Environmental and metabolic control of LHCII protein phosphorylation:: revealing the mechanisms for dual regulation of the LHCII kinase

Light-harvesting complex II (LHCII) protein phosphorylation in plant chloroplasts is under complex regulation. Combination of the in vivo monitoring of LHCII protein phosphorylation (by immunoblotting) with the in vitro [gamma-P-32]ATP phosphorylation assays revealed that the basic activation/deactivation model of the LHCII kinase, regulated by reversible occupation/release of plastoquinol at the plastoquinol oxidation (Q(o)) site of the cytochrome b(6)f (cyt b(6)f) complex, is consistent with, but not sufficient to explain the data obtained with isolated chloroplasts, leaf discs or intact leaves. Not only the light conditions but also the metabolic state of the entire plant, particularly the sugar metabolism, exerted a control over LHCII protein phosphorylation. Feeding of leaves with glucose (also with glutathione) activated the LHCII kinase in darkness. On the other hand, independently of the basic activation/deactivation mechanism of the kinase, a strong inhibition of LHCII protein phosphorylation occurred in vivo at increasing irradiances and even at low light conditions, depending on the metabolic state of the plant. Both the experiments with intact chloroplasts and the reconstitution experiments with isolated thylakoids to mimic LHCII kinase inhibition, disclosed that the kinase in its activated state (plastoquinol at the Q(o) site of cyt b(6)f complex) is protected against inhibition by thiol reductants. However, directly upon deactivation of the kinase (release of plastoquinol from the Q(o) site) it becomes a target for inhibition by thiol reductants. Thus the two interdependent regulatory systems of the LHCII kinase, the constantly occurring activation and deactivation on the one hand and the inhibition by thiol reductants on the other, are strongly dependent on the concentration of reducing equivalents in the chloroplast stroma. A scheme demonstrating the interconversion of activated, deactivated and inhibited states of the LHCII kinase in the chloroplast environment of intact leaves is presented.