Calredoxin regulates the chloroplast NADPH-dependent thioredoxin reductase in Chlamydomonas reinhardtii.

Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast of Chlamydomonas (Chlamydomonas reinhardtii) with a largely unclear physiological role. We elucidated the CRX functionality by performing in-depth quantitative proteomics of wild-type cells compared with a crx insertional mutant (IMcrx), two CRISPR/Cas9 KO mutants, and CRX rescues. These analyses revealed that the chloroplast NADPH-dependent TRX reductase (NTRC) is co-regulated with CRX. Electron transfer measurements revealed that CRX inhibits NADPH-dependent reduction of oxidized chloroplast 2-Cys peroxiredoxin (PRX1) via NTRC and that the function of the NADPH-NTRC complex is under strict control of CRX. Via non-reducing SDS-PAGE assays and mass spectrometry, our data also demonstrated that PRX1 is more oxidized under high light (HL) conditions in the absence of CRX. The redox tuning of PRX1 and control of the NADPH-NTRC complex via CRX interconnect redox control with active photosynthetic electron transport and metabolism, as well as Ca2+ signaling. In this way, an economic use of NADPH for PRX1 reduction is ensured. The finding that the absence of CRX under HL conditions severely inhibited light-driven CO2 fixation underpins the importance of CRX for redox tuning, as well as for efficient photosynthesis.

Bi-directional electron transfer between H2 and NADPH mitigates light fluctuation responses in green algae.

The metabolism of green algae has been the focus of much research over the last century. These photosynthetic organisms can thrive under various conditions and adapt quickly to changing environments by concomitant usage of several metabolic apparatuses. The main electron coordinator in their chloroplasts, nicotinamide adenine dinucleotide phosphate (NADPH), participates in many enzymatic activities and is also responsible for inter-organellar communication. Under anaerobic conditions, green algae also accumulate molecular hydrogen (H2), a promising alternative for fossil fuels. However, to scale-up its accumulation, a firm understanding of its integration in the photosynthetic apparatus is still required. While it is generally accepted that NADPH metabolism correlates to H2 accumulation, the mechanism of this collaboration is still vague and relies on indirect measurements. Here, we investigated this connection in Chlamydomonas reinhardtii using simultaneous measurements of both dissolved gases concentration, NADPH fluorescence and electrochromic shifts at 520-546 nm. Our results indicate that energy transfer between H2 and NADPH is bi-directional and crucial for the maintenance of redox balance under light fluctuations. At light onset, NADPH consumption initially eventuates in H2 evolution, which initiates the photosynthetic electron flow. Later on, as illumination continues the majority of NADPH is diverted to the Calvin-Benson-Bassham cycle. Dark onset triggers re-assimilation of H2, which produces NADPH and so, enables initiation of dark fermentative metabolism.

Phylloquinone is the principal Mehler reaction site within photosystem I in high light.

Photosynthesis is a vital process, responsible for fixing carbon dioxide, and producing most of the organic matter on the planet. However, photosynthesis has some inherent limitations in utilizing solar energy, and a part of the energy absorbed is lost in the reduction of O2 to produce the superoxide radical (O2•-) via the Mehler reaction, which occurs principally within photosystem I (PSI). For decades, O2 reduction within PSI was assumed to take place solely in the distal iron-sulfur clusters rather than within the two asymmetrical cofactor branches. Here, we demonstrate that under high irradiance, O2 photoreduction by PSI primarily takes place at the phylloquinone of one of the branches (the A-branch). This conclusion derives from the light dependency of the O2 photoreduction rate constant in fully mature wild-type PSI from Chlamydomonas reinhardtii, complexes lacking iron-sulfur clusters, and a mutant PSI, in which phyllosemiquinone at the A-branch has a significantly longer lifetime. We suggest that the Mehler reaction at the phylloquinone site serves as a release valve under conditions where both the iron-sulfur clusters of PSI and the mobile ferredoxin pool are highly reduced.

Juggling Lightning: How Chlorella ohadii handles extreme energy inputs without damage.

The green alga Chlorella ohadii was isolated from a desert biological soil crust, one of the harshest environments on Earth. When grown under optimal laboratory settings it shows the fastest growth rate ever reported for a photosynthetic eukaryote and a complete resistance to photodamage even under unnaturally high light intensities. Here we examined the energy distribution along the photosynthetic pathway under four light and carbon regimes. This was performed using various methodologies such as membrane inlet mass spectrometer with stable O2 isotopes, variable fluorescence, electrochromic shift and fluorescence assessment of NADPH level, as well as the use of specific inhibitors. We show that the preceding illumination and CO2 level during growth strongly affect the energy dissipation strategies employed by the cell. For example, plastid terminal oxidase (PTOX) plays an important role in energy dissipation, particularly in high light- and low-CO2-grown cells. Of particular note is the reliance on PSII cyclic electron flow as an effective and flexible dissipation mechanism in all conditions tested. The energy management observed here may be unique to C. ohadii, as it is the only known organism to cope with such conditions. However, the strategies demonstrated may provide an insight into the processes necessary for photosynthesis under high-light conditions.

Green Algal Hydrogenase Activity Is Outcompeted by Carbon Fixation before Inactivation by Oxygen Takes Place.

