In nature, environmental conditions strongly fluctuate, frequently subjecting plants to periods of immediate photo-oxidative stress. The small molecule ascorbate allows plants to cope with such stress conditions. Ascorbate scavenges reactive oxygen species and enables the rapid and full induction of photoprotective non-photochemical quenching (NPQ). NPQ is dependent on zeaxanthin, which requires ascorbate as the electron donor during its synthesis by the violaxanthin de-epoxidase. The VTC2 gene encodes one of two isoforms of GDP-l-galactose phosphorylase, the rate-controlling enzyme of ascorbate biosynthesis. In the current study, by including a newly identified vtc2 allele, we found that loss of VTC2 depleted ascorbate mainly from the mature leaves and thereby limited NPQ specifically in this tissue. Growth in fluctuating light and controlled climate suppressed the slow NPQ induction phenotype of vtc2 mature leaves to some degree. This was concurrent with a constitutively higher accumulation of zeaxanthin under this condition. When plants were shifted to natural conditions, with strongly fluctuating light and temperature, the ascorbate-deficient mature leaves of vtc2 bleached. Together, our results reveal developmental and environmental effects on VTC2-dependent ascorbate accumulation and function. The VTC2 enzyme controls ascorbate accumulation primarily in the mature leaves of Arabidopsis thaliana . The resulting developmental effects on photoprotection and stress resilience are environmentally modulated.

Photosynthesis provides energy and organic substrates to most life. In plants, photosynthesis dominates chloroplast physiology but represents only a fraction of the tightly interconnected metabolic network that spans the entire cell. Here, we explore how photosynthetic activity affects energy physiology within and beyond the chloroplast. We developed a new standard for the live-monitoring of subcellular energy physiology by combining confocal imaging of genetically encoded fluorescent protein biosensors with advanced on-stage illumination technology to investigate pH, MgATP[2][1]- and NADH/NAD+ dynamics at dark-light transitions in Arabidopsis mesophyll cells. Our findings reveal a stromal alkalinization signature induced by photosynthetic proton pumping, extending to the cytosol and mitochondria as an ’alkalinization wave’. Photosynthesis leads to increased MgATP[2][1]- levels in both the stroma and cytosol. Additionally, we observed reduction of the NAD pool driven by photosynthesis-derived electron export. Arabidopsis lines defective in chloroplast NADP- and mitochondrial NAD-dependent malate dehydrogenases show more reduced cytosolic NAD redox status even in darkness, highlighting the involvement of chloroplasts and mitochondria in shaping cytosolic redox metabolism via malate metabolism. Our study sets a novel methodological standard for precision live-monitoring of photosynthetic cell physiology. Applying this technology reveals signatures of photosynthetic physiology within and beyond the chloroplast with unprecedented resolution. Those signatures link photosynthetic activity and the fundamental biochemical functions of phototrophic cells. Significance statement By applying novel live microscopy monitoring using fluorescent protein biosensors in plant cells, we reveal that dark-light transitions trigger profound re-orchestration of subcellular pH, ATP and NAD redox physiology not limited to chloroplasts but extending into the cytosol and the mitochondria. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 386512654, 507704013, 289357231 China Scholarship Council, 202006910019 [1]: #ref-2

The amino acid cysteine is the precursor for a wide range of sulfur-containing functional molecules in plants, including enzyme cofactors and defence compounds. Due to its redox active thiol group cysteine is highly reactive. Synthesis and degradation pathways are present in several subcellular compartments to adjust the intracellular cysteine concentration. However, stress conditions can lead to a transient increase in local cysteine levels. Here we investigate links between cysteine homeostasis and metabolic signalling in Arabidopsis thaliana. The systemic proteome response to cysteine feeding strongly suggests that Arabidopsis seedlings interpret accumulation of cysteine above a certain threshold as a signal for a biotic threat. Cysteine supplementation of Arabidopsis plants via the roots increases their resistance to the hemibiotrophic bacterium Pseudomonas syringae confirming the protective function of the cysteine induced defence pathways. Analysis of mutant plants reveals that the balance of cysteine synthesis between the cytosol and organelles is crucial during Arabidopsis immune response to Pseudomonas syringae. The induction profile of pathogen responsive proteins by cysteine provides insight into potential modes of action. Our results highlight the role of cysteine as a metabolic signal in the plant immune response and add evidence to the emerging concept of intracellular organelles as important players in plant stress signalling.

