Glucose‐signalled inhibition of cysteine‐proteases involved in nitrate reductase degradation in oat leaf segments

Abstract

Regulation of nitrate reductase (NR; EC 1.6.6.1) breakdown, measured as loss of maximal activity (MNRA), was studied in leaf segments of 7‐day‐old oat plants in the light for up to 4 h. In segments floating on 1 mM tungstate, NR lost more than 40% of its initial maximal activity. Cycloheximide, high (300 mM) glucose (Glc) and inhibitors of cysteine proteases stabilized NR in situ, suggesting that MNRA decrease was due to the hydrolysis of NR by a short‐lived, glucose‐modulated cysteine protease. Loss of MNRA was accelerated by cantharidin (CTHR) and inhibited by staurosporine, suggesting that NR breakdown required continuous phosphorylation. High glucose inhibited any further MNRA decrease when supplied after a 30‐min pretreatment with CTHR, suggesting that a phosphorylated protein was its target. Isoosmolar polyethylene glycol also stabilized NR but not in the presence of CTHR. Low (30 mM) Glc stabilized NR only in the presence of Ca2+, and CTHR inhibited its effect. EGTA and LaCl3 completely arrested the effects of both high‐ and low‐ Glc. Like low D‐Glc, low L‐Glc (glucose analog not transported) inhibited NR breakdown in the presence of Ca2+, but at high concentration only 2‐deoxyglucose, that is phosphorylated but not further metabolized, and glucose‐6P were effective in the presence of CTHR, suggesting that receptors for high‐ and low‐ Glc were located in different cell compartments. It is proposed that high‐ and low‐ Glc trigger different signalling pathways, with calcium as a common upstream secondary messenger and protein kinases and protein phosphatases being downstream components in the cascade of reactions that modulates NR proteolysis.