Photoproduction of hydrogen by green algae is considered a transitory release valve of excess reducing power and a potential carbon-free source of sustainable energy. It is generally accepted that the transitory production of hydrogen is governed by fast inactivation of hydrogenase by oxygen. However, our data suggest that photosynthetic electron loss to competing processes, mainly carbon fixation, stops hydrogen production, supports hydrogen uptake, and precedes the inevitable inactivation by oxygen. Here, we show that when transitioning from dark anaerobiosis to light, hydrogen production ceases within 2 min, regardless of the presence of oxygen. Simultaneous monitoring of the active hydrogenase pool size shows that it remains entirely intact up to 4 min after illumination and is inactivated only later. Thus, our data reveal a window of 4 min in which the hydrogenase pool is not being degraded by oxygen. Furthermore, we show that electron loss, prominently to carbon fixation, outcompetes hydrogen production and leads to hydrogen uptake. Indeed, supplying additional reducing power to hydrogenase at the cessation point regenerates the accumulation of hydrogen. Our results imply the fast cessation of hydrogen production is governed by electron loss rather than oxygen inactivation, which takes place minutes later.

Microoxic Niches within the Thylakoid Stroma of Air-Grown Chlamydomonas reinhardtii Protect [FeFe]-Hydrogenase and Support Hydrogen Production under Fully Aerobic Environment.

Photosynthetic hydrogen production in the microalga Chlamydomonas reinhardtii is catalyzed by two [FeFe]-hydrogenase isoforms, HydA1 and HydA2, both irreversibly inactivated upon a few seconds exposure to atmospheric oxygen. Until recently, it was thought that hydrogenase is not active in air-grown microalgal cells. In contrast, we show that the entire pool of cellular [FeFe]-hydrogenase remains active in air-grown cells due to efficient scavenging of oxygen. Using membrane inlet mass spectrometry, (18)O2 isotope, and various inhibitors, we were able to dissect the various oxygen uptake mechanisms. We found that both chlororespiration, catalyzed by plastid terminal oxidase, and Mehler reactions, catalyzed by photosystem I and Flavodiiron proteins, significantly contribute to oxygen uptake rate. This rate is considerably enhanced with increasing light, thus forming local anaerobic niches at the proximity of the stromal face of the thylakoid membrane. Furthermore, we found that in transition to high light, the hydrogen production rate is significantly enhanced for a short duration (100 s), thus indicating that [FeFe]-hydrogenase functions as an immediate sink for surplus electrons in aerobic as well as in anaerobic environments. In summary, we show that an anaerobic locality in the chloroplast preserves [FeFe]-hydrogenase activity and supports continuous hydrogen production in air-grown microalgal cells.

A PSII photosynthetic control is activated in anoxic cultures of green algae following illumination.

Photosynthetic hydrogen production from microalgae is considered to have potential as a renewable energy source. Yet, the process has two main limitations holding it back from scaling up; (i) electron loss to competing processes, mainly carbon fixation and (ii) sensitivity to O2 which diminishes the expression and the activity of the hydrogenase enzyme catalyzing H2 production. Here we report a third, hitherto unknown challenge: We found that under anoxia, a slow-down switch is activated in photosystem II (PSII), diminishing the maximal photosynthetic productivity by three-fold. Using purified PSII and applying in vivo spectroscopic and mass spectrometric techniques on Chlamydomonas reinhardtii cultures, we show that this switch is activated under anoxia, within 10 s of illumination. Furthermore, we show that the recovery to the initial rate takes place following 15 min of dark anoxia, and propose a mechanism in which, modulation in electron transfer at the acceptor site of PSII diminishes its output. Such insights into the mechanism broaden our understanding of anoxic photosynthesis and its regulation in green algae and inspire new strategies to improve bio-energy yields.

The dual effect of a ferredoxin-hydrogenase fusion protein in vivo: successful divergence of the photosynthetic electron flux towards hydrogen production and elevated oxygen tolerance.

BACKGROUND: Hydrogen photo-production in green algae, catalyzed by the enzyme [FeFe]-hydrogenase (HydA), is considered a promising source of renewable clean energy. Yet, a significant increase in hydrogen production efficiency is necessary for industrial scale-up. We have previously shown that a major challenge to be resolved is the inferior competitiveness of HydA with NADPH production, catalyzed by ferredoxin-NADP(+)-reductase (FNR). In this work, we explored the in vivo hydrogen production efficiency of Fd-HydA, where the electron donor ferredoxin (Fd) is fused to HydA and expressed in the model organism Chlamydomonas reinhardtii. RESULTS: We show that once the Fd-HydA fusion gene is expressed in micro-algal cells of C. reinhardtii, the fusion enzyme is able to intercept photosynthetic electrons and use them for efficient hydrogen production, thus supporting the previous observations made in vitro. We found that Fd-HydA has a ~4.5-fold greater photosynthetic hydrogen production rate standardized for hydrogenase amount (PHPRH) than that of the native HydA in vivo. Furthermore, we provide evidence suggesting that the fusion protein is more resistant to oxygen than the native HydA. CONCLUSIONS: The in vivo photosynthetic activity of the Fd-HydA enzyme surpasses that of the native HydA and shows higher oxygen tolerance. Therefore, our results provide a solid platform for further engineering efforts towards efficient hydrogen production in microalgae through the expression of synthetic enzymes.