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Temporal patterns of hydrogen peroxide (H2O2) levels and total catalase activity are presented for post-imbibition scutella from six maize inbred lines expressing variable catalase activity. In all lines examined, H2O2 levels were highest during the initial days post-imbibition (1-2 dpi) and decreased thereafter, while total catalase activity was lowest during early dpi (1-2 dpi) and reached maximal activity at 4-6 dpi. In three of the six lines tested, a simple inverse correlation between catalase activity and H2O2 level was significant by Spearman's rank (P < 0.01). In addition to the general decline in H2O2 level throughout the dpi period, a reproducible increase in H2O2 level was observed at 4-5 dpi in five of six lines examined. Mutant lines lacking CAT-3 activity demonstrated a temporal shift in the occurrence of this increase. The role of total catalase (and individual isozymes) in controlling H2O2 levels during germination and the role of H2O2 as a potential regulator of catalase expression during germination are discussed.

Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with ['-32P]ATP Arch Biochem Biophys 270: 681-690). Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 250C before assay. The "spontaneous" inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg2+ and was relatively insensitive to Ca2+ and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with [32PJPi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 250C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate. We postulate that highly activated SPS contains phosphorylated residue(s) that increase activation state, and that spontaneous inactivation occurs by removal of these phosphate group(s). Inactivation of SPS in vivo caused by feeding uncouplers to darkened leaf tissue that had previously been fed mannose in the dark, may occur by this mechanism. However, there is no evidence that this mechanism is involved in light-dark regulation of SPS in vivo.Regulation of SPS2 activity is thought to be one of the components that controls the flux of carbon into sucrose, and hence translocation, in situ (for review see ref. 19 (9). Thus, phosphorylation of SPS occurred both in vivo and in vitro, and in both systems, the more highly phosphorylated enzyme had a lower activation state. Attempts to study the biochemical mechanism involved have been successful because the spinach leaf enzyme is often relatively stable (for short periods of time) at room temperature. The work we report here focused on the highly activated enzyme extracted from illuminated leaves that were fed mannose. We compared this SPS form with the relatively inactive (extracted from darkened leaves) and intermediate enzymeforms (extracted from leaves illuminated without mannose). We observed that highly activated enzyme was not stable when desalted crude extracts were preincubated at 25°C. Our study of this loss in activation state indicates that the "spontaneous" inactivation is mediated by an endogenous phosphatase(s), which constitutes evidence for a novel regulatory covalent modification of SPS.

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