted in increased protein amounts in tonoplast fractions and a shift in protein distribution to other membrane fractions, suggesting aquaporin relocalization. Indirect immunofluorescence labeling also supports a change in membrane distribution for McTIP1;2 and the appearance of a unique compartment where McTIP1;2 is expressed. Mannitol-induced redistribution of McTIP1;2 was arrested by pretreatment with brefeldin A, wortmannin, and cytochalasin D, inhibitors of vesicle trafficking-related processes. Evidence suggests a role for glycosylation and involvement of a cAMP-dependent signaling pathway in McTIP1;2 redistribution. McTIP1;2 redistribution to endosomal compartments may be part of a homeostatic process to restore and maintain cellular osmolarity under osmotic-stress conditions.

In plants, Na ϩ /H ϩ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na ϩ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na ϩ /H ϩ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na ϩ /H ϩ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H ϩ-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na ϩ /H ϩ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent K m) of the transporter for Na ϩ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na ϩ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na ϩ /H ϩ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na ϩ /H ϩ exchanger and its relative contribution to the maintenance of cellular Na ϩ homeostasis during plant growth in salt.

We have characterized transcripts for nine major intrinsic proteins (MIPs), some of which function as water channels (aquaporins), from the ice plant Mesembryanthemum crystallinum. To determine the cellular distribution and expression of these MIPs, oligopeptide-based antibodies were generated against MIP-A, MIP-B, MIP-C, or MIP-F, which, according to sequence and functional characteristics, are located in the plasma membrane (PM) and tonoplast, respectively. MIPs were most abundant in cells involved in bulk water flow and solute flux. The tonoplast MIP-F was found in all cells, while signature cell types identified different PM-MIPs: MIP-A predominantly in phloem-associated cells, MIP-B in xylem parenchyma, and MIP-C in the epidermis and endodermis of immature roots. Membrane protein analysis confirmed MIP-F as tonoplast located. MIP-A and MIP-B were found in tonoplast fractions and also in fractions distinct from either the tonoplast or PM. MIP-C was most abundant but not exclusive to PM fractions, where it is expected based on its sequence signature. We suggest that within the cell, MIPs are mobile, which is similar to aquaporins cycling through animal endosomes. MIP cycling and the differential regulation of these proteins observed under conditions of salt stress may be fundamental for the control of tissue water flux.

Salinity is considered one of the major limiting factors for plant growth and agricultural productivity. We are using salt cress (Thellungiella halophila) to identify biochemical mechanisms that enable plants to grow in saline conditions. Under salt stress, the major site of Na 1 accumulation occurred in old leaves, followed by young leaves and taproots, with the least accumulation occurring in lateral roots. Salt treatment increased both the H 1 transport and hydrolytic activity of salt cress tonoplast (TP) and plasma membrane (PM) H 1 -ATPases from leaves and roots. TP Na 1 /H 1 exchange was greatly stimulated by growth of the plants in NaCl, both in leaves and roots. Expression of the PM H 1 -ATPase isoform AHA3, the Na 1 transporter HKT1, and the Na 1 /H 1 exchanger SOS1 were examined in PMs isolated from control and salt-treated salt cress roots and leaves. An increased expression of SOS1, but no changes in levels of AHA3 and HKT1, was observed. NHX1 was only detected in PM fractions of roots, and a saltinduced increase in protein expression was observed. Analysis of the levels of expression of vacuolar H 1 -translocating ATPase subunits showed no major changes in protein expression of subunits VHA-A or VHA-B with salt treatment; however, VHA-E showed an increased expression in leaf tissue, but not in roots, when the plants were treated with NaCl. Salt cress plants were able to distribute and store Na 1 by a very strict control of ion movement across both the TP and PM.

