EFFECTS OF UVB RADIATION ON LIGHT-DEPENDENT AND LIGHT-INDEPENDENT PROTEIN-PHOSPHORYLATION IN THYLAKOID PROTEINS

Protein phosphorylation in thylakoids can be divided into light-depend ent and light-independent types. The phosphorylation patterns of these two types of process show about 30 and 40 phosphorylated polypeptides respectively in two-dimensional (2-D) protein separation (molecular m ass discrimination in one dimension, isoelectric point (Ip) in the oth er), It is suggested that each type involves its own signal switch sys tem and kinases responsible for signal transduction from the input dom ain to the final substrates. A thylakoid protein having a molecular ma ss of 45 kDa and focusing at Ip 5.0 in the 2-D protein pattern is sugg ested to be a kinase-like protein and its N-terminal sequence is repor ted. Freshly prepared spinach thylakoids were exposed to UVB (280-315 nm) radiation emitted from a 4 W Philips fluorescent TL12 lamp for 60 min (0.21 W m(-2) plant weighted radiation). The proteins were extract ed and subjected to 2-D gel electrophoresis, There was no distinct dif ference in the 2-D protein patterns between UVB-irradiated thylakoids and non-irradiated thylakoids. The experiment was repeated under condi tions of light-dependent protein phosphorylation with [gamma-P-31]ATP as phosphate donor. The phosphorylation of a group of polypeptides hav ing molecular masses around 60-65 kDa and isoelectric points around 5- 6, thought to include LHCII kinase, was suppressed by UVB pretreatment . The phosphorylation of LHCII polypeptides and other polypeptides was also decreased by UVB treatment. A similar experiment was performed w ith phosphorylation taking place in the dark after 1 h UVB (or control ) treatment. In this case, the UVB treatment had an even greater impac t on the phosphorylation pattern. The phosphorylation of a 45 kDa poly peptide, thought to be a kinase-like thylakoid protein active under ox idizing conditions, was almost eliminated, and that of a 60-65 kDa pol ypeptide group, thought to include LHCII kinase, was strongly suppress ed; however, some polypeptides, e.g. 9 kDa and 10 kDa polypeptides foc using at Ip 5.7 and 7.3 respectively, became strongly phosphorylated i n UVB-irradiated thylakoids. They may contribute to the protection aga inst UVB radiation, A preliminary model is proposed for the action mec hanism of both the enhanced phosphorylated proteins. We believe that a ltered kinase activity and protein phosphorylation may be important ea rly steps in the impact of UVB radiation on plants. (C) 1997 Elsevier Science S.A.