Jeanne M. Erickson

Herbicide resistance and cross-resistance: changes at three distinct sites in the herbicide-binding protein.

Plants and algae resistant to the commonly used s-triazine herbicides display a wide spectrum of cross-resistance to other herbicides that act in a similar manner. Analysis of uniparental mutants of the green alga Chlamydomonas reinhardi showed that three different amino acid residues in the 32-kilodalton thylakoid membrane protein can be independently altered to produce three different patterns of resistance to s-triazine and urea-type herbicides. These results clarify the molecular basis for herbicide resistance and cross-resistance. Two of the mutations do not alter normal electron transport and thus may have applications of agronomic interest.

Chlamydomonas reinhardii gene for the 32 000 mol. wt. protein of photosystem II contains four large introns and is located entirely within the chloroplast inverted repeat.

The chloroplast psbA gene from the green unicellular alga Chlamydomonas reinhardii has been localized, cloned and sequenced. This gene codes for the rapidly‐labeled 32‐kd protein of photosystem II, also identified as as herbicide‐binding protein. Unlike psbA in higher plants which is found in the large single copy region of the chloroplast genome and is uninterrupted, psbA in C. reinhardii is located entirely within the inverted repeat, hence present in two identical copies per circular chloroplast genome, and contains four large introns. These introns range from 1.1 to 1.8 kb in size and fall into the category of Group I introns. Two of the introns contain open reading frames which are in‐frame with the preceding exon sequences. We present the nucleotide sequence for the C. reinhardii psbA 5′‐and 3′ ‐flanking sequences, the coding region contained in five exons and the deduced amino acid sequence. The algal gene codes for a protein of 352 amino acid residues which is 95% homologous, excluding the last eight amino acid residues, with the higher plant protein.

Lack of the D2 protein in a Chlamydomonas reinhardtii psbD mutant affects photosystem II stability and D1 expression

D1 and D2, two chloroplast proteins with apparent mol. wt of 32 000‐34 000, play an important role in the photosynthetic reactions mediated by the membrane‐bound protein complex of photosystem II (PSII). We have isolated and characterized an uniparental, non‐photosynthetic mutant of Chlamydomonas reinhardtii and show that the mutation is in the chloroplast gene psbD, coding for D2. A 46 bp direct DNA duplication in the coding region of the mutant gene causes a frame‐shift which results in a psbD transcript coding for 186 amino acid residues instead of the normal 352. The truncated D2 peptide is never seen, even after pulse‐labeling, suggesting that the mutant protein is very unstable. In addition, little or no D1 protein is detected in this mutant although the gene and normal levels of mRNA for D1 are present in mutant cells. All other core PSII proteins are synthesized and inserted into the membrane fraction, but never accumulate. These results suggest that D2 contributes not only to the stabilization of the PSII complex in the membrane, but also may play a specific role in the regulation of the D1 protein, either at the translational or post‐translational level.

Function and assembly of photosystem II: genetic and molecular analysis

The isolation and molecular characterization of mutants that are resistant to herbicides and deficient in photosystem II activity in higher plants, eukaryotic algae and cyanobacteria, has greatly improved our understanding of the structure, function and assembly of this important photosynthetic complex.

Characterization of photosystem II mutants of Chlamydomonas reinhardii lacking the psbA gene

SummaryWe have examined 78 chloroplast mutants of Chlamydomonas reinhardii lacking photosystem II activity. Most of them are unable to synthesize the 32 Kdalton protein. Analysis of 22 of these mutants reveals that they have deleted both copies of the psbA gene (which codes for the 32 Kdalton protein) in their chloroplast genome. Although these mutants are able to synthesize and to integrate the other photosystem II polypeptides in the thylakoid membranes, they are unable to assemble a stable functional photosystem II complex. The 32 Kprotein appears therefore to play an important role not only in photosystem II function, but also in stabilizing this complex.

Molecular and biophysical analysis of herbicide-resistant mutants of Chlamydomonas reinhardtii: structure-function relationship of the photosystem II D1 polypeptide.

Plants and green algae can develop resistance to herbicides that block photosynthesis by competing with quinones in binding to the chloroplast photosystem II (PSII) D1 polypeptide. Because numerous herbicide-resistant mutants of Chlamydomonas reinhardtii with different patterns of resistance to such herbicides are readily isolated, this system provides a powerful tool for examining the interactions of herbicides and endogenous quinones with the photosynthetic membrane, and for studying the structure-function relationship of the D1 protein with respect to PSII electron transfer. Here we report the results of DNA sequence analysis of the D1 gene from four mutants not previously characterized at the molecular level, the correlation of changes in specific amino acid residues of the D1 protein with levels of resistance to the herbicides atrizine, diuron, and bromacil, and the kinetics of fluorescence decay for each mutant, which show that changes at two different amino acid residues dramatically slow PSII electron transfer. Our analyses, which identify a region of 57 amino acids of the D1 polypeptide involved in herbicide binding and which define a D1 binding niche for the second quinone acceptor, QB of PSII, provide a strong basis of support for structural and functional models of the PSII reaction center.

