Calcium-dependent regulation of cyclic photosynthetic electron transfer by a CAS, ANR1, and PGRL1 complex.

Cyclic photosynthetic electron flow (CEF) is crucial to photosynthesis because it participates in the control of chloroplast energy and redox metabolism, and it is particularly induced under adverse environmental conditions. Here we report that down-regulation of the chloroplast localized Ca(2+) sensor (CAS) protein by an RNAi approach in Chlamydomonas reinhardtii results in strong inhibition of CEF under anoxia. Importantly, this inhibition is rescued by an increase in the extracellular Ca(2+) concentration, inferring that CEF is Ca(2+)-dependent. Furthermore, we identified a protein, anaerobic response 1 (ANR1), that is also required for effective acclimation to anaerobiosis. Depletion of ANR1 by artificial microRNA expression mimics the CAS-depletion phenotype, and under anaerobic conditions the two proteins coexist within a large active photosystem I-cytochrome b(6)/f complex. Moreover, we provide evidence that CAS and ANR1 interact with each other as well as with PGR5-Like 1 (PGRL1) in vivo. Overall our data establish a Ca(2+)-dependent regulation of CEF via the combined function of ANR1, CAS, and PGRL1, associated with each other in a multiprotein complex.

Control of hydrogen photoproduction by the proton gradient generated by cyclic electron flow in Chlamydomonas reinhardtii.

Hydrogen photoproduction by eukaryotic microalgae results from a connection between the photosynthetic electron transport chain and a plastidial hydrogenase. Algal H2 production is a transitory phenomenon under most natural conditions, often viewed as a safety valve protecting the photosynthetic electron transport chain from overreduction. From the colony screening of an insertion mutant library of the unicellular green alga Chlamydomonas reinhardtii based on the analysis of dark-light chlorophyll fluorescence transients, we isolated a mutant impaired in cyclic electron flow around photosystem I (CEF) due to a defect in the Proton Gradient Regulation Like1 (PGRL1) protein. Under aerobiosis, nonphotochemical quenching of fluorescence (NPQ) is strongly decreased in pgrl1. Under anaerobiosis, H2 photoproduction is strongly enhanced in the pgrl1 mutant, both during short-term and long-term measurements (in conditions of sulfur deprivation). Based on the light dependence of NPQ and hydrogen production, as well as on the enhanced hydrogen production observed in the wild-type strain in the presence of the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, we conclude that the proton gradient generated by CEF provokes a strong inhibition of electron supply to the hydrogenase in the wild-type strain, which is released in the pgrl1 mutant. Regulation of the trans-thylakoidal proton gradient by monitoring pgrl1 expression opens new perspectives toward reprogramming the cellular metabolism of microalgae for enhanced H2 production.

Hydrogen production by photoautotrophic sulfur-deprived Chlamydomonas reinhardtii pre-grown and incubated under high light.

We have previously demonstrated that Chlamydomonas reinhardtii can produce hydrogen under strictly photoautotrophic conditions during sulfur deprivation [Tsygankov et al. (2006); Int J Hydrogen Energy 3:1574-1584]. The maximum hydrogen photoproduction was achieved by photoautotrophic cultures pre-grown under a low light regime (25 microE m(-2) s(-1)). We failed to establish sustained hydrogen production from cultures pre-grown under high light (100 microE m(-2) s(-1)). A new approach for sustained hydrogen production by these cultures is presented here. Assuming that stable and reproducible transition to anerobiosis as well as high starch accumulation are important for hydrogen production, the influence of light intensity and dissolved oxygen concentration during the oxygen evolving stage of sulfur deprivation were investigated in cultures pre-grown under high light. Results showed that light higher than 175 microE m(-2) s(-1) during sulfur deprivation induced reproducible transition to anerobiosis, although the total amount of starch accumulation and hydrogen production were insignificant. The potential PSII activity measured in the presence of an artificial electron acceptor (DCBQ) and an inhibitor of electron transport (DBMIB) did not change in cultures pre-grown under 20 microE m(-2) s(-1) and incubated under 150 microE m(-2) s(-1) during sulfur deprivation. In contrast, the potential PSII activity decreased in cultures pre-grown under 100 microE m(-2) s(-1) and incubated under 420 microE m(-2) s(-1). This indicates that cultures grown under higher light experience irreversible inhibition of PSII in addition to reversible down regulation. High dissolved O(2) content during the oxygen evolving stage of sulfur deprivation has a negative regulatory role on PSII activity. To increase hydrogen production by C. reinhardtii pre-grown under 100 microE m(-2) s(-1), cultures were incubated under elevated PFD and decreased oxygen pressure during the oxygen evolving stage. These cultures reproducibly reached anaerobic stage, accumulated significant quantities of starch and produced significant quantities of H(2). It was found that elevation of pH from 7.4 to 7.7 during the oxygen producing stage of sulfur deprivation led to a significant increase of accumulated starch. Thus, control of pH during sulfur deprivation is a possible way to further optimize hydrogen production by photoautotrophic cultures.