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The highly phosphorylated nucleotide, guanosine tetraphosphate (ppGpp), functions as a secondary messenger in bacteria and chloroplasts. The accumulation of ppGpp alters plastidial gene expression and metabolism, which are required for proper photosynthetic regulation and robust plant growth. However, because four plastid-localized ppGpp synthases/hydrolases function redundantly, the impact of the loss of ppGpp-dependent stringent response on plant physiology remains unclear. We used CRISPR/Cas9 technology to generate an Arabidopsis thaliana mutant lacking all four ppGpp synthases/hydrolases and characterized its phenotype. The mutant showed over 20-fold less ppGpp levels than the wild type under normal growth conditions and exhibited leaf chlorosis and increased expression of defense-related genes as well as salicylic acid and jasmonate levels upon transition to nitrogen-starvation conditions. These results demonstrate that proper levels of ppGpp in plastids are required for controlling not only plastid metabolism but also phytohormone signaling, which is essential for plant defense., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In bacteria, the hyper-phosphorylated nucleotide, guanosine 3',5'-bis(pyrophosphate) (ppGpp), functions as a secondary messenger under stringent conditions. ppGpp levels are controlled by two distinct enzymes, namely RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3 and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2 and RSH3 were undertaken, which showed that the ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here, to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase in ppGpp was observed for 30 min in the wild type (WT) after the light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyses were performed with the rsh2-rsh3 double and rsh1-rsh2-rsh3 triple mutants and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase in ppGpp in the WT and rsh2 rsh3 accompanied decrements in the mRNA levels of some plastidial genes transcribed by the plastid-encoded plastid RNA polymerase. These results indicate that the transient increase in ppGpp at night is due to CRSH-dependent ppGpp synthesis and that the ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2 and RSH3 to accustom plastidial gene expression to darkness., (� The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Precise control of the proton concentration gradient across thylakoid membranes (ΔpH) is essential for photosynthesis and its regulation because the gradient contributes to the generation of the proton motive force used for ATP synthesis and also for the fast and reversible induction of non-photochemical quenching (NPQ) to avoid photoinhibition and photodamage. However, the regulatory mechanism(s) controlling ΔpH in response to fluctuating light has not been fully elucidated. We previously described a new NPQ-regulatory chloroplastic protein, Fluctuating-Light-Acclimation Protein1 (FLAP1), which is important for plant growth and modulation of ΔpH under fluctuating light conditions. For this report, we further characterized FLAP1 activity by individually crossing an Arabidopsis flap1 mutant with npq4 and pgr5 plants; npq4 is defective in PsbS-dependent NPQ, and pgr5 is defective in induction of steady-state proton motive force (pmf) and energy-dependent quenching (qE). Both npq4 and npq4 flap1 exhibited similar NPQ kinetics and other photosynthetic parameters under constant or fluctuating actinic light. Conversely, pgr5 flap1 had recovered NPQ, photosystem II quantum yield and growth under fluctuating light, each of which was impaired in pgr5. Together with other data, we propose that FLAP1 activity controls proton homeostasis under steady-state photosynthesis to manipulate luminal acidification levels appropriately to balance photoprotection and photochemical processes.

The proton motive force (PMF) across the chloroplast thylakoid membrane that is generated by electron transport during photosynthesis is the driving force for ATP synthesis in plants. The PMF mainly arises from the oxidation of water in photosystem II and from electron transfer within the cytochrome b 6 f complex. There are two electron transfer pathways related to PMF formation: linear electron flow and cyclic electron flow. Proton gradient regulation 5 (PGR5) is a major component of the cyclic electron flow pathway, and the Arabidopsis pgr5 mutant shows a substantial reduction in the PMF. How the PGR5-dependent cyclic electron flow contributes to ATP synthesis has not, however, been fully delineated. In this study, we monitored in vivo ATP levels in Arabidopsis chloroplasts in real time using a genetically encoded bioluminescence-based ATP indicator, Nano-lantern(ATP1). The increase in ATP in the chloroplast stroma of pgr5 leaves upon illumination with actinic light was significantly slower than in wild type, and the decrease in ATP levels when this illumination stopped was significantly faster in pgr5 leaves than in wild type. These results indicated that PGR5-dependent cyclic electron flow around photosystem I helps to sustain the rate of ATP synthesis, which is important for growth under fluctuating light conditions.

