Exceptional Quantum Efficiency Powers Biomass Production in Halotolerant Algae Picochlorum sp.

The green algal genus Picochlorum is of biotechnological interest because of its robust response to multiple environmental stresses. We compared the metabolic performance of P. SE3 and P. oklahomense to diverse microbial phototrophs and observed exceptional performance of photosystem II (PSII) in light energy conversion in both Picochlorum species. The quantum yield (QY) for O2 evolution is the highest of any phototroph yet observed, 32% (20%) by P. SE3 (P. okl) when normalized to total PSII subunit PsbA (D1) protein, and 80% (75%) normalized per active PSII, respectively. Three factors contribute: (1) an efficient water oxidizing complex (WOC) with the fewest photochemical misses of any organism; (2) faster reoxidation of reduced (PQH2)B in P. SE3 than in P. okl. (period-2 Fourier amplitude); and (3) rapid reoxidation of the plastoquinol pool by downstream electron carriers (Cyt b6f/PETC) that regenerates PQ faster in P. SE3. This performance gain is achieved without significant residue changes around the QB site and thus points to a pull mechanism involving faster PQH2 reoxidation by Cyt b6f/PETC that offsets charge recombination. This high flux in P. SE3 may be explained by genomically encoded plastoquinol terminal oxidases 1 and 2, whereas P. oklahomense has neither. Our results suggest two distinct types of PSII centers exist, one specializing in linear electron flow and the other in PSII-cyclic electron flow. Several amino acids within D1 differ from those in the low-light-descended D1 sequences conserved in Viridiplantae, and more closely match those in cyanobacterial high-light D1 isoforms, including changes near tyrosine Yz and a water/proton channel near the WOC. These residue changes may contribute to the exceptional performance of Picochlorum at high-light intensities by increasing the water oxidation efficiency and the electron/proton flux through the PSII acceptors (QAQB).

Regulation of light energy conversion between linear and cyclic electron flow within photosystem II controlled by the plastoquinone/quinol redox poise.

Ultrapurified Photosystem II complexes crystalize as uniform microcrystals (PSIIX) of unprecedented homogeneity that allow observation of details previously unachievable, including the longest sustained oscillations of flash-induced O2 yield over > 200 flashes and a novel period-4.7 water oxidation cycle. We provide new evidence for a molecular-based mechanism for PSII-cyclic electron flow that accounts for switching from linear to cyclic electron flow within PSII as the downstream PQ/PQH2 pool reduces in response to metabolic needs and environmental input. The model is supported by flash oximetry of PSIIX as the LEF/CEF switch occurs, Fourier analysis of O2 flash yields, and Joliot-Kok modeling. The LEF/CEF switch rebalances the ratio of reductant energy (PQH2) to proton gradient energy (H+o/H+i) created by PSII photochemistry. Central to this model is the requirement for a regulatory site (QC) with two redox states in equilibrium with the dissociable secondary electron carrier site QB. Both sites are controlled by electrons and protons. Our evidence fits historical LEF models wherein light-driven water oxidation delivers electrons (from QA-) and stromal protons through QB to generate plastoquinol, the terminal product of PSII-LEF in vivo. The new insight is the essential regulatory role of QC. This site senses both the proton gradient (H+o/H+i) and the PQ pool redox poise via e-/H+ equilibration with QB. This information directs switching to CEF upon population of the protonated semiquinone in the Qc site (Q-H+)C, while the WOC is in the reducible S2 or S3 states. Subsequent photochemical primary charge separation (P+QA-) forms no (QH2)B, but instead undergoes two-electron backward transition in which the QC protons are pumped into the lumen, while the electrons return to the WOC forming (S1/S2). PSII-CEF enables production of additional ATP needed to power cellular processes including the terminal carboxylation reaction and in some cases PSI-dependent CEF.

Proximity-based proteomics reveals the thylakoid lumen proteome in the cyanobacterium Synechococcus sp. PCC 7002.

