Induction of alternative pathway respiration by nitrate in Chlamydomonas

Abstract Besides the ubiquitous cytochrome pathway of mitochondrial respiration, the green algae Chlamydomonas reinhardtii possesses an alternative pathway of respiration, which is comprised of a single protein, alternative oxidase (AOX). AOX is induced when C. reinhardtii cells are shifted from a growth medium containing ammonium as the nitrogen source to one with nitrate. The primary aim of this thesis was to understand the metabolic connections between nitrate assimilation and the induction of the alternative pathway. That these two metabolic processes are closely linked is supported by the fact that a gene encoding AOX (AOX1) is clustered with the genes required for nitrate assimilation (NAR genes). To investigate if the clustering of AOX1 with NAR genes occurs in other green algae, publicly available genome databases were searched. It was found that while the clustering of NAR genes is widespread in many lineages of green algae, the presence of AOX1 within a NAR cluster is a characteristic of only a single group of green algae, the chlorophytes. Interestingly, it was found that lineages that lack NAR gene clustering seem to have a preference for importing amino acids instead of nitrate as their dominant source of nitrogen. The role of AOX in nitrate assimilation was investigated by constitutively blocking AOX1 expression. AOX knockdown cells displayed a slightly decreased rate of growth and distinctly different photosynthetic electron transport characteristics. Whole-cell metabolite analysis showed that the lack of AOX in the mitochondrion in knockdown cells suppressed the stimulatory effect of nitrate on oxidative pentose phosphate pathway (oxPPP) activity within the chloroplast. Interestingly, this regulation also occurred in the opposite direction. In wild-type cells, the biochemical inhibition of oxPPP by glycerol in the chloroplast decreased the accumulation of AOX in the mitochondrion. Overall, the data suggests that the induction of AOX by nitrate relieves respiratory electron transport from adenylate control. During nitrate assimilation increased demand by the chloroplast for ATP would decrease the transfer of ADP and Pi to the mitochondria restricting the rate of the cytochrome pathway. Up regulation of the alternative pathway would allow for continued upstream respiratory carbon flow under limiting adenylate conditions. Keywords Alternative oxidase, nitrate, Chlamydomonas, respiration, oxidative pentose phosphate pathwa