The regulatory nucleotides, guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp), were originally identified in Escherichia coli, and control a large set of gene expression and enzyme activities. The (p)ppGpp-dependent control of cell activities is referred to as the stringent response. A growing number of (p)ppGpp synthase/hydrolase homologs have been identified in plants, which are localized in plastids in Arabidopsis thaliana. We recently reported that the Arabidopsis mutant overproducing ppGpp in plastids showed dwarf chloroplasts, and transcript levels in the mutant plastids were significantly suppressed. Furthermore, the mutant showed more robust growth than the wild type (WT), especially under nutrient-deficient conditions, although the mechanisms are unclear. To better understand the impact of the ppGpp accumulation on plant responses to nutrient deficiency, photosynthetic activities and metabolic changes in the ppGpp-overproducing mutant were characterized here. Upon transition to the nitrogen-deficient conditions, the mutant showed reduction of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) contents, and effective and maximum quantum yield of photosystem II compared with WT. The mutant also showed more obvious changes in key metabolite levels including some amino acid contents than WT; similar metabolic change is known to be critical for plants to maintain carbon-nitrogen balance in their cells. These results suggest that artificially overproducing ppGpp modulates the organelle functions that play an important role in controlling photosynthetic performance and metabolite balance during nitrogen starvation.

Plants have mechanisms allowing them to acclimate to intense light conditions, which involves the dissipation of excess light energy. These mechanisms allow plants to perform photosynthesis efficiently and, therefore, must be accurately and precisely controlled. However, how plants dissipate excess light energy has yet to be fully elucidated. Herein we report the identification of a gene, which we named Fluctuating-Light-Acclimation Protein1 (FLAP1), that is conserved in oxygenic phototrophs. We show that Arabidopsis FLAP1 is associated with chloroplast thylakoid and envelope membranes and that the flap1 mutant shows delayed non-photochemical quenching (NPQ) relaxation during induction of photosynthesis at moderate light intensity. Under fluctuating light conditions, NPQ levels in the flap1 mutant were higher than those in the wild type during the high light period, and the mutant exhibited a pale-green phenotype. These findings suggest that FLAP1 is involved in NPQ control, which is important for an acclimation response to fluctuating light., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The chloroplast, an essential organelle for plants, performs a wide variety of metabolic processes for host cells, which include photosynthesis as well as amino acid and fatty acid biosynthesis. The organelle conserves many bacterial systems in its functions, implicating its origin from symbiosis of a photosynthetic bacterium. In bacterial cells, the stringent response acts as a global regulatory system for gene expression mediated by a small nucleotide, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), that is necessary for cell adaptation to diverse environmental stimuli such as amino acid starvation. Recent studies indicated that proteins similar to the bacterial ppGpp synthase/hydrolyase are conserved in plants, although their precise roles are not known. Here we show that the stringent response in chloroplasts is crucial for normal plant fertilization. Specifically, one of the Arabidopsis ppGpp synthase homologs, CRSH (Ca(2+)-activated RelA/SpoT homolog), exhibits calcium-dependent ppGpp synthesis activity in vitro, and is localized in chloroplasts in vivo. A knockdown mutation of CRSH in Arabidopsis results in a significant reduction in silique size and seed production, indicating that plant reproduction is under the control of chloroplast function through a ppGpp-mediated stringent response.

The genetic system of chloroplasts, including the machinery for transcription, translation, and DNA replication, exhibits substantial similarity to that of eubacteria. Chloroplasts are also thought to possess a system for generating guanosine 5'-triphosphate ((p)ppGpp), which triggers the stringent response in eubacteria, with genes encoding chloroplastic (p)ppGpp synthetase having been identified. We now describe the identification and characterization of genes (OsCRSH1, OsCRSH2, and OsCRSH3) for a novel type of (p)ppGpp synthetase in rice. The proteins encoded by these genes contain a putative chloroplast transit peptide at the NH(2) terminus, a central RelA-SpoT-like domain, and two EF-hand motifs at the COOH terminus. The recombinant OsCRSH1 protein was imported into chloroplasts in vitro, and genetic complementation analysis revealed that expression of OsCRSH1 suppressed the phenotype of an Escherichia coli mutant deficient in the RelA and SpoT enzymes. Biochemical analysis showed that the OsCRSH proteins possess (p)ppGpp synthetase activity that is dependent both on Ca(2+) and on the EF-hand motifs. A data base search identified a CRSH homolog in the dicotyledon Arabidopsis thaliana, indicating that such genes are conserved among both monocotyledonous and dicotyledonous land plants. CRSH proteins thus likely function as Ca(2+)-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca(2+) and (p)ppGpp signaling in regulation of the genetic system of these organelles.

