Critical role of cyclic electron transport around photosystem I in the maintenance of photosystem I activity.

In angiosperms, cyclic electron transport around photosystem I (PSI) is mediated by two pathways that depend on the PROTON GRADIENT REGULATION 5 (PGR5) protein and the chloroplast NADH dehydrogenase-like (NDH) complex, respectively. In the Arabidopsis double mutants defective in both pathways, plant growth and photosynthesis are impaired. The pgr5-1 mutant used in the original study is a missense allele and accumulates low levels of PGR5 protein. In this study, we generated two knockout (KO) alleles, designated as pgr5-5 and pgr5-6, using the CRISPR-Cas9 technology. Although both KO alleles showed a severe reduction in P700 similar to the pgr5-1 allele, NPQ induction was less severely impaired in the KO alleles than in the pgr5-1 allele. In the pgr5-1 allele, the second mutation affecting NPQ size was mapped to ~21 cM south of the pgr5-1 locus. Overexpression of the pgr5-1 allele, encoding the glycine130-to-serine change, complemented the pgr5-5 phenotype, suggesting that the pgr5-1 mutation destabilizes PGR5 but that the mutant protein retains partial functionality. Using two KO alleles, we created the double mutants with two chlororespiratory reduction (crr) mutants defective in the NDH complex. The growth of the double mutants was notably impaired. In the double mutant seedlings that survived on the medium containing sucrose, PSI activity evaluated by the P700 oxidation was severely impaired, whereas PSII activity was only mildly impaired. Cyclic electron transport around PSI is required to maintain PSI activity.

Molecular Genetic Dissection of the Regulatory Network of Proton Motive Force in Chloroplasts.

The proton motive force (pmf) generated across the thylakoid membrane rotates the Fo-ring of ATP synthase in chloroplasts. The pmf comprises two components: membrane potential (∆Ψ) and proton concentration gradient (∆pH). Acidification of the thylakoid lumen resulting from ∆pH downregulates electron transport in the cytochrome b6f complex. This process, known as photosynthetic control, is crucial for protecting photosystem I (PSI) from photodamage in response to fluctuating light. To optimize the balance between efficient photosynthesis and photoprotection, it is necessary to regulate pmf. Cyclic electron transport around PSI and pseudo-cyclic electron transport involving flavodiiron proteins contribute to the modulation of pmf magnitude. By manipulating the ratio between the two components of pmf, it is possible to modify the extent of photosynthetic control without affecting the pmf size. This adjustment can be achieved by regulating the movement of ions (such as K+ and Cl-) across the thylakoid membrane. Since ATP synthase is the primary consumer of pmf in chloroplasts, its activity must be precisely regulated to accommodate other mechanisms involved in pmf optimization. Although fragments of information about each regulatory process have been accumulated, a comprehensive understanding of their interactions is lacking. Here, I summarize current knowledge of the network for pmf regulation, mainly based on genetic studies.

FZL, a dynamin-like protein localized to curved grana edges, is required for efficient photosynthetic electron transfer in Arabidopsis.

Photosynthetic electron transfer and its regulation processes take place on thylakoid membranes, and the thylakoid of vascular plants exhibits particularly intricate structure consisting of stacked grana and flat stroma lamellae. It is known that several membrane remodeling proteins contribute to maintain the thylakoid structure, and one putative example is FUZZY ONION LIKE (FZL). In this study, we re-evaluated the controversial function of FZL in thylakoid membrane remodeling and in photosynthesis. We investigated the sub-membrane localization of FZL and found that it is enriched on curved grana edges of thylakoid membranes, consistent with the previously proposed model that FZL mediates fusion of grana and stroma lamellae at the interfaces. The mature fzl thylakoid morphology characterized with the staggered and less connected grana seems to agree with this model as well. In the photosynthetic analysis, the fzl knockout mutants in Arabidopsis displayed reduced electron flow, likely resulting in higher oxidative levels of Photosystem I (PSI) and smaller proton motive force (pmf). However, nonphotochemical quenching (NPQ) of chlorophyll fluorescence was excessively enhanced considering the pmf levels in fzl, and we found that introducing kea3-1 mutation, lowering pH in thylakoid lumen, synergistically reinforced the photosynthetic disorder in the fzl mutant background. We also showed that state transitions normally occurred in fzl, and that they were not involved in the photosynthetic disorders in fzl. We discuss the possible mechanisms by which the altered thylakoid morphology in fzl leads to the photosynthetic modifications.