Cyanobacteria possess unique intracellular organization. Many proteomic studies have examined different features of cyanobacteria to learn about the intracellular structures and their respective functions. While these studies have made great progress in understanding cyanobacterial physiology, the conventional fractionation methods used to purify cellular structures have limitations; specifically, certain regions of cells cannot be purified with existing fractionation methods. Proximity-based proteomics techniques were developed to overcome the limitations of biochemical fractionation for proteomics. Proximity-based proteomics relies on spatiotemporal protein labeling followed by mass spectrometry of the labeled proteins to determine the proteome of the region of interest. We performed proximity-based proteomics in the cyanobacterium Synechococcus sp. PCC 7002 with the APEX2 enzyme, an engineered ascorbate peroxidase. We determined the proteome of the thylakoid lumen, a region of the cell that has remained challenging to study with existing methods, using a translational fusion between APEX2 and PsbU, a lumenal subunit of photosystem II. Our results demonstrate the power of APEX2 as a tool to study the cell biology of intracellular features and processes, including photosystem II assembly in cyanobacteria, with enhanced spatiotemporal resolution.

Photosystem II-cyclic electron flow powers exceptional photoprotection and record growth in the microalga Chlorella ohadii.

The desert microalga Chlorella ohadii was reported to grow at extreme light intensities with minimal photoinhibition, tolerate frequent de/re-hydrations, yet minimally employs antenna-based non-photochemical quenching for photoprotection. Here we investigate the molecular mechanisms by measuring Photosystem II charge separation yield (chlorophyll variable fluorescence, Fv/Fm) and flash-induced O2 yield to measure the contributions from both linear (PSII-LEF) and cyclic (PSII-CEF) electron flow within PSII. Cells grow increasingly faster at higher light intensities (µE/m2/s) from low (20) to high (200) to extreme (2000) by escalating photoprotection via shifting from PSII-LEF to PSII-CEF. This shifts PSII charge separation from plastoquinone reduction (PSII-LEF) to plastoquinol oxidation (PSII-CEF), here postulated to enable proton gradient and ATP generation that powers photoprotection. Low light-grown cells have unusually small antennae (332 Chl/PSII), use mainly PSII-LEF (95%) and convert 40% of PSII charge separations into O2 (a high O2 quantum yield of 0.06mol/mol PSII/flash). High light-grown cells have smaller antenna and lower PSII-LEF (63%). Extreme light-grown cells have only 42 Chl/PSII (no LHCII antenna), minimal PSII-LEF (10%), and grow faster than any known phototroph (doubling time 1.3h). Adding a synthetic quinone in excess to supplement the PQ pool fully uncouples PSII-CEF from its natural regulation and produces maximum PSII-LEF. Upon dark adaptation PSII-LEF rapidly reverts to PSII-CEF, a transient protection mechanism to conserve water and minimize the cost of antenna biosynthesis. The capacity of the electron acceptor pool (plastoquinone pool), and the characteristic times for exchange of (PQH2)B with PQpool and reoxidation of (PQH2)pool were determined.

The strontium inorganic mutant of the water oxidizing center (CaMn4O5) of PSII improves WOC efficiency but slows electron flux through the terminal acceptors.

Herein we extend prior studies of biosynthetic strontium replacement of calcium in PSII-WOC core particles to characterize whole cells. Previous studies of Thermosynechococcus elongatus found a lower rate of light-saturated O2 from isolated PSII-WOC(Sr) cores and 5-8× slower rate of oxygen release. We find similar properties in whole cells, and show it is due to a 20% larger Arrhenius activation barrier for O2 evolution. Cellular adaptation to the sluggish PSII-WOC(Sr) cycle occurs in which flux through the QAQB acceptor gate becomes limiting for turnover rate in vivo. Benzoquinone derivatives that bind to QB site remove this kinetic chokepoint yielding 31% greater O2 quantum yield (QY) of PSII-WOC(Sr) vs. PSII-WOC(Ca). QY and efficiency of the WOC(Sr) catalytic cycle are greatly improved at low light flux, due to fewer misses and backward transitions and 3-fold longer lifetime of the unstable S3 state, attributed to greater thermodynamic stabilization of the WOC(Sr) relative to the photoactive tyrosine YZ. More linear and less cyclic electron flow through PSII occurs per PSII-WOC(Sr). The organismal response to the more active PSII centers in Sr-grown cells at 45°C is to lower the number of active PSII-WOC per Chl, producing comparable oxygen and energy per cell. We conclude that redox and protonic energy fluxes created by PSII are primary determinants for optimal growth rate of T. elongatus. We further conclude that the (Sr-favored) intermediate-spin S=5/2 form of the S2 state is the active form in the catalytic cycle relative to the low-spin S=1/2 form.

