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I present my personal reminiscence of Paul Levine—a highly innovative scientist who did seminal work in photosynthesis. He was among the first to initiate and use a genetic approach toward photosynthesis. He greatly helped in establishing the green unicellular alga Chlamydomonas reinhardtii as a powerful model system not only for understanding the function of the photosynthetic apparatus but also for studying its biogenesis and regulation. During the period he spent at Harvard, he made several ground-breaking contributions such as identifying and establishing the order of some components of the photosynthetic electron transport chain as well as determining their genetic origin. He trained many students and post-doctoral fellows several of whom later became prominent in this field and in other areas of plant science.

Most chloroplast proteins are nucleus-encoded, translated on cytoplasmic ribosomes as precursor proteins, and imported into chloroplasts through TOC and TIC, the translocons of the outer and inner chloroplast envelope membranes. While the composition of the TOC complex is well established, there is still some controversy about the importance of a recently identified TIC complex consisting of Tic20, Tic214, Tic100, and Tic56. TOC and TIC form a supercomplex with a protein channel at the junction of the outer and inner envelope membranes through which preproteins are pulled into the stroma by the ATP-powered Ycf2 complex consisting of several FtsH-like ATPases and/or by chloroplast Hsp proteins. Several components of the TOC/TIC system are moonlighting proteins with additional roles in chloroplast gene expression and metabolism. Chaperones and co-chaperones, associated with TOC and TIC on the cytoplasmic and stromal side of the chloroplast envelope, participate in the unfolding and folding of the precursor proteins and act together with the ubiquitin-proteasome system in protein quality control. Chloroplast protein import is also intimately linked with retrograde signaling, revealing altogether an unsuspected complexity in the regulation of this process.

The leaf veins of higher plants contain a highly specialized vascular system comprised of xylem and phloem cells that transport water, organic compounds and mineral nutrients. The development of the vascular system is controlled by phytohormones that interact with complex transcriptional regulatory networks. Before the emergence of true leaves, the cotyledons of young seedlings perform photosynthesis that provides energy for the sustainable growth and survival of seedlings. However, the mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood, in part due to the complex cellular composition of this tissue. To better understand the development of leaf veins, we analyzed 14 117 single cells from 3-day-old cotyledons using single-cell RNA sequencing. Based on gene expression patterns, we identified 10 clusters of cells and traced their developmental trajectories. We discovered multiple new marker genes and developmental features of leaf veins. The transcription factor networks of some cell types indicated potential roles of CYCLING DOF FACTOR 5 (CDF5) and REPRESSOR OF GA (RGA) in the early development and function of the leaf veins in cotyledons. These new findings lay a foundation for understanding the early developmental dynamics of cotyledon veins. The mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood. In this study, we comprehensively characterized the early differentiation and development of leaf veins in 3-day-old cotyledons based on single-cell transcriptome analysis. We identified the cell types and novel marker genes of leaf veins and characterized the novel regulators of leaf vein.

Restricted genetic diversity can supply only a limited number of elite genes for modern plant cultivation and transgenesis. In this study, we demonstrate that rational design enables the engineering of geranylgeranyl diphosphate synthase (NtGGPPS), an enzyme of the methylerythritol phosphate pathway (MEP) in the model plant Nicotiana tabacum. As the crucial bottleneck in carotenoid biosynthesis, NtGGPPS1 interacts with phytoene synthase (NtPSY1) to channel GGPP into the production of carotenoids. Loss of this enzyme in the ntggpps1 mutant leads to decreased carotenoid accumulation. With the aim of enhancing NtGGPPS1 activity, we undertook structure-guided rational redesign of its substrate binding pocket in combination with sequence alignment. The activity of the designed NtGGPPS1 (a pentuple mutant of five sites V154A/I161L/F218Y/I209S/V233E, d-NtGGPPS1) was measured by a high-throughput colorimetric assay. d-NtGGPPS1 exhibited significantly higher conversion of IPP and each co-substrate (DMAPP ~1995.5-fold, GPP ~25.9-fold, and FPP ~16.7-fold) for GGPP synthesis compared with wild-type NtGGPPS1. Importantly, the transient and stable expression of d-NtGGPPS1 in the ntggpps1 mutant increased carotenoid levels in leaves, improved photosynthetic efficiency, and increased biomass relative to NtGGPPS1. These findings provide a firm basis for the engineering of GGPPS and will facilitate the development of quality and yield traits. Our results open the door for the structure-guided rational design of elite genes in higher plants.

