Chloroplastic ascorbate modifies plant metabolism and may act as a metabolite signal regardless of oxidative stress.

Ascorbate (Asc) is a major plant metabolite that plays crucial roles in various processes, from reactive oxygen scavenging to epigenetic regulation. However, to what extent and how Asc modulates metabolism is largely unknown. We investigated the consequences of chloroplastic and total cellular Asc deficiencies by studying chloroplastic Asc transporter mutant lines lacking PHOSPHATE TRANSPORTER 4; 4 and the Asc-deficient vtc2-4 mutant of Arabidopsis (Arabidopsis thaliana). Under regular growth conditions, both Asc deficiencies caused minor alterations in photosynthesis, with no apparent signs of oxidative damage. In contrast, metabolomics analysis revealed global and largely overlapping alterations in the metabolome profiles of both Asc-deficient mutants, suggesting that chloroplastic Asc modulates plant metabolism. We observed significant alterations in amino acid metabolism, particularly in arginine metabolism, activation of nucleotide salvage pathways, and changes in secondary metabolism. In addition, proteome-wide analysis of thermostability revealed that Asc may interact with enzymes involved in arginine metabolism, the Calvin-Benson cycle, and several photosynthetic electron transport components. Overall, our results suggest that, independent of oxidative stress, chloroplastic Asc modulates the activity of diverse metabolic pathways in vascular plants and may act as an internal metabolite signal.

Function of iron-stress-induced protein A in cyanobacterial cells with monomeric and trimeric photosystem I.

The acclimation of cyanobacteria to iron deficiency is crucial for their survival in natural environments. In response to iron deficiency, many cyanobacterial species induce the production of a pigment-protein complex called iron-stress-induced protein A (IsiA). IsiA proteins associate with photosystem I (PSI) and can function as light-harvesting antennas or dissipate excess energy. They may also serve as chlorophyll storage during iron limitation. In this study, we examined the functional role of IsiA in cells of Synechocystis sp. PCC 6803 grown under iron limitation conditions by measuring the cellular IsiA content and its capability to transfer energy to PSI. We specifically tested the effect of the oligomeric state of PSI by comparing wild-type (WT) Synechocystis sp. PCC 6803 with mutants lacking specific subunits of PSI, namely PsaL/PsaI (PSI subunits XI/VIII) and PsaF/PsaJ (PSI subunits III/IX). Time-resolved fluorescence spectroscopy revealed that IsiA formed functional PSI3-IsiA18 supercomplexes, wherein IsiA effectively transfers energy to PSI on a timescale of 10 ps at room temperature-measured in isolated complexes and in vivo-confirming the primary role of IsiA as an accessory light-harvesting antenna to PSI. However, a notable fraction (40%) remained unconnected to PSI, supporting the notion of a dual functional role of IsiA. Cells with monomeric PSI under iron deficiency contained, on average, only 3 to 4 IsiA complexes bound to PSI. These results show that IsiA can transfer energy to trimeric and monomeric PSI but to varying degrees and that the acclimatory production of IsiA under iron stress is controlled by its ability to perform its light-harvesting function.

Chloroplast phosphate transporter CrPHT4-7 regulates phosphate homeostasis and photosynthesis in Chlamydomonas.

In eukaryotic cells, phosphorus is assimilated and utilized primarily as phosphate (Pi). Pi homeostasis is mediated by transporters that have not yet been adequately characterized in green algae. This study reports on PHOSPHATE TRANSPORTER 4-7 (CrPHT4-7) from Chlamydomonas reinhardtii, a member of the PHT4 transporter family, which exhibits remarkable similarity to AtPHT4;4 from Arabidopsis (Arabidopsis thaliana), a chloroplastic ascorbate transporter. Using fluorescent protein tagging, we show that CrPHT4-7 resides in the chloroplast envelope membrane. Crpht4-7 mutants, generated by the CRISPR/Cas12a-mediated single-strand templated repair, show retarded growth, especially in high light, reduced ATP level, strong ascorbate accumulation, and diminished non-photochemical quenching in high light. On the other hand, total cellular phosphorous content was unaffected, and the phenotype of the Crpht4-7 mutants could not be alleviated by ample Pi supply. CrPHT4-7-overexpressing lines exhibit enhanced biomass accumulation under high light conditions in comparison with the wild-type strain. Expressing CrPHT4-7 in a yeast (Saccharomyces cerevisiae) strain lacking Pi transporters substantially recovered its slow growth phenotype, demonstrating that CrPHT4-7 transports Pi. Even though CrPHT4-7 shows a high degree of similarity to AtPHT4;4, it does not display any substantial ascorbate transport activity in yeast or intact algal cells. Thus, the results demonstrate that CrPHT4-7 functions as a chloroplastic Pi transporter essential for maintaining Pi homeostasis and photosynthesis in C. reinhardtii.

