Ascorbate-mediated LHCII protein phosphorylation--LHCII kinase regulation in light and in darkness.
A freeze-thaw cycle of isolated thylakoids in darkness in the presence of ascorbate was employed as a novel experimental system to activate the light-harvesting complex (LHC) II kinase. Under these conditions ascorbate reduces Q(A), the primary quinone electron acceptor of photosystem (PS) II, and the subsequent reduction of plastoquinone and the cytochrome (cyt) b(6)f complex results in the activation of the LHCII kinase. Using this activation system, several facets of regulation of LHCII protein phosphorylation were unravelled. (i) Myxothiazol inhibited the activation of LHCII protein phosphorylation, thus being a potent inhibitor of electron flow not only in cyt bc complexes but in darkness also in cyt b(6)f complexes. (ii) Oxygen, the only electron acceptor in darkness, was required for LHCII kinase activation demonstrating that after a full reduction of the cyt b(6)f complex, an additional plastoquinol oxidation cycle in the quinol oxidation (Qo) site is required for LHCII kinase activation. (iii) In the absence of electron flow, when the intersystem electron carriers are reduced, the activated LHCII kinase has a half-life of 40 min, whereas the fully activated LHCII kinase becomes deactivated in a time scale of seconds upon oxidation of the cyt b(6)f complex, indicating that the kinase constantly reads the redox poise of the cyt b(6)f complex. (iv) The LHCII kinase is more tightly bound to the thylakoid membrane than the PS II core protein kinase(s). It is concluded that oxidation of plastoquinol at the Qo site of the reduced cyt b(6)f complex is required for LHCII kinase activation, while rapid reoccupation of the Qo site with plastoquinol is crucial for sustenance of the active state of the LHCII kinase.
Hou
Cai-Xia
CX
Department of Biology, University of Turku, FIN-20014 Turku, Finland.
Rintamäki
Eevi
E
Aro
Eva-Mari
EM
D000255:Adenosine Triphosphate / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism*; D003624:Darkness; D004579:Electron Transport; D004789:Enzyme Activation; D005615:Freezing; D008027:Light; D045342:Light-Harvesting Protein Complexes; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D018532:Pisum sativum / Q000378:metabolism / Q000528:radiation effects; D010766:Phosphorylation; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects*; D010971:Plastoquinone / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D020524:Thylakoids / Q000378:metabolism / Q000528:radiation effects
D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D009249:NADP; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C120337:light-harvesting complex II kinase; D010971:Plastoquinone; D001205:Ascorbic Acid