Thioredoxins (TRXs) play an important role in controlling photosynthetic acclimation to fluctuating light (FL), but the underlying mechanisms remain unclear. Here, we used Arabidopsis mutants lacking NADPH-dependent TRX-reductase C (NTRC) or parts of the light-dependent TRX system to investigate the specific functions of this network in FL. In the wild type, photosynthetic acclimation required 3 d to develop and stabilized after 5 d of growth in FL. In the ntrc mutant, these acclimation responses were strongly attenuated, leading to decreased PSII acceptor availability, increased non-photochemical quenching (NPQ), and impaired PSII quantum efficiency. Moreover, PSI performance decreased, while acceptor-side limitation of PSI increased. This was particularly due to a strong decline in the reduction states of 2-Cys-peroxiredoxins and fructose-1,6-bisphosphatase, resulting in decreased CO2-fixation rates and delayed relaxation of NPQ in the low-light phases of FL. Since this was not reflected by changes in de-epoxidation of violaxanthin, state transition, or chlorophyll a level, the ntrc mutants showed an apparent lack of photoprotective responses that might explain their increased vulnerability under prolonged growth in FL. Our results show that NADPH-dependent NTRC balances chloroplast redox-systems to optimize the activity of the Calvin-Benson cycle during prolonged light variability in order to optimize PSI, PSII, and water-use efficiency, while having no direct effect on photoprotective mechanisms.

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 β-sheet region, and the conserved Motif III in the C-terminal regions of Alb3/4.

Plant yields heavily depend on proper macro- and micronutrient supply from the soil. In the leaf cells, nutrient ions fulfill specific roles in biochemical reactions, especially photosynthesis housed in the chloroplast. Here, a well-balanced ion homeostasis is maintained by a number of ion transport proteins embedded in the envelope and thylakoid membranes. Ten years ago, the first alkali metal transporters from the K+ EFFLUX ANTIPORTER family were discovered in the model plant Arabidopsis. Since then, our knowledge about the physiological importance of these carriers and their substrates has greatly expanded. New insights into the role of alkali ions in plastid gene expression and photoprotective mechanisms, both prerequisites for plant productivity in natural environments, were gained. The discovery of a Cl- channel in the thylakoid and several additional plastid alkali and alkali metal transport proteins have advanced the field further. Nevertheless, scientists still have long ways to go before a complete systemic understanding of the chloroplast's ion transportome will emerge. In this Tansley review, we highlight and discuss the achievements of the last decade. More importantly, we make recommendations on what areas to prioritize, so the field can reach the next milestones. One area, laid bare by our similarity-based comparisons among phototrophs is our lack of knowledge what ion transporters are used by cyanobacteria to buffer photosynthesis fluctuations.

Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed C-terminus of the thylakoid K+-exchange antiporter KEA3, which regulates the ΔpH across the thylakoid membrane and therefore pH-dependent photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the KEA3 C-terminus senses the energy state of the chloroplast in a pH-dependent manner and regulates transport activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via multi-level regulation of KEA3.

During photosynthesis, plants must manage strong fluctuations in light availability on different time scales, leading to long-term acclimation and short-term responses. However, little is known about the regulation and coordination of these processes and the modulators involved. In this study, we used proteomics, metabolomics, and reverse genetics to investigate how different light environmental factors, such as intensity or variability, affect long-term and short-term acclimation responses of Arabidopsis (Arabidopsis thaliana) and the importance of the chloroplast redox network in their regulation. In the wild type, high light, but not fluctuating light, led to large quantitative changes in the proteome and metabolome, accompanied by increased photosynthetic dynamics and plant growth. This finding supports light intensity as a stronger driver for acclimation than variability. Deficiencies in NADPH-thioredoxin reductase C (NTRC) or thioredoxins m1/m2, but not thioredoxin f1, almost completely suppressed the reengineering of the proteome and metabolome, with both the induction of proteins involved in stress and redox responses and the repression of those involved in cytosolic and plastid protein synthesis and translation being strongly attenuated. Moreover, the correlations of protein or metabolite levels with light intensity were severely disturbed, suggesting a general defect in the light-dependent acclimation response, resulting in impaired photosynthetic dynamics. These results indicate a previously unknown role of NTRC and thioredoxins m1/m2 in modulating light acclimation at proteome and metabolome levels to control dynamic light responses. NTRC, but not thioredoxins m1/m2 or f1, also improves short-term photosynthetic responses by balancing the Calvin-Benson cycle in fluctuating light.

