5-aminoimidazole-4-carboxamide riboside activates nitrate reductase in darkened spinach and pea leaves

Nitrate reductase (NR) activity is modulated in vivo by phosphorylation (inactivation)/dephosphorylation (activation) in response to light/dark signals. The dephosphorylation of phospho-NR in vitro, catalyzed by endogenous protein phosphatases, is known to be stimulated by 5′-AMP suggesting that this metabolite may be an important regulator of the activity of NR, e.g. under anoxia. To determine whether 5′-AMP might be a regulatory metabolite in vivo, excised spinach (Spinacia oleracea) and pea (Pisum sativum) leaves were provided 5-aminoimidazole-4-carboxamide riboside (AICAR) via the transpiration stream, and the apparent phosphorylation status of NR was assessed by assay of activity in the presence of free Mg²⁺. NR was activated in darkened spinach leaves in a time- and concentration-dependent manner when leaves were fed AICAR; there was also an accumulation of nitrite in treated leaves in the dark. The activation by AICAR could be blocked by several type 2A protein phosphatase inhibitors (microcystin-LR, okadaic acid and cantharidin), and was not the result of a reduction of kinase activity by lack of ATP because cellular adenylates were unaffected. It was confirmed that AICAR-P, but not AICAR, mimicked 5′-AMP in the activation of phospho-NR in vitro. Our results are consistent with the notion that AICAR is converted to the monophosphorylated derivative, which accumulates in cells and acts as a structural analog of 5′-AMP. Our results suggest that a rise in cytosolic [5′-AMP] may be sufficient to activate NR in vivo. AICAR should be a useful compound for identifying AMP-regulated processes in plant systems.