DNA insertional mutagenesis and screening of the green alga Chlamydomonas reinhardtii was employed to isolate tla1, a stable transformant having a truncated light-harvesting chlorophyll antenna size. Molecular analysis showed a single plasmid insertion into an open reading frame of the nuclear genome corresponding to a novel gene (Tla1) that encodes a protein of 213 amino acids. Genetic analysis showed co-segregation of plasmid and tla1 phenotype. Biochemical analyses showed the tla1 mutant to be chlorophyll deficient, with a functional chlorophyll antenna size of photosystem I and photosystem II being about 50% and 65% of that of the wild type, respectively. It contained a correspondingly lower amount of light-harvesting proteins than the wild type and had lower steady-state levels of Lhcb mRNA. The tla1 strain required a higher light intensity for the saturation of photosynthesis and showed greater solar conversion efficiencies and a higher photosynthetic productivity than the wild type under mass culture conditions. Results are discussed in terms of the tla1 mutation, its phenotype, and the role played by the Tla1 gene in the regulation of the photosynthetic chlorophyll antenna size in C. reinhardtii.
KeywordsChlamydomonas AE DNA insertional mutagenesis AE Chlorophyll-deficient mutant AE Light-harvesting antenna AE Photosynthesis AE Solar conversion efficiency Abbreviations Chl: chlorophyll AE CP26 and CP29: chlorophyll proteins 26 and 29 AE PS I and PS II: photosystems I and II AE LHC I and LHC II: light harvesting complex of PS I and PS II, respectively AE PMSF: phenylmethylsulfonyl fluoride AE tla: truncated light-harvesting chlorophyll antenna
DNA insertional transformants of Chlamydomonas reinhardtii were screened chemochromically for attenuated H 2 production. One mutant, displaying low H 2 gas photoproduction, has a nonfunctional copy of a gene that shows high homology to the family of isoamylase genes found in several photosynthetic organisms. DNA gel blotting and gene complementation were used to link this isoamylase gene to previously characterized nontagged sta7 mutants. This mutant is therefore denoted sta7-10. In C. reinhardtii, the STA7 isoamylase gene is important for the accumulation of crystalline starch, and the sta7-10 mutant reported here contains <3% of the glucose found in insoluble starch when compared with wild-type control cells. Hydrogen photoproduction rates, induced after several hours of dark, anaerobic treatment, are attenuated in sta7 mutants. RNA gel blot analysis indicates that the mRNA transcripts for both the HydA1 and HydA2 [Fe]-hydrogenase genes are expressed in the sta7-10 mutant at greater than wild-type levels 0.5 h after anaerobic induction. However, after 1.5 h, transcript levels of both HydA1 and HydA2 begin to decline rapidly and reach nearly undetectable levels after 7 h. In wildtype cells, the hydrogenase transcripts accumulate more slowly, reach a plateau after 4 h of anaerobic treatment, and maintain the same level of expression for >7 h under anaerobic incubation. Complementation of mutant cells with genomic DNA corresponding to the STA7 gene restores both the starch accumulation and H 2 production phenotypes. The results indicate that STA7 and starch metabolism play an important role in C. reinhardtii H 2 photoproduction. Moreover, the results indicate that mere anaerobiosis is not sufficient to maintain hydrogenase gene expression without the underlying physiology, an important aspect of which is starch metabolism.
One of the most notable contrasts between the photorespiratory pathway of higher plants and that of many of the green algae including Chlamydomonas reinhardtii lies in the enzymes that serve for oxidation of glycolate to glyoxylate. The gene disrupted by insertional mutagenesis in a high-CO2-requiring mutant, HCR89, of C. reinhardtii was determined to encode glycolate dehydrogenase (EC 1.1.99.14), which serves as the counterpart of glycolate oxidase (EC 1.1.3.15) in classical higher plant photorespiration. Neither glycolate nor D-lactate oxidation from the membrane fraction of HCR89 was detected. Excretion of over-accumulated glycolate into media due to the absence of glycolate dehydrogenase activity was observed for HCR89 under both high- and low-CO2 conditions. Chlamydomonas glycolate dehydrogenase, CrGDH, with a molecular mass of 118 851 Da, comprises a relatively hydrophobic N-terminal region, a FAD-containing domain homologous to the D subunit of the glycolate oxidase complex from Escherischia coli, and an ironsulfur cluster containing domain homologous to the C subunit of anaerobic glycerol-3-phosphate dehydrogenase complex from Escherichia coli. The second Cys residue in the second ironsulfur cluster motif of CrGDH is replaced by Asp, as CxxDxxCxxxCP, indicating the second ironsulfur cluster coordinates most likely 3Fe4S instead of 4Fe4S. The membrane association of the glycolate dehydrogenase activity agrees with three predicted transmembrane regions on the ironsulfur domain.Key words: algae, Chlamydomonas, CO2, glycolate, lactate, mitochondria, photorespiration, photosynthesis.
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