SummaryChloroplasts rely on the translocon complexes in the outer and inner envelope membranes (termed TOC and TIC, respectively) to import thousands of different nuclear-encoded proteins from the cytosol1–4. While previous studies indicated that the TOC and TIC complexes may assemble into larger supercomplexes5–7, the overall architectures of the TOC-TIC supercomplexes and the mechanism of preprotein translocation are elusive. Here we report the cryo-electron microscopy (cryo-EM) structure of the TOC-TIC supercomplex fromChlamydomonas reinhardtiiat an overall resolution of 2.8 Å. The major subunits of the TOC complex (Toc75, Toc90 and Toc34) and TIC complex (Tic214, Tic20, Tic100 and Tic56), three chloroplast translocon-associated proteins (Ctap3, Ctap4 and Ctap5) and three newly-identified small inner-membrane proteins (Simp1-3) have been located in the supercomplex. As the largest protein, Tic214 traverses the inner membrane, the intermembrane space and the outer membrane, connecting the TOC complex with the TIC proteins. An inositol hexaphosphate (InsP6 or I6P) molecule is located at the Tic214-Toc90 interface and stabilizes their assembly. Moreover, four lipid molecules are located within or above an inner-membrane funnel formed by Tic214, Tic20, Simp1 and Ctap5. Furthermore, multiple potential pathways found in the TOC-TIC supercomplex may support translocation of different substrate preproteins into chloroplasts.

Plastid retrograde signaling plays a key role in coordinating the expression of plastid genes and photosynthesis-associated nuclear genes ( PhANGs). Although plastid retrograde signaling can be substantially compromised by mitochondrial dysfunction, it is not yet clear whether specific mitochondrial factors are required to regulate plastid retrograde signaling. Here, we show that mitochondrial ATP synthase β-subunit mutants with decreased ATP synthase activity are impaired in plastid retrograde signaling. Transcriptome analysis revealed that the expression levels of PhANGs were significantly higher in seedlings affected in AT5G08670, the gene of the β-subunit of mitochondrial ATP synthase than in wild-type (WT) seedlings upon treatment with lincomycin (LIN) and norflurazon (NF). Further studies showed that the expression of nuclear genes involved in chloroplast and mitochondrial retrograde signaling was affected in AT5G08670 mutant seedlings treated with LIN. These changes might be associated with the repression of some transcriptional factors (TFs), such as ARF3/ETTIN ( ETT), PLASTID TRANSCRIPTION FACTOR 1 ( PTF1), CYCLOIDEA AND PCF TRANSCRIPTION FACTOR 2 ( TCP2), and KOW DOMAIN-CONTAINING TRANSCRIPTION FACTOR 1 ( KTF1). These findings indicate that the activity of mitochondrial ATP synthase affects plastid retrograde signaling.

The reactive oxygen species (ROS) are continuously produced and are essential for mediating the growth and development of plants. However too much accumulation of ROS can result in the oxidative damage to cells, especially under the adverse environmental conditions. Plants have evolved sophisticated strategies to regulate the homeostasis of H2O2. In this study, we generated transgenic Arabidopsis plants in the Ws ecotype (Ws) background in which WRKY33 is co-suppressed (csWRKY33/Ws). Compared with Ws, csWRKY33/Ws plants accumulate more H2O2. RNA-seq analysis indicated that in csWRKY33/Ws plants, expression of oxidative stress related genes such as ascorbate peroxidase 2 (APX2) is affected. Over-expression of APX2 can rescue the phenotype of csWRKY33/Ws, suggesting that the changes in the growth of csWRKY33/Ws is duo to the higher accumulation of H2O2. Analysis of the CHIP-seq data suggested that WRKY33 can directly regulate the expression of PIF4, vice versa. qPCR analysis also confirmed that the mutual regulation between WRKY33 and PIF4. Similar to that of csWRKY33/Ws, and the accumulation of H2O2 in pif4 also increased. Taken together, our results reveal a WRKY33-PIF4 regulatory loop that appears to play an important role in regulating the growth and development of seedlings by mediating H2O2 homeostasis.

