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Nitrate reductase expression in maize leaves ( Zea mays ) during dark‐light transitions. Complex effects of protein phosphatase inhibitors on enzyme activity, protein synthesis and transcript levels
Author(s) -
Redinbaugh Margaret G.,
Huber Steven C.,
Huber Joan L.,
Hendrix Keith W.,
Campbell Wilbur H.
Publication year - 1996
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1996.tb00676.x
Subject(s) - cycloheximide , okadaic acid , phosphatase , nitrate reductase , protein biosynthesis , biochemistry , enzyme assay , enzyme , chemistry , specific activity , biology , microbiology and biotechnology
The effects of cytoplasmic protein synthesis and protein phosphatase activity on NADH:nitrate reductase (NR) activity, protein and transcript were examined in maize ( Zea mays L.) seedling leaves. A rapid increase in NR activity, measured in the presence of 5 m M Mg 2+ , was found upon exposure of excised leaves to light. Inhibitors of protein phosphatase activity (okadaic acid [OKA] and microcystin [MC]‐LR) completely prevented the increase in NR activity. The cytoplasmic protein synthesis inhibitor, cycloheximide (CHX), did not affect Mg 2+ inhibition of NR activity during the dark‐to‐light transition. V max NR activity, measured in the presence of 5m M Mg 2+ , was found upon exposure of excised leaves to light. Inhibitors of protein phosphatase activity (okadaic acid [OKA] and microcystin [MC]‐LR) completely prevented the increase in NR activity. The cytoplasmic protein synthesis inhibitor, cycloheximide (CHX), did not affect Mg 2+ inhibition of NR activity during the dark‐to‐light transition. V max NR activity, measured in the presence of P i and EDTA, remained constant or increased slightly in maize leaves during the first 2 h of the light period. OKA, MC‐LR or CHX treatment caused a 40 to 50% reduction in V max NR activity during this time. Incorporation of 35 S‐Met into NR protein was reduced more than 90% by CHX and 80% by OKA. The inhibition of NR protein synthesis by CHX and OKA correlated with a 50 to 60% decrease in 35 S‐Met incorporation into total soluble protein over the treatment period. The increase in NR mRNA levels early in the light period was prevented by OKA and MC‐LR, but not by CHX. OKA had a similar effect on sucrose phosphate synthase mRNA levels, but did not affect Catalasel or Catalase3 mRNA accumulation. The data suggest that light‐induced decreases in Mg 2+ inhibition of NR activity and transcript levels are independent of new protein synthesis. The effects of OKA and MC‐LR indicate that protein phosphatase activities could be involved, directly or indirectly, in the regulation of NR activity, protein synthesis and transcript accumulation.