STIC2 selectively binds ribosome-nascent chain complexes in the cotranslational sorting of Arabidopsis thylakoid proteins.

Chloroplast-encoded multi-span thylakoid membrane proteins are crucial for photosynthetic complexes, yet the coordination of their biogenesis remains poorly understood. To identify factors that specifically support the cotranslational biogenesis of the reaction center protein D1 of photosystem (PS) II, we generated and affinity-purified stalled ribosome-nascent chain complexes (RNCs) bearing D1 nascent chains. Stalled RNCs translating the soluble ribosomal subunit uS2c were used for comparison. Quantitative tandem-mass spectrometry of the purified RNCs identified around 140 proteins specifically associated with D1 RNCs, mainly involved in protein and cofactor biogenesis, including chlorophyll biosynthesis, and other metabolic pathways. Functional analysis of STIC2, a newly identified D1 RNC interactor, revealed its cooperation with chloroplast protein SRP54 in the de novo biogenesis and repair of D1, and potentially other cotranslationally-targeted reaction center subunits of PSII and PSI. The primary binding interface between STIC2 and the thylakoid insertase Alb3 and its homolog Alb4 was mapped to STIC2's ß-sheet region, and the conserved Motif III in the C-terminal regions of Alb3/4.

Assignment of the slowly exchanging substrate water of nature's water-splitting cofactor.

Identifying the two substrate water sites of nature's water-splitting cofactor (Mn4CaO5 cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S1 state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved 17O-Electron-electron Double resonance detected NMR (TR-17O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H218O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn4CaO5 cofactor after mixing with H217O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (WS) in the TR-MIMS experiments and ii) that the exchange rates of O5 and WS are both enhanced by Ca2+→Sr2+ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being WS. This strongly restricts options for the mechanism of water oxidation.

Mutation-induced shift of the photosystem II active site reveals insight into conserved water channels.

Photosystem II (PSII) is the water-plastoquinone photo-oxidoreductase central to oxygenic photosynthesis. PSII has been extensively studied for its ability to catalyze light-driven water oxidation at a Mn4CaO5 cluster called the oxygen-evolving complex (OEC). Despite these efforts, the complete reaction mechanism for water oxidation by PSII is still heavily debated. Previous mutagenesis studies have investigated the roles of conserved amino acids, but these studies have lacked a direct structural basis that would allow for a more meaningful interpretation. Here, we report a 2.14-Å resolution cryo-EM structure of a PSII complex containing the substitution Asp170Glu on the D1 subunit. This mutation directly perturbs a bridging carboxylate ligand of the OEC, which alters the spectroscopic properties of the OEC without fully abolishing water oxidation. The structure reveals that the mutation shifts the position of the OEC within the active site without markedly distorting the Mn4CaO5 cluster metal-metal geometry, instead shifting the OEC as a rigid body. This shift disturbs the hydrogen-bonding network of structured waters near the OEC, causing disorder in the conserved water channels. This mutation-induced disorder appears consistent with previous FTIR spectroscopic data. We further show using quantum mechanics/molecular mechanics methods that the mutation-induced structural changes can affect the magnetic properties of the OEC by altering the axes of the Jahn-Teller distortion of the Mn(III) ion coordinated to D1-170. These results offer new perspectives on the conserved water channels, the rigid body property of the OEC, and the role of D1-Asp170 in the enzymatic water oxidation mechanism.

Improved capacity for the repair of photosystem II via reinforcement of the translational and antioxidation systems in Synechocystis sp. PCC 6803.

In the cyanobacterium Synechocystis sp. PCC 6803, translation factor EF-Tu is inactivated by reactive oxygen species (ROS) via oxidation of Cys82 and the oxidation of EF-Tu enhances the inhibition of the repair of photosystem II (PSII) by suppressing protein synthesis. In our present study, we generated transformants of Synechocystis that overexpressed a mutated form of EF-Tu, designated EF-Tu (C82S), in which Cys82 had been replaced by a Ser residue, and ROS-scavenging enzymes individually or together. Expression of EF-Tu (C82S) alone in Synechocystis enhanced the repair of PSII under strong light, with the resultant mitigation of PSII photoinhibition, but it stimulated the production of ROS. However, overexpression of superoxide dismutase and catalase, together with the expression of EF-Tu (C82S), lowered intracellular levels of ROS and enhanced the repair of PSII more significantly under strong light, via facilitation of the synthesis de novo of the D1 protein. By contrast, the activity of photosystem I was hardly affected in wild-type cells and in all the lines of transformed cells under the same strong-light conditions. Furthermore, transformed cells that overexpressed EF-Tu (C82S), superoxide dismutase, and catalase were able to survive longer under stronger light than wild-type cells. Thus, the reinforced capacity for both protein synthesis and ROS scavenging allowed both photosynthesis and cell proliferation to tolerate strong light.

