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C4 plants assimilate CO2 more efficiently than C3 plants because of their C4 cycle that concentrates CO2. However, the C4 cycle requires additional ATP molecules, which may be supplied by cyclic electron flow (CEF) around photosystem I. One CEF route, which depends on a chloroplast NADH dehydrogenase-like (NDH) complex, is suggested to be crucial for C4 plants despite the low activity in C3 plants. The other route depends on proton gradient regulation 5 (PGR5) and PGR5-like photosynthetic phenotype 1 (PGRL1) and is considered a major CEF route to generate the proton gradient across the thylakoid membrane in C3 plants. However, its contribution to C4 photosynthesis is still unclear. In this study, we investigated the contribution of the two CEF routes to the NADP-malic enzyme subtype of C4 photosynthesis in Flaveria bidentis. We observed that suppressing the NDH-dependent route drastically delayed growth and decreased the CO2 assimilation rate to approximately 30% of the wild-type rate. On the other hand, suppressing the PGR5/PGRL1-dependent route did not affect plant growth and resulted in a CO2 assimilation rate that was approximately 80% of the wild-type rate. Our data indicate that the NDH-dependent CEF substantially contributes to the NADP-malic enzyme subtype of C4 photosynthesis and that the PGR5/PGRL1-dependent route cannot complement the NDH-dependent route in F. bidentis. These findings support the fact that during C4 evolution, photosynthetic electron flow may have been optimized to provide the energy required for C4 photosynthesis.

Cyclic electron transport (CET) around Photosystem I (PSI) acidifies the thylakoid lumen and downregulates electron transport at the cytochrome b6f complex. This photosynthetic control is essential for oxidizing special pair chlorophylls (P700) of PSI for PSI photoprotection. In addition, CET depending on the PROTON GRADIENT REGULATION 5 (PGR5) protein oxidizes P700 by moving a pool of electrons from the acceptor side of PSI to the plastoquinone pool. This model of the acceptor-side regulation was proposed on the basis of the phenotype of the Arabidopsis (Arabidopsis thaliana) pgr5-1 mutant expressing Chlamydomonas (Chlamydomonas reinhardtii) plastid terminal oxidase (CrPTOX2). In this study, we extended the research including the Arabidopsis chlororespiratory reduction 2-2 (crr2-2) mutant defective in another CET pathway depending on the chloroplast NADH dehydrogenase-like (NDH) complex. Although the introduction of CrPTOX2 did not complement the defect in the acceptor-side regulation by PGR5, the function of the NDH complex was complemented except for its reverse reaction during the induction of photosynthesis. We evaluated the impact of CrPTOX2 under fluctuating light intensity in the wild-type, pgr5-1 and crr2-2 backgrounds. In the high-light period, both PGR5- and NDH-dependent CET were involved in the induction of photosynthetic control, whereas PGR5-dependent CET preferentially contributed to the acceptor-side regulation. On the contrary, the NDH complex probably contributed to the acceptor-side regulation in the low-light period but not in the high-light period. We evaluated the sensitivity of PSI to fluctuating light and clarified that acceptor-side regulation was necessary for PSI photoprotection by oxidizing P700 under high light.

Plastid terminal oxidase (PTOX) accepts electrons from plastoquinol to reduce molecular oxygen to water. We introduced the gene encoding Chlamydomonas reinhardtii (Cr)PTOX2 into the Arabidopsis (Arabidopsis thaliana) wild-type (WT) and proton gradient regulation5 (pgr5) mutant defective in cyclic electron transport around photosystem I (PSI). The accumulation of CrPTOX2 only mildly affected photosynthetic electron transport in the WT background during steady-state photosynthesis but partly complemented the induction of nonphotochemical quenching (NPQ) in the pgr5 background. During the induction of photosynthesis by actinic light (AL) of 130 µmol photons m−2 s−1, the high level of PSII yield (Y(II)) was induced immediately after the onset of AL in WT plants accumulating CrPTOX2. NPQ was more rapidly induced in the transgenic plants than in WT plants. P700 was also oxidized immediately after the onset of AL. Although CrPTOX2 does not directly induce a proton concentration gradient (ΔpH) across the thylakoid membrane, the coupled reaction of PSII generated ΔpH to induce NPQ and the downregulation of the cytochrome b6f complex. Rapid induction of Y(II) and NPQ was also observed in the pgr5 plants accumulating CrPTOX2. In contrast to the WT background, P700 was not oxidized in the pgr5 background. Although the thylakoid lumen was acidified by CrPTOX2, PGR5 was essential for oxidizing P700. In addition to acidification of the thylakoid lumen to downregulate the cytochrome b6f complex (donor-side regulation), PGR5 may be required for draining electrons from PSI by transferring them to the plastoquinone pool. We propose a reevaluation of the contribution of this acceptor-side regulation by PGR5 in the photoprotection of PSI.

RNA-binding pentatricopeptide repeat (PPR) proteins catalyze hundreds of cytidine to uridine RNA editing events in plant organelles; these editing events are essential for proper gene expression. More than half of the PPR-type RNA editing factors, however, lack the DYW cytidine deaminase domain. Genetic analyses have suggested that their cytidine deaminase activity arises by association with a family of DYW1-like proteins that contain an N-terminally truncated DYW domain, but their molecular mechanism has been unclear. Here, we report the crystal structure of the Arabidopsis thaliana DYW1 deaminase domain at 1.8 Å resolution. DYW1 has a cytidine deaminase fold lacking the PG box. The internal insertion within the deaminase fold shows an α-helical fold instead of the β-finger reported for the gating domain of the A. thaliana ORGANELLE TRANSCRIPT PROCESSING 86. The substrate-binding pocket is incompletely formed and appears to be complemented in the complex by the E2 domain and the PG box of the interacting PPR protein. In vivo RNA editing assays corroborate the activation model for DYW1 deaminase. Our study demonstrates the common activation mechanism of the DYW1-like proteins by molecular complementation of the DYW domain and reconstitution of the substrate-binding pocket.

Bacterial cytidine deaminase fused to the DNA binding domains of transcription activator-like effector nucleases was recently reported to transiently substitute a targeted C to a T in mitochondrial DNA of mammalian cultured cells1. We applied this system to targeted base editing in the Arabidopsis thaliana plastid genome. The targeted Cs were homoplasmically substituted to Ts in some plantlets of the T1 generation and the mutations were inherited by their offspring independently of their nuclear-introduced vectors., This study used bacterial cytidine deaminase fused to the DNA binding domains of transcription activator-like effector nucleases to enable targeted base editing in the Arabidopsis thaliana plastid genome, generating T1 plants with inheritable homoplasmic mutations.

C-to-U RNA editing sites in plant organelles show a strong bias for neighboring nucleotides. The nucleotide upstream of the target cytidine is typically C or U, whereas A and G are less common and rare, respectively. In pentatricopeptide repeat (PPR)-type RNA editing factors, the PPR domain specifically binds to the 5' sequence of target cytidines, whereas the DYW domain catalyzes the C-to-U deamination. We comprehensively analyzed the effects of neighboring nucleotides of the target cytidines using an Escherichia coli orthogonal system. Physcomitrium PPR56 efficiently edited target cytidines when the nucleotide upstream was U or C, whereas it barely edited when the position was G or the nucleotide downstream was C. This preference pattern, which corresponds well with the observed nucleotide bias for neighboring nucleotides in plant organelles, was altered when the DYW domain of OTP86 or DYW1 was adopted. The PPR56 chimeric proteins edited the target sites even when the -1 position was G. Our results suggest that the DYW domain possesses a distinct preference for the neighboring nucleotides of the target sites, thus contributing to target selection in addition to the existing selection determined by the PPR domain.

