Complementation to identify novel nuclear genes implicated in photosynthesis

Xenie Johnson, Richard Kuras, Giovanni Finazzi, Francis-André Wollman and Olivier Vallon

Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, Paris 75005

Non-photosynthetic mutants of Chlamydomonas reinhardtii fall into two classes of nuclear genes: those directly involved in photosynthesis, and those that participate in biogenesis of the photosynthetic apparatus (for example involved in co-factor assembly or in the expression of a specific chloroplast gene). We have devised a technique to rapidly identify unknown nuclear genes belonging to these two classes. In a genetic background optimised for transformation efficiency and genetic analysis, we have generated a library of 34 acetate requiring (ac) non-photosynthetic mutants using insertional mutagenesis with the ble and aphVIII antibiotics resistance markers. Using fluorescence induction, absorption spectroscopy or biochemical analysis, they were categorized as lacking PSII (8), cytochrome b6f (11), plastocyanin (1), PSI (9) or ATP synthase (1), or residing in steps downstream from the electron transfer chain (4). In order to identify the deleted gene, we transform the mutants in groups of four with an indexed cosmid library. We are currently analysing mutants with a b6f signature, falling into 4 phenotypic classes. A number of these mutants have been complemented and we are pursuing the identification of the affected genes. We believe that this approach will be useful to rapidly identify novel Chlamydomonas genes implicated in photosynthetic processes. We will present in detail two independent mutants with WT fluorescence that are photosensitive and low oxygen-evolving. Western blotting reveals an absence of Rubisco Large and Small Subunits, and the rbcL mRNA is undetectable. The two mutants are complemented by the same gene encoding a Pentatricopeptide Repeat protein (PPR). Because it regulates the stability of the mRNA for RbcL, it has been called MRL1. Transformation with reporter constructs containing the 5' or 3' UTRs of rbcL indicates that the target of MRL1 resides in the 5' UTR. This is the second time that a Chlamydomonas PPR protein has been identified as stabilizing a chloroplast transcript via its 5' UTR.

e-mail address of presenting author: xenie.johnson@ibpc.fr