The Oxygen quantum yield in diverse algae and cyanobacteria is controlled by partitioning of flux between linear and cyclic electron flow within photosystem II.

We have measured flash-induced oxygen quantum yields (O2-QYs) and primary charge separation (Chl variable fluorescence yield, Fv/Fm) in vivo among phylogenetically diverse microalgae and cyanobacteria. Higher O2-QYs can be attained in cells by releasing constraints on charge transfer at the Photosystem II (PSII) acceptor side by adding membrane-permeable benzoquinone (BQ) derivatives that oxidize plastosemiquinone QB(-) and QBH2. This method allows uncoupling PSII turnover from its natural regulation in living cells, without artifacts of isolating PSII complexes. This approach reveals different extents of regulation across species, controlled at the QB(-) acceptor site. Arthrospira maxima is confirmed as the most efficient PSII-WOC (water oxidizing complex) and exhibits the least regulation of flux. Thermosynechococcus elongatus exhibits an O2-QY of 30%, suggesting strong downregulation. WOC cycle simulations with the most accurate model (VZAD) show that a light-driven backward transition (net addition of an electron to the WOC, distinct from recombination) occurs in up to 25% of native PSIIs in the S2 and S3 states, while adding BQ prevents backward transitions and increases the lifetime of S2 and S3 by 10-fold. Backward transitions occur in PSIIs that have plastosemiquinone radicals in the QB site and are postulated to be physiologically regulated pathways for storing light energy as proton gradient through direct PSII-cyclic electron flow (PSII-CEF). PSII-CEF is independent of classical PSI/cyt-b6f-CEF and provides an alternative proton translocation pathway for energy conversion. PSII-CEF enables variable fluxes between linear and cyclic electron pathways, thus accommodating species-dependent needs for redox and ion-gradient energy sources powered by a single photosystem.

Analysis of Gambierdiscus transcriptome data supports ancient origins of mixotrophic pathways in dinoflagellates.

Toxic dinoflagellates pose serious threats to human health and to fisheries. The genus Gambierdiscus is significant in this respect because its members produce ciguatoxin that accumulates in predominantly tropical marine food webs and leads to ciguatera fish poisoning. Understanding the biology of toxic dinoflagellates is crucial to developing control strategies. To this end, we generated a de novo transcriptome library from G. caribaeus and studied its growth under different culture conditions to elucidate pathways of carbon (C) and nitrogen (N) utilization. We also gathered available dinoflagellate transcriptome data to trace the evolutionary history of C and N pathways in this phylum. We find that rather than being specific adaptations to the epiphytic lifestyle in G. caribaeus, the majority of dinoflagellates share a large array of genes that putatively confer mixotrophy and the ability to use N via the ornithine-urea cycle and nitric oxide synthase production. These results suggest that prior to plastid endosymbiosis, the dinoflagellate ancestor possessed complex pathways that linked metabolism, intercellular signaling, and stress responses to environmental cues that have been maintained by extant photosynthetic species. This metabolic flexibility likely explains the success of dinoflagellates in marine ecosystems and may presage difficulties in controlling the spread of toxic species.

Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002.

To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations--both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))--were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d-lactate, by 6-fold (2.8% yield) and 2-fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7-fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49-fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways.

Low CO2 results in a rearrangement of carbon metabolism to support C4 photosynthetic carbon assimilation in Thalassiosira pseudonana.

