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Article Dans Une Revue Biochemical Journal Année : 2010

RNA silencing of hydrogenase(-like) genes and investigation of their physiological roles in the green alga Chlamydomonas reinhardtii

James E Godman
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Attila Molnár
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David C Baulcombe
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Janneke Balk
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Résumé

The genome of the green alga Chlamydomonas reinhardtii encodes two [FeFe]-hydrogenases, HydA1 and HydA2, and the hydrogenase-like protein HYD3. The unique combination of these proteins in one eukaryotic cell allows for direct comparison of their in-vivo functions, which have not been established for HydA2 and HYD3. Using a recently developed artificial miRNA silencing method, the expression of HydA1, HydA2 and HYD3 was specifically down-regulated. Silencing of HydA1 resulted in 4-fold lower hydrogenase protein and activity under anaerobe conditions. In contrast, silencing of HydA2 or HYD3 did not affect hydrogen production. Cell lines with strongly (<90%) decreased HYD3 transcript levels grew more slowly than wild-type. The activity of aldehyde oxidase, a cytosolic iron-sulphur (Fe-S) enzyme, was decreased in HYD3 knockdown lines, while Fe-S dependent activities in the chloroplast and mitochondria were unaffected. In addition, the HYD3 knockdown lines grew poorly on hypoxanthine, indicating impaired function of xanthine dehydrogenase, another cytosolic Fe-S enzyme. The expression levels of selected genes in response to hypoxia were unaltered upon HYD3 silencing. Together, our data clearly distinguish the cellular roles of HydA1 and HYD3, and indicate that HYD3, like its yeast and human homologues, has an evolutionary conserved role in the biogenesis or maintenance of cytosolic Fe-S proteins.

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Dates et versions

hal-00525047 , version 1 (11-10-2010)

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James E Godman, Attila Molnár, David C Baulcombe, Janneke Balk. RNA silencing of hydrogenase(-like) genes and investigation of their physiological roles in the green alga Chlamydomonas reinhardtii. Biochemical Journal, 2010, 431 (3), pp.345-351. ⟨10.1042/BJ20100932⟩. ⟨hal-00525047⟩

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