Epidermal cells are the main avenue for signal and material exchange between plants and the environment. Leaf epidermal cells primarily include pavement cells (PCs), guard cells, and trichomes cells (TCs), which differentiate from protodermal cells or meristemoids. The development and distribution of different epidermal cells are tightly regulated by a complex transcriptional regulatory network mediated by phytohormones, including jasmonic acid (JA), and transcription factors. Understanding how the fate of leaf epidermal cells is determined, however, is still largely unknown due to the diversity of cell types and the complexity of its regulation. Here, we characterized the transcriptional profiles of epidermal cells in 3-day-old true leaves of Arabidopsis thaliana using single-cell RNA-sequencing. We identified two genes encoding BASIC LEUCINE-ZIPPER (bZIP) transcription factors, namely the bZIP25 and bZIP53, which are highly expressed in PCs and early-stage meristemoid cells. Densities of PCs and TCs were found to increase and decrease, respectively, in bzip25 and bzip53 mutants, compared with wild-type plants. This trend was more pronounced in the presence of JA, suggesting that these transcription factors regulate the development of TCs and PCs in response to JA.IN A NUTSHELLBackgroundLeaf epidermal cells, comprised of trichome cells (TCs), guard cells (GCs), and pavement cells (PCs), are responsible for exchanging materials and information between plants and the surrounding aerial environment. Many genes have been identified in Arabidopsis thaliana and confirmed to be involved in the initiation and differentiation of TCs and PCs. The fate determination of TCs and PCs is tightly regulated by positive and negative regulators at the cellular level. The precise underlying molecular mechanisms responsible for the fate determination of TCs and PCs, however, are still unclear at this time.QuestionWhat are the transcriptomic profiles of different leaf epidermal cell types? Can we dissect the genes that are specifically expressed in certain epidermal cell types? What kinds of transcription factors are involved in regulating the fate determination of TCs and PCs?FindingsWe performed single cell RNA-seq to investigate the transcriptomic profiles of different leaf epidermal cell types and identified differentially expressed genes in each cell type. We found that genes that are involved in jasmonic acid signaling are highly expressed in early-stage meristemoid (EM) cells which can act as the precursor of PCs and perhaps of TCs. To investigate the regulatory mechanisms underlying EM development, we identified the transcription factors (TFs) in EM cells and found that two bZIP TF genes, bZIP25 and bZIP53, are highly expressed in EMs. Further analyses of these two genes using both loss-of-function and gain-of-function approaches indicated that bZIP25 and bZIP53 are functionally involved in promoting trichome formation but inhibit pavement cell development in response to jasmonic acid.Next stepsBesides of bZIP25 and bZIP53, we also identified other key genes, for example FES1B, in leaf epidermal cells. Our next step will be to explore the regulation of other key genes involved in the fate determination of different cell types in leaf epidermis.

Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes approximately 100 proteins. The remaining chloroplast proteins, close to 3,000, are encoded by nuclear genes whose products are translated in the cytosol and imported into chloroplasts. However, there is still no consensus on the composition of the protein import machinery including its motor proteins and on how newly imported chloroplast proteins are refolded. In this study, we have examined the function of orf2971, the largest chloroplast gene of Chlamydomonas reinhardtii. The depletion of Orf2971 causes the accumulation of protein precursors, partial proteolysis and aggregation of proteins, increased expression of chaperones and proteases, and autophagy. Orf2971 interacts with the TIC (translocon at the inner chloroplast envelope) complex, catalyzes ATP (adenosine triphosphate) hydrolysis, and associates with chaperones and chaperonins. We propose that Orf2971 is intimately connected to the protein import machinery and plays an important role in chloroplast protein quality control.