Psb28 protein is indispensable for stable accumulation of PSII core complexes in Arabidopsis.

Enhancing the efficiency of photosynthesis represents a promising strategy to improve crop yields, with keeping the steady state of PSII being key to determining the photosynthetic performance. However, the mechanisms whereby the stability of PSII is maintained in oxygenic organisms remain to be explored. Here, we report that the Psb28 protein functions in regulating the homeostasis of PSII under different light conditions in Arabidopsis thaliana. The psb28 mutant is much smaller than the wild-type plants under normal growth light, which is due to its significantly reduced PSII activity. Similar defects were seen under low light and became more pronounced under photoinhibitory light. Notably, the amounts of PSII core complexes and core subunits are specifically decreased in psb28, whereas the abundance of other representative components of photosynthetic complexes remains largely unaltered. Although the PSII activity of psb28 was severely reduced when subjected to high light, its recovery from photoinactivation was not affected. By contrast, the degradation of PSII core protein subunits is dramatically accelerated in the presence of lincomycin. These results indicate that psb28 is defective in the photoprotection of PSII, which is consistent with the observation that the overall NPQ is much lower in psb28 compared to the wild type. Moreover, the Psb28 protein is associated with PSII core complexes and interacts mainly with the CP47 subunit of PSII core. Taken together, these findings reveal an important role for Psb28 in the protection and stabilization of PSII core in response to changes in light environments.

Plastid HSP90C C-terminal extension region plays a regulatory role in chaperone activity and client binding.

HSP90Cs are essential molecular chaperones localized in the plastid stroma that maintain protein homeostasis and assist the import and thylakoid transport of chloroplast proteins. While HSP90C contains all conserved domains as an HSP90 family protein, it also possesses a unique feature in its variable C-terminal extension (CTE) region. This study elucidated the specific function of this HSP90C CTE region. Our phylogenetic analyses revealed that this intrinsically disordered region contains a highly conserved DPW motif in the green lineages. With biochemical assays, we showed that the CTE is required for the chaperone to effectively interact with client proteins PsbO1 and LHCB2 to regulate ATP-independent chaperone activity and to effectuate its ATP hydrolysis. The CTE truncation mutants could support plant growth and development reminiscing the wild type under normal conditions except for a minor phenotype in cotyledon when expressed at a level comparable to wild type. However, higher HSP90C expression was observed to correlate with a stronger response to specific photosystem II inhibitor DCMU, and CTE truncations dampened the response. Additionally, when treated with lincomycin to inhibit chloroplast protein translation, CTE truncation mutants showed a delayed response to PsbO1 expression repression, suggesting its role in chloroplast retrograde signaling. Our study therefore provides insights into the mechanism of HSP90C in client protein binding and the regulation of green chloroplast maturation and function, especially under stress conditions.

High-resolution cryo-electron microscopy structure of photosystem II from the mesophilic cyanobacterium, Synechocystis sp. PCC 6803.

Photosystem II (PSII) enables global-scale, light-driven water oxidation. Genetic manipulation of PSII from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 has provided insights into the mechanism of water oxidation; however, the lack of a high-resolution structure of oxygen-evolving PSII from this organism has limited the interpretation of biophysical data to models based on structures of thermophilic cyanobacterial PSII. Here, we report the cryo-electron microscopy structure of PSII from Synechocystis sp. PCC 6803 at 1.93-Å resolution. A number of differences are observed relative to thermophilic PSII structures, including the following: the extrinsic subunit PsbQ is maintained, the C terminus of the D1 subunit is flexible, some waters near the active site are partially occupied, and differences in the PsbV subunit block the Large (O1) water channel. These features strongly influence the structural picture of PSII, especially as it pertains to the mechanism of water oxidation.

From antenna to reaction center: Pathways of ultrafast energy and charge transfer in photosystem II.

