Ascorbate deficiency does not limit non-photochemical quenching in Chlamydomonas reinhardtii

Ascorbate (Asc, vitamin C) plays essential roles in development, signaling, hormone biosynthesis, regulation of gene expression, stress resistance and photoprotection. In vascular plants, violaxanthin de-epoxidase (VDE) requires Asc as reductant, thereby it is required for the energy-dependent component of non-photochemical quenching (NPQ). To assess the role of Asc in NPQ in green algae, which are known to contain low amounts of Asc, we searched for an insertional Chlamydomonas reinhardtii mutant affected in the VTC2 gene encoding GDP-L-galactose phosphorylase, which catalyzes the first committed step in the biosynthesis of Asc.. The Crvtc2-1 knockout mutant was viable and, depending on the growth conditions, contained 10 to 20% Asc relative to its wild type. When C. reinhardtii was grown photomixotrophically at moderate light, the zeaxanthin-dependent component of NPQ emerged upon strong red illumination both in the Crvtc2-1 mutant and in its wild type. Deepoxidation was unaffected by Asc deficiency, demonstrating the Chlorophycean VDE found in C. reinhardtii does not require Asc as a reductant. The rapidly induced, energy-dependent NPQ component characteristic of photoautotrophic C. reinhardtiicultures grown at high light was not limited by Asc deficiency either. On the other hand, a reactive oxygen species-induced photoinhibitory NPQ component was greatly enhanced upon Asc deficiency, both under photomixotrophic and photoautotrophic conditions. These results demonstrate Asc has distinct roles in NPQ formation in C. reinhardtiithan in vascular plants

Water-splitting-based, sustainable and efficient H2 production in green algae as achieved by substrate limitation of the Calvin-Benson-Bassam cycle

Abstract Background Photobiological H2 production has the potential of becoming a carbon-free renewable energy source, because upon the combustion of H2, only water is produced. The [Fe–Fe]-type hydrogenases of green algae are highly active, although extremely O2-sensitive. Sulphur deprivation is a common way to induce H2 production, which, however, relies substantially on organic substrates and imposes a severe stress effect resulting in the degradation of the photosynthetic apparatus. Results We report on the establishment of an alternative H2 production method by green algae that is based on a short anaerobic induction, keeping the Calvin–Benson–Bassham cycle inactive by substrate limitation and preserving hydrogenase activity by applying a simple catalyst to remove the evolved O2. Cultures remain photosynthetically active for several days, with the electrons feeding the hydrogenases mostly derived from water. The amount of H2 produced is higher as compared to the sulphur-deprivation procedure and the process is photoautotrophic. Conclusion Our protocol demonstrates that it is possible to sustainably use algal cells as whole-cell catalysts for H2 production, which enables industrial application of algal biohydrogen production

The mechanism of photosystem II inactivation during sulphur deprivation-induced H2 production in Chlamydomonas reinhardtii

Sulphur limitation may restrain cell growth and viability. In the green alga, Chlamydomonas reinhardtii, sulphur limitation may induce H2 production lasting for several days, to be exploited as a renewable energy source. Sulphur limitation causes a large number of physiological changes, including the inactivation of photosystem II (PSII), leading to the establishment of hypoxia, essential for the increase in hydrogenase expression and activity. The inactivation of PSII has been long assumed to be caused by the sulphur-limited turnover of its reaction center protein, PsbA. Here we reinvestigated this issue in detail and show that i) upon transferring Chlamydomonas cells to sulphur-free media, the amount of cellular sulphur content decreases only by about 25%, ii) as demonstrated by lincomycin treatments, PsbA has a significant turnover and other photosynthetic subunits, namely RbcL and CP43, are degraded more rapidly than PsbA. On the other hand, sulphur limitation imposes oxidative stress early on, most probably involving the formation of singlet oxygen in PSII, which leads to an increase in the expression of GDP-L-galactose phosphorylase, playing an essential role in ascorbate biosynthesis. When accumulated to the millimolar concentration range, ascorbate may inactivate the oxygen-evolving complex and provide electrons to PSII albeit at a low rate. In the absence of a functional donor side and sufficient electron transport, PSII reaction centers get inactivated and degraded. We therefore demonstrate that the inactivation of PSII is a complex and multistep process, which may serve to mitigate the damaging effects of sulphur limitation. This article is protected by copyright. All rights reserved