The mechanisms of carbon concentration in marine diatoms are controversial. At low CO2 , decreases in O2 evolution after inhibition of phosphoenolpyruvate carboxylases (PEPCs), and increases in PEPC transcript abundances, have been interpreted as evidence for a C4 mechanism in Thalassiosira pseudonana, but the ascertainment of which proteins are responsible for the subsequent decarboxylation and PEP regeneration steps has been elusive. We evaluated the responses of T. pseudonana to steady-state differences in CO2 availability, as well as to transient shifts to low CO2 , by integrated measurements of photosynthetic parameters, transcript abundances and quantitative proteomics. On shifts to low CO2 , two PEPC transcript abundances increased and then declined on timescales consistent with recoveries of Fv /Fm , non-photochemical quenching (NPQ) and maximum chlorophyll a-specific carbon fixation (Pmax ), but transcripts for archetypical decarboxylation enzymes phosphoenolpyruvate carboxykinase (PEPCK) and malic enzyme (ME) did not change. Of 3688 protein abundances measured, 39 were up-regulated under low CO2 , including both PEPCs and pyruvate carboxylase (PYC), whereas ME abundance did not change and PEPCK abundance declined. We propose a closed-loop biochemical model, whereby T. pseudonana produces and subsequently decarboxylates a C4 acid via PEPC2 and PYC, respectively, regenerates phosphoenolpyruvate (PEP) from pyruvate in a pyruvate phosphate dikinase-independent (but glycine decarboxylase (GDC)-dependent) manner, and recuperates photorespiratory CO2 as oxaloacetate (OAA).

How chloride functions to enable proton conduction in photosynthetic water oxidation: Time-resolved kinetics of intermediates (S-states) in vivo and bromide substitution.

Chloride (Cl-) is essential for O2 evolution during photosynthetic water oxidation. Two chlorides near the water-oxidizing complex (WOC) in Photosystem II (PSII) structures from Thermosynechococcus elongatus (and T. vulcanus) have been postulated to transfer protons generated from water oxidation. We monitored four criteria: primary charge separation flash yield (P* â†’ P+QA-), rates of water oxidation steps (S-states), rate of proton evolution, and flash O2 yield oscillations by measuring chlorophyll variable fluorescence (P* quenching), pH-sensitive dye changes, and oximetry. Br-substitution slows and destabilizes cellular growth, resulting from lower light-saturated O2 evolution rate (-20 %) and proton release (-36 % ΔpH gradient). The latter implies less ATP production. In Br- cultures, protonogenic S-state transitions (S2 â†’ S3 â†’ S0') slow with increasing light intensity and during O2/water exchange (S0' â†’ S0 â†’ S1), while the non-protonogenic S1 â†’ S2 transition is kinetically unaffected. As flash rate increases in Cl- cultures, both rate and extent of acidification of the lumen increase, while charge recombination is suppressed relative to Br-. The Cl- advantage in rapid proton escape from the WOC to lumen is attributed to correlated ion-pair movement of H3O+Cl- in dry water channels vs. separated Br- and H+ ion movement through different regions (>200-fold difference in Bronsted acidities). By contrast, at low flash rates a previously unreported reversal occurs that favors Br- cultures for both proton evolution and less PSII charge recombination. In Br- cultures, slower proton transfer rate is attributed to stronger ion-pairing of Br- with AA residues lining the water channels. Both anions charge-neutralize protons and shepherd them to the lumen using dry aqueous channels.

Zam Is a Redox-Regulated Member of the RNB-Family Required for Optimal Photosynthesis in Cyanobacteria.