There are numerous exchanges of signals and materials between leaves and roots, including nitrogen, which is one of the essential nutrients for plant growth and development. In this study we identified and characterized the Chlorophyll A/B-Binding Protein (CAB) (named coe2 for CAB overexpression 2) mutant, which is defective in the development of chloroplasts and roots under normal growth conditions. The phenotype of coe2 is caused by a mutation in the Nitric Oxide Associated (NOA1) gene that is implicated in a wide range of chloroplast functions including the regulation of metabolism and signaling of nitric oxide (NO). A transcriptome analysis reveals that expression of genes involved in metabolism and lateral root development are strongly altered in coe2 seedlings compared with WT. COE2 is expressed in hypocotyls, roots, root hairs, and root caps. Both the accumulation of NO and the growth of lateral roots are enhanced in WT but not in coe2 under nitrogen limitation. These new findings suggest that COE2-dependent signaling not only coordinates gene expression but also promotes chloroplast development and function by modulating root development and absorption of nitrogen compounds.

Although numerous studies have been carried out on chloroplast development and biogenesis, the underlying regulatory mechanisms are still largely elusive. Here, we characterized a chloroplast stromal protein Chloroplast Development and Biogenesis1 (CDB1). The knockout

Members of the mitochondrial transcription terminator factor (mTERF) family, originally identified in vertebrate mitochondria, are involved in the termination of organellular transcription. In plants, mTERF proteins are mainly localized in chloroplasts and mitochondria. In Arabidopsis (Arabidopsis thaliana), mTERF8/pTAC15 was identified in the plastid-encoded RNA polymerase (PEP) complex, the major RNA polymerase of chloroplasts. In this work, we demonstrate that mTERF8 is associated with the PEP complex. An mTERF8 knockout line displayed a wild-type–like phenotype under standard growth conditions, but showed impaired efficiency of photosystem II electron flow. Transcription of most chloroplast genes was not substantially affected in the mterf8 mutant; however, the level of the psbJ transcript from the psbEFLJ polycistron was increased. RNA blot analysis showed that a larger transcript accumulates in mterf8 than in the wild type. Thus, abnormal transcription and/or RNA processing occur for the psbEFLJ polycistron. Circular reverse transcription PCR and sequence analysis showed that the psbJ transcript terminates 95 nucleotides downstream of the translation stop codon in the wild type, whereas its termination is aberrant in mterf8. Both electrophoresis mobility shift assays and chloroplast chromatin immunoprecipitation analysis showed that mTERF8 specifically binds to the 3′ terminal region of psbJ. Transcription analysis using the in vitro T7 RNA polymerase system showed that mTERF8 terminates psbJ transcription. Together, these results suggest that mTERF8 is specifically involved in the transcription termination of the chloroplast gene psbJ.

The green unicellular alga Chlamydomonas reinhardtii has emerged as a very attractive model system for chloroplast genetic engineering. Algae can be transformed readily at the chloroplast level through bombardment of cells with a gene gun and transformants can be selected using antibiotic resistance or phototrophic growth. An inducible chloroplast gene expression system could be very useful for several reasons. First, it could be used to elucidate the function of essential chloroplast genes required for cell growth and survival. Second, it could be very helpful for expressing proteins which are toxic to the algal cells. Third, it would allow for the reversible depletion of photosynthetic complexes, thus making it possible to study their biogenesis in a controlled fashion. Fourth, it opens promising possibilities for hydrogen production in Chlamydomonas. Here we describe an inducible/ repressible chloroplast gene expression system in Chlamydomonas in which the copper-regulated Cyc

