Plant Physiol. (1 996) 1 1 O: 561-569
Aluminum lnteractions with Voltage-Dependent Calcium Transport in Plasma Membrane Vesicles lsolated from Roots of Aluminum-Sensitive and -Resistant Wheat Cultivars’ Jianwei W. Huang’, Didier M. Pellet, Lisa A. Papernik, and Leon V. Kochian*
United States Plant, Soil and Nutrition Laboratory, United States Department of Agriculture-Agricultura1 Research Service, Cornell University, Ithaca, New York 14853 the root growth medium, which is the case for most acid soils, the AI-induced inhibition of Ca2+ influx can be Loosely correlated with A1 resistance and sensitivity both in wheat (Triticum aestivum L.) cultivars and in near-isogeneic pairs of wheat lines (Huang et al., 1992a, 1992b; Ryan and Kochian, 1993). However, when the A13+/Ca2+ ratio is relatively low, or in the presence of high levels of other cations that ameliorate A1 toxicity, A1 inhibition of . root growth can be decoupled from A1 inhibition of Ca2+ uptake, suggesting that A1 inhibition of Ca2+ transport is not a primary cause of root growth inhibition (Ryan et al., 1994). Nonetheless, the possibility exists that the general correlation behveen A1 sensitivity and inhibition of Ca2+ influx into root cells observed at high Al/Ca ratios results from A1 disruption of Ca2+ homeostasis in the cell cytoplasm. There is circumstantial evidence indicating that A1 disruption of Ca2+ homeostasis may trigger A1 toxicity in both plant and animal cells (Rengel, 1992; Shi and Haug, 1992). Based on the nature of A1/Ca2+ transport interactions, the most likely scenario for A1 inhibition of Ca2+influx into roots is via A1 blockage of CaZt channels at the outer surface of the root-cell PM or A1 transport into the cell via Ca2+ channels (Huang et al., 1992a, 199213; Rengel and Elliott, 1992; Pifieros and Tester, 1993,1995). Ca2+ channels in the plant cell PM are presumed to play important roles in signal transduction processes, as well as in the provision of Ca2+ for plant mineral nutrition. Because Ca2+ translocation within the plants to the root apex is limited (Huang et al., 1993b), most of the Ca2+ needed for normal cellular function in the apex must be absorbed from the externa1 solution. Thus, continuous A1 disruption of Ca2+ absorption into cells of the root apex, which is the site of the A1 toxicity (Bennet et al., 1985; Ryan et al., 1993), could alter Ca2+ homeostasis in these cells. Until recently, detailed information concerning plant PM Ca2+ channels has been lacking. Using current- and voltage-clamp techniques, Shiina and Tazawa (1987) demonstrated the presence of a Ca2+ channel in the PM of tonoplast-free Nitellopsis obtusa cells. There is also evidence of stretch-activated Ca2+ channels in the PM of Vi& faba
The role of AI interactions with root-cell plasma memhrane (PM) Ca2+ channels in AI toxicity and resistance was studied. l h e experimental approach involved the imposition of a transmembrane electrical potential (via K+ diffusion) in right-side-out PM vesicles derived from roots of two wheat (Trificum aestivum L.) cultivars (AI-sensitive Scout 66 and AI-resistant Atlas 66). W e previously used this technique to characterize a voltage-dependent Ca” channel in the wheat root PM (J.W. Huang, D.L. Crunes, L.V. Kochian 119941 Proc Natl Acad Sci USA 91: 3473-3477). W e found that AI3+ effectively blocked this P M Cazf channel; however, AI3+ blocked this Ca2+ channel equally well in both the AI-sensitive and -resistant cultivars. I t was found that the differential genotypic sensitivity of this Ca2+ transport system to AI in intact roots versus isolated PM vesicles was due to AI-induced malate exudation localized to the root apex in AI-resistant Atlas but not in AI-sensitive Scout. Because malate can effectively chelate AI3+ in the rhizosphere and exclude it from the root apex, the differential sensitivity of Ca2+ influx to AI in intact roots of AI-resistant versus AI-sensitive wheat cultivars is probahly dueto the maintenance of lower AI3+ activities in the root apical rhizosphere of the resistant cultivar.
