Figure 7.

BES1 phosphorylation activity in the bin2-3bil1bil2 mutant is likely catalyzed by other GSK3s. A, Li⁺ treatment rescued the short hypocotyl phenotype of etiolated bin2-1 seedlings. Shown are five bin2-1 seedlings grown in the dark on half-strength MS medium supplemented with or without 10 mm LiCl. B, Li⁺ treatment rescued the petiole length defect of the bin2-1 mutation in the light. Phenotypic comparison between 2-week-old bin2-1 mutants grown in the light on half-strength MS medium supplemented with or without 10 mm LiCl. C, Complete elimination of phosphorylated BES1 by treatment with 100 mm LiCl. D, Treatment of 100 mm KCl had no effect on the BES1 phosphorylation status. E, Li⁺ treatment led to rapid inhibition of CPD gene expression. Northern-blot analysis of CPD expression in Ws wild-type seedlings treated with 100 mm LiCl for 0, 10, 30, or 60 min. The top panel shows an autoradiograph of ³²P-labeled CPD hybridization, and the bottom panel shows an ethidium bromide-stained RNA gel showing the amounts of rRNAs of each sample for the control of equal loading of total RNAs. F, Treatment with the inositol monophosphatase inhibitor L690330 had no effect on the in vivo BES1 phosphorylation activity. G, The BES1 phosphorylation activity in the bin2bil1bil2 mutant can be completely inhibited by treatment with BL or Li⁺. For C, D, F, and G, total protein crude extracts from treated seedlings were separated by 10% SDS-PAGE and analyzed by immunoblotting with an anti-BES1 antibody. BES1-P and BES1 indicate phosphorylated and nonphosphorylated forms of BES1 on western blots, and asterisks denote nonspecific bands used for loading controls.