PGR3 is a P-class pentatricopeptide repeat (PPR) protein required for the stabilization of petL operon RNA and the translation of the petL gene in plastids. Irrespective of its important roles in plastids, key questions have remained unanswered, including how PGR3 protein promotes translation and which plastid mRNA PGR3 activates the translation. Here, we show that PGR3 facilitates the translation from ndhG, in addition to petL, through binding to their 5′ untranslated regions (UTRs). Ribosome profiling and RNA sequencing in pgr3 mutants revealed that translation from petL and ndhG was specifically suppressed. Harnessing small RNA fragments protected by PPR proteins in vivo, we probed the PGR3 recruitment to the 5′ UTRs of petL and ndhG. The putative PGR3-bound RNA segments per se repress the translation possibly with a strong secondary structure and thereby block ribosomes’ access. However, the PGR3 binding antagonizes the effects and facilitates the protein synthesis from petL and ndhG in vitro. The prediction of the 3-dimensional structure of PGR3 suggests that the 26th PPR motif plays important roles in target RNA binding. Our data show the specificity of a plastidic RNA-binding protein and provide a mechanistic insight into translational control.

In angiosperms, the NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around PSI (CET). K(+) Efflux Antiporter3 (KEA3) is a putative thylakoid H(+)/K(+) antiporter and allows an increase in membrane potential at the expense of the ∆pH component of the proton motive force. In this study, we discovered that the chlororespiratory reduction2-1 (crr2-1) mutation, which abolished NDH-dependent CET, enhanced the kea3-1 mutant phenotypes in Arabidopsis (Arabidopsis thaliana). The NDH complex pumps protons during CET, further enhancing ∆pH, but its physiological function has not been fully clarified. The observed effect only took place upon exposure to light of 110 µmol photons m(−2) s(−1) after overnight dark adaptation. We propose two distinct modes of NDH action. In the initial phase, within 1 min after the onset of actinic light, the NDH-dependent CET engages with KEA3 to enhance electron transport efficiency. In the subsequent phase, in which the ∆pH-dependent down-regulation of the electron transport is relaxed, the NDH complex engages with KEA3 to relax the large ∆pH formed during the initial phase. We observed a similar impact of the crr2-1 mutation in the genetic background of the PROTON GRADIENT REGULATION5 overexpression line, in which the size of ∆pH was enhanced. When photosynthesis was induced at 300 µmol photons m(−2) s(−1), the contribution of KEA3 was negligible in the initial phase and the ∆pH-dependent down-regulation was not relaxed in the second phase. In the crr2-1 kea3-1 double mutant, the induction of CO(2) fixation was delayed after overnight dark adaptation.

In response to light, plants efficiently induce photosynthesis. Light activation of thiol enzymes by the thioredoxin (Trx) systems and cyclic electron transport by the PROTON GRADIENT REGULATION5 (PGR5)-dependent pathway contribute substantially to regulation of photosynthesis. Arabidopsis (Arabidopsis thaliana) mutants lacking f-type Trxs (trx f1f2) show delayed activation of carbon assimilation due to impaired photoreduction of Calvin-Benson cycle enzymes. To further study regulatory mechanisms that contribute to efficiency during the induction of photosynthesis, we analyzed the contributions of PSI donor- and acceptor-side regulation in the trx f1f2 mutant background. The cytochrome b(6)f complex is involved in PSI donor-side regulation, whereas PGR5-dependent PSI cyclic electron transport is required for both donor and acceptor functions. Introduction of the pgr1 mutation, which is conditionally defective in cytochrome b(6)f complex activity, into the trx f1f2 mutant background did not further affect the induction of photosynthesis, but the combined deficiency of Trx f and PGR5 severely impaired photosynthesis and suppressed plant growth under long-day conditions. In the pgr5 trx f1f2 mutant, the acceptor-side of PSI was almost completely reduced, and quantum yields of PSII and PSI hardly increased during the induction of photosynthesis. We also compared the photoreduction of thiol enzymes between the trx f1f2 and pgr5 trxf1f2 mutants. The pgr5 mutation did not result in further impaired photoreduction of Calvin-Benson cycle enzymes or ATP synthase in the trx f1f2 mutant background. These results indicated that acceptor-side limitations in the pgr5 trx f1f2 mutant suppress photosynthesis initiation, suggesting that PGR5 is required for efficient photosynthesis induction.

Despite generating an obvious mutant phenotype, whether the Arabidopsis (Arabidopsis thaliana) proton gradient regulation5 (pgr5) mutation influences cyclic electron transport (CET) around PSI is a topic of debate. Results of electrochromic shift analysis show that proton conductivity across the thylakoid membrane (g(H)(+)) in the pgr5 mutant is enhanced at high light intensity. Given this observation, PGR5 was proposed to regulate ATP synthase activity rather than mediating CET. The originally reported pgr5 phenotype reflects a smaller proton motive force (pmf) and could be explained by this H(+) leakage model. In this study, we genetically reexamined the high-g(H)(+) phenotype of the pgr5 mutant. Transgenic lines in which flavodiiron protein-dependent pseudo-CET replaced PGR5-dependent CET had wild-type levels of g(H)(+), suggesting that the high-g(H)(+) phenotype in pgr5 plants is caused secondarily by the low pmf. The pgr1 mutant shows a similar reduction in pmf because of enhanced sensitivity of its cytochrome b(6)f complex to lumenal acidification. In contrast to the pgr5 mutant, g(H)(+) was lower in the pgr1 mutant than in the wild type. In the pgr1 pgr5 double mutants, g(H)(+) was intermediate to g(H)(+) values of the respective single mutants. It is unlikely that g(H)(+) is upregulated simply in response to a low pmf. We did not observe uncoupling of the thylakoid membrane in the pgr5 mutant upon monitoring the quenching of 9-aminoacridine fluorescence. We conclude that the g(H)(+) parameter may be influenced by other factors not related to the H(+) leakage through ATP synthase. It is unlikely that the pgr5 mutant leaks protons from the thylakoid membrane.

C4 plants can fix CO2 efficiently using CO2 -concentrating mechanisms (CCMs), but they require additional ATP. To supply the additional ATP, C4 plants operate at higher rates of cyclic electron transport around photosystem I (PSI), in which electrons are transferred from ferredoxin to plastoquinone. Recently, it has been reported that the NAD(P)H dehydrogenase-like complex (NDH) accumulated in the thylakoid membrane in leaves of C4 plants, making it a candidate for the additional synthesis of ATP used in the CCM. In addition, C4 plants have higher levels of PROTON GRADIENT REGULATION 5 (PGR5) expression, but it has been unknown how PGR5 functions in C4 photosynthesis. In this study, PGR5 was overexpressed in a C4 dicot, Flaveria bidentis. In PGR5-overproducing (OP) lines, PGR5 levels were 2.3- to 3.0-fold greater compared with wild-type plants. PGR5-like PHOTOSYNTHETIC PHENOTYPE 1 (PGRL1), which cooperates with PGR5, increased with PGR5. A spectroscopic analysis indicated that in the PGR5-OP lines, the acceptor side limitation of PSI was reduced in response to a rapid increase in photon flux density. Although it did not affect CO2 assimilation, the overproduction of PGR5 contributed to an enhanced electron sink downstream of PSI.

The proton concentration gradient (ΔpH) and membrane potential (Δψ) formed across the thylakoid membrane contribute to ATP synthesis in chloroplasts. Additionally, ΔpH downregulates photosynthetic electron transport via the acidification of the thylakoid lumen. K+ exchange antiporter 3 (KEA3) relaxes this downregulation by substituting ΔpH with Δψ in response to fluctuation of light intensity. In the Arabidopsis (Arabidopsis thaliana) line overexpressing KEA3 (KEA3ox), the rate of electron transport is elevated by accelerating the relaxation of ΔpH after a shift from high light (HL) to low light. However, the plant cannot control electron transport toward photosystem I (PSI), resulting in PSI photodamage. In this study, we crossed the KEA3ox line with the line (Flavodiiron [Flv]) expressing the Flv proteins of Physcomitrium patens. In the double transgenic line (Flv-KEA3ox), electrons overloading toward PSI were pumped out by Flv proteins. Consequently, photodamage of PSI was alleviated to the wild-type level. The rate of CO2 fixation was enhanced in Flv and Flv-KEA3ox lines during HL periods of fluctuating light, although CO2 fixation was unaffected in any transgenic lines in constant HL. Upregulation of CO2 fixation was accompanied by elevated stomatal conductance in fluctuating light. Consistent with the results of gas exchange experiments, the growth of Flv and Flv-KEA3ox plants was better than that of WT and KEA3ox plants under fluctuating light.

