Altered Patterns of Sucrose Synthase Phosphorylation and Localization Precede Callose Induction and Root Tip Death in Anoxic Maize Seedlings

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Abstract
Root extracts made from maize (Zea mays) seedlings submerged for 2 h showed an increased (32)P-labeling of a 90-kD polypeptide in a Ca(2+)-dependent manner. This protein was identified as sucrose synthase (SS) by immunoprecipitation and mutant analysis. Metabolic labeling with (32)P(i) indicated that the aerobic levels of SS phosphorylation were maintained up to 2 h of anoxia. In contrast, during prolonged anoxia the protein was under-phosphorylated, and by 48 h most of the protein existed in the unphosphorylated form. In seedlings submerged for 2 h or longer, a part of SS became associated with the microsomal fraction and this membrane localization of SS was confined only to the root tip. This redistribution of SS in the root tip preceded callose induction, an indicator of cell death. The sh1 mutants showed sustained SS phosphorylation and lacked the anoxia-induced relocation of SS, indicating that it was the SH1 form of the enzyme that was redistributed during anoxia. The sh1 mutants also showed less callose deposition and greater tolerance to prolonged anoxia than their non-mutant siblings. EGTA accentuated anoxic effects on membrane localization of SS and callose accumulation, whereas Ca(2+) addition reversed the EGTA effects. These results indicate that the membrane localization of SS is an important early event in the anoxic root tip, probably associated with the differential anoxic tolerance of the two SS mutants. We propose that beside the transcriptional control of genes encoding SS, the reversible phosphorylation of SS provides a potent regulatory mechanism of sugar metabolism in response to developmental and environmental signals.