Chloroplast development in cotyledons differs in a number of ways from that in true leaves, but the cotyledon-specific program of chloroplast biogenesis has not been clarified. The cyo1 mutant in Arabidopsis thaliana has albino cotyledons but normal green true leaves. Chloroplasts develop abnormally in cyo1 mutant plants grown in the light, but etioplasts are normal in mutants grown in the dark. We isolated CYO1 by T-DNA tagging and verified that the mutant allele was responsible for the albino cotyledon phenotype by complementation. CYO1 has a C(4)-type zinc finger domain similar to that of Escherichia coli DnaJ. CYO1 is expressed mainly in young plants under light conditions, and the CYO1 protein localizes to the thylakoid membrane in chloroplasts. Transcription of nuclear photosynthetic genes is generally unaffected by the cyo1 mutation, but the level of photosynthetic proteins is decreased in cyo1 mutants. Recombinant CYO1 accelerates disulfide bond reduction in the model substrate insulin and renatures RNase A, indicating that CYO1 has protein disulfide isomerase activity. These results suggest that CYO1 has a chaperone-like activity required for thylakoid biogenesis in cotyledons.

The control of pH in chloroplasts is important to regulate photosynthesis, although details of the precise regulatory mechanisms of H+ homeostasis in chloroplasts are not fully understood. We recently found that the cyanobacterial PxcA homolog DLDG1 is involved in plastidial pH control. PxcA and DLDG1 have been thought to control light‐dependent H+ extrusion across the cyanobacterial cytoplasmic and chloroplast envelope membranes, respectively. To investigate DLDG1‐dependent pH control in chloroplasts, we crossed the dldg1 mutant with various mutants lacking known non‐photochemical quenching (NPQ)‐related proteins, such as fluctuating‐light acclimation protein 1 (FLAP1), PsbS/NPQ4, and proton gradient regulation 5 (PGR5). Phenotypes of these double mutants revealed that PsbS works upstream of DLDG1, PGR5 affects NPQ independently from DLDG1, and the ΔpH regulation by FLAP1 and DLDG1 are independent of each other. [ABSTRACT FROM AUTHOR]

Guanosine tetraphosphate (ppGpp) is known as an alarmone that mediates bacterial stress responses. In plants, ppGpp is synthesized in chloroplasts from GTP and ATP and functions as a regulator of chloroplast gene expression to affect photosynthesis and plant growth. This observation indicates that ppGpp metabolism is closely related to chloroplast function, but the regulation of ppGpp and its role in chloroplast differentiation are not well understood. In rice, ppGpp directly inhibits plastidial guanylate kinase (GKpm), a key enzyme in GTP biosynthesis. GKpm is highly expressed during early leaf development in rice, and the GKpm-deficient mutant, virescent-2 (v2), develops chloroplast-deficient chlorotic leaves under low-temperature conditions. To examine the relationship between GTP synthesis and ppGpp homeostasis, we generated transgenic rice plants over-expressing RSH3, a protein known to act as a ppGpp synthase. When RSH3 was overexpressed in v2 , the leaf chlorosis was more severe. Although the RSH3 overexpression in the wild type caused no visible effects, pulse amplitude modulation fluorometer measurements indicated that photosynthetic rates were reduced in this line. This finding implies that the regulation of ppGpp synthesis in rice is involved in the maintenance of the GTP pool required to regulate plastid gene expression during early chloroplast biogenesis. We further investigated changes in the expressions of RelA/SpoT Homolog (RSH) genes encoding ppGpp synthases and hydrolases during the same period. Comparing the expression of these genes with the cellular ppGpp content suggests that the basal ppGpp level is determined by the antagonistic action of multiple RSH enzymatic activities during early leaf development in rice. [ABSTRACT FROM AUTHOR]