Two cyclic electron flows around photosystem I differentially participate in C4 photosynthesis.

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.

Impact of engineering the ATP synthase rotor ring on photosynthesis in tobacco chloroplasts.

The chloroplast ATP synthase produces the ATP needed for photosynthesis and plant growth. The trans-membrane flow of protons through the ATP synthase rotates an oligomeric assembly of c subunits, the c-ring. The ion-to-ATP ratio in rotary F1F0-ATP synthases is defined by the number of c-subunits in the rotor c-ring. Engineering the c-ring stoichiometry is, therefore, a possible route to manipulate ATP synthesis by the ATP synthase and hence photosynthetic efficiency in plants. Here, we describe the construction of a tobacco (Nicotiana tabacum) chloroplast atpH (chloroplastic ATP synthase subunit c gene) mutant in which the c-ring stoichiometry was increased from 14 to 15 c-subunits. Although the abundance of the ATP synthase was decreased to 25% of wild-type (WT) levels, the mutant lines grew as well as WT plants and photosynthetic electron transport remained unaffected. To synthesize the necessary ATP for growth, we found that the contribution of the membrane potential to the proton motive force was enhanced to ensure a higher proton flux via the c15-ring without unwanted low pH-induced feedback inhibition of electron transport. Our work opens avenues to manipulate plant ion-to-ATP ratios with potentially beneficial consequences for photosynthesis.

Structural insight into the activation of an Arabidopsis organellar C-to-U RNA editing enzyme by active site complementation.

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 ß-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.

Distinct contribution of two cyclic electron transport pathways to P700 oxidation.

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.

DYW deaminase domain has a distinct preference for neighboring nucleotides of the target RNA editing sites.

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.

Expression of flavodiiron protein rescues defects in electron transport around PSI resulting from overproduction of Rubisco activase in rice.

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.

Flavodiiron proteins enhance the rate of CO2 assimilation in Arabidopsis under fluctuating light intensity.

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.

PTOX-dependent safety valve does not oxidize P700 during photosynthetic induction in the Arabidopsis pgr5 mutant.

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.

Targeted base editing in the plastid genome of Arabidopsis thaliana.

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.

Evolution of an assembly factor-based subunit contributed to a novel NDH-PSI supercomplex formation in chloroplasts.

Chloroplast NADH dehydrogenase-like (NDH) complex is structurally related to mitochondrial Complex I and forms a supercomplex with two copies of Photosystem I (the NDH-PSI supercomplex) via linker proteins Lhca5 and Lhca6. The latter was acquired relatively recently in a common ancestor of angiosperms. Here we show that NDH-dependent Cyclic Electron Flow 5 (NDF5) is an NDH assembly factor in Arabidopsis. NDF5 initiates the assembly of NDH subunits (PnsB2 and PnsB3) and Lhca6, suggesting that they form a contact site with Lhca6. Our analysis of the NDF5 ortholog in Physcomitrella and angiosperm genomes reveals the subunit PnsB2 to be newly acquired via tandem gene duplication of NDF5 at some point in the evolution of angiosperms. Another Lhca6 contact subunit, PnsB3, has evolved from a protein unrelated to NDH. The structure of the largest photosynthetic electron transport chain complex has become more complicated by acquiring novel subunits and supercomplex formation with PSI.

HBD1 protein with a tandem repeat of two HMG-box domains is a DNA clip to organize chloroplast nucleoids in Chlamydomonas reinhardtii.

Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA-protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9-mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids.

The Pentatricopeptide Repeat Protein PGR3 Is Required for the Translation of petL and ndhG by Binding Their 5' UTRs.

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.