Fe-S clusters are ancient, ubiquitous and highly essential prosthetic groups for numerous fundamental processes of life. The biogenesis of Fe-S clusters is a multistep process including iron acquisition, sulfur mobilization, and cluster formation. Extensive studies have provided deep insights into the mechanism of the latter two assembly steps. However, the mechanism of iron utilization during chloroplast Fe-S cluster biogenesis is still unknown. Here we identified two Arabidopsis DnaJ proteins, DJA6 and DJA5, that can bind iron through their conserved cysteine residues and facilitate iron incorporation into Fe-S clusters by interactions with the SUF (sulfur utilization factor) apparatus through their J domain. Loss of these two proteins causes severe defects in the accumulation of chloroplast Fe-S proteins, a dysfunction of photosynthesis, and a significant intracellular iron overload. Evolutionary analyses revealed that DJA6 and DJA5 are highly conserved in photosynthetic organisms ranging from cyanobacteria to higher plants and share a strong evolutionary relationship with SUFE1, SUFC, and SUFD throughout the green lineage. Thus, our work uncovers a conserved mechanism of iron utilization for chloroplast Fe-S cluster biogenesis.

In response to changing light quantity and quality, photosynthetic organisms perform state transitions, a process which optimizes photosynthetic yield and mitigates photo-damage. The serine/threonine-protein kinase STN7 phosphorylates the light-harvesting complex of photosystem II (PSII; light-harvesting complex II), which then migrates from PSII to photosystem I (PSI), thereby rebalancing the light excitation energy between the photosystems and restoring the redox poise of the photosynthetic electron transport chain. Two conserved cysteines forming intra- or intermolecular disulfide bonds in the lumenal domain (LD) of STN7 are essential for the kinase activity although it is still unknown how activation of the kinase is regulated. In this study, we show lumen thiol oxidoreductase 1 (LTO1) is co-expressed with STN7 in Arabidopsis (Arabidopsis thaliana) and interacts with the LD of STN7 in vitro and in vivo. LTO1 contains thioredoxin (TRX)-like and vitamin K epoxide reductase domains which are related to the disulfide-bond formation system in bacteria. We further show that the TRX-like domain of LTO1 is able to oxidize the conserved lumenal cysteines of STN7 in vitro. In addition, loss of LTO1 affects the kinase activity of STN7 in Arabidopsis. Based on these results, we propose that LTO1 helps to maintain STN7 in an oxidized active state in state 2 through redox interactions between the lumenal cysteines of STN7 and LTO1.

The green unicellular alga Chlamydomonas reinhardtii has emerged as a very attractive model system for chloroplast genetic engineering. Algae can be transformed readily at the chloroplast level through bombardment of cells with a gene gun and transformants can be selected using antibiotic resistance or phototrophic growth. An inducible chloroplast gene expression system could be very useful for several reasons. First, it could be used to elucidate the function of essential chloroplast genes required for cell growth and survival. Second, it could be very helpful for expressing proteins which are toxic to the algal cells. Third, it would allow for the reversible depletion of photosynthetic complexes, thus making it possible to study their biogenesis in a controlled fashion. Fourth, it opens promising possibilities for hydrogen production in Chlamydomonas. Here we describe an inducible/ repressible chloroplast gene expression system in Chlamydomonas in which the copper-regulated Cyc6 promoter or the vitamin-controlled MetE promoter and TPP riboswitch drive the expression of the nuclear Nac2 gene encoding a protein which is targeted to the chloroplast where it acts specifically on the chloroplast psbD 5' untranslated region and is required for the stable accumulation of the psbD mRNA and photosystem II. The system can be used for any chloroplast gene or trans-gene by placing it under the control of the psbD 5'untranslated region.

Significance Chloroplasts are of vital importance in photosynthetic eukaryotic organisms. Like mitochondria, they contain their own genomes. Nevertheless, most chloroplast proteins are encoded by nuclear genes, translated in the cytosol, and must cross the chloroplast envelope membranes to reach their proper destinations inside the organelle. Despite its fundamental role, our knowledge of the machinery catalyzing chloroplast protein import is incomplete and controversial. Here, we address the evolutionary conservation, composition, and function of the chloroplast protein import machinery using the green alga Chlamydomonas reinhardtii as a model system. Our findings help clarify the current debate regarding the composition of the chloroplast protein import machinery, provide evidence for cross-compartmental coordination of its biogenesis, and open promising avenues for its structural characterization., In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons—termed TOC and TIC—located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.