A1 toxicity is the primary environmental stress limiting crop productivity on acid soils. One of the proposed mechanisms of A1 toxicity involves A1 interaction with ion transport systems functioning at the root-cell PM (Taylor, 1988; Kochian, 1995). The role of A1/Ca2+ transport interactions in the mechanisms of A1 toxicity has recently received considerable attention, because Ca2+ plays a central role in the regulation of many plant cellular processes, including mitosis and cytokinesis, gravitropism, polar growth, and cytoplasmic streaming (Williamson and Ashley, 1982; Hepler and Wayne, 1985). A1 inhibition of Ca2+ influx into plant cells is rapid and reversible, and the A1 blockage of Caz+ influx precedes visible symptoms of A1 toxicity (Huang et al., 1992a, 199213; Rengel and Elliott, 1992; Ryan and Kochian, 1993). When the A13+/Ca2+ ratio is high in This work was supported by U.S. Department of Agriculture/ National Research Initiative Competitive Grant No. 93-371008874 to L.V.K. and a Swiss National Science Foundation Postdoctoral Fellowship to D.M.P. Present address: DuPont Central Research and Development, Environmental Biotechnology, Glasgow-301, Newark, DE 19714. * Corresponding author; e-mail
[email protected];fax 1-607255-2459.
Abbreviations: E,, membrane potential; MF, microsomal fraction; PM, plasma membrane; U, phase, upper phase from aqueous two-phase partitioning. 561
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guard cells and onion leaf epidermal cells (Cosgrove and Hedrich, 1991; Ding and Pickard, 1993). Using photoaffinity labeling of membrane proteins from carrot suspensioncell protoplasts with Ca2+ channel blockers, Thuleau et al. (1990) identified a 75-kD protein that might be a component of a PM Ca2+ channel. This purified protein, when reconstituted into liposomes, behaves as an unstable Ca2+permeable channel that is also permeable to C1- (Thuleau et al., 1993). Subsequently, Thuleau et al. (1994) used the patch-clamp technique with protoplasts isolated from carrot suspension cells to characterize a depolarization-activated Ca2+ channel in the PM. Most recently, Pifieros and Tester (1995) characterized a voltage-dependent Ca2+ channel in wheat root PM vesicles incorporated into artificial planar lipid bilayers. They found that the channel was also permeable to the essential nutrients Mg2+ and Mn2+ and was effectively blocked by micromolar concentrations of A13+. Because Caz+ channels functioning in the membranes of higher plant cells exist in low density and have a very low open probability (Gelli and Blumwald, 1993), in comparison with K+ channels, they have proven to be difficult to identify and characterize using patch-clamp techniques, which have contributed significant information concerning K + and anion channels. We recently developed a technique to study Ca2+ influx into voltage-clamped right-side-out PM vesicles isolated from wheat roots. Using this technique, we identified and characterized a voltage-dependent Ca2+ channel at the root-cell plasma membrane (Huang et al., 1994). This Ca2+ channel opens upon depolarization of E,, with Ca”+ influx increasing to a maximum at approximately -100 mV. Upon further depolarization of E,, Ca2+ influx declines, thus yielding a bell-shaped current-voltage curve. The channel was found to be selective for Ca2+ over Mg2+, Sr2+, K+, and Na+, is blocked by very low concentrations of La3+, and is not affected by high concentrations of the K+ channel blocker tetraethylammonium. Marshall et al. (1994) also used the same technique to characterize a similar voltage-dependent Ca2+ transporter in the maize root PM. The objectives of this study were (a) to investigate whether A1 blocks this voltage-dependent Ca2+ channel; (b) to determine whether A1 preferentially blocks Ca2+ channels in PM vesicles derived from Al-sensitive Scout 66 in comparison with Al-resistant Atlas 66; (c) to examine the A1 effects on voltage-dependent Ca2+ influx into the PM vesicles isolated from the root apices versus mature root regions; and (d) to investigate the effect of A1 exposure on organic acid exudation in the Al-resistant and -sensitive wheat genotypes, to determine whether differences in A1 inhibition of Ca2+ influx at the root leve1 involve an A1 exclusion mechanism that reduces the A13+ activity in the rhizosphere of the Al-resistant wheat genotype. MATERIALS A N D METHODS Plant Material
Seeds of winter wheat (Triticum aestivum L.) cultivars (Al-resistant Atlas 66 and Al-sensitive Scout 66) were obtained from Dr. J Peterson (University of Nebraska, Lin-
Plant Physiol. Vol. 11 O, 1996
coln). Selected seeds were germinated on filter paper saturated with 0.1 mM CaC1, solution at pH 4.5. For isolation of PM vesicles, seedlings were grown in complete nutrient solutions as previously described (Huang et al., 1993a). Roots of 20- to 25-d-old plants were harvested and briefly washed (
