INDUCTION AND SEGREGATION OF CHLOROPLAST MUTATIONS IN VEGETATIVE CELL CULTURES OF CHLAMYDOMONAS REINHARDTII

ABSTRACT The single chloroplast of the alga Chlamydomonas reinhardtii contains at least 100 copies of the chloroplast chromosome. It is not known how the chloroplast (or cell) becomes homoplasmic for a mutation that arises in one of these copies. Under suitable selection conditions, clones with chloroplast mutations for streptomycin resistance induced by methyl methanesulfonate can be recovered with direct plating after mutagenesis. Using an adaptation of the Luria-Delbrück fluctuation test, mutagenized cultures grown on nonselective liquid medium for seven to nine doublings show negligible proliferation of cells capable of forming such mutant colonies. In contrast, cells among the same cultures with reduced nuclear mutations conferring streptomycin resistance reveal considerable clonal propagation prior to plating on selection medium. Reconstruction growth-rate experiments show no reduced growth of cells with chloroplast mutations relative to either wild-type cells or to those with nuclear mutations. We propose that newly arising chloroplast mutations and their copies are usually transmitted to only one daughter cell for several cell generations by reductional divisions of the chloroplast genome. In the absence of recombination and mixing, such a reductional partition of chloroplast alleles would readily permit the formation of homoplasmic lines without the need for selection.

A METHOD FOR COMPLEMENTATION ANALYSIS OF NUCLEAR AND CHLOROPLAST MUTANTS OF PHOTOSYNTHESIS IN CHLAMYDOMONAS

ABSTRACT The photosynthetic properties of young zygotes of Chlamydomonas reinhardi were analyzed. In heterozygotes for two nuclear or two chloroplast mutations affecting photosynthesis, recovery of photosynthetic activity was observed that is most likely the result of intergenic complementation.—We observed that chloramphenicol inhibited the recovery of activity in double heterozygotes for mutants lacking at least one thylakoid polypeptide of chloroplast origin, while it had no effect on wild-type homozygotes. This indicates that the recovery of activity in double heterozygotes could result from the repair of existing thylakoid membranes by de nouo synthesis of the missing polypeptides.