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Anion transporters sustain a variety of physiological states in cells. Bestrophins (BSTs) belong to a Cl− and/or HCO3− transporter family conserved in bacteria, animals, algae, and plants. Recently, putative BSTs were found in the green alga Chlamydomonas reinhardtii, where they are upregulated under low CO2 (LC) conditions and play an essential role in the CO2-concentrating mechanism (CCM). The putative BST orthologs are also conserved in diatoms, secondary endosymbiotic algae harboring red-type plastids, but their physiological functions are unknown. Here, we characterized the subcellular localization and expression profile of BSTs in the marine diatoms Phaeodactylum tricornutum (PtBST1 to 4) and Thalassiosira pseudonana (TpBST1 and 2). PtBST1, PtBST2, and PtBST4 were localized at the stroma thylakoid membrane outside of the pyrenoid, and PtBST3 was localized in the pyrenoid. Contrarily, TpBST1 and TpBST2 were both localized in the pyrenoid. These BST proteins accumulated in cells grown in LC but not in 1% CO2 (high CO2 [HC]). To assess the physiological functions, we generated knockout mutants for the PtBST1 gene by genome editing. The lack of PtBST1 decreased photosynthetic affinity for dissolved inorganic carbon to the level comparable with the HC-grown wild type. Furthermore, non-photochemical quenching in LC-grown cells was 1.5 to 2.0 times higher in the mutants than in the wild type. These data suggest that HCO3− transport at the stroma thylakoid membranes by PtBST1 is a critical part of the CO2-evolving machinery of the pyrenoid in the fully induced CCM and that PtBST1 may modulate photoprotection under CO2-limited environments in P. tricornutum. Bestrophin transports HCO3− at stroma thylakoid membranes, maintaining the CO2-evolving machinery in the pyrenoid of marine diatoms under low CO2. [ABSTRACT FROM AUTHOR]

SUMMARY: The plastid terminal oxidase PTOX controls the oxidation level of the plastoquinone pool in the thylakoid membrane and acts as a safety valve upon abiotic stress, but detailed characterization of its role in protecting the photosynthetic apparatus is limited. Here we used PTOX mutants in two model plants Arabidopsis thaliana and Marchantia polymorpha. In Arabidopsis, lack of PTOX leads to a severe defect in pigmentation, a so‐called variegated phenotype, when plants are grown at standard light intensities. We created a green Arabidopsis PTOX mutant expressing the bacterial carotenoid desaturase CRTI and a double mutant in Marchantia lacking both PTOX isoforms, the plant‐type and the alga‐type PTOX. In both species, lack of PTOX affected the redox state of the plastoquinone pool. Exposure of plants to high light intensity showed in the absence of PTOX higher susceptibility of photosystem I to light‐induced damage while photosystem II was more stable compared with the wild type demonstrating that PTOX plays both, a pro‐oxidant and an anti‐oxidant role in vivo. Our results shed new light on the function of PTOX in the protection of photosystem I and II. Significance Statement: In photosynthesis, PTOX activity is important for keeping the plastoquinone pool oxidized upon light stress and acts as a safety valve protecting photosystem I against photodamage. In mutants lacking PTOX electron flow to photosystem I is not restricted, preventing partially the accumulation of P700+. As a consequence photosystem I is more easily damaged by excess light. [ABSTRACT FROM AUTHOR]

Following the principle of oxygenic photosynthesis, electron transport in the thylakoid membranes (i.e., light reaction) generates ATP and NADPH from light energy, which is subsequently utilized for CO2 fixation in the Calvin-Benson-Bassham cycle (i.e., dark reaction). However, light and dark reactions could discord when an alternative electron flow occurs with a rate comparable to the linear electron flow. Here, we quantitatively monitored O2 and total dissolved inorganic carbon (DIC) during photosynthesis in the pennate diatom Phaeodactylum tricornutum, and found that evolved O2 was larger than the consumption of DIC, which was consistent with 14CO2 measurements in literature. In our measurements, the stoichiometry of O2 evolution to DIC consumption was always around 1.5 during photosynthesis at different DIC concentrations. The same stoichiometry was observed in the cells grown under different CO2 concentrations and nitrogen sources except for the nitrogen-starved cells showing O2 evolution 2.5 times larger than DIC consumption. An inhibitor to nitrogen assimilation did not affect the extra O2 evolution. Further, the same physiological phenomenon was observed in the centric diatom Thalassiosira pseudonana. Based on the present dataset, we propose that the marine diatoms possess the metabolic pathway(s) functioning as the O2-independent electron sink under steady state photosynthesis that reaches nearly half of electron flux of the Calvin-Benson-Bassham cycle. [ABSTRACT FROM AUTHOR]

Marine diatoms are responsible for up to 20% of the annual global primary production by performing photosynthesis in seawater where CO2 availability is limited while HCO3- is abundant. Our previous studies have demonstrated that solute carrier 4 proteins at the plasma membrane of the diatom Phaeodactylum tricornutum facilitate the use of the abundant seawater HCO3-. There has been an unconcluded debate as to whether such HCO3- use capacity may itself supply enough dissolved inorganic carbon (DIC) to saturate the enzyme Rubisco. Here, we show that the 1-type carbonic anhydrase, Pt1-CA1, a luminal factor of the pyrenoid-penetrating thylakoid membranes, plays an essential role in saturating photosynthesis of P. tricornutum. We isolated and analyzed genome-edited mutants of P. tricornutum defective in Pt1-CA1. The mutants showed impaired growth in seawater aerated with a broad range of CO2 levels, from atmospheric to 1%. Independently of growth CO2 conditions, the photosynthetic affinity measured as K0.5 for DIC in mutants reached around 2 mM, which is about 10 times higher than K0.5[DIC] of high-CO2-grown wild-type cells that have repressed CO2-concentrating mechanism levels. The results clearly indicate that diatom photosynthesis is not saturated with either seawater-level DIC or even under a highly elevated CO2 environment unless the CO2-evolving machinery is at the core of the pyrenoid. [ABSTRACT FROM AUTHOR]

Diatoms are one of the most important phytoplankton on Earth. They comprise at least ten thousand species and contribute to up to 20% of the global primary production. Because of serial endosymbiotic events and horizontal gene transfers, diatoms have developed a "secondary plastid" bounded by four membranes containing a large phase‐separated compartment, termed the pyrenoid. However, the physiological significance of this unique chloroplast morphology is poorly understood. Characterization of fundamental physiological parameters such as local pH in various subcellular compartments should facilitate a greater understanding of the physiological roles of the unique structure of the secondary plastid. A promising method to estimate local pH is the in situ expression of the pH‐sensitive green fluorescent protein. Here, we first developed the molecular tool for the mapping of in situ local pH in the diatom Phaeodactylum tricornutum by heterologously expressing pHluorin2 in the cytosol, periplastidal compartment (PPC; the space in between two sets of outer and inner chloroplast envelopes), chloroplast stroma, and the pyrenoid matrix. Our data suggested that PPC and the pyrenoid matrix are more acidic than the adjacent areas, the cytosol and the chloroplast stroma. Finally, absolute pH values at each compartment were estimated from the ratiometric fluorescence of a recombinant pHluorin2 protein, giving pH values of approximately 7.9, 6.8, 8.0, and 7.5 respectively, for the cytosol, PPC, stroma, and pyrenoid of the P. tricornutum cells, indicating the occurrence of pH gradients and the associated electrochemical potentials at their boundary. [ABSTRACT FROM AUTHOR]