LHC (light-harvesting complex) proteins of plants and algae are known to be involved both in collecting light energy for driving the primary photochemical reactions of photosynthesis and in photoprotection when the absorbed light energy exceeds the capacity of the photosynthetic apparatus. These proteins usually contain three transmembrane (TM) helices which span the thylakoid membranes and bind several chlorophyll, carotenoid and lipid molecules. In addition, the LHC protein family includes LHC-like proteins containing one, two, three or even four TM domains. One-helix proteins are not only present in eukaryotic photosynthetic organisms but also in cyanobacteria where they have been named high light-inducible proteins. These small proteins are probably the ancestors of the members of the extant LHC protein family which arouse through gene duplications, deletions and fusions. During evolution, some of these proteins have diverged and acquired novel functions. In most cases, LHC-like proteins are induced in response to various stress conditions including high light, high salinity, elevated temperature and nutrient limitation. Many of these proteins play key roles in photoprotection, notably in non-photochemical quenching of absorbed light energy. Moreover, some of these proteins appear to be involved in the regulation of chlorophyll synthesis and in the assembly and repair of Photosystem II and also of Photosystem I possibly by mediating the insertion of newly synthesized pigments into the photosynthetic reaction centers.

F-type ATP synthases produce nearly all of the ATP found in cells. The catalytic module F1 commonly comprises an α3β3 hexamer surrounding a γ/ε stalk. However, it is unclear how these subunits assemble to form a catalytic motor. In this work, we identified and characterized a chloroplast protein that interacts with the CF1β, γ, and ε subunits of the chloroplast ATP synthase and is required for assembly of its F1 module. We named this protein BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE1 (BFA1) and determined its crystal structure at 2.8-Å resolution. BFA1 is comprised primarily of two interacting β-barrels that are oriented nearly perpendicularly to each other. The contact region between BFA1 and the CF1β and γ subunits was further mapped by yeast two-hybrid assays. An in silico molecular docking analysis was performed and revealed close fitting contact sites without steric conflicts between BFA1 and CF1β/γ. We propose that BFA1 acts mainly as a scaffold protein promoting the association of a CF1α/β heterodimer with CF1γ. The subsequent assembly of other CF1α/β heterodimers may shift the position of the CF1γ subunit to complete assembly of the CF1 module. This CF1 assembly process is likely to be valid for other F-type ATP synthases, as their structures are highly conserved.

Singlet oxygen (1 O2 ) is a prime cause of photo-damage of the photosynthetic apparatus. The chlorophyll molecules in the photosystem II reaction center and in the light-harvesting antenna complex are major sources of 1 O2 generation. It has been thought that the generation of 1 O2 mainly takes place in the appressed regions of the thylakoid membranes, namely, the grana core, where most of the active photosystem II complexes are localized. Apart from being a toxic molecule, new evidence suggests that 1 O2 significantly contributes to chloroplast-to-nucleus retrograde signalling that primes acclimation and cell death responses. Interestingly, recent studies reveal that chloroplasts operate two distinct 1 O2 -triggered retrograde signalling pathways in which β-carotene and a nuclear-encoded chloroplast protein EXECUTER1 play essential roles as signalling mediators. The coexistence of these mediators raises several questions: their crosstalk, source(s) of 1 O2 , downstream signalling components, and the perception and reaction mechanism of these mediators towards 1 O2 . In this review, we mainly discuss the molecular genetic basis of the mode of action of these two putative 1 O2 sensors and their corresponding retrograde signalling pathways. In addition, we also propose the possible existence of an alternative source of 1 O2 , which is spatially and functionally separated from the grana core.

In this issue of Cell, Singharoy et al. present a rate kinetic model for the chromatophore of photosynthetic purple bacteria generated from molecular dynamics simulations. It shows that the chromatophore's architecture is optimized for producing bioenergy under low light typical of the natural bacterial habitat while minimizing photodamage under stress conditions.