The photosystem II core complex (PSII-CC) is the smallest subunit of the oxygenic photosynthetic apparatus that contains core antennas and a reaction center, which together allow for rapid energy transfer and charge separation, ultimately leading to efficient solar energy conversion. However, there is a lack of consensus on the interplay between the energy transfer and charge separation dynamics of the core complex. Here, we report the application of two-dimensional electronic-vibrational (2DEV) spectroscopy to the spinach PSII-CC at 77 K. The simultaneous temporal and spectral resolution afforded by 2DEV spectroscopy facilitates the separation and direct assignment of coexisting dynamical processes. Our results show that the dominant dynamics of the PSII-CC are distinct in different excitation energy regions. By separating the excitation regions, we are able to distinguish the intraprotein dynamics and interprotein energy transfer. Additionally, with the improved resolution, we are able to identify the key pigments involved in the pathways, allowing for a direct connection between dynamical and structural information. Specifically, we show that C505 in CP43 and the peripheral chlorophyll ChlzD1 in the reaction center are most likely responsible for energy transfer from CP43 to the reaction center.

Rubredoxin 1 promotes the proper folding of D1 and is not required for heme b559 assembly in Chlamydomonas photosystem II.

Photosystem II (PSII), the water:plastoquinone oxidoreductase of oxygenic photosynthesis, contains a heme b559 iron whose axial ligands are provided by histidine residues from the α (PsbE) and ß (PsbF) subunits. PSII assembly depends on accessory proteins that facilitate the step-wise association of its protein and pigment components into a functional complex, a process that is challenging to study due to the low accumulation of assembly intermediates. Here, we examined the putative role of the iron[1Fe-0S]-containing protein rubredoxin 1 (RBD1) as an assembly factor for cytochrome b559, using the RBD1-lacking 2pac mutant from Chlamydomonas reinhardtii, in which the accumulation of PSII was rescued by the inactivation of the thylakoid membrane FtsH protease. To this end, we constructed the double mutant 2pac ftsh1-1, which harbored PSII dimers that sustained its photoautotrophic growth. We purified PSII from the 2pac ftsh1-1 background and found that α and ß cytochrome b559 subunits are still present and coordinate heme b559 as in the WT. Interestingly, immunoblot analysis of dark- and low light-grown 2pac ftsh1-1 showed the accumulation of a 23-kDa fragment of the D1 protein, a marker typically associated with structural changes resulting from photodamage of PSII. Its cleavage occurs in the vicinity of a nonheme iron which binds to PSII on its electron acceptor side. Altogether, our findings demonstrate that RBD1 is not required for heme b559 assembly and point to a role for RBD1 in promoting the proper folding of D1, possibly via delivery or reduction of the nonheme iron during PSII assembly.

Structural insights into the action mechanisms of artificial electron acceptors in photosystem II.

Photosystem II (PSII) utilizes light energy to split water, and the electrons extracted from water are transferred to QB, a plastoquinone molecule bound to the D1 subunit of PSII. Many artificial electron acceptors (AEAs) with molecular structures similar to that of plastoquinone can accept electrons from PSII. However, the molecular mechanism by which AEAs act on PSII is unclear. Here, we solved the crystal structure of PSII treated with three different AEAs, 2,5-dibromo-1,4-benzoquinone, 2,6-dichloro-1,4-benzoquinone, and 2-phenyl-1,4-benzoquinone, at 1.95 to 2.10 Å resolution. Our results show that all AEAs substitute for QB and are bound to the QB-binding site (QB site) to receive electrons, but their binding strengths are different, resulting in differences in their efficiencies to accept electrons. The acceptor 2-phenyl-1,4-benzoquinone binds most weakly to the QB site and showed the highest oxygen-evolving activity, implying a reverse relationship between the binding strength and oxygen-evolving activity. In addition, a novel quinone-binding site, designated the QD site, was discovered, which is located in the vicinity of QB site and close to QC site, a binding site reported previously. This QD site is expected to play a role as a channel or a storage site for quinones to be transported to the QB site. These results provide the structural basis for elucidating the actions of AEAs and exchange mechanism of QB in PSII and also provide information for the design of more efficient electron acceptors.

Structure of a dimeric photosystem II complex from a cyanobacterium acclimated to far-red light.