The pH of various chloroplast compartments, such as the thylakoid lumen and stroma, is light-dependent. Light illumination induces electron transfer in the photosynthetic apparatus, coupled with proton translocation across the thylakoid membranes, resulting in acidification and alkalization of the thylakoid lumen and stroma, respectively. Luminal acidification is crucial for inducing regulatory mechanisms that protect photosystems against photodamage caused by the overproduction of reactive oxygen species (ROS). Stromal alkalization activates enzymes involved in the Calvin–Benson–Bassham (CBB) cycle. Moreover, proton translocation across the thylakoid membranes generates a proton gradient (ΔpH) and an electric potential (ΔΨ), both of which comprise the proton motive force (pmf) that drives ATP synthase. Then, the synthesized ATP is consumed in the CBB cycle and other chloroplast metabolic pathways. In the dark, the pH of both the chloroplast stroma and thylakoid lumen becomes neutral. Despite extensive studies of the above-mentioned processes, the molecular mechanisms of how chloroplast pH can be maintained at proper levels during the light phase for efficient activation of photosynthesis and other metabolic pathways and return to neutral levels during the dark phase remain largely unclear, especially in terms of the precise control of stromal pH. The transient increase and decrease in chloroplast pH upon dark-to-light and light-to-dark transitions have been considered as signals for controlling other biological processes in plant cells. Forward and reverse genetic screening approaches recently identified new plastid proteins involved in controlling ΔpH and ΔΨ across the thylakoid membranes and chloroplast proton/ion homeostasis. These proteins have been conserved during the evolution of oxygenic phototrophs and include putative photosynthetic protein complexes, proton transporters, and/or their regulators. Herein, we summarize the recently identified protein players that control chloroplast pH and influence photosynthetic efficiency in plants. [ABSTRACT FROM AUTHOR]

pH homeostasis in the chloroplast is crucial for the control of photosynthesis and other metabolic processes in plants. Recently, nuclear‐encoded Day‐Length‐dependent Delayed Greening1 (DLDG1) and Fluctuating‐Light Acclimation Protein1 (FLAP1) that are required for the light‐inducible optimization of plastidial pH in Arabidopsis thaliana were identified. DLDG1 and FLAP1 homologs are specifically conserved in oxygenic phototrophs, and a DLDG1 homolog, Ycf10, is encoded in the chloroplast genome in plant cells. However, the function of Ycf10 and its physiological significance are unknown. To address this, we constructed ycf10 tobacco Nicotiana tabacum mutants and characterized their phenotypes. The ycf10 tobacco mutants grown under continuous‐light conditions showed a pale‐green phenotype only in developing leaves, and it was suppressed in short‐day conditions. The ycf10 mutants also induced excessive non‐photochemical quenching (NPQ) compared with those in the wild‐type at the induction stage of photosynthesis. These phenotypes resemble those of Arabidopsis dldg1 mutants, suggesting that they have similar functions. However, there are distinct differences between the two mutant phenotypes: The highly induced NPQ in tobacco ycf10 and the Arabidopsis dldg1 mutants are diminished and enhanced, respectively, with increasing duration of the fluctuating actinic‐light illumination. Ycf10 and DLDG1 were previously shown to localize in chloroplast envelope‐membranes, suggesting that Ycf10 and DLDG1 differentially control H+ exchange across these membranes in a light‐dependent manner to control photosynthesis. [ABSTRACT FROM AUTHOR]

Plants convert solar energy into chemical energy through photosynthesis, which supports almost all life activities on earth. Because the intensity and quality of sunlight can change dramatically throughout the day, various regulatory mechanisms help plants adjust their photosynthetic output accordingly, including the regulation of light energy accumulation to prevent the generation of damaging reactive oxygen species. Non-photochemical quenching (NPQ) is a regulatory mechanism that dissipates excess light energy, but how it is regulated is not fully elucidated. In this study, we report a new NPQ-regulatory protein named Day-Length-dependent Delayed-Greening1 (DLDG1). The Arabidopsis DLDG1 associates with the chloroplast envelope membrane, and the dldg1 mutant had a large NPQ value compared with wild type. The mutant also had a pale-green phenotype in developing leaves but only under continuous light; this phenotype was not observed when dldg1 was cultured in the dark for ≥8 h/d. DLDG1 is a homolog of the plasma membrane-localizing cyanobacterial proton-extrusion-protein A that is required for light-induced H+ extrusion and also shows similarity in its amino-acid sequence to that of Ycf10 encoded in the plastid genome. Arabidopsis DLDG1 enhances the growth-retardation phenotype of the Escherichia coli K+/H+ antiporter mutant, and the everted membrane vesicles of the E. coli expressing DLDG1 show the K+/H+ antiport activity. Our findings suggest that DLDG1 functionally interacts with Ycf10 to control H+ homeostasis in chloroplasts, which is important for the light-acclimation response, by optimizing the extent of NPQ. [ABSTRACT FROM AUTHOR]

