The presence of phosphorylation form of D1 protein in its cross-linked aggregates in high light treated spinach leaves in vivo

In the present study, using specific antibody against D1 protein, we detected four aggregates of D1 protein in thylakoid membranes from spinach leaves illuminated at high light (800–2500 μmol photons·m 2·s 1) for 3 h. Their accumulations were dependent on the light intensity to which the leaves had been subjected. Further immunoblot analysis indicated that 70 kD aggregate was a product of D1 protein cross-linked with CP43, 65 and 60 kD aggregate were two cross-linked products between D1 and D2 proteins, and 41 kD aggregate was one cross-linked D1 with α-subunit of cytochrome b 559 (Cyt b 559). This result provided the evidence for the existence of the aggregation of the D1 protein in vivo. The maximal level of D1/Cyt b 559 aggregate occurred at 1000 μmol photons·m 2·s 1 but drastically decreased with further increasing light intensity. Immunoblot analysis with phosphothreonine (Thr (P)) antibody indicated that D1/CP43 and D1/Cyt b 559 aggregates contained the phosphorylated protein(s). In vitro dephosphorylation experiment also showed that D1/Cyt b 559 and D1/CP43 aggregates lost the immunoreactivity with Thr (P) antibody after the phosphatase treatment of the membranes from high-light-illuminated leaves. Our results demonstrated that strong illumination of spinach leaves induced cross-linked products of D1 protein with its nearby polypeptides of PS, some of which co.n-tained the phosphorylated D1 protein.