Peter Bernard Heifetz

Genetic engineering of the chloroplast.

Transformation of the plastid genome has a number of inherent advantages for the engineering of gene expression in plants. These advantages include: 10-50 times higher transgene expression levels; the absence of gene silencing and position effect variation; the ability to express polycistronic messages from a single promoter; uniparental plastid gene inheritance in most crop plants that prevents pollen transmission of foreign DNA; integration via a homologous recombination process that facilitates targeted gene replacement and precise transgene control; and sequestration of foreign proteins in the organelle which prevents adverse interactions with the cytoplasmic environment. It is now 12 years since the first conclusive demonstration of stable introduction of cloned DNA into the Chlamydomonas chloroplast by the Boynton and Gillham laboratory, and 10 years since the laboratory of Pal Maliga successfully extended these approaches to tobacco. Since then, technical developments in plastid transformation and advances in our understanding of the rules of plastid gene expression have facilitated tremendous progress towards the goal of establishing the chloroplast as a feasible platform for genetic modification of plants.

Herbicide Resistance and Growth of D1 Ala251 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. Ala251 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 Ala251 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 Ala251 mutations differed markedly from an Ala mutant (dr-1) at the well characterized Ser264 D1 residue in terms of herbicide resistance and photoautotrophic growth