Recently, a bacterial second messenger, guanosine 5′-diphosphate 3′-diphosphate (ppGpp), has been detected in chloroplasts. However, because ppGpp concentration in plants is much lower than that in bacteria, detailed analysis of ppGpp in plants has not been performed. A highly sensitive quantification method is required for further characterization of ppGpp function in chloroplasts. Here, we report a new method that allows for the highly sensitive and selective high-throughput quantification of ppGpp by ultra-performance liquid chromatography (UPLC) coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an electrospray interface (ESI). This method requires only ~100 mg of plant tissue for ppGpp quantification. We used this method to measure ppGpp levels in Arabidopsis thaliana under different light conditions. A. thaliana accumulated ppGpp during dark periods. This method will be helpful to further characterize the stringent response in higher plants. [ABSTRACT FROM AUTHOR]

Monogalactosyldiacylglycerol (MGDG), which is conserved in almost all photosynthetic organisms, is the most abundant natural polar lipid on Earth. In plants, MGDG is highly accumulated in the chloroplast membranes and is an important bulk constituent of thylakoid membranes. However, precise functions of MGDG in photosynthesis have not been well understood. Here, we report a novel MGDG synthase from the green sulfur bacterium Chlorobaculum tepidum. This enzyme, MgdA, catalyzes MGDG synthesis using UDP-Gal as a substrate. The gene encoding MgdA was essential for this bacterium; only heterozygous mgdA mutants could be isolated. An mgdA knockdown mutation affected in vivo assembly of bacteriochlorophyll c aggregates, suggesting the involvement of MGDG in the construction of the light-harvesting complex called chlorosome. These results indicate that MGDG biosynthesis has been independently established in each photosynthetic organism to perform photosynthesis under different environmental conditions. We complemented an Arabidopsis thaliana MGDG synthase mutant by heterologous expression of MgdA. The complemented plants showed almost normal levels of MGDG, although they also had abnormal morphological phenotypes, including reduced chlorophyll content, no apical dominance in shoot growth, atypical flower development, and infertility. These observations provide new insights regarding the importance of regulated MGDG synthesis in the physiology of higher plants. [ABSTRACT FROM AUTHOR]

Upon a dark-to-light transition, multiple species of cyanobacteria release a certain amount of H+ from the inside to the outside of their cells. Previous studies revealed the plasma membrane-localizing Proton exchange A (PxcA) is involved in the light-induced H+ extrusion in the cyanobacterium Synechocystis sp. PCC6803. Among oxygenic phototrophs, two PxcA homologs are conserved; they are the nuclear-encoded Day-length-dependent delayed-greening1 (DLDG1) and the plastid-encoded Ycf10 in Arabidopsis thaliana. We previously identified the putative DLDG1/Ycf10-interacting protein, Fluctuating-light acclimation protein1 (FLAP1), required for pH regulation in Arabidopsis chloroplasts. Synechocystis has PxcA and FLAP1 homologs designated here as PxcA like (PxcL) and FLAP1 homolog A (FlpA). Synechocystis mutants lacking pxcA , pxcL, and flpA were constructed and characterized to gain more insight into regulatory mechanisms of light-induced H+ extrusion in cyanobacteria. pH change kinetics of the extracellular solvent after shifting Synechocystis cells from dark to light indicated that PxcA is essential for the light-induced H+ extrusion, and both PxcA and PxcL were involved in H+ uptake. Mutational loss of flpA resulted in altered PxcA- and PxcL-dependent H+ efflux/influx activities, and the flpA -null mutant showed inhibited growth under dark–light cycles, indicating the importance of FlpA function for photosynthetic growth under fluctuating light. Collectively, these data suggest that PxcA is involved in H+ efflux immediately after light irradiation for the rapid formation of the H+ concentration gradient across the thylakoid membranes, PxcL is involved in H+ influx for activation of the Calvin-Benson-Bassham cycle, and FlpA controls the H+ transport under fluctuating light. Unlabelled Image • pH homeostasis is critical for regulation of oxygenic photosynthesis. • Cyanobacteria extrude and uptake H+ during light illumination • Three proteins conserved in oxygenic phototrophs are involved in the H+ translocation. • A schematic model of the control of H+ translocation in cyanobacteria is proposed. [ABSTRACT FROM AUTHOR]