The lifetime of the oxygen-evolving complex subunit PSBO depends on light intensity and carbon availability in Chlamydomonas.

PSBO is essential for the assembly of the oxygen-evolving complex in plants and green algae. Despite its importance, we lack essential information on its lifetime and how it depends on the environmental conditions. We have generated nitrate-inducible PSBO amiRNA lines in the green alga Chlamydomonas reinhardtii. Transgenic strains grew normally under non-inducing conditions, and their photosynthetic performance was comparable to the control strain. Upon induction of the PSBO amiRNA constructs, cell division halted. In acetate-containing medium, cellular PSBO protein levels decreased by 60% within 24 h in the dark, by 75% in moderate light, and in high light, the protein completely degraded. Consequently, the photosynthetic apparatus became strongly damaged, probably due to 'donor-side-induced photoinhibition', and cellular ultrastructure was also severely affected. However, in the absence of acetate during induction, PSBO was remarkably stable at all light intensities and less substantial changes occurred in photosynthesis. Our results demonstrate that the lifetime of PSBO strongly depends on the light intensity and carbon availability, and thus, on the metabolic status of the cells. We also confirm that PSBO is required for photosystem II stability in C. reinhardtii and demonstrate that its specific loss also entails substantial changes in cell morphology and cell cycle.

Thin cell layer cultures of Chlamydomonas reinhardtii L159I-N230Y, pgrl1 and pgr5 mutants perform enhanced hydrogen production at sunlight intensity.

Photobiological hydrogen (H2) production is a promising renewable energy source. HydA hydrogenases of green algae are efficient but O2-sensitive and compete for electrons with CO2-fixation. Recently, we established a photoautotrophic H2 production system based on anaerobic induction, where the Calvin-Benson cycle is inactive and O2 scavenged by an absorbent. Here, we employed thin layer cultures, resulting in a three-fold increase in H2 production relative to bulk CC-124 cultures (50 µg chlorophyll/ml, 350 µmol photons m-2 s-1). Productivity was maintained when increasing the light intensity to 1000 µmol photons m-2s-1 and the cell density to 150 µg chlorophyll/ml. Remarkably, the L159I-N230Y photosystem II mutant and the pgrl1 photosystem I cyclic electron transport mutant produced 50% more H2 than CC-124, while the pgr5 mutant generated 250% more (1.2 ml H2/ml culture in six days). The photosynthetic apparatus of the pgr5 mutant and its in vitro HydA activity remained remarkably stable.

Characterization of a new class of blue-fluorescent lipid droplet markers for live-cell imaging in plants.

KEY MESSAGE: The present work demonstrates the use and advantages of novel, live cell permeable, lipid droplet localizing, non toxic, blue fluorochromes for use in live plant cells. Lipid droplets (LDs) are ubiquitous components of both animal and plant cells. They consist of a core of neutral lipids surrounded by a monolayer of phospholipids, glycolipids and/or sterols with embedded amphipathic proteins. Although initially considered to be simple energy depots, they have recently emerged as organelles that serve important regulatory functions. Here we report three new fluorochromes as markers for LDs in plants. These bright blue fluorochromes with their unique spectral properties can easily be combined with other green and red fluorescent reporters for multicolor fluorescence imaging. The fluorochromes are non-toxic and photo-stable. All in all, they represent a reliable tool to use, for the investigation of dynamic LD biology within living plant cells using fluorescence microscopy.

Combination of small molecule microarray and confocal microscopy techniques for live cell staining fluorescent dye discovery.

Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS) methods. In the present study a combination of small molecule microarray (SMM) prescreening and confocal laser scanning microscopy (CLSM) was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted) fluorochromes were identified. Their cytotoxicity was tested and found that between 1-10 micromolar range, they were non-toxic even during long-term incubations.