Fragility of photosystem I has been observed in transgenic rice plants that overproduce Rubisco activase (RCA). In this study, we examined the effects of RCA overproduction on the sensitivity of PSI to photoinhibition in three lines of plants overexpressing RCA (RCA-ox). In all the RCA-ox plants the quantum yield of PSI [Y(I)] decreased whilst in contrast the quantum yield of acceptor-side limitation of PSI [Y(NA)] increased, especially under low light conditions. In the transgenic line with the highest RCA content (RCA-ox 1), the quantum yield of PSII [Y(II)] and CO2 assimilation also decreased under low light. When leaves were exposed to high light (2000 μmol photon m−2 s−1) for 60 min, the maximal activity of PSI (Pm) drastically decreased in RCA-ox 1. These results suggested that overproduction of RCA disturbs PSI electron transport control, thus increasing the susceptibility of PSI to photoinhibition. When flavodiiron protein (FLV), which functions as a large electron sink downstream of PSI, was expressed in the RCA-ox 1 background (RCA-FLV), PSI and PSII parameters, and CO2 assimilation were recovered to wild-type levels. Thus, expression of FLV restored the robustness of PSI in RCA-ox plants.

The thylakoid K(+) efflux antiporter 3 (KEA3) is required for regulating components of the proton motive force (pmf), proton concentration gradient (ΔpH), and membrane potential (Δψ). The Arabidopsis (Arabidopsis thaliana) disturbed proton gradient regulation mutant (dpgr) is a dominant allele of KEA3, conferring disturbed transport activity. Here, we show that overexpressing the DPGR-type KEA3 (DPGRox) retarded plant growth, whereas overexpressing the wild-type KEA3 (KEA3ox) did not. In KEA3ox lines, the contribution of Δψ to pmf was enhanced, but in DPGRox lines, the size of pmf was reduced. In DPGRox plants, proton conductivity of the thylakoid membrane (g(H)(+)) was elevated under high light, implying disturbed stoichiometry of H(+)/K(+) antiport through DPGR-type KEA3 rather than simply enhanced activity. The ΔpH-dependent regulation consisting of thermal dissipation of excessively absorbed light energy and downregulation of cytochrome b(6)f complex activity was severely and mildly affected in DPGRox and KEA3ox plants, respectively. Consequently, photosystem I was sensitive to fluctuating light in both transgenic plants. Both photosystems were sensitive to constant high light and were slightly photodamaged even at standard growth light intensity in DPGRox plants. KEA3 regulates the components of pmf and optimizes the operation of ∆pH-dependent regulation of electron transport.

Amborella trichopoda is placed close to the base of the angiosperm lineage (basal angiosperm). By genome-wide RNA sequencing, we identified 184C-to-U RNA editing sites in the plastid genome of Amborella. This number is much higher than that observed in other angiosperms including maize (44 sites), rice (39 sites) and grape (115 sites). Despite the high frequency of RNA editing, the biased distribution of RNA editing sites in the genome, target codon preference and nucleotide preference adjacent to the edited cytidine are similar to that in other angiosperms, suggesting a common editing machinery. Consistent with this idea, the Amborella nuclear genome encodes 2–3 times more of the E- and DYW-subclass members of pentatricopeptide repeat proteins responsible for RNA editing site recognition in plant organelles. Among 165 editing sites in plastid protein coding sequences in Amborella, 100 sites were conserved at least in one out of 38 species selected to represent key branching points of the angiosperm phylogenetic tree. We assume these 100 sites represent at least a subset of the sites in the plastid editotype of ancestral angiosperms. We then mapped the loss and gain of editing sites on the phylogenetic tree of angiosperms. Our results support the idea that the evolution of angiosperms has led to the loss of RNA editing sites in plastids.

SQUAMOSA promoter-binding protein-like 7 mediates copper deficiency response in the presence of high nitrogen even with the sufficient level of copper in Arabidopsis thaliana. Under copper (Cu) deficiency, accumulation of mRNA encoding two Cu/Zn superoxide dismutases, CSD1 and CSD2, is downregulated to save Cu for plastocyanin. This downregulation depends on miR398 and is under the control of SQUAMOSA promoter-binding protein-like7 (SPL7). Arabidopsis seedlings are routinely cultured on Murashige and Skoog medium. However, the high nitrogen (N) content of the medium (60 mM) has been shown to induce a similar response to Cu deficiency. The mRNA and protein levels of CSD1 and CSD2 are reduced under high N conditions, even if the Cu concentration in the medium is sufficient (0.1-0.5 µM). In this study, we show that this symptom, similar to the Cu deficiency, occurred in the presence of high N largely depending on SPL7, suggesting that plants actually sensed Cu deficiency. However, a change in N concentration in the medium did not influence the total Cu concentration in shoots or roots. High N did not increase the protein content in leaves but facilitated rapid seedling growth. We speculate that this rapid growth causes a continuous Cu deficiency mainly because of high Cu uptake by mesophyll cells in the leaves. This idea was supported by the observation that plastocyanin did not overaccumulate at the range of 0.1-0.5 µM Cu with 30 mM N. In contrast, in the presence of 5 µM Cu with 30 mM N, plants accumulate more Cu in plastocyanin in the thylakoid lumen, resulting in a slight Cu deficiency in the chloroplast stroma. This process is independent of SPL7.

Chloroplast gene expression is controlled by numerous nuclear-encoded RNA-binding proteins. Among these, pentatricopeptide repeat (PPR) proteins are known to be key players in post-transcriptional regulation in chloroplasts. However, the functions of many PPR proteins remain unknown. In this study, we characterized the function of a chloroplast-localized P-class PPR protein PpPPR_21 in Physcomitrella patens. Knockout (KO) mutants of PpPPR_21 exhibited reduced protonemata growth and lower photosynthetic activity. Immunoblot analysis and blue-native gel analysis showed a remarkable reduction of the photosystem II (PSII) reaction center protein and poor formation of the PSII supercomplexes in the KO mutants. To assess whether PpPPR_21 is involved in chloroplast gene expression, chloroplast genome-wide microarray analysis and Northern blot hybridization were performed. These analyses indicated that the psbI-ycf12 transcript encoding the low molecular weight subunits of PSII did not accumulate in the KO mutants while other psb transcripts accumulated at similar levels in wild-type and KO mutants. A complemented PpPPR_21KO moss transformed with the cognate full-length PpPPR_21cDNA rescued the level of accumulation of psbI-ycf12 transcript. RNA-binding experiments showed that the recombinant PpPPR_21 bound efficiently to the 5' untranslated and translated regions of psbImRNA. The present study suggests that PpPPR_21 may be essential for the accumulation of a stable psbI-ycf12mRNA.

In response to a sudden increase in light intensity, plants must cope with absorbed excess photon energy to protect photosystems from photodamage. Under fluctuating light, PSI is severely photodamaged in the Arabidopsis (Arabidopsis thaliana) proton gradient regulation5 (pgr5) mutant defective in the main pathway of PSI cyclic electron transport (CET). Here, we aimed to determine how PSI is protected by two proposed regulatory roles of CET via transthylakoid ΔpH formation: (1) reservation of electron sink capacity by adjusting the ATP/NADPH production ratio (acceptor-side regulation) and (2) down-regulation of the cytochrome b(6)f complex activity called photosynthetic control for slowing down the electron flow toward PSI (donor-side regulation). We artificially enhanced donor- and acceptor-side regulation in the wild-type and pgr5 backgrounds by introducing the pgr1 mutation conferring the hypersensitivity of the cytochrome b(6)f complex to luminal acidification and moss Physcomitrella patens flavodiiron protein genes, respectively. Enhanced photosynthetic control partially alleviated PSI photodamage in the pgr5 mutant background but restricted linear electron transport under constant high light, suggesting that the strength of photosynthetic control should be optimized. Flavodiiron protein-dependent oxygen photoreduction formed a large electron sink and alleviated PSI photoinhibition, accompanied by the induction of photosynthetic control. Thus, donor-side regulation is essential for PSI photoprotection but acceptor-side regulation also is important to rapidly induce donor-side regulation. In angiosperms, PGR5-dependent CET is required for both functions.

