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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.

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 the cyclic electron flow around photosystem I. One cyclic electron flow 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), which is considered a major cyclic electron flow 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 cyclic electron flow routes to the NADP-malic enzyme subtype of C4 photosynthesis in Flaveria bidentis. We observed that the suppression of 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, the suppression of 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 cyclic electron flow 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 the C4 evolution, the photosynthetic electron flow may be optimized to provide the energy required for C4 photosynthesis.

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., The chloroplast NDH complex interacts with Photosystem I to form the NDH-PSI supercomplex. Here the authors show that Arabidopsis NDF5 shares a common ancestor with the NDH subunit PnsB2 and acts as an NDH assembly factor initiating the assembly of PnsB2 and the evolutionarily distinct PnsB3.

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.

Significance Compaction of bulky DNA is a universal issue for all DNA-based life forms. Both chloroplasts and mitochondria maintain their own multicopy genomes organized as nucleoids, but the mechanism of DNA compaction remains obscure. Here, we discovered a chloroplast nucleoid protein (HBD1) that is highly similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p) in terms of possessing two DNA-binding high mobility group box (HMG-box) domains. Our analyses of HBD1 based on DNA origami/atomic force microscopy showed that HBD1 is capable of compacting DNA by introducing U-turns and cross-strand bridges with the two HMG-box domains, indicating that proteins with two HMG-box domains could compact DNA in both mitochondrial and chloroplast nucleoids., 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.

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 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 most sexual eukaryotes, mitochondrial (mt) DNA is uniparentally inherited, although the detailed mechanisms underlying this phenomenon remain controversial. The most widely accepted explanations include the autophagic elimination of paternal mitochondria in the fertilized eggs and the active degradation of paternal mitochondrial DNA. To decode the precise program for the uniparental inheritance, we focused on Cryptococcus neoformans as a model system, in which mtDNA is inherited only from the a-parent, although gametes of a- and α-cells are of equal size and contribute equal amounts of mtDNA to the zygote. In this research, the process of preferential elimination of the mitochondria contributed by the α-parent (α-mitochondria) was studied by fluorescence microscopy and single cell analysis using optical tweezers, which revealed that α-mitochondria are preferentially reduced by the following three steps: (1) preferential reduction of α-mitochondrial (mt) nucleoids and α-mtDNA, (2) degradation of the α-mitochondrial structure and (3) proliferation of remaining mt nucleoids during the zygote development. Furthermore, AUTOPHAGY RELATED GENE (ATG) 8 and the gene encoding mitochondrial endonuclease G (NUC1) were disrupted, and the effects of their disruption on the uniparental inheritance were scrutinized. Disruption of ATG8 (ATG7) and NUC1 did not have severe effects on the uniparental inheritance, but microscopic examination revealed that α-mitochondria lacking mt nucleoids persisted in Δatg8 zygotes, indicating that autophagy is not critical for the uniparental inheritance per se but is responsible for the clearance of mitochondrial structures after the reduction of α-mt nucleoids.

Cyclic electron transport (CET) around photosystem I (PSI) was discovered more than 60 years ago but it was 2004 when two pathways of CET were proposed. The main pathway depends on the PROTON GRADIENT REGULATION 5 (PGR5) protein, whereas the minor pathway is mediated by the chloroplast NADH dehydrogenase-like (NDH) complex. Although it is still a topic of debate, as to whether PGR5 is really involved in CET, the Arabidopsis pgr5 mutant is severely defective in the ΔpH-dependent regulation of photosynthetic electron transport. PGR5 is necessary to induce a ΔpH-dependent component of nonphotochemical quenching (NPQ) of chlorophyll fluorescence at a high light intensity and this qE mechanism dissipates excessively absorbed light energy safely as heat from photosystem II antennae. The lumenal acidification also downregulates the electron transport at the cytochrome b6f complex. The latter is essential for protecting PSI and the pgr5 mutant cannot survive the fluctuating light intensity. Although the contribution of the NDH complex to the pmf formation is much smaller than that of the PGR5-dependent CET during steady-state photosynthesis, the NDH complex efficiently pumps protons probably in the absence of large pmf. In addition to the size of pmf, it is also necessary to regulate the partitioning pmf components, ΔpH, and membrane potential (ΔΨ) across the thylakoid membrane. Ion channels and transporters localized to the thylakoid membrane involve the regulation and form a regulatory network of photosynthesis with the CET machinery.

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.

Resolution achieved Chloroplasts possess multiple copies of their own chloroplast DNA that are packaged into DNA-protein complexes known as nucleoids. The shape, number, and distribution of chloroplast nucleoids change markedly depending on the cell cycle, developmental stage, and nutritional environment. Kobayashi et al. identified Holliday junction resolvase as a key factor in the dynamism of chloroplast nucleoids in the unicellular green algae Chlamydomonas reinhardtii . The gene encoding the resolvase is ubiquitously conserved among green plants. Disruption or down-regulation of this gene also disturbed chloroplast nucleoid organization and segregation in the land plant Arabidopsis thaliana . Science , this issue p. 631

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.