An immunological approach using a polyclonal phosphothreonine antibody is introduced for the analysis of thylakoid protein phosphorylation in vivo. Virtually the same photosystem II (PSII) core phosphoproteins (D1, D2, CP43, and the psbH gene product) and the lightharvesting chlorophyll a/b complex II (LHCII) phosphopolypeptides (LHCB1 and LHCB2), as earlier identified by radiolabeling experiments, were recognized in both pumpkin and spinach leaves. Notably, the PSII core proteins and LHCII polypeptides were found to have a different phosphorylation pattern in vivo with respect to increasing irradiance. Phosphorylation of the PSII core proteins in leaf discs attained the saturation level at the growth light intensity, and this level was also maintained at high irradiances. Maximal phosphorylation of LHCII polypeptides only occurred at low light intensities, far below the growth irradiance, and then drastically decreased at higher irradiances. These observations are at variance with traditional studies in vitro, where LHCII shows a light-dependent increase in phosphorylation, which is maintained even at high irradiances. Only a slow restoration of the phosphorylation capacity for LHCII polypeptides at the low light conditions occurred in vivo after the high light-induced inactivation. Furthermore, if thylakoid membranes were isolated from the high light-inactivated leaves, no restoration of LHCII phosphorylation took place in vitro. However, both the high light-induced inactivation and low light-induced restoration of LHCII phosphorylation seen in vivo could be mimicked in isolated thylakoid membranes by incubating with reduced and oxidized dithiothreitol, respectively. We propose that stromal components are involved in the regulation of LHCII phosphorylation in vivo, and inhibition of LHCII phosphorylation under increasing irradiance results from reduction of the thiol groups in the LHCII kinase. Photosystem (PS)1 II is a multiprotein complex of the thyla- (14) protein phosphatases, active in dephosphorylation of thylakoid phosphoproteins, are reported to be present in plant chloroplasts. Distinct physiological roles have been established for the phosphorylation of LHCII polypeptides, being implicated in the regulation of excitation energy distribution between PSII and PSI (1, 2), and in the protection of PSII against photoinhibition (15,16). Under conditions where PSII becomes more excited than PSI, the redox-controlled kinase is activated and phosphorylates LHCII polypeptides. The outer LHCII subcomplex, consisting of LHCB1 and LHCB2 proteins (see, e.g., Ref. 17), then detaches from the PSII complex and migrates out from the appressed region of the thylakoid membrane, thereby reducing the excitation of PSII (1). Dephosphorylation of this mobile LHCII subcomplex allows its subsequent migration back to the stacked grana region and reassociation with PSII (16).The only function so far established for reversible phosphorylation of PSII core proteins is the regulation of the degradation of photodamaged D1 protein ...

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