Protease-stable integration of Lhcb1 into thylakoid membranes is dependent on chlorophyll b in allelic chlorina-f 2 mutants of barley (Hordeum vulgare L.)

Abstract.

Allelic chlorina-f2 mutants of barley (Hordeum vulgare L.) growing under different light and temperature conditions demonstrated that the chlorophyll b-free chlorina-f 2^{f  2} and chlorina-f 2¹⁰¹ express a stable phenotype. Only 3 out of 10 light-harvesting chlorophyll a/b-binding proteins, Lhca4 (photosystem I), and Lhcb1 and Lhcb6 (photosystem II), required chlorophyll b for accumulation. The other light-harvesting proteins were found in all chlorina-f2 alleles, indicating that the integration pathway of these proteins into mutant thylakoid membranes was not affected. Chlorina-f2 alleles with a thylakoid membrane capable of fullfilling photosynthesis and transport demands, but with various amounts of chlorophyll b: chlorina-f 2¹⁰¹ (chlorophyll b-free), chlorina-f2¹²³ (27% of chlorophyll b compared with the wild type) and chlorina-f 2¹²² (70% chlorophyll b), were chosen to investigate whether chlorophyll b is necessary for the protease-stable insertion of Lhcb l into mutant thylakoid membranes. The Lhcb1 was affected in almost all alleles and was most sensitive to chlorophyll b deficiency. The Lhcb1 antibody confirmed the heterogeneity of the polypeptides of the light-harvesting chlorophyll a/b-binding protein II (LHCII) and detected in wild-type membranes, two protease-resistant, mature forms of Lhcb1 with apparent molecular masses of 28 and 29 kDa. Only one band reacting with the Lhcb1 antibody could be detected in chlorophyll b-free chlorina-f 2 ^{f  2}. It co-migrated with the 29-kDa band, but was completely digested after treatment of the isolated mutant membranes with exogenous protease. This showed that in chlorina-f 2 f  ² the Lhcb1 precursor was processed at one cleavage site only. The resulting 29-kDa Lhcb1 was not provided with chlorophyll b, and, consequently, not properly folded and inserted into the membrane. It remained susceptible to protease and was inconvertable to a 28-kDa form.