First published online July 21, 2004; 10.1105/tpc.104.021972
The Plant Cell 16:2151-2163 (2004)
© 2004 American Society of Plant Biologists
Hydrogen Photoproduction Is Attenuated by Disruption of an Isoamylase Gene in Chlamydomonas reinhardtii
Matthew C. Posewitza,
Sharon L. Smolinskia,
Saradadevi Kanakagirib,
Anastasios Melisb,
Michael Seiberta and
Maria L. Ghirardia,1
a National Renewable Energy Laboratory, Golden, Colorado 80401
b University of California, Berkeley, California 94720
1 To whom correspondence should be addressed. E-mail maria_ghirardi{at}nrel.gov; fax 303-384-6150.
DNA insertional transformants of Chlamydomonas reinhardtii were screened chemochromically for attenuated H2 production. One mutant, displaying low H2 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 wild-type 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 H2 production phenotypes. The results indicate that STA7 and starch metabolism play an important role in C. reinhardtii H2 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.
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