Rapid and highly specific monitoring of reversible thylakoid protein phosphorylation by polyclonal antibody to phosphothreonine-containing proteins

Reversible thylakoid protein phosphorylation in plant chloroplast probably plays an important role in acclimation of the photosynthetic apparatus to changes in environmental conditions. Studies of regulation and the significance of these reactions have in vivo greatly benefited from recent availability of phosphothreonine antibodies. To verify the specificity of these polyclonal antibodies, leaves were treated in conditions expected to induce either phosphorylation or dephosphorylation of thylakoid proteins D1, D2 and CP43 of the photosystem II core as well as light harvesting polypeptides of 27 and 25 kDa; subsequently, the proteins were isolated to homogeneity. Immunoreactions of these purified proteins with phosphothreonine antibodies were very similar to those observed with intact thylakoids. Moreover, their positive immunoresponse could be totally abolished by treating the thylakoid samples or purified photosystem II core preparations with acid phosphatase before immunoblotting. We conclude that the analytical method of using polyclonal phosphothreonine antibodies will turn out to be a highly specific and valuable tool in monitoring changes in the phosphorylation patterns of individual thylakoid phosphoproteins, both in vivo and in vitro.