Elucidating Functional Aspects of LHCII Phosphorylation by DNA Insertional Mutagenesis in the Green Alga Chlamydomonas Reinhardth

Abstract

The reversible phosphorylation of two peptides of the light harvesting complex II (Lhcbl and Lhcb2) (1) and of the core peptides D1, D2, CP43 and psbH of the PSII complex (2, 3) in thylakoids of higher plants is well established. However, functional aspects of this covalent modification have not yet been fully understood (see: 4,5). Recently, a model was published supporting the idea that under light stress phosphorylation plays an important role in the regulation of the PSII dimer to monomer conversion (6). Earlier LHCII protein phosphorylation was postulated to be involved in the regulation of the distribution of light energy between the two photosystems by state2 to statel transition (7,8,9). Up to date no functional aspects of the reversible phosphorylation of the LHC complex have been verified. In order to identify participating redox components and enzymes like the putative LHCII kinase, the generation of a library of photoautotrophic mutants, incapable of implementing state2 to statel transition and influenced in their ability of phosphorylating LHC peptides appears to be a promising attempt. By localising participating genes in the nuclear genome of a photoautotrophic organism with the help of specific probes the identification of proteins and enzymes involved in the process of reversible LHCII phosphorylation is possible. With the powerful techniques of gene tagging in the green alga Chlamydomonas reinhardtii using transfor-ming DNA like the ARG7 gene (encoding argininosuccinate lyase) which complements the arginine-requiring mutant (arg7cw15,mt), a number of non-homologous recombinants can be achieved by random integration of transforming DNA (10). Whereas the phosphorylation of certain PSII core proteins in Chlamydomonas reinhardtii is still under doubt (11), LHCII phosphorylation and its importance for state transition is similar to higher plants. The present work focuses on the generation of a library of ARG7 tagged nuclear Chlamydomonas mutants by insertional mutagenesis. Screening methods have been established resulting in the identification of a photoautotrophic clone that shows no state2 to statel transition and appears to be drastically affected in the process of reversible phosphorylation of its LHCII peptides.