Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation.

Area of Science:

• Plant Physiology
• Biochemistry
• Enzyme Regulation

Background:

• Nitrate reductase (NR) is crucial for nitrate assimilation in plants.
• NR activity in spinach leaves exhibits dynamic changes in response to light and dark conditions.

Purpose of the Study:

• To investigate the rapid modulation of spinach leaf nitrate reductase (NR) activity during light/dark transitions.
• To elucidate the role of covalent modification, specifically phosphorylation, in regulating NR activity and magnesium sensitivity.

Main Methods:

• Measuring NR activity using NADH and methyl viologen as electron donors.
• Investigating magnesium inhibition of NR activity.
• In vivo phosphorylation studies using [32P]Pi feeding.
• Tryptic phosphopeptide mapping.
• Analyzing the effect of light, mannose, and okadaic acid on NR phosphorylation and activity.

Main Results:

• A magnesium-sensitive form of NR predominates in the dark and is rapidly lost in the light.
• Spinach NR is phosphorylated in vivo on seryl residues, with specific phosphopeptides (Pp1 and Pp3) correlating with Mg2+-inhibited activity.
• Light or mannose feeding in darkness leads to dephosphorylation and activation of NR.
• Okadaic acid blocks the light-induced activation, implicating protein phosphatases.

Conclusions:

• Spinach leaf NR activity is dynamically regulated by light/dark cycles through a phosphorylation/dephosphorylation mechanism.
• This covalent modification alters NR's sensitivity to magnesium, suggesting a key regulatory process in nitrate assimilation.
• Protein phosphatases (type 1 and/or 2A) are involved in the light-mediated dephosphorylation and activation of NR.