SummaryMesembryanthemum crystallinum (ice plant) exhibits extreme tolerance to salt. Epidermal bladder cells (EBCs), developing on the surface of aerial tissues and specialized in sodium sequestration and other protective functions, are critical for the plant's stress adaptation. We present the first transcriptome analysis of EBCs isolated from intact plants, to investigate cell type-specific responses during plant salt adaptation.We developed a de novo assembled, nonredundant EBC reference transcriptome. Using RNAseq, we compared the expression patterns of the EBC-specific transcriptome between control and salt-treated plants.The EBC reference transcriptome consists of 37 341 transcript-contigs, of which 7% showed significantly different expression between salt-treated and control samples. We identified significant changes in ion transport, metabolism related to energy generation and osmolyte accumulation, stress signalling, and organelle functions, as well as a number of lineagespecific genes of unknown function, in response to salt treatment.The salinity-induced EBC transcriptome includes active transcript clusters, refuting the view of EBCs as passive storage compartments in the whole-plant stress response. EBC transcriptomes, differing from those of whole plants or leaf tissue, exemplify the importance of cell typespecific resolution in understanding stress adaptive mechanisms.

Extensive research in the last few years has disclosed a sequence of biochemical events that appear to participate in activation of plant disease defense reactions. Early in the interaction between the plant host and the pathogen, signals are produced that induce responses in the respective partners. This process involves the interaction between pathogenassociated molecules (elicitors) and putative plant receptors. This recognition is followed by a signal transduction cascade resulting in defense gene activation and the expression of disease resistance in the plant host. Specific recognition between the plant host and the fungal pathogen determines the outcome of the interaction (Keen, 1990). The resistance or susceptibility of host plants to different races of a fungal pathogen is determined by the match of dominant resistance genes in the plant with dominant avirulence genes (avr genes) in the pathogen (De Wit, 1992). The avr genes, in at least a few well-studied systems, have been shown to code directly or indirectly for elicitor molecules; resistance genes are suggested to code directly or indirectly for receptor molecules (De Wit, 1992).

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na + sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H + -ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H + -pump activity.

Intercellular fluid (IF) obtained from tomato (Lycopersicon esculentum L.) leaflets colonized by Cladosporium fulvum Cooke contains specific elicitors that induce necrosis in tomato cultivars resistant to the race of C. fulvum used to produce the IF. The responses of cell-suspension cultures produced from tomato lines near-isogenic for resistance genes Cf 4 and Cf 5 to IF produced from leaves infected by races 4 (virulent on Cf 4 but not Cf 5 plants), 2.4.5, and 2.4.5.9 (both virulent on Cf 4 and Cf 5 plants) were used to investigate the possibility that active oxygen (AO) species were involved in the initial host reaction to these elicitors. Concurrently, the same assays were used to determine if the cell lines retained the elicitor specificity of the original plants. An response. IF2 obtained from plants infected with a virulent race of the fungus and injected into tomato cultivars resistant to that race cause necrosis and/or chlorosis. This host response, which appears to duplicate the localized cell death (hypersensitive response) occurring in incompatible race/ cultivar combinations, is not observed on injection of susceptible cultivars (9). To date, all studies support the hypothesis that each avr gene in C fulvum codes for a different elicitor molecule and that virulence on a specific cultivar results when the fungus lacks a functional avr gene (8,26). One of these elicitors, the putative product of the avr9 gene (i.e. it elicits necrosis on cultivars with the Cf 9 gene), has been characterized as a small peptide (21,26), and cDNA of the avr9 gene was recently cloned (26).Little is known about how C fulvum elicitors, or elicitors from other pathogens, interact with the plant cell to initiate the cascade of events involved in the defense response. Recently, the production of AO species, particularly hydrogen peroxide (H202), superoxide (02-), and hydroxyl radicals (OH), in the early events following elicitor treatment, has received considerable experimental support. Systems in which a role for AO has been well documented include Phytophthora infestans/potato (11), Pseudomonas spp./tobacco (17), Colletotrichum lindemuthianum/bean (2), and Verticillium dahliae/soybean (3). In tomato leaves, treatment with C. fulvum-specific elicitors caused electrolyte leakage, lipid peroxidation, and increased lipoxygenase activity (20). As reviewed by Siedow (22), lipoxygenases and their products have been shown to generate AO species, and both lipoxygenases and AO species, particularly OH, cause lipid peroxidation. Thus, this investigation of AO production by tomato cells in the period immediately following elicitor treatment was initiated.In the present study, the effects of specific elicitors of C fulvum were tested on cell-suspension cultures derived from lines of tomato near-isogenic for resistance genes Cf 4 or Cf 5. Our objective was to investigate the possibility that AO