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

The genome of the green alga Chlamvdomonas 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 an artificial microRNA silencing method developed recently, the expression of HydA1, HydA2 and HYD3 was specifically down-regulated Silencing of HydA1 resulted in 4-fold lower hydrogenase protein and activity under anaerobic conditions In contrast silencing of liydA2 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 Fe-S enzyme was decreased in HYD3-knockdown lines, whereas 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 results 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