Photorespiration (PR) is the pathway that detoxifies the product of the oxygenation reaction of Rubisco. It has been hypothesized that in dynamic light environments, PR provides a photoprotective function. To test this hypothesis, we characterized plants with varying PR enzyme activities under fluctuating and non-fluctuating light conditions. Contrasting our expectations, growth of mutants with decreased PR enzyme levels was least affected in fluctuating light compared with wild type. Results for growth, photosynthesis and metabolites combined with thermodynamics-based flux analysis revealed two main causal factors for this unanticipated finding: reduced rates of photosynthesis in fluctuating light and complex re-routing of metabolic fluxes. Only in non-fluctuating light, mutants lacking the glutamate:glyoxylate aminotransferase 1 re-routed glycolate processing to the chloroplast, resulting in photooxidative damage through H2O2 production. Our results reveal that dynamic light environments buffer plant growth and metabolism against photorespiratory perturbations.

The initial step of oxygenic photosynthesis is the thermodynamically challenging extraction of electrons from water and the release of molecular oxygen. This light-driven process, which is the basis of life on Earth, is catalyzed by the photosystem II (PSII) within the thylakoid membrane of photosynthetic organisms. The biogenesis of PSII requires a controlled step-wise assembly process of which the early steps are considered to be highly conserved between plants and their cyanobacterial progenitors. This assembly process involves auxiliary proteins, which are likewise conserved. In the present work, we show that in plants, the early assembly step, in which the PSII reaction center (RC) is associated with the intrinsic antenna protein CP47 to form the RC47 intermediate, is facilitated by a novel eukaryote-exclusive assembly factor. This factor, we named DEAP2 for DECREASED ELECTRON TRANSPORT AT PSII, works in concert with the conserved PAM68 assembly factor. The deap2 and pam68 mutants showed similar defects in PSII accumulation and assembly of the RC47 intermediate. The combined lack of both proteins results in a loss of functional PSII and the inability of plants to grow photoautotrophically on soil. While overexpression of DEAP2 partially rescued the pam68 PSII accumulation phenotype, this effect was not reciprocal. DEAP2 accumulates at 20-fold higher levels than PAM68, together suggesting that both proteins have distinct functions. In summary, our results uncover eukaryotic adjustments to the PSII assembly process, which involve the addition of DEAP2 for the rapid progression from RC to RC47.

The initial step of oxygenic photosynthesis is the thermodynamically challenging extraction of electrons from water and the release of molecular oxygen. This light-driven process, which is the basis for most life on Earth, is catalyzed by photosystem II (PSII) within the thylakoid membrane of photosynthetic organisms. The biogenesis of PSII requires a controlled step-wise assembly process of which the early steps are considered to be highly conserved between plants and their cyanobacterial progenitors. This assembly process involves auxiliary proteins, which are likewise conserved. In the present work, we used Arabidopsis (Arabidopsis thaliana) as a model to show that in plants, a eukaryote-exclusive assembly factor facilitates the early assembly step, during which the intrinsic antenna protein CP47 becomes associated with the PSII reaction center (RC) to form the RC47 intermediate. This factor, which we named DECREASED ELECTRON TRANSPORT AT PSII (DEAP2), works in concert with the conserved PHOTOSYNTHESIS AFFECTED MUTANT 68 (PAM68) assembly factor. The deap2 and pam68 mutants showed similar defects in PSII accumulation and assembly of the RC47 intermediate. The combined lack of both proteins resulted in a loss of functional PSII and the inability of plants to grow photoautotrophically on the soil. While overexpression of DEAP2 partially rescued the pam68 PSII accumulation phenotype, this effect was not reciprocal. DEAP2 accumulated at 20-fold higher levels than PAM68, together suggesting that both proteins have distinct functions. In summary, our results uncover eukaryotic adjustments to the PSII assembly process, which involve the addition of DEAP2 for the rapid progression from RC to RC47.

The thylakoid protein Proton Gradient Regulation5 (PGR5) is thought to be a key component of cyclic electron flux around photosystem I. The pgr5 mutant is characterized by impaired proton motive force (pmf) formation across the thylakoid membrane, decreased photoprotective non-photochemical quenching (NPQ), and an over-reduction of the PSI acceptor side. This over-reduction has been attributed to impaired photosynthetic control, which down-regulates plastoquinol re-oxidation at the cytochrome b 6 f complex when the lumen is strongly acidified. Here, using the cgl160 ATP synthase assembly mutant, we show that in cgl160 pgr5 double mutants, both the pmf across the thylakoid membrane and NPQ are fully restored to wild-type levels. However, the acceptor-side limitation of PSI in the double mutants stays comparable to the single pgr5 mutant. This demonstrates that impaired photosynthetic control is not causal for the over-reduction of the PSI acceptor side in pgr5 . Instead, we show that both in pgr5 and the clg160 pgr5 mutants, the entire high-potential chain from cytochrome f to PSI remains strongly reduced in high light. This leads to insufficient oxidizing power for plastoquinol re-oxidation by the cytochrome b 6 f complex, thus impairing pmf formation. We conclude that PGR5 plays a critical role in electron partitioning downstream of PSI.