Photosystem II (PSII) is the water-splitting enzyme central to oxygenic photosynthesis. To drive water oxidation, light is harvested by accessory pigments, mostly chlorophyll (Chl) a molecules, which absorb visible light (400-700 nm). Some cyanobacteria facultatively acclimate to shaded environments by altering their photosynthetic machinery to additionally absorb far-red light (FRL, 700-800 nm), a process termed far-red light photoacclimation or FaRLiP. During far-red light photoacclimation, FRL-PSII is assembled with FRL-specific isoforms of the subunits PsbA, PsbB, PsbC, PsbD, and PsbH, and some Chl-binding sites contain Chls d or f instead of the usual Chl a. The structure of an apo-FRL-PSII monomer lacking the FRL-specific PsbH subunit has previously been determined, but visualization of the dimeric complex has remained elusive. Here, we report the cryo-EM structure of a dimeric FRL-PSII complex. The site assignments for Chls d and f are consistent with those assigned in the previous apo-FRL-PSII monomeric structure. All sites that bind Chl d or Chl f at high occupancy exhibit a FRL-specific interaction of the formyl moiety of the Chl d or Chl f with the protein environment, which in some cases involves a phenylalanine sidechain. The structure retains the FRL-specific PsbH2 subunit, which appears to alter the energetic landscape of FRL-PSII, redirecting energy transfer from the phycobiliprotein complex to a Chl f molecule bound by PsbB2 that acts as a bridge for energy transfer to the electron transfer chain. Collectively, these observations extend our previous understanding of the structure-function relationship that allows PSII to function using lower energy FRL.

Comparative proteomics in tall fescue to reveal underlying mechanisms for improving Photosystem II thermotolerance during heat stress memory.

BACKGROUND: The escalating impacts of global warming intensify the detrimental effects of heat stress on crop growth and yield. Among the earliest and most vulnerable sites of damage is Photosystem II (PSII). Plants exposed to recurring high temperatures develop heat stress memory, a phenomenon that enables them to retain information from previous stress events to better cope with subsequent one. Understanding the components and regulatory networks associated with heat stress memory is crucial for the development of heat-resistant crops. RESULTS: Physiological assays revealed that heat priming (HP) enabled tall fescue to possess higher Photosystem II photochemical activity when subjected to trigger stress. To investigate the underlying mechanisms of heat stress memory, we performed comparative proteomic analyses on tall fescue leaves at S0 (control), R4 (primed), and S5 (triggering), using an integrated approach of Tandem Mass Tag (TMT) labeling and Liquid Chromatography-Mass Spectrometry. A total of 3,851 proteins were detected, with quantitative information available for 3,835 proteins. Among these, we identified 1,423 differentially abundant proteins (DAPs), including 526 proteins that were classified as Heat Stress Memory Proteins (HSMPs). GO and KEGG enrichment analyses revealed that the HSMPs were primarily associated with the "autophagy" in R4 and with "PSII repair", "HSP binding", and "peptidase activity" in S5. Notably, we identified 7 chloroplast-localized HSMPs (HSP21, DJC77, EGY3, LHCA4, LQY1, PSBR and DEGP8, R4/S0 > 1.2, S5/S0 > 1.2), which were considered to be effectors linked to PSII heat stress memory, predominantly in cluster 4. Protein-protein interaction (PPI) analysis indicated that the ubiquitin-proteasome system, with key nodes at UPL3, RAD23b, and UCH3, might play a role in the selective retention of memory effectors in the R4 stage. Furthermore, we conducted RT-qPCR validation on 12 genes, and the results showed that in comparison to the S5 stage, the R4 stage exhibited reduced consistency between transcript and protein levels, providing additional evidence for post-transcriptional regulation in R4. CONCLUSIONS: These findings provide valuable insights into the establishment of heat stress memory under recurring high-temperature episodes and offer a conceptual framework for breeding thermotolerant crops with improved PSII functionality.

The Effect of Removal of External Proteins PsbO, PsbP and PsbQ on Flash-Induced Molecular Oxygen Evolution and Its Biphasicity in Tobacco PSII.