A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chlamydomonas reinhardtii.

The light‐independent pathway of chlorophyll synthesis which occurs in some lower plants and algae is still largely unknown. We have characterized a chloroplast mutant, H13, of Chlamydomonas reinhardtii which is unable to synthesize chlorophyll in the dark and is also photosystem I deficient. The mutant has a 2.8 kb deletion as well as other rearrangements of its chloroplast genome. By performing particle gun mediated chloroplast transformation of H13 with defined wild‐type chloroplast DNA fragments, we have identified a new chloroplast gene, chlN, coding for a 545 amino acid protein which is involved in the light‐independent accumulation of chlorophyll, probably at the step of reduction of protochlorophyllide to chlorophyllide. The chlN gene is also found in the chloroplast genomes of liverwort and pine, but is absent from the chloroplast genomes of tobacco and rice.

Chloroplast Transformation: Current Results and Future Prospects

Genetic engineering of proteins is a powerful tool used in both basic and applied research. In vitro alteration of the primary structure of a gene and subsequent introduction of the mutated DNA into the genome of a living cell allows the directed manipulation of protein structure. Such an approach, often termed ‘reverse genetics,’ has been widely used to investigate the complex relationship between the structure of a protein and its function, and to explore the intricacies of biochemical and developmental pathways. An obvious prerequisite for genetic engineering is the ability to introduce DNA into a living cell in such a way that it is stably maintained and properly expressed in the appropriate genome of the host cell. The genome of a prokaryote is in the cell cytoplasm and generally consists of one or a few copies of a large DNA molecule. In contrast, photosynthetic eukaryotes contain three distinct genomes, each located within a subcellular organelle enveloped by one or more membranes and hence separated from the cytoplasm. The three plant cell genomes are those of the nucleus, the mitochondrion, and the plastid. The genome of the chloroplast, the plastid type found in photosynthetic cells, presents a complex genetic target because there are often hundreds of copies of the circular chloroplast DNA molecule per chloroplast, and often hundreds of chloroplasts per cell. Obtaining a plant cell in which every resident copy of a given chloroplast gene has been replaced by an engineered, mutant gene copy is an essential step in experiments involving DNA-mediated chloroplast transformation. An ideal model organism for such studies is provided by the unicellular green alga Chlamydomonas reinhardtii, which contains a single large chloroplast. This chapterpresents current results and future prospects for chloroplast transformation, both in Chlamydomonas and in plants of agronomic interest.

Chloroplast Gene Function: Combined Genetic and Molecular Approach in Chlamydomonas Reinhardii

Chlamydanonas reinhardii is a green unicellular algae that is particularly well-suited for the study of chloroplast gene function and regulation. It contains a single, large chloroplast which comprises about 40% of the cell volume. Moreover, the life cycle of this alga involves sexual recanbination in which haploid gametes of opposite mating type (+ and −) fuse to form a diploid zygote (Fig. 1). At this point, the chloroplasts from both gametes fuse, but the chloroplast genaue is not inherited in mendelian fashion. Instead, the zygote retains only the chloroplast genome from the mating type + or maternal parent (Gillham, 1978). Fortunately, for those interested in chloroplast genetics, this uniparental maternal inheritance occurs about 95% of the time, so that approximately 5% of the zygotes are either uniparental paternal or biparental with respect to the chloroplast genome. And it is in these exceptional, biparental zygotes that recanbination occurs between the maternal and paternal chloroplast genes, at a frequency that allows for genetic mapping of chloroplast DNA. Thus, coupling the classical genetic approach and the ability to obtain and map mutants with the techniques of molecular biology and the ability to isolate, analyze and in some cases modify individual genes, their messages and their protein products, provides a powerful framework for studying the function and expression of chloroplast genes.

Chlamydomonas Reinhardii, A Potential Model System for Chloroplast Gene Manipulation

Studies on the structure, function and regulation of genes coding for chloroplast proteins are important for understanding the biosynthesis of the photosynthetic apparatus and the integration of chloroplasts within plant cells. Chlamydomonas reinhardii is particularly well suited for solving these problems because this green unicellular alga can be manipulated with ease both at the biochemical and genetic level. Several genes have been identified on the physical map of the chloroplast genome. They include genes coding for ribosomal RNA, tRNA and several proteins including the large subunit of ribulose 1,5 bisphosphate carboxylase (RubisCo) and several thylakoid polypeptides. The nuclear gene for the small subunit of RubisCo has also been cloned. Because chloroplast DNA recombination occurs in C. reinhardii, a rare property among plants, chloroplast genes can be analyzed by genetic means. Numerous chloroplast photosynthetic mutations have been isolated and several of them have been shown to be part of a single linkage group (Gillham, 1978). We have reached the stage where the genetic and biochemical approaches can be coupled efficiently in C. reinhardii; in particular, it has been possible to correlate the physical and genetic chloroplast DNA maps at a few sites.