The zam gene mediating resistance to acetazolamide in cyanobacteria was discovered thirty years ago during a drug tolerance screen. We use phylogenetics to show that Zam proteins are distributed across cyanobacteria and that they form their own unique clade of the ribonuclease II/R (RNB) family. Despite being RNB family members, multiple sequence alignments reveal that Zam proteins lack conservation and exhibit extreme degeneracy in the canonical active site-raising questions about their cellular function(s). Several known phenotypes arise from the deletion of zam, including drug resistance, slower growth, and altered pigmentation. Using room-temperature and low-temperature fluorescence and absorption spectroscopy, we show that deletion of zam results in decreased phycocyanin synthesis rates, altered PSI:PSII ratios, and an increase in coupling between the phycobilisome and PSII. Conserved cysteines within Zam are identified and assayed for function using in vitro and in vivo methods. We show that these cysteines are essential for Zam function, with mutation of either residue to serine causing phenotypes identical to the deletion of Zam. Redox regulation of Zam activity based on the reversible oxidation-reduction of a disulfide bond involving these cysteine residues could provide a mechanism to integrate the 'central dogma' with photosynthesis in cyanobacteria.

Why Did Nature Choose Manganese over Cobalt to Make Oxygen Photosynthetically on the Earth?

All contemporary oxygenic phototrophs─from primitive cyanobacteria to complex multicellular plants─split water using a single invariant cluster comprising Mn4CaO5 (the water oxidation catalyst) as the catalyst within photosystem II, the universal oxygenic reaction center of natural photosynthesis. This cluster is unstable outside of PSII and can be reconstituted, both in vivo and in vitro, using elemental aqueous ions and light, via photoassembly. Here, we demonstrate the first functional substitution of manganese in any oxygenic reaction center by in vitro photoassembly. Following complete removal of inorganic cofactors from cyanobacterial photosystem II microcrystal (PSIIX), photoassembly with free cobalt (Co2+), calcium (Ca2+), and water (OH-) restores O2 evolution activity. Photoassembly occurs at least threefold faster using Co2+ versus Mn2+ due to a higher quantum yield for PSIIX-mediated charge separation (P*): Co2+ → P* → Co3+QA-. However, this kinetic preference for Co2+ over native Mn2+ during photoassembly is offset by significantly poorer catalytic activity (∼25% of the activity with Mn2+) and ∼3- to 30-fold faster photoinactivation rate. The resulting reconstituted Co-PSIIX oxidizes water by the standard four-flash photocycle, although they produce 4-fold less O2 per PSII, suggested to arise from faster charge recombination (Co3+QA ← Co4+QA-) in the catalytic cycle. The faster photoinactivation of reconstituted Co-PSIIX occurs under anaerobic conditions during the catalytic cycle, suggesting direct photodamage without the involvement of O2. Manganese offers two advantages for oxygenic phototrophs, which may explain its exclusive retention throughout Darwinian evolution: significantly slower charge recombination (Mn3+QA ← Mn4+QA-) permits more water oxidation at low and fluctuating solar irradiation (greater net energy conversion) and much greater tolerance to photodamage at high light intensities (Mn4+ is less oxidizing than Co4+). Future work to identify the chemical nature of the intermediates will be needed for further interpretation.

Realtime kinetics of the light driven steps of photosynthetic water oxidation in living organisms by "stroboscopic" fluorometry.

We develop a rapid "stroboscopic" fluorescence induction method, using the fast repetition rate fluorometry (FRRF) technique, to measure changes in the quantum yield of light emission from chlorophyll in oxygenic photosynthesis arising from competition with primary photochemical charge separation (P680* âž” P680+QA-). This method determines the transit times of electrons that pass through PSII during the successive steps in the catalytic cycle of water oxidation/O2 formation (S states) and plastoquinone reduction in any oxygenic phototroph (in vivo or in vitro). We report the first measurements from intact living cells, illustrated by a eukaryotic alga (Nannochloropsis oceanica). We demonstrate that S state transition times depend strongly on the redox state of the PSII acceptor side, at both QB and the plastoquinone pool which serve as the major locus of regulation of PSII electron flux. We provide evidence for a kinetic intermediate S3' state (lifetime 220 µs) following formation of S3 and prior to the release of O2. We compare the FRRF-detected kinetics to other previous spectroscopic methods (optical absorbance, EPR, and XES) that are applicable only to in vitro samples.