Recently, the liverwort Marchantia polymorpha has received increasing attention as a basal plant model for multicellular studies. Its ease of handling, well-characterized plastome and proven protocols for biolistic plastid transformation qualify M. polymorpha as an attractive platform to study the evolution of chloroplasts during the transition from water to land. In addition, chloroplasts of M. polymorpha provide a convenient test-bed for the characterization of genetic elements involved in plastid gene expression due to the absence of mechanisms for RNA editing. While reporter genes have proven valuable to the qualitative and quantitative study of gene expression in chloroplasts, expression of green fluorescent protein (GFP) in chloroplasts of M. polymorpha has proven problematic. We report the design of a codon-optimized gfp varian, mturq2cp, which allowed successful expression of a cyan fluorescent protein under control of the tobacco psbA promoter from the chloroplast genome of M. polymorpha. We demonstrate the utility of mturq2cp in (i) early screening for transplastomic events following biolistic transformation of M. polymorpha spores; (ii) visualization of stromules as elements of plastid structure in Marchantia; and (iii) quantitative microscopy for the analysis of promoter activity.

Background Plastid electron transport systems are essential not only for photosynthesis but also for dissipating excess reducing power and sinking excess electrons generated by various redox reactions. Although numerous organisms with plastids have lost their photoautotrophic lifestyles, there is a spectrum of known functions of remnant plastids in non-photosynthetic algal/plant lineages; some of non-photosynthetic plastids still retain diverse metabolic pathways involving redox reactions while others, such as apicoplasts of apicomplexan parasites, possess highly reduced sets of functions. However, little is known about underlying mechanisms for redox homeostasis in functionally versatile non-photosynthetic plastids and thus about the reductive evolution of plastid electron transport systems. Results Here we demonstrated that the central component for plastid electron transport systems, plastoquinone/plastoquinol pool, is still retained in a novel strain of an obligate heterotrophic green alga lacking the photosynthesis-related thylakoid membrane complexes. Microscopic and genome analyses revealed that the Volvocales green alga, chlamydomonad sp. strain NrCl902, has non-photosynthetic plastids and a plastid DNA that carries no genes for the photosynthetic electron transport system. Transcriptome-based in silico prediction of the metabolic map followed by liquid chromatography analyses demonstrated carotenoid and plastoquinol synthesis, but no trace of chlorophyll pigments in the non-photosynthetic green alga. Transient RNA interference knockdown leads to suppression of plastoquinone/plastoquinol synthesis. The alga appears to possess genes for an electron sink system mediated by plastid terminal oxidase, plastoquinone/plastoquinol, and type II NADH dehydrogenase. Other non-photosynthetic algae/land plants also possess key genes for this system, suggesting a broad distribution of an electron sink system in non-photosynthetic plastids. Conclusion The plastoquinone/plastoquinol pool and thus the involved electron transport systems reported herein might be retained for redox homeostasis and might represent an intermediate step towards a more reduced set of the electron transport system in many non-photosynthetic plastids. Our findings illuminate a broadly distributed but previously hidden step of reductive evolution of plastid electron transport systems after the loss of photosynthesis.

The chloroplast NADH dehydrogenase-like (NDH) complex mediates ferredoxin-dependent plastoquinone reduction in the thylakoid membrane. In angiosperms, chloroplast NDH is composed of five subcomplexes and further forms a supercomplex with photosystem I (PSI). Subcomplex A (SubA) mediates the electron transport and consists of eight subunits encoded by both plastid and nuclear genomes. The assembly of SubA in the stroma has been extensively studied, but it is unclear how SubA is incorporated into the membrane-embedded part of the NDH complex. Here, we isolated a novel Arabidopsis mutant chlororespiratory reduction 16 (crr16) defective in NDH activity. CRR16 encodes a chloroplast-localized P-class pentatricopeptide repeat protein conserved in angiosperms. Transcript analysis of plastid-encoded ndh genes indicated that CRR16 was responsible for the efficient splicing of the group II intron in the ndhA transcript, which encodes a membrane-embedded subunit localized to the connecting site between SubA and the membrane subcomplex (SubM). To analyze the roles of NdhA in the assembly and stability of the NDH complex, the homoplastomic knockout plant of ndhA (ΔndhA) was generated in tobacco (Nicotiana tabacum). Biochemical analyses of crr16 and ΔndhA plants indicated that NdhA was essential for stabilizing SubA and SubE but not for the accumulation of the other three subcomplexes. Furthermore, the crr16 mutant accumulated the SubA assembly intermediates in the stroma more than that in the wild type. These results suggest that NdhA biosynthesis is essential for the incorporation of SubA into the membrane-embedded part of the NDH complex at the final assembly step of the NDH–PSI supercomplex.

In Arabidopsis, a central regulator of copper (Cu) homeostasis is the transcription factor SQUAMOSA promoter binding protein-like7 (SPL7). Under Cu deficiency, SPL7 induces the expression of miR398, which suppresses the expression of the genes CSD1 and CSD2, which encode cytosolic and chloroplastic isoforms of Cu/Zn superoxide dismutase, respectively. Consequently, the limited Cu is preferentially assigned to plastocyanin, which is essential for photosynthetic electron transport. Consistent with this function of miR398 related to photosynthesis, its expression is strongly induced in leaves. In this study, however, we showed that SPL7 was transcribed mainly around the vasculature in roots, where Cu levels were likely sensed. To test the possible long-distance signaling of Cu availability from roots to shoots, we conducted a series of grafting experiments using spl7 mutant and wild-type (WT) plants. Expression of Cu-responsive microRNAs and the resulting suppression of CSD1 and CSD2 mRNAs were observed in leaves only when the aerial part was from WT plants, in which a low level of SPL7 was transcribed also in the vascular tissues. Although local sensing of Cu was disturbed in the spl7 mutant, the Cu level was not affected in the shoots. SPL7 is expressed in specific cell layers in both roots and shoots and locally senses Cu availability, transmitting the information to surrounding cells.

In Arabidopsis, the chloroplast NADH-dehydrogenase-like (NDH) complex is sandwiched between two copies of photosystem I (PSI) supercomplex, consisting of a PSI core and four light-harvesting complex I (LHCI) proteins (PSI-LHCI) to form the NDH-PSI supercomplex. Two minor LHCI proteins, Lhca5 and Lhca6, contribute to the interaction of each PSI-LHCI copy with the NDH complex. Here, large-pore blue-native gel electrophoresis revealed that, in addition to this complex, there were at least two types of higher-order association of more LHCI copies with the NDH complex. In single-particle images, this higher-order association of PSI-LHCI preferentially occurs at the left side of the NDH complex when viewed from the stromal side, placing subcomplex A at the top (Yadav et al., Biochim. Biophys. Acta - Bioenerg., 1858, 2017, 12). The association was impaired in the lhca6 mutant but not in the lhca5 mutant, suggesting that the left copy of PSI-LHCI was linked to the NDH complex via Lhca6. From an analysis of subunit compositions of the NDH-PSI supercomplex in lhca5 and lhca6 mutants, we propose that Lhca6 substitutes for Lhca2 in the left copy of PSI-LHCI, whereas Lhca5 substitutes for Lhca4 in the right copy. In the lhca2 mutant, Lhca3 was specifically stabilized in the NDH-PSI supercomplex through heterodimer formation with Lhca6. In the left copy of PSI-LHCI, subcomplex B, Lhca6 and NdhD likely formed the core of the supercomplex interaction. In contrast, a larger protein complex, including at least subcomplexes B and L and NdhB, was needed to form the contact site with Lhca5 in the right copy of PSI-LHCI.