Understanding photosynthesis in natural, dynamic light environments requires knowledge of long-term acclimation, short-term responses, and their mechanistic interactions. To approach the latter, we systematically determined and characterized light-environmental effects on thylakoid ion transport-mediated short-term responses during light fluctuations. For this, Arabidopsis thaliana wild-type and mutants of the Cl- channel VCCN1 and the K+ exchange antiporter KEA3 were grown under eight different light environments and characterized for photosynthesis-associated parameters and factors in steady state and during light fluctuations. For a detailed characterization of selected light conditions, we monitored ion flux dynamics at unprecedented high temporal resolution by a modified spectroscopy approach. Our analyses reveal that daily light intensity sculpts photosynthetic capacity as a main acclimatory driver with positive and negative effects on the function of KEA3 and VCCN1 during high-light phases, respectively. Fluctuations in light intensity boost the accumulation of the photoprotective pigment zeaxanthin (Zx). We show that KEA3 suppresses Zx accumulation during the day, which together with its direct proton transport activity accelerates photosynthetic transition to lower light intensities. In summary, both light-environment factors, intensity and variability, modulate the function of thylakoid ion transport in dynamic photosynthesis with distinct effects on lumen pH, Zx accumulation, photoprotection, and photosynthetic efficiency.

In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize photosynthetic output while protecting components of the light-dependent reaction and CO2 fixation pathways. Plant phenotyping of mutant collections has become a powerful tool to unveil the genetic loci involved in environmental acclimation. Here, we describe the phenotyping of the transfer-DNA (T-DNA) insertion mutant line SALK_008491, previously known as nhd1-1. Growth in a fluctuating light regime caused a loss in growth rate accompanied by a spike in photosystem (PS) II damage and increased non-photochemical quenching (NPQ). Interestingly, an independent nhd1 null allele did not recapitulate the NPQ phenotype. Through bulk sequencing of a backcrossed segregating F2 pool, we identified an ~14-kb large deletion on chromosome 3 (Chr3) in SALK_008491 affecting five genes upstream of NHD1. Besides NHD1, which encodes for a putative plastid Na+/H+ antiporter, the stromal NAD-dependent D-3-phosphoglycerate dehydrogenase 3 (PGDH3) locus was eradicated. Although some changes in the SALK_008491 mutant's photosynthesis can be assigned to the loss of PGDH3, our follow-up studies employing respective single mutants and complementation with overlapping transformation-competent artificial chromosome (TAC) vectors reveal that the exacerbated fluctuating light sensitivity in SALK_008491 mutants result from the simultaneous loss of PGDH3 and NHD1. Altogether, the data obtained from this large deletion-carrying mutant provide new and unintuitive insights into the molecular mechanisms that function to protect the photosynthetic machinery. Moreover, our study renews calls for caution when setting up reverse genetic studies using T-DNA lines. Although second-site insertions, indels, and SNPs have been reported before, large deletion surrounding the insertion site causes yet another problem. Nevertheless, as shown through this research, such unpredictable genetic events following T-DNA mutagenesis can provide unintuitive insights that allow for understanding complex phenomena such as the plant acclimation to dynamic high light stress.

Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed regulatory C-terminus (RCT) of the thylakoid K + -exchange antiporter KEA3. RCT-mediated control of KEA3 contributes to the balance between light capture and photoprotection. By combining in silico, in vitro , and in vivo approaches, we demonstrate that the RCT senses the energy state of the chloroplast in form of both, phosphorylation and redox potential, in a pH-dependent manner and regulates KEA3 activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via KEA3.

Due to a production error, a typo was introduced in the title of this article after the authors reviewed the proof. The title “Functional characterization of protonantiport regulation in the thylakoid membrane” has been corrected to “Functional characterization of proton antiport regulation in the thylakoid membrane.” We apologize to the authors for the error.

