Immunological characterization of chlorophyll a/b-binding proteins of barley thylakoids

Abstract

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCI) using a photosystem (PS) I preparation (PSI-200) wild-type from barley (Hordeum vulgare L. cv. Svaløf's Bonus) as the antigen. These antibodies cross-reacted with a minor light-harvesting chlorophyll a/b-protein of PSII (Chla/b-P1=CP29), but not with the major one, LHCII (=Chla/b-P2(**)). Similarly, a monoclonal antibody to Chla/b-P1, elicited by a PSII preparation as the antigen, cross-reacted with LHCI, but not LHCII. This explains why an antigen consisting of LHCII, free of LHCI, but contaminated with Chla/b-P1, can elicit antibodies which cross-react with LHCI. Immunoblot assays showed that LHCI and Chla/b-P1 have at least two epitopes in common. Immunogold labelling of thin-sectioned wild-type thylakoids confirmed a preferential localisation of Chla/b-P1 in grana partition membranes and LHCI in stroma lamellae. The presence of LHCI was demonstrated in barley mutants lacking the PSI reaction centre (viridis-zb (63)) and chlorophyll b (chlorina-f2), and was correlated with the presence of long-wavelength (730 nm) fluorescence emission at 77 K. The mutant viridis-k (23), which has a 77 K long-wavelength fluorescence peak at 720 nm, was shown by immune-blot assay to lack LHCI, although Chla/b-P1 was present.

Probing in vitro translation products with monoclonal antibodies to chlorophylla/b-binding proteins of barley thylakoids

“…What the change in mobility is due to is not clear, but it is always observed (4,14). Figure 2 shows that CMpLHCI:2 reacts with the Thylakoid polypeptides from wild-type seedlings (20 lag Chl) illuminated for 6, 9 or 12 hours were separated on 11-15% SDS-PAGE prior to electroblotting.…”

“…Immobilized antigens were visualized using peroxidase-coupled rabbit antimouse immunoglobulins (Dakopatts A/S, Copenhagen, Denmark, P161). The antibodies to LHCI and chlorophyll a/b-protein 1 (CP29) were CMp-LHCI:2 and CMpChla/b-Pl:l. They are described earlier (14).…”

“…The major chlorophyll a/b-binding protein, LHCII, is absent from the thylakoids of the chlorophyll b-less barley mutant, chlorina-f2 (18) even though the polypeptides can be detected with an immunoblot assay (20). Interestingly, the polypeptides corresponding to those in Chla/~-P1 and LHCI are present in chlorina-f2 and bind chlorophyll a (14). A contrasting phenotype with the absence of LHCI is exhibited by the barley nuclear gene mutant viridis-k 23.…”

“…There have been reported polyclonal antibodies to LHCI that react with LHCII (6) and monoclonal antibodies raised to LHCII that reacts with LHCI (5). There have also been raised both mono-and polycional antibodies to LHCI, that react with Chla/h-Pl and vice versa (14,26).…”

“…We have previously described monoclonal antibodies to LHCI and Chic/h-P1 that show mutual cross-reactivity (14). In this study we have employed these antibodies for immunoprecipitation of in vitro translated polypeptides, using polyA + RNA isolated from both wild-type and the LHCI-less mutant viridis-k :3.…”