Flavodiiron protein (FLV) mediates photoreduction of O2 to H2O. It is conserved from cyanobacteria to gymnosperms but not in angiosperms. The introduction of a moss (Physcomitrella patens) FLV (PpFLV) gene into Arabidopsis (Arabidopsis thaliana) made photosystem I (PSI) resistant to fluctuating light. Here, we used the same strategy with three rice (Oryza sativa) genotypes. PpFLV in the wild-type rice background functioned as an efficient PSI electron sink and increased resistance to PSI photodamage under fluctuating light. The introduction of PpFLV into the PGR5-RNAi mutant [defective in PROTON GRADIENT REGULATION5 (PGR5)-dependent cyclic electron transport around PSI, CET-PSI], the crr6 mutant [defective in chloroplast NAD(P)H-dehydrogenase-like complex (NDH)-dependent CET-PSI], and the PGR5-RNAi crr6 double mutant (double defective in CET-PSI activity) alleviated PSI photodamage under fluctuating light. Furthermore, PpFLV substituted for the function of PGR5- and NDH-dependent CET-PSI without competing for CO2 assimilation under constant light, as there was no difference in CO2 assimilation per Rubisco content and biomass production was recovered to the wild-type level. Thus, the exogenous FLV system could act not only as a safety valve under fluctuating light, but also generate a proton motive force for balancing the ATP/NADPH production ratio during steady-state photosynthesis.

The chloroplast NADH dehydrogenase-like (NDH) complex is structurally similar to respiratory complex I and mediates PSI cyclic electron flow. In Arabidopsis (Arabidopsis thaliana), chloroplast NDH is composed of at least 29 subunits and associates with two copies of PSI to form the NDH-PSI supercomplex. Here, we found that CHLORORESPIRATORY REDUCTION3 (CRR3) is an assembly factor required for the accumulation of subcomplex B (SubB) of chloroplast NDH. In Suc density gradient centrifugation, CRR3 was detected in three protein complexes. Accumulation of the largest peak III complex was impaired in mutants defective in the SubB subunits PnsB2-PnsB5. The oligomeric form of CRR3 likely functions to assemble the core of SubB to form the peak III complex as an assembly intermediate. A defect in the PnsL3 subunit increased the level of the peak III complex, suggesting that CRR3 was released from the assembly intermediate after PnsL3 binding. Unlike PnsB2-PnsB5 and PnsL3, PnsB1 was not absolutely necessary for stabilizing SubB. PnsB1 is likely incorporated into the intermediate at the final step during SubB assembly. Lhca6 is a linker protein mediating NDH-PSI supercomplex formation, and its site of contact with NDH was suggested to be SubB. In the lhca6 mutant, accumulation of the peak III complex was impaired, suggesting that SubB interacted with Lhca6 during the step of SubB assembly. The process of supercomplex formation was triggered before the completion of the NDH assembly. Consistent with its predicted function, CRR3 accumulated in young leaves, where the NDH complex was assembled.

Photo-reduction of O2 to water mediated by flavodiiron proteins (FDPs) represents a safety valve for the photosynthetic electron transport chain in fluctuating light. So far, the FDP-mediated O2 photo-reduction has been evidenced only in cyanobacteria and the moss Physcomitrella; however, a recent phylogenetic analysis of transcriptomes of photosynthetic organisms has also revealed the presence of FDP genes in several nonflowering plant groups. What remains to be clarified is whether the FDP-dependent O2 photo-reduction is actually operational in these organisms. We have established a simple method for the monitoring of FDP-mediated O2 photo-reduction, based on the measurement of redox kinetics of P700 (the electron donor of photosystem I) upon dark-to-light transition. The O2 photo-reduction is manifested as a fast re-oxidation of P700. The validity of the method was verified by experiments with transgenic organisms, namely FDP knock-out mutants of Synechocystis and Physcomitrella and transgenic Arabidopsis plants expressing FDPs from Physcomitrella. We observed the fast P700 re-oxidation in representatives of all green plant groups excluding angiosperms. Our results provide strong evidence that the FDP-mediated O2 photo-reduction is functional in all nonflowering green plant groups. This finding suggests a major change in the strategy of photosynthetic regulation during the evolution of angiosperms.

The chloroplast (cp) genome is organized as nucleoids that are dispersed throughout the cp stroma. Previously, a cp homolog of bacterial recombinase RecA (cpRECA) was shown to be involved in the maintenance of cp genome integrity by repairing damaged chloroplast DNA and by suppressing aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. Here, overexpression and knockdown analysis of cpRECA in the green alga Chlamydomonas reinhardtii revealed that cpRECA was involved in cp nucleoid dynamics as well as having a role in maintaining cp genome integrity. Overexpression of cpRECA tagged with yellow fluorescent protein or hemagglutinin resulted in the formation of giant filamentous structures that colocalized exclusively to chloroplast DNA and cpRECA localized to cp nucleoids in a heterogenous manner. Knockdown of cpRECA led to a significant reduction in cp nucleoid number that was accompanied by nucleoid enlargement. This phenotype resembled those of gyrase inhibitor-treated cells and monokaryotic chloroplast mutant cells and suggested that cpRECA was involved in organizing cp nucleoid dynamics. The cp genome also was destabilized by induced recombination between short dispersed repeats in cpRECA-knockdown cells and gyrase inhibitor-treated cells. Taken together, these results suggest that cpRECA and gyrase are both involved in nucleoid dynamics and the maintenance of genome integrity and that the mechanisms underlying these processes may be intimately related in C. reinhardtii cps.

Grana are stacked thylakoid membrane structures in land plants that contain PSII and light-harvesting complex II proteins (LHCIIs). We isolated two Arabidopsis thaliana mutants, reduced induction of non-photochemical quenching1 (riq1) and riq2, in which stacking of grana was enhanced. The curvature thylakoid 1a (curt1a) mutant was previously shown to lack grana structure. In riq1 curt1a, the grana were enlarged with more stacking, and in riq2 curt1a, the thylakoids were abnormally stacked and aggregated. Despite having different phenotypes in thylakoid structure, riq1, riq2, and curt1a showed a similar defect in the level of nonphotochemical quenching of chlorophyll fluorescence (NPQ). In riq curt1a double mutants, NPQ induction was more severely affected than in either single mutant. In riq mutants, state transitions were inhibited and the PSII antennae were smaller than in wild-type plants. The riq defects did not affect NPQ induction in the chlorophyll b-less mutant. RIQ1 and RIQ2 are paralogous and encode uncharacterized grana thylakoid proteins, but despite the high level of identity of the sequence, the functions of RIQ1 and RIQ2 were not redundant. RIQ1 is required for RIQ2 accumulation, and the wild-type level of RIQ2 did not complement the NPQ and thylakoid phenotypes in riq1. We propose that RIQ proteins link the grana structure and organization of LHCIIs.

Eukaryotes harbor mitochondria obtained via ancient symbiosis events. The successful evolution of energy production in mitochondria has been dependent on the control of mitochondrial gene expression by the nucleus. In flowering plants, the nuclear-encoded pentatricopeptide repeat (PPR) superfamily proteins are widely involved in mitochondrial RNA metabolism. Here, we show that an Arabidopsis nuclear-encoded RNA-binding protein, Restorer-of-fertility-like PPR protein 2 (RFL2), is required for RNA degradation of the mitochondrial orf291 transcript via endonucleolytic cleavage of the transcript in the middle of its reading frame. Both in vivo and in vitro, this RNA cleavage requires the activity of mitochondrial proteinaceous RNase P, which is possibly recruited to the site by RFL2. The site of RNase P cleavage likely forms a tRNA-like structure in the orf291 transcript. This study presents an example of functional collaboration between a PPR protein and an endonuclease in RNA cleavage. Furthermore, we show that the RFL2-binding region within the orf291 gene is hypervariable in the family Brassicaceae, possibly correlated with the rapid evolution of the RNA-recognition interfaces of the RFL proteins.