The oxygen evolution within photosystem II (PSII) is one of the most enigmatic processes occurring in nature. It is suggested that external proteins surrounding the oxygen-evolving complex (OEC) not only stabilize it and provide an appropriate ionic environment but also create water channels, which could be involved in triggering the ingress of water and the removal of O2 and protons outside the system. To investigate the influence of these proteins on the rate of oxygen release and the efficiency of OEC function, we developed a measurement protocol for the direct measurement of the kinetics of oxygen release from PSII using a Joliot-type electrode. PSII-enriched tobacco thylakoids were used in the experiments. The results revealed the existence of slow and fast modes of oxygen evolution. This observation is model-independent and requires no specific assumptions about the initial distribution of the OEC states. The gradual removal of exogenous proteins resulted in a slowdown of the rapid phase (~ms) of O2 release and its gradual disappearance while the slow phase (~tens of ms) accelerated. The role of external proteins in regulating the biphasicity and efficiency of oxygen release is discussed based on observed phenomena and current knowledge.

Phe265 of the D1 protein is required to stabilize plastoquinone binding in the QB-binding site of photosystem II in Synechocystis sp. PCC 6803.

In Photosystem II electrons from water splitting pass through a primary quinone electron acceptor (QA) to the secondary plastoquinone (QB). The D2 protein forms the QA-binding site and the D1 protein forms the QB-binding site. A non-heme iron sits between QA and QB resulting in a quinone-Fe-acceptor complex that must be activated before assembly of the oxygen-evolving complex can occur. An extended loop (residues 223-266) between the fourth (helix D) and fifth (helix E) helices of the D1 protein activates forward electron transfer via a conformational change that stabilizes a bidentate bicarbonate ligand to the non-heme iron while simultaneously stabilizing the binding of QB. We show that positioning of D1:Phe265 to provide a hydrogen bond to the distal oxygen of QB is required for forward electron transfer. In addition, mutations targeting D1:Phe265, resulted in a 50 mV decrease in the QB/QB- midpoint potential.

Mutagenesis of Ile184 in the cd-loop of the photosystem II D1 protein modifies acceptor-side function via spontaneous mutation of D1-His252 in Synechocystis sp. PCC 6803.

The Photosystem II water-plastoquinone oxidoreductase is a multi-subunit complex which catalyses the light-driven oxidation of water to molecular oxygen in oxygenic photosynthesis. The D1 reaction centre protein exists in multiple forms in cyanobacteria, including D1FR which is expressed under far-red light. We investigated the role of Phe184 that is found in the lumenal cd-loop of D1FR but is typically an isoleucine in other D1 isoforms. The I184F mutant in Synechocystis sp. PCC 6803 was similar to the control strain but accumulated a spontaneous mutation that introduced a Gln residue in place of His252 located on the opposite side of the thylakoid membrane. His252 participates in the protonation of the secondary plastoquinone electron acceptor QB. The I184F:H252Q double mutant exhibited reduced high-light-induced photodamage and an altered QB-binding site that impaired herbicide binding. Additionally, the H252Q mutant had a large increase in the variable fluorescence yield although the number of photochemically active PS II centres was unchanged. In the I184F:H252Q mutant the extent of the increased fluorescence yield decreased. Our data indicates substitution of Ile184 to Phe modulates PS II-specific variable fluorescence in cells with the His252 to Gln substitution by modifying the QB-binding site.

Deciphering desiccation tolerance in wild eggplant species: insights from chlorophyll fluorescence dynamics.

BACKGROUND: Climate change exacerbates abiotic stresses, which are expected to intensify their impact on crop plants. Drought, the most prevalent abiotic stress, significantly affects agricultural production worldwide. Improving eggplant varieties to withstand abiotic stress is vital due to rising drought from climate change. Despite the diversity of wild eggplant species that thrive under harsh conditions, the understanding of their drought tolerance mechanisms remains limited. In the present study, we used chlorophyll fluorescence (ChlaF) imaging, which reveals a plant's photosynthetic health, to investigate desiccation tolerance in eggplant and its wild relatives. Conventional fluorescence measurements lack spatial heterogeneity, whereas ChlaF imaging offers comprehensive insights into plant responses to environmental stresses. Hence, employing noninvasive imaging techniques is essential for understanding this heterogeneity. RESULTS: Desiccation significantly reduced the leaf tissue moisture content (TMC) across species. ChlaF and TMC displayed greater photosystem II (PSII) efficiency after 54 h of desiccation in S. macrocarpum, S. torvum, and S. indicum, with S. macrocarpum demonstrating superior efficiency due to sustained fluorescence. PSII functions declined gradually in S. macrocarpum and S. torvum, unlike those in other species, which exhibited abrupt declines after 54 h of desiccation. However, after 54 h, PSII efficiency remained above 50% of its initial quantum yield in S. macrocarpum at 35% leaf RWC (relative water content), while S. torvum and S. indicum displayed 50% decreases at 31% and 33% RWC, respectively. Conversely, the susceptible species S. gilo and S. sisymbriifolium exhibited a 50% reduction in PSII function at an early stage of 50% RWC, whereas in S. melongena, this reduction occurred at 40% RWC. CONCLUSION: Overall, our study revealed notably greater leaf desiccation tolerance, especially in S. macrocarpum, S. torvum, and S. indicum, attributed to sustained PSII efficiency at low TMC levels, indicating that these species are promising sources of drought tolerance.