During photosynthesis, electrons travel from light-excited chlorophyll molecules along the electron transport chain to the final electron acceptor nicotinamide adenine dinucleotide phosphate (NADP) to form NADPH, which fuels the Calvin-Benson-Bassham cycle (CBBC). To allow photosynthetic reactions to occur flawlessly, a constant resupply of the acceptor NADP is mandatory. Several known stromal mechanisms aid in balancing the redox poise, but none of them utilizes the structurally highly similar coenzyme NAD(H). Using Arabidopsis (Arabidopsis thaliana) as a C-3-model, we describe a pathway that employs the stromal enzyme PHOSPHOGLYCERATE DEHYDROGENASE 3 (PGDH3). We showed that PGDH3 exerts high NAD(H)-specificity and is active in photosynthesizing chloroplasts. PGDH3 withdrew its substrate 3-PGA directly from the CBBC. As a result, electrons become diverted from NADPH via the CBBC into the separate NADH redox pool. pgdh3 loss-of-function mutants revealed an overreduced NADP(H) redox pool but a more oxidized plastid NAD(H) pool compared to wild-type plants. As a result, photosystem I acceptor side limitation increased in pgdh3. Furthermore, pgdh3 plants displayed delayed CBBC activation, changes in nonphotochemical quenching, and altered proton motive force partitioning. Our fluctuating light-stress phenotyping data showed progressing photosystem II damage in pgdh3 mutants, emphasizing the significance of PGDH3 for plant performance under natural light environments. In summary, this study reveals an NAD(H)-specific mechanism in the stroma that aids in balancing the chloroplast redox poise. Consequently, the stromal NAD(H) pool may provide a promising target to manipulate plant photosynthesis.

Abstract During photosynthesis, energy is transiently stored as an electrochemical proton gradient across the thylakoid membrane. The resulting proton motive force (pmf) is composed of a membrane potential (ΔΨ) and a proton concentration gradient (ΔpH) and powers the synthesis of ATP. Light energy availability for photosynthesis can change very rapidly and frequently in nature. Thylakoid ion transport proteins buffer the effects that light fluctuations have on photosynthesis by adjusting pmf and its composition. Ion channel activities dissipate ΔΨ, thereby reducing charge recombinations within photosystem II. The dissipation of ΔΨ allows for increased accumulation of protons in the thylakoid lumen, generating the signal that activates feedback downregulation of photosynthesis. Proton export from the lumen via the thylakoid K+ exchange antiporter 3 (KEA3), instead, decreases the ΔpH fraction of the pmf and thereby reduces the regulatory feedback signal. Here, we reveal that the Arabidopsis (Arabidopsis thaliana) KEA3 protein homo-dimerizes via its C-terminal domain. This C-terminus has a regulatory function, which responds to light intensity transients. Plants carrying a C-terminus-less KEA3 variant show reduced feed-back downregulation of photosynthesis and suffer from increased photosystem damage under long-term high light stress. However, during photosynthetic induction in high light, KEA3 deregulation leads to an increase in carbon fixation rates. Together, the data reveal a trade-off between long-term photoprotection and a short-term boost in carbon fixation rates, which is under the control of the KEA3 C-terminus.

The unparalleled performance ofChlorella ohadiiunder irradiances of twice full sunlight underlines the gaps in our understanding of how the photosynthetic machinery operates, and what sets its upper functional limit. Rather than succumbing to photodamage under extreme irradiance, unique features of photosystem II function allowC. ohadiito maintain high rates of photosynthesis and growth, accompanied by major changes in composition and cellular structure. This remarkable resilience allowed us to investigate the systems response of photosynthesis and growth to extreme illumination in a metabolically active cell. Using redox proteomics, transcriptomics, metabolomics and lipidomics, we explored the cellular mechanisms that promote dissipation of excess redox energy, protein S-glutathionylation, inorganic carbon concentration, lipid and starch accumulation, and thylakoid stacking.C. ohadiipossesses a readily available capacity to utilize a sudden excess of reducing power and carbon for growth and reserve formation, and post-translational redox regulation plays a pivotal role in this rapid response. Frequently the response inC. ohadiideviated from that of model species, reflecting its life history in desert sand crusts. Comparative global and case-specific analyses provided insights into the potential evolutionary role of effective reductant utilization in this extreme resistance ofC. ohadiito extreme irradiation. The green alga,Chlorella ohadii, can survive under desert conditions of extreme irradiance. Redox proteomics, transcriptomics, metabolomics and lipidomics uncover howC. ohadiican absorb and use the sudden excesses of reducing power and carbon.