Mutagenesis and suppression of a light-regulated group I intron in the chloroplast psbA gene of Chlamydomonas reinhardtii

Four chloroplast psbA introns in the green alga Chlamydomonas reinhardtii (Cr.psbA1-Cr.psbA4) are self-splicing group I introns and their splicing is lightpromoted. Because evidence indicates that some chloroplast mRNAs are in great excess over what is needed to sustain translation rates, the physiological significance of light-promoted psbA splicing has not been clear. Also, splicing factor(s) for these introns have not been identified. I have generated several point mutations in the core helices of the Cr.psbA4 intron, and tested their effects on self-splicing in vitro. I also replaced the wild-type intron in vivo with each mutant, and compared the in vitro self-splicing data to the effects of these mutations in vivo. The results indicate that destabilizing mutations in the intron core have less effect in vivo than in vitro, suggesting that there is considerable stabilization of the intron’s active structure in vivo. Interestingly, however, there were approximate correlations between in vivo Cr.psbA4 splicing efficiency, synthesis of full-length D1, and autotrophic growth rates. For example, the P4′-3,4 mutant, which showed a 45 % reduction in psbA mRNA, had a 28 % decline in synthesis of full–length D1, and a 18 % decline in photoautotrophic growth rate. These results indicate that psbA mRNA is not in great excess, and that efficient splicing of the psbA introns, which is light-dependent, is essential for maintaining robust autotrophic growth. I have also found that three nuclear suppressors of a mutated 23S rRNA intron (7120, 7151, and 71N1) also suppressed the P4′-3,4 mutation in the Cr.psbA4 intron. This was accomplished by replacing the wild-type Cr.psbA4 intron in the suppressors with the P4′-3,4 mutant; higher steady-state levels of spliced psbA mRNA and less precursor was observed in the suppressors. This result indicates that these genes play a role in splicing of multiple group I introns in the chloroplast. In addition, further genetic analysis was performed with the 7120 suppressor, which had been only partially characterized previously. Tetrad analysis of a cross of 7120 (mt+) with wild-type (mt-) indicated that it is a single nuclear gene mutation. Complementation analysis in diploids demonstrated that the suppressor mutation is dominant. We propose to call this gene css2 (for chloroplast splicing suppressor).