Oxygenic photosynthesis converts solar energy into chemical energy in the chloroplasts of plants and microalgae as well as in prokaryotic cyanobacteria using a complex machinery composed of two photosystems and both membrane-bound and soluble electron carriers. In addition to the major photosynthetic complexes photosystem II (PSII), cytochrome b6f, and photosystem I (PSI), chloroplasts also contain minor components, including a well-conserved type I NADH dehydrogenase (NDH-1) complex that functions in close relationship with photosynthesis and likewise originated from the endosymbiotic cyanobacterial ancestor. Some plants and many microalgal species have lost plastidial ndh genes and a functional NDH-1 complex during evolution, and studies have suggested that a plastidial type II NADH dehydrogenase (NDH-2) complex substitutes for the electron transport activity of NDH-1. However, although NDH-1 was initially thought to use NAD(P)H as an electron donor, recent research has demonstrated that both chloroplast and cyanobacterial NDH-1s oxidize reduced ferredoxin. We discuss more recent findings related to the biochemical composition and activity of NDH-1 and NDH-2 in relation to the physiology and regulation of photosynthesis, particularly focusing on their roles in cyclic electron flow around PSI, chlororespiration, and acclimation to changing environments.

In angiosperms, such as Arabidopsis and barley, the chloroplast NADH dehydrogenase-like (NDH) complex associates with two copies of photosystem I (PSI) supercomplex to form an NDH-PSI supercomplex for the stabilization of the NDH complex. Two linker proteins, Lhca5 and Lhca6, are members of the light-harvesting complex I (LHCI) family and mediate this supercomplex formation. The liverwort Marchantia polymorpha has branched from the basal land plant lineage and has neither Lhca5 nor Lhca6. Consequently, the NDH complex does not form a supercomplex with PSI in this plant. The Lhca6 gene does not seem to exist also in the moss Physcomitrella patens (Physcomitrella). Conversely, the Lhca5 gene has been found in Physcomitrella, although experimental evidence is still lacking for its contribution to NDH-PSI supercomplex formation as a linker. Here, we biochemically characterized the Lhca5 knock-out mutant (lhca5) in Physcomitrella. The NDH-PSI supercomplex observed in wild-type Physcomitrella was absent in the lhca5 mutant. Lhca5 protein was detected in this NDH-PSI supercomplex. Some PSI and NDH subunits were co-immunoprecipitated with Lhca5-HA. These results indicate that the Physcomitrella gene is the functional ortholog of Lhca5 reported in Arabidopsis. Between Physcomitrella and Arabidopsis, the stromal loop region is highly conserved in Lhca5 proteins but not in other LHCI members. We found that Lhca5 contributed to the stable accumulation of the NDH complex, but part of the NDH complex was still sensitive to high light intensity, even in the wild-type. We considered that angiosperms acquired another linker protein, Lhca6, to further stabilize the NDH complex.

Arabidopsis proton gradient regulation (pgr) mutants have high chlorophyll fluorescence and reduced non-photochemical quenching (NPQ) caused by defects in photosynthetic electron transport. Here, we identify PGR6 as the chloroplast lipid droplet (plastoglobule, PG) kinase ABC1K1 (activity of bc1 complex kinase 1). The members of the ABC1/ADCK/UbiB family of atypical kinases regulate ubiquinone synthesis in bacteria and mitochondria, and impact various metabolic pathways in plant chloroplasts. Here, we demonstrate that abc1k1 has a unique photosynthetic and metabolic phenotype that is distinct from that of the abc1k3 homolog. The abc1k1/pgr6 single mutant is specifically deficient in the electron carrier plastoquinone, as well as in ß–carotene and the xanthophyll lutein, and is defective in membrane antioxidant tocopherol metabolism. After 2 days of continuous high light stress, abc1k1/pgr6 plants suffer extensive photosynthetic and metabolic perturbations, strongly affecting carbohydrate metabolism. Remarkably, however, the mutant acclimates to high light after 7 days together with a recovery of carotenoid levels and a drastic alteration in the starch-to-sucrose ratio. Moreover, ABC1K1 behaves as an active kinase and phosphorylates VTE1, a key enzyme of tocopherol (vitamin E) metabolism in vitro. Our results indicate that the ABC1K1 kinase constitutes a new type of regulatory link between photosynthetic activity and chloroplast metabolism.

International audience; KEA3 is a thylakoid membrane localized K(+) /H(+) antiporter that regulates photosynthesis by modulating two components of proton motive force (pmf), the proton gradient (∆pH) and the electric potential (∆ψ). We identified a mutant allele of KEA3, disturbed proton gradient regulation (dpgr) based on its reduced non-photochemical quenching (NPQ) in artificial (CO2 -free with low O2 ) air. This phenotype was enhanced in the mutant backgrounds of PSI cyclic electron transport (pgr5 and crr2-1). In ambient air, reduced NPQ was observed during induction of photosynthesis in dpgr, the phenotype that was enhanced after overnight dark adaptation. In contrast, the knockout allele of kea3-1 exhibited a high-NPQ phenotype during steady state in ambient air. Consistent with this kea3-1 phenotype in ambient air, the membrane topology of KEA3 indicated a proton efflux from the thylakoid lumen to the stroma. The dpgr heterozygotes showed a semidominant and dominant phenotype in artificial and ambient air, respectively. In dpgr, the protein level of KEA3 was unaffected but the downregulation of its activity was probably disturbed. Our findings suggest that fine regulation of KEA3 activity is necessary for optimizing photosynthesis.

The light-harvesting complex I (LHCI) proteins in Arabidopsis thaliana are encoded by six genes. Major LHCI proteins (Lhca1-Lhca4) harvest light energy and transfer the resulting excitation energy to the PSI core by forming a PSI supercomplex. In contrast, the minor LHCI proteins Lhca5 and Lhca6 contribute to supercomplex formation between the PSI supercomplex and the chloroplast NADH dehydrogenase-like (NDH) complex, although Lhca5 is also solely associated with the PSI supercomplex. Lhca6 was branched from Lhca2 during the evolution of land plants. In this study, we focused on the molecular evolution involved in the transition from a major LHCI, Lhca2, to the linker protein Lhca6. To elucidate the domains of Lhca6 responsible for linker function, we systematically swapped domains between the two LHCI proteins. To overcome problems due to the low stability of chimeric proteins, we employed sensitive methods to evaluate supercomplex formation: we monitored NDH activity by using Chl fluorescence analysis and detected NDH-PSI supercomplex formation by using protein blot analysis in the form of two-dimensional blue-native (BN)/SDS-PAGE. The stromal loop of Lhca6 was shown to be necessary and sufficient for linker function. Chimeric Lhca6, in which the stromal loop was substituted by that of Lhca2, was not functional as a linker and was detected at the position of the PSI supercomplex in the BN-polyacrylamide gel. The stromal loop of Lhca6 is likely to be necessary for the interaction with chloroplast NDH, rather than for the association with the PSI supercomplex.

The PGR5 (PROTON GRADIENT REGULATION 5) gene that is required for PSI cyclic electron transport in Arabidopsis was knocked down in rice (Oryza sativa). In three PGR5 knockdown (KD) lines, the PGR5 protein level was reduced to 5-8% of that in the wild type, resulting in a 50% reduction in PGRL1 (PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE 1) protein levels. In ruptured chloroplasts, ferredoxin-dependent plastoquinone reduction activity was partially impaired; the phenotype was mimicked by addition of antimycin A to wild-type chloroplasts. As occurred in the Arabidopsis pgr5 mutant, non-photochemical quenching of Chl fluorescence (NPQ) induction was impaired in the leaves, but the electron transport rate (ETR) was only mildly affected at high light intensity. The P700(+) level was reduced even at low light intensity, suggesting that the PGR5 function was severely disturbed as in the Arabidopsis pgr5 mutant and that the other alternative routes of electrons could not compensate the stromal redox balance. The amplitude of the light-dark electrochromic shift (ECS) signal (ECSt), which reflects the total size of the proton motive force in steady-state photosynthesis, was reduced by 13-25% at approximately the growth light intensity. The CO(2) fixation rate was only slightly reduced in the PGR5 KD lines. Despite the drastic reduction in NPQ and P700(+) levels, total biomass was only slightly reduced in PGR5 KD lines grown at 370 µmol photons m(-2) s(-1). These results suggest that CO(2) fixation and growth rate are very robust in the face of alterations in the fundamental reactions of photosynthesis under constant light conditions in rice.

