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In many organisms, an increasing number of proteins seem to play two or more unrelated roles. Here we report that maize sucrose synthase (SUS) is distributed in organelles not involved in sucrose metabolism and may have novel roles beyond sucrose degradation. Bioinformatics analysis predicts that among the three maize SUS isoforms, SH1 protein has a putative mitochondrial targeting peptide (mTP). We validated this prediction by the immunodetection of SUS in mitochondria. Analysis with isoform-specific antisera revealed that both SH1 and SUS1 are represented in mitochondria, although the latter lacks a canonical mTP. The SUS2 isoform is not detectable in mitochondria, despite its presence in the cytosol. In maize primary roots, the mitochondrion-associated SUS (mtSUS; which includes SH1 and SUS1) is present mostly in the root tip, indicating tissue-specific regulation of SUS compartmentation. Unlike the glycolytic enzymes that occur attached to the outside of mitochondria, SH1 and SUS1 are intramitochondrial. The low abundance of SUS in mitochondria, its high Km value for sucrose, and the lack of sucrose in mitochondria suggest that mtSUS plays a non-sucrolytic role. Co-immunoprecipitation studies indicate that SUS interacts with the voltage-dependent anion channel in an isoform-specific and anoxia-enhanced manner and may be involved in the regulation of solute fluxes into and out of mitochondria. In several plant species, at least one of the SUS proteins possesses a putative mTP, indicating the conservation of the noncatalytic function across plant species. Taken together, these observations suggest that SUS has a novel noncatalytic function in plant cells.

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.

Ca-mediated processes are known to be involved in transducing many developmental, hormonal, and environmental cues in plant cells. In this study, the role of Ca in the perception of anoxic stress signals by maize (Zea mays L. cv B73) roots was assessed by studying the effect of various Ca antagonists on the induction of alcohol dehydrogenase (ADH) and sucrose synthase mRNA as well as ADH activity under anoxia. The effect of these compounds on the poststress recovery of the seedlings was also monitored. Ruthenium red (RR), an inhibitor of organellar Ca fluxes, repressed the anoxic activation of the alcohol dehydrogenase1 and shrunken1 genes as measured by their transcript levels as well as ADH activity. Furthermore, RR-treated seedlings could not recover even after only 2 h of flooding, in contrast to untreated B73 seedlings that survived 72 h of submergence. Ca, when supplied along with RR, allowed normal anoxic gene expression and also prevented the RR-imposed death of the seedlings from short-term anoxia. Ca (45Ca) fluxes were measured in maize roots to elucidate the mode of action of RR. RR abolished anoxia-stimulated 45Ca influx into maize roots but did not affect aerobic Ca2+ uptake, unlike a few other antagonists that blocked both the aerobic and anoxic fluxes. However, Ca uptake across the plasma membrane was not necessary for the adaptive response to anoxia, since chelation of extracellular Ca by ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid did not affect the induction of ADH activity or poststress survival of flooded seedlings. The data suggest that RR may act on one of the intracellular stores of Ca and the Ca mobilized from this source is a physiological transducer of anoxic stress signals in maize roots.