Thylakoid ultrastructural variations in chlorophyll-deficient wheat: aberrations or structural acclimation?

MAIN CONCLUSION: A structural re-modeling of the thylakoid system, including granum size and regularity, occurs in chlorophyll-deficient wheat mutants affected by photosynthetic membrane over-reduction. In the chloroplast of land plants, the thylakoid system is defined by appressed grana stacks and unstacked stroma lamellae. This study focuses on the variations of the grana organization occurring in outdoor-grown wheat mutants characterized by low chlorophyll content and a tendency for photosynthetic membrane over-reduction. Triticum aestivum ANK-32A and Triticum durum ANDW-7B were compared to their corresponding WT lines, NS67 and LD222, respectively. Electron micrographs of chloroplasts were used to calculate grana ultrastructural parameters. Photosynthetic parameters were obtained by modulated chlorophyll fluorescence and applying Light Curves (LC) and Rapid Light Curves (RLC) protocols. For each photosynthetic parameter, the difference Δ(RLC-LC) was calculated to evaluate the flexible response to light in the examined lines. In the mutants, fewer and smaller disks formed grana stacks characterized by a marked increase in lateral and cross-sectional irregularity, both negatively correlated with the number of layers per granum. A relationship was found between membrane over-reduction and granum structural irregularity. The possible acclimative significance of a greater proportion of stroma-exposed grana domains in relieving the excess electron pressure on PSI is discussed.

Heat tolerance of a tropical-subtropical rainforest tree species Polyscias elegans: time-dependent dynamic responses of physiological thermostability and biochemistry.

Heat stress interrupts physiological thermostability and triggers biochemical responses that are essential for plant survival. However, there is limited knowledge on the speed plants adjust to heat in hours and days, and which adjustments are crucial. Tropical-subtropical rainforest tree species (Polyscias elegans) were heated at 40°C for 5 d, before returning to 25°C for 13 d of recovery. Leaf heat tolerance was quantified using the temperature at which minimal chl a fluorescence sharply rose (Tcrit ). Tcrit , metabolites, heat shock protein (HSP) abundance and membrane lipid fatty acid (FA) composition were quantified. Tcrit increased by 4°C (48-52°C) within 2 h of 40°C exposure, along with rapid accumulation of metabolites and HSPs. By contrast, it took > 2 d for FA composition to change. At least 2 d were required for Tcrit , HSP90, HSP70 and FAs to return to prestress levels. The results highlight the multi-faceted response of P. elegans to heat stress, and how this response varies over the scale of hours to days, culminating in an increased level of photosynthetic heat tolerance. These responses are important for survival of plants when confronted with heat waves amidst ongoing global climate change.

Bicarbonate is a key regulator but not a substrate for O2 evolution in Photosystem II.

Photosystem II (PSII) uses light energy to oxidize water and to reduce plastoquinone in the photosynthetic electron transport chain. O2 is produced as a byproduct. While most members of the PSII research community agree that O2 originates from water molecules, alternative hypotheses involving bicarbonate persist in the literature. In this perspective, we provide an overview of the important roles of bicarbonate in regulating PSII activity and assembly. Further, we emphasize that biochemistry, spectroscopy, and structural biology experiments have all failed to detect bicarbonate near the active site of O2 evolution. While thermodynamic arguments for oxygen-centered bicarbonate oxidation are valid, the claim that bicarbonate is a substrate for photosynthetic O2 evolution is challenged.

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).