In vascular plants, the chloroplast NADH dehydrogenase-like (NDH) complex, a homolog of respiratory NADH:quinone oxidoreductase (Complex I), mediates plastoquinone reduction using ferredoxin as an electron donor in cyclic electron transport around PSI in the thylakoid membrane. In angiosperms, chloroplast NDH is composed of five subcomplexes and forms a supercomplex with PSI. The modular assembly of stroma-protruded subcomplex A, which corresponds to the Q module of Complex I, was recently reported. However, the factors involved in the specific assembly steps have not been completely identified. Here, we isolated an Arabidopsis mutant, chlororespiratory reduction 9 (crr9), defective in NDH activity. The CRR9 gene encodes a novel stromal protein without any known functional domains or motifs. CRR9 is highly conserved in cyanobacteria and land plants but not in green algae, which do not have chloroplast NDH. Blue native-PAGE and immunoblot analyses of thylakoid proteins indicated that formation of subcomplex A was impaired in crr9 CRR9 was specifically required for the accumulation of NdhK, a subcomplex A subunit, in NDH assembly intermediates in the stroma. Furthermore, two-dimensional clear native/SDS-PAGE analysis of the stroma fraction indicated that incorporation of NdhM into NDH assembly intermediate complex 400 was impaired in crr9 These results suggest that CRR9 is a novel factor required for the formation of NDH subcomplex A.

Photosynthetic electron transport is coupled to proton translocation across the thylakoid membrane, resulting in the formation of a trans-thylakoid proton gradient (ΔpH) and membrane potential (Δψ). Ion transporters and channels localized to the thylakoid membrane regulate the contribution of each component to the proton motive force (pmf). Although both ΔpH and Δψ contribute to ATP synthesis as pmf, only ΔpH downregulates photosynthetic electron transport via the acidification of the thylakoid lumen by inducing thermal dissipation of excessive absorbed light energy from photosystem II antennae and slowing down of the electron transport through the cytochrome b6f complex. To optimize the tradeoff between efficient light energy utilization and protection of both photosystems against photodamage, plants have to regulate the pmf amplitude and its components, ΔpH and Δψ. Cyclic electron transport around photosystem I (PSI) is a major regulator of the pmf amplitude by generating pmf independently of the net production of NADPH by linear electron transport. Chloroplast ATP synthase relaxes pmf for ATP synthesis, and its activity should be finely tuned for maintaining the size of the pmf during steady-state photosynthesis. Pseudo-cyclic electron transport mediated by flavodiiron protein (Flv) forms a large electron sink, which is essential for PSI photoprotection in fluctuating light in cyanobacteria. Flv is conserved from cyanobacteria to gymnosperms but not in angiosperms. The Arabidopsis proton gradient regulation 5 (pgr5) mutant is defective in the main pathway of PSI cyclic electron transport. By introducing Physcomitrella patens genes encoding Flvs, the function of PSI cyclic electron transport was substituted by that of Flv-dependent pseudo-cyclic electron transport. In transgenic plants, the size of the pmf was complemented to the wild-type level but the contribution of ΔpH to the total pmf was lower than that in the wild type. In the pgr5 mutant, the size of the pmf was drastically lowered by the absence of PSI cyclic electron transport. In the mutant, ΔpH occupied the majority of pmf, suggesting the presence of a mechanism for the homeostasis of luminal pH in the light. To avoid damage to photosynthetic electron transport by periods of excess solar energy, plants employ an intricate regulatory network involving alternative electron transport pathways, ion transporters/channels, and pH-dependent mechanisms for downregulating photosynthetic electron transport.

The light reactions in photosynthesis drive both linear and cyclic electron transport around photosystem I (PSI). Linear electron transport generates both ATP and NADPH, whereas PSI cyclic electron transport produces ATP without producing NADPH. PSI cyclic electron transport is thought to be essential for balancing the ATP/NADPH production ratio and for protecting both photosystems from damage caused by stromal overreduction. Two distinct pathways of cyclic electron transport have been proposed in angiosperms: a major pathway that depends on the PROTON GRADIENT REGULATION 5 (PGR5) and PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE 1 (PGRL1) proteins, which are the target site of antimycin A, and a minor pathway mediated by the chloroplast NADH dehydrogenase–like (NDH) complex. Recently, the regulation of PSI cyclic electron transport has been recognized as essential for photosynthesis and plant growth. In this review, we summarize the possible functions and importance of the two pathways of PSI cyclic electron transport.

ALADIN is a component of the nuclear pore complex in higher eukaryotes. An Arabidopsis knockout line that had a T-DNA insertion in the ALADIN gene was defective in plant growth and thylakoid development and had reduced photosynthetic activity resulting from lower chlorophyll accumulation. The mutation appeared to decrease the level of chloroplast RuBisCO subunits and PSBA and PGL35 proteins. Unexpectedly, the T-DNA insertion in the ALADIN gene decreased the expression of the neighboring gene PSRP5, which functions in translation in chloroplasts. The mutant phenotype was rescued by expressing PSRP5, but not by expressing ALADIN. The abnormal phenotypes were also detected in an artificial microRNA (amiRNA)-mediated PSRPS5 knockdown, but not in an amiRNA-mediated ALADIN knockdown line. Thus, users of T-DNA insertions should be aware that a T-DNA insertion in one gene can have effects on the expression of neighboring genes.

†These authors contributed equally to this work. SUMMARY The chloroplast NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport and chlororespiration and consists of five sub-omplexes, which in angiosperms further associate with photosystem I (PSI) to form a super-complex. In Marchantia polymorpha, 11 plastid-encoded subunits and all the nuclearencoded subunits of the A, B, membrane and ferredoxin-binding sub-complexes are conserved. However, it is unlikely that the genome of this liverwort encodes Lhca5 and Lhca6, both of which mediate NDH–PSI supercomplex formation. It is also unlikely that the subunits of the lumen sub-complex, PnsL1–L4, are encoded by the genome. Consistent with this in silico prediction, the results of blue-native gel electrophoresis showed that NDH subunits were detected in a protein complex with lower molecular mass in Marchantia than the NDH–PSI super-complex in Arabidopsis. Using the plastid transformation technique, we knocked out the ndhB gene in Marchantia. Although the wild-type genome copies were completely segregated out, the DndhB lines grew like the wild-type photoautotrophically. A post-illumination transient increase in chlorophyll fluorescence, which reflects NDH activity in vivo in angiosperms, was absent in the thalli of the DndhB lines. In ruptured chloroplasts, antimycin A-insensitive, and ferredoxin-dependent plastoquinone reduction was impaired, suggesting that chloroplast NDH mediates similar electron transport in Marchantia and Arabidopsis, despite its possible difference in structure. As in angiosperms, linear electron transport was not strongly affected in the DndhB lines. However, the plastoquinone pool was slightly more reduced at low light intensity, suggesting that chloroplast NDH functions in redox balancing of the inter system, especially under low light conditions.

The isogamous green alga Chlamydomonas reinhardtii has emerged as a premier model for studying the genetic regulation of fertilization and sexual development. A key regulator is known to be a homeoprotein gene, GAMETE-SPECIFIC PLUS1 (GSP1), which triggers the zygotic program. In this study, we isolated a mutant, biparental31 (bp31), which lacks GSP1. bp31 mt+ gametes fuse normally to form zygotes, but the sexual development of the resulting diploid cell is arrested and pellicle/zygospore/tetrad formation is abolished. The uniparental inheritance of chloroplast (cp) and mitochondrial (mt) DNA (cytoplasmic inheritance) was also impaired. bp31 has a deletion of ∼60 kb on chromosome 2, including GSP1. The mutant phenotype was not rescued by transformation with GSP1 alone but could be rescued by the cotransformation with GSP1 and another gene, INOSITOL MONOPHOSPHATASE-LIKE1, which is involved in various cellular processes, including the phosphatidylinositol signaling pathway. This study confirms the importance of Gsp1 in mediating the zygotic program, including the uniparental inheritance of cp/mtDNA. Moreover, the results also suggest a role for inositol metabolism in the sexual developmental program.

