Herbicide Resistance and Growth of D1 Ala 251 Mutants in Chlamydomonas

We elucidated the effects of substituting seven amino acids for Ala at residue 251 of the Chlamydomonas reinhardtii D1 protein on herbicide resistance and photoautotrophic growth. Ala 251 has been suggested to play a key role in the structural integrity and function of the stromal loop between transmembrane helices IV and V of D1 and has previously been shown to affect resistance to “classical” PSII specific herbicides. Sensitive and rapid microtiter assays were employed to compare herbicide resistance and photoautotrophic growth in the various mutants. Substitution of Ala 251 by Ile, Leu or Val conferred resistance to the PSII herbicides atrazine, bromacil and metribuzin but not to DCMU, and impaired photoautotrophic growth in high and low light. Compared to an otherwise isogenic wildtype strain, the lie and Val mutants exhibited nearly identical levels of herbicide resistance and reduced growth while the Leu mutant had even slower growth and higher levels of herbicide resistance. In contrast Cys, Pro, Ser and Gly mutants were phenotypically indistinguishable from wildtype in terms of herbicide sensitivity and photoautotrophic doubling times. Collectively the seven Ala 251 mutations differed markedly from an Ala mutant (dr-1) at the well characterized Ser 264 D1 residue in terms of herbicide resistance and photoautotrophic growth