We have investigated transcript level changes of CO(2)-concentrating mechanism (CCM) genes during light-dark (12 h:12 h) cycles in synchronized Chlamydomonas reinhardtii at air-level CO(2). CCM gene transcript levels vary at various times of light-dark cycles, even at same air-level CO(2). Transcripts of inorganic carbon transporter genes (HLA3, LCI1, CCP1, CCP2 and LCIA) and mitochondrial carbonic anhydrase genes (CAH4 and CAH5) are up regulated in light, following which their levels decline in dark. Contrastingly, transcripts of chloroplast carbonic anhydrases namely CAH6, CAH3 and LCIB are up regulated in dark. CAH3 and LCIB transcript levels reached maximum by the end of dark, followed by high expression into early light period. In contrast, CAH6 transcript level stayed high in dark, followed by high level even in light. Moreover, the up regulation of transcripts in dark was undone by high CO(2), suggesting that the dark induced CCM transcripts were regulated by CO(2) even in dark when CCM is absent. Thus while the CAH3 transcript level modulations appear not to positively correlate with that of CCM, the protein regulation matched with CCM status: in spite of high transcript levels in dark, CAH3 protein reached peak level only in light and localized entirely to pyrenoid, a site functionally relevant for CCM. Moreover, in dark, CAH3 protein level not only reduced but also the protein localized as a diffused pattern in chloroplast. We propose that transcription of most CCM genes, followed by protein level changes including their intracellular localization of a subset is subject to light-dark cycles.

UVI31+ is an evolutionarily conserved BolA family protein. In this study we examine the presence, localization and possible functions of this protein in the context of a unicellular alga, Chlamydomonas reinhardtii. UVI31+ in C. reinhardtii exhibits DNA endonuclease activity and is induced upon UV stress. Further, UVI31+ that normally localizes to the cell wall and pyrenoid regions gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV stress. The observed induction upon UV-stress as well as the endonuclease activity suggests plausible role of this protein in DNA repair. We have also observed that UV31+ is induced in C. reinhardtii grown in dark conditions, whereby the protein localization is enhanced in the pyrenoid. Biomolecular interaction between the purified pyrenoids and UVI31+ studied by NMR demonstrates the involvement of the disordered loop domain of the protein in its interaction.

Carbon concentrating mechanism (CCM) and photorespiration (PR) are interlinked and co-regulated in Chlamydomonas reinhardtii, but conditions where co-regulation alters are not sufficiently explored. Here, we uncover that PR gene transcripts, like CCM transcripts, are induced even in the dark when both processes are not active. Such diurnal cycles show that transcript levels peak in the middle of 12 h day, decline by early part of 12-h dark followed by their onset again at mid-dark. Interestingly, the onset in the mid-dark phase is sensitive to high CO, implying that the active carbon sensing mechanism operates even in the dark. The rhythmic alterations of both CCM and PR transcript levels are unlinked to circadian clock: the "free-running state" reveals no discernible rhythmicity in transcript changes. Only continuous light leads to high transcript levels but no detectable transcripts were observed in continuous dark. Asynchronous continuous light cultures, upon shifting to low from high CO exhibit only transient induction of PR transcripts/proteins while CCM transcript induction is stable, indicating the loss of co-regulation between PR and CCM gene transcription. Lastly, we also describe that both CCM and PR transcripts/proteins are induced in low CO even in mixotrophic cultures, but only in high light, the same being attenuated in high CO, implying that high light is a mandatory "trigger" for CCM and PR induction in low CO mixotrophy. Our study provides comprehensive analyses of conditions where CCM and PR were differently regulated, setting a paradigm for a detailed mechanistic probing of these responses.