Cyclic electron transport around photosystem I (PSI) generates ∆pH across the thylakoid membrane without net production of NADPH. In angiosperms, two pathways of PSI cyclic electron transport operate. The main pathway depends on PGR5/PGRL1 proteins and is likely identical to the historical Arnon's pathway. The minor pathway depends on chloroplast NADH dehydrogenase-like (NDH) complex. In assays of their rates in vivo, the two independent pathways are often mixed together. Theoretically, linear electron transport from water to NADP(+) cannot satisfy the ATP/NADPH production ratio required by the Calvin-Benson cycle and photorespiration. PGR5/PGRL1-dependent PSI cyclic electron transport contributes substantially to the supply of ATP for CO2 fixation, as does linear electron transport. Also, the contribution of chloroplast NDH cannot be ignored, especially at low light intensity, although the extent of the contribution depends on the plant species. An increase in proton conductivity of ATP synthase may compensate ATP synthesis to some extent in the pgr5 mutant. Combined with the decreased rate of ∆pH generation, however, this mechanism sacrifices homeostasis of the thylakoid lumen pH, seriously disturbing the pH-dependent regulation of photosynthetic electron transport, induction of qE, and downregulation of the cytochrome b 6 f complex. PGR5/PGRL1-dependent PSI cyclic electron transport produces sufficient proton motive force for ATP synthesis and the regulation of photosynthetic electron transport.

In photosynthesis, linear electron transport from water to nicotinamide adenine dinucleotide phosphate (NADP(+)) cannot satisfy the ATP/NADPH production stoichiometry required by the Calvin-Benson cycle. Cyclic electron transport (CET) around photosystem I (PSI) and pseudocyclic electron transport (pseudoCET) can produce ATP without the accumulation of NADPH. Flavodiiron proteins (Flv) are the main mediator of pseudoCET in photosynthetic organisms, spanning cyanobacteria to gymnosperms. However, their genes are not conserved in angiosperms. Here we explore the possibility of complementing CET with Flv-dependent pseudoCET in the angiosperm Arabidopsis thaliana. We introduced FlvA and FlvB genes from the moss Physcomitrella patens into both wild-type (WT) Arabidopsis and the proton gradient regulation 5 (pgr5) mutant, which is defective in the main pathway of CET. We measured rates of pseudoCET using membrane inlet mass spectrometry, along with several photosynthetic parameters. Flv expression significantly increased rates of pseudoCET in the mutant plants, particularly at high light intensities, and partially restored the photosynthetic phenotype. In WT plants, Flv did not compete with PGR5-dependent CET during steady-state photosynthesis, but did form a large electron sink in fluctuating light. We conclude that flavodiiron proteins can help to protect the photosystems in Arabidopsis under fluctuating light, even in the presence of CET.

Summary The role of NAD(P)H dehydrogenase (NDH)-dependent cyclic electron flow around photosystem I in photosynthetic regulation and plant growth at several temperatures was examined in rice (Oryza sativa) that is defective in CHLORORESPIRATORY REDUCTION 6 (CRR6), which is required for accumulation of sub-complex A of the chloroplast NDH complex (crr6). NdhK was not detected by Western blot analysis in crr6 mutants, resulting in lack of a transient post-illumination increase in chlorophyll fluorescence, and confirming that crr6 mutants lack NDH activity. When plants were grown at 28 or 35°C, all examined photosynthetic parameters, including the CO2 assimilation rate and the electron transport rate around photosystems I and II, at each growth temperature at light intensities above growth light (i.e. 800 μmol photons m−2 sec−1), were similar between crr6 mutants and control plants. However, when plants were grown at 20°C, all the examined photosynthetic parameters were significantly lower in crr6 mutants than control plants, and this effect on photosynthesis caused a corresponding reduction in plant biomass. The Fv/Fm ratio was only slightly lower in crr6 mutants than in control plants after short-term strong light treatment at 20°C. However, after long-term acclimation to the low temperature, impairment of cyclic electron flow suppressed non-photochemical quenching and promoted reduction of the plastoquinone pool in crr6 mutants. Taken together, our experiments show that NDH-dependent cyclic electron flow plays a significant physiological role in rice during photosynthesis and plant growth at low temperature.

PROTON GRADIENT REGULATION 3 (PGR3) contains 27 pentatricopeptide repeat (PPR) motifs and belongs to the P-subfamily. Previous studies have suggested that PGR3 functions in the stabilization of petL operon RNA and also in the translation of petL and one, or some, of the 11 plastid ndh genes encoding subunits of chloroplast NADH dehydrogenase-like complex (NDH). The pgr3-3 allele has been suggested to be specifically defective in the putative PGR3 function of translation. Herein, we show that the polysome association of the monocistronic petL transcript is impaired in pgr3-3. We detected sequences weakly conserved in the 5' untranslated regions (UTRs) of petL and ndhA, and these putative elements were recognized by recombinant PGR3 in vitro. Previously, pgr3-2 was shown to be specifically defective in stabilizing petL operon RNA and to accumulate NDH at wild-type levels. Consistent with this pgr3-2 phenotype, we show here that a recombinant protein carrying the pgr3-2 mutation in the 12th PPR motif bound to the 5' UTR of ndhA but not of petL. This indicates that a single amino acid alteration changes the binding specificity of PGR3. In contrast, the recombinant protein carrying the pgr3-3 mutation in the final, 27th, incomplete PPR motif can bind to both petL and ndhA 5' UTRs, suggesting that the C-terminal end of PGR3 is not required for binding to targets but is essential for translation of petL and probably also ndhA. Our results fully support the model in which PGR3 recognizes two target sequences and is involved in multiple functions, i.e. stabilizing RNA and activating translation.

Some subunits of chloroplast NAD(P)H dehydrogenase (NDH) are related to those of the respiratory complex I, and NDH mediates photosystem I (PSI) cyclic electron flow. Despite extensive surveys, the electron donor and its binding subunits have not been identified. Here, we identified three novel components required for NDH activity. CRRJ and CRRL are J- and J-like proteins, respectively, and are components of NDH subcomplex A. CRR31 is an Src homology 3 domain-like fold protein, and its C-terminal region may form a tertiary structure similar to that of PsaE, a ferredoxin (Fd) binding subunit of PSI, although the sequences are not conserved between CRR31 and PsaE. Although CRR31 can accumulate in thylakoids independently of NDH, its accumulation requires CRRJ, and CRRL accumulation depends on CRRJ and NDH. CRR31 was essential for the efficient operation of Fd-dependent plastoquinone reduction in vitro. The phenotype of crr31 pgr5 suggested that CRR31 is required for NDH activity in vivo. We propose that NDH functions as a PGR5-PGRL1 complex-independent Fd:plastoquinone oxidoreductase in chloroplasts and rename it the NADH dehydrogenase-like complex.

In higher plants, the chloroplast NADH dehydrogenase-like complex (NDH) interacts with photosystem I (PSI) to form the NDH-PSI supercomplex via two minor light-harvesting complex I (LHCI) proteins, Lhca5 and Lhca6. Previously, we showed that in lhca5 and lhca6, NDH still associates with PSI to form smaller versions of the NDH-PSI supercomplex, although their molecular masses are far smaller than that of the full-size NDH-PSI supercomplex. In this study, we show that the NDH complex is present in the monomeric form in Arabidopsis (Arabidopsis thaliana) lhca5 lhca6, implying that NDH interacts with multiple copies of PSI. NDH subunit levels were slightly reduced in immature leaves and more drastically (approximately 50%) in mature leaves of the lhca5 lhca6 double mutant compared with the wild type. Chlorophyll fluorescence analyses detected NDH activity of lhca5 lhca6, suggesting that the supercomplex formation is not essential for NDH activity. However, the severe phenotypes of the lhca5 lhca6 proton gradient regulation5 triple mutant in both plant growth rate and photosynthesis suggest that the function of NDH was impaired in this mutant in vivo. Accumulation of NDH subunits was drastically reduced in lhca5 lhca6 when the light intensity was shifted from 50 to 500 μmol photons m−2 s−1. Furthermore, the half-life of NDH subunits, especially that of NDH18, was shorter in monomeric NDH than in the NDH-PSI supercomplex under the high-light conditions. We propose that NDH-PSI supercomplex formation stabilizes NDH and that the process is especially required under stress conditions.