Al-Induced, 51-Kilodalton, Membrane-Bound Proteins Are Associated with Resistance to Al in a Segregating Population of Wheat

Abstract

~~ lncorporation of 35S into protein i s reduced by exposure t o AI i n wheat (Trificum aestivum), but the effects are genotype-specific.Exposure to 10 t o 75 p~ AI had little effect on 35S incorporation into total protein, nuclear and mitochondrial protein, microsomal protein, and cytosolic protein in the AI-resistant cultivar PT741. I n contrast, 10 p~ AI reduced incorporation by 21 t o 38% i n the AI-sensitive cultivar Katepwa, with effects becoming more pronounced (31-6270) as concentrations of AI increased. We previously reported that a pair of 51 -kD membrane-bound proteins accumulated i n root tips of PT741 under conditions of AI stress.We now report that the 51-kD band is labeled with 35S after 24 h of exposure t o 75 p~ AI. The specific induction of the 51-kD band i n PT741 suggested a potential role of one or both of these proteins i n mediating resistance t o AI. Therefore, we analyzed their expression in single plants from an F, population arising from a cross between the PT741 and Katepwa cultivars. Accumulation of 1,3-/3-glucans (callose) i n root tips after 24 h of exposure t o 1 O0 p~ AI indicated that this population segregated for AI resistance i n about a 3:l ratio. A close correlation between resistance t o AI (low callose content of root tips) and accumulation of the 51-kD band was observed, indicating that at least one of these proteins cosegregates with the AI-resistance phenotype. As a first step i n identifying a possible function, we have demonstrated that the 51-kD band i s most clearly associated with the tonoplast. Whereas AI has been reported to stimulate the activity of the tonoplast H+-ATPase and H+-PPase, antibodies raised against these proteins did not cross-react with the 51-kD band. Efforts are now under way t o purify this protein from tonoplast-enriched fractions.

Induction of Vacuolar ATPase and Mitochondrial ATP Synthase by Aluminum in an Aluminum-Resistant Cultivar of Wheat

“…Antibodies raised against the V-ATPase B subunit from mung bean (Vigna radiata) and the F 1 F 0 -ATPase ␣-and ␤-subunits from Brewer's yeast (Saccharomyces cerevisiae) all cross-react with RMP51, suggesting that all three proteins are present. It is interesting that this contrasts with previous observations (Taylor et al, 1997) that antibodies raised against the B subunit of V-ATPase did not cross-react with RMP51. This inconsistency is most likely due to the more stringent binding conditions (37°C) used here or misalignment of the blots with protein gels in previous experiments, where alignment of cross-reacting species with a single band in a complex mixture had to be determined.…”

“…To test this hypothesis, V-ATPase, F 1 F 0 -ATPase, and P-ATPase activities were measured in three Alresistant and two Al-sensitive cultivars of wheat in control (0 m AlCl 3 ) conditions, conditions stressful to sensitive cultivars (20 m AlCl 3 ), and conditions stressful to resistant cultivars (100 m AlCl 3 ; Taylor et al, 1997). In cv PT741, V-ATPase activity increased by 2.1ϫ, even at 20 m AlCl 3 (Fig.…”

“…Because RMP51 protein levels increase in a dosedependent manner with Al treatment (Taylor et al, 1997), levels of V-ATPase and F 1 F 0 -ATPase activity in Al-treated and control seedlings were compared. Activities were measured in total microsomal membrane fractions isolated from 5-d-old seedlings of wheat cv PT741 exposed to 0 to 150 m AlCl 3 for 2 d (Fig.…”

“…Only three Al-induced proteins have been shown to co-segregate with the Al-resistance phenotype. One of these is a 23-kD root exudate protein (Basu et al, 1997), whereas the other two are 51-kD, tonoplast associated proteins (RMP51), which are the focus of this study. These 51-kD proteins are specifically induced by Al in root tips of an Al-resistant cultivar of wheat (cv PT741).…”

“…Al-induced proteins include membrane-bound (Basu et al, 1994a;CruzOrtega et al, 1997;Taylor et al, 1997), cytosolic (Richards et al, 1998), cytoskeletal (Cruz-Ortega et al, 1997), and exudate (Basu et al, 1994b(Basu et al, , 1997 proteins. Many of these have been implicated as general stress response proteins (Snowden et al, 1995;Hamel et al, 1998).…”