Jan J. Slaski

Effect of Aluminium on Calmodulin-Dependent and Calmodulin-Independent NAD Kinase Activity in Wheat (Triticum aestivum L.) Root Tips

Abstract Total NAD kinase activity is twice as high in the Al tolerant wheat strain (BHG) than in the Al sensitive variety (Grana). In the former the calmodulin-dependent enzyme constitutes about 50 % of the total activity, whereas in the latter it does not exceed 30 %. Aluminium induced NAD kinase activity 2.5 fold in the sensitive variety Grana and six fold in the Al tolerant BHG upon 10 h incubation in 0.74 mM Al. The induction, abolished by cycloheximide, involves in Grana both calmodulin-dependent and calmodulin-independent enzymes, whereas in the Al tolerant genotype the induction involves only the calmodulin-independent form, and the activity of the calmodulin-dependent enzyme becomes marginal. Changes in the activity of NAD kinase are paralleled by the shift in the NADP/NAD ratio. It is suggested that the mechanism of Al tolerance is manifested in proportion to calmodulin-dependent and calmodulin-independent NAD kinases and in the ability to replace the former by the latter enzyme in the tolerant genotype under Al stress.

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

Incorporation of 35S into protein is reduced by exposure to Al in wheat (Triticum aestivum), but the effects are genotype-specific. Exposure to 10 to 75 [mu]M Al had little effect on 35S incorporation into total protein, nuclear and mitochondrial protein, microsomal protein, and cytosolic protein in the Al-resistant cultivar PT741. In contrast, 10 [mu]M Al reduced incorporation by 21 to 38% in the Al-sensitive cultivar Katepwa, with effects becoming more pronounced (31–62%) as concentrations of Al increased. We previously reported that a pair of 51-kD membrane-bound proteins accumulated in root tips of PT741 under conditions of Al stress. We now report that the 51-kD band is labeled with 35S after 24 h of exposure to 75 [mu]M Al. The specific induction of the 51-kD band in PT741 suggested a potential role of one or both of these proteins in mediating resistance to Al. Therefore, we analyzed their expression in single plants from an F2 population arising from a cross between the PT741 and Katepwa cultivars. Accumulation of 1,3-[beta]-glucans (callose) in root tips after 24 h of exposure to 100 [mu]M Al indicated that this population segregated for Al resistance in about a 3:1 ratio. A close correlation between resistance to Al (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 Al-resistance phenotype. As a first step in identifying a possible function, we have demonstrated that the 51-kD band is most clearly associated with the tonoplast. Whereas Al 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 to purify this protein from tonoplast-enriched fractions.

Effects of Protein Synthesis Inhibitors on Uptake of Aluminium in Aluminium-Resistant and Aluminium-Sensitive Cultivars of Wheat

Summary The effects of two inhibitors of protein synthesis on the kinetics of Al uptake by excised roots of Al-resistant cultivars (Atlas 66 and PT 741) and Al-sensitive cultivars (Neepawa and Scout 66) of wheat were investigated. Treatment of intact roots of the Al-sensitive cultivar, Neepawa, with cycloheximide (0 to 5 mM) showed that this inhibitor was more effective in reducing incorporation of 35 S into microsomal membrane proteins than into total proteins. Cycloheximide at 0.25 mM reduced incorporation of 35 S into microsomal membrane proteins by 68 %, while a 21 % reduction into total proteins was observed. Cycloheximide at 1.0 mM was sufficient to induce maximal inhibition of 35 S incorporation into both microsomal membrane (72 %) and total (40 %) proteins. At this concentration, cycloheximide caused quantitative differences in short-term (3 h) Al uptake between Al-resistant and Al-sensitive genotypes. A significant decrease in rates of Al uptake was observed in both Al-sensitive cultivars (24 and 29 %), while no significant effect was observed in the Al-resistant cultivars. These results are in contrast to previous results from long-term experiments (6 and 12 h), where cycloheximide stimulated Al uptake in an Al-resistant cultivar, Atlas 66 (Aniol, 1984; Rincon and Gonzales, 1992). In experiments where Al uptake was measured after a 4-h pretreatment with 1.0 mM cycloheximide (7 h total exposure), a stimulation of uptake (45 %) was observed in the Al-sensitive cultivar, Neepawa. Thus, time of exposure is an important experimental variable that can account for contradicting results in the literature. In both resistant and sensitive cultivars, treatment with chloramphenicol, an inhibitor of prokaryotic protein synthesis, had no effect on the kinetics of Al uptake. Posible mechanisms whereby protein synthesis might affect Al uptake in Al-resistant and Al-sensitive genotypes are discused.