Although chloroplasts contain their own genetic system and are semi-autonomous cell organelles, plastid biogenesis and homeostasis are heavily dependent on the nucleo-cytosolic compartment. These two cellular compartments are closely co-ordinated through a complex signaling network comprising both anterograde and retrograde signaling chains. Developmental changes or any perturbation in the chloroplast system induced by a particular stress resulting from changes in environmental conditions such as excess light, elevated temperature, nutrient limitation, pathogen infection, give rise to specific signals. They migrate out of the chloroplast and are perceived by the nucleus where they elicit changes in expression of particular genes that allow for the maintenance of plastid homeostasis toward environmental cues. These genes mainly include those of photosynthesis-associated proteins, chaperones, proteases, nucleases and immune/defense proteins. Besides this transcriptional response, a chloroplast quality control system exists that is involved in the repair and turnover of damaged plastid proteins. This system degrades aggregated or damaged proteins and it can even remove entire chloroplasts when they have suffered heavy damage. This response comprises several processes such as plastid autophagy and ubiquitin–proteasome mediated proteolysis that occurs on the plastid envelope through the action of the ubiquitin–proteasome system.

The regulation of stomatal lineage cell development has been extensively investigated. However, a comprehensive characterization of this biological process based on single-cell transcriptome analysis has not yet been reported. In this study, we performed RNA sequencing on 12 844 individual cells from the cotyledons of 5-day-old Arabidopsis seedlings. We identified 11 cell clusters corresponding mostly to cells at specific stomatal developmental stages using a series of marker genes. Comparative analysis of genes with the highest variable expression among these cell clusters revealed transcriptional networks that regulate development from meristemoid mother cells to guard mother cells. Examination of the developmental dynamics of marker genes via pseudo-time analysis revealed potential interactions between these genes. Collectively, our study opens the door for understanding how the identified novel marker genes participate in the regulation of stomatal lineage cell development.

The regulation of stomatal lineage cell development has been extensively investigated. However a comprehensive characterization of this biological process based on single-cell transcriptome analysis has not yet been reported. Here, we performed RNA-seq on over 12,844 individual cells from the cotyledons of five-day-old Arabidopsis seedlings. We identified 11 cell clusters corresponding mostly to cells at specific stomatal developmental stages with a series of new marker genes. Comparative analysis of genes with the highest variable expression in these cell clusters revealed three transcriptional networks that regulate the development of mesophyll and guard cells, as well as the differentiation from protodermal to guard mother cells. We investigated the developmental dynamics of marker genes via pseudo-time analysis which revealed potential interactions between them. The identification of several novel marker genes suggests new regulatory mechanisms during development of stomatal cell lineage.

Chloroplast ATP synthase belongs to the F-type ATP synthases. It is localized in the thylakoid membrane and consists of nine subunits with a stoichiometry of α3β3γ1ɛ1δ1a1b1b′1c14. Since its subunits are encoded by both nuclear and chloroplast genomes, its biogenesis requires an intricate coordination of gene expression between these two genetic systems as well as a tight regulation of assembly by various chloroplast protein chaperones. Isolation of mutants defective in the biogenesis of chloroplast ATP synthase and detailed characterization of the corresponding genes revealed the existence of several pentatricopeptide repeat (PPR) proteins that are required for proper expression of chloroplast atp genes at various post-transcriptional steps including RNA splicing, RNA editing, RNA stabilization as well as protein translation. As other F-type ATP synthases, assembly of the chloroplast ATP synthase occurs in a stepwise manner through the formation of several transient intermediates. Several chaperones facilitate the assembly of chloroplast ATP synthase by interacting with specific subunits at different stages. In particular, structural studies of some assembly factors have provided many new insights into the assembly of the chloroplast ATP synthase at the molecular level. Because the structure and the assembly pathway of the F-type ATP synthase are highly conserved among most species, some of the assembly mechanisms of chloroplast ATP synthase are likely to be valid for other ATP synthases.

In order to withstand high light (HL) stress, plants have evolved both short-term defense and repair mechanisms and long-term acclimation responses. At present, however, the underlying signaling events and molecular mechanisms are still poorly understood. Analysis of the mutants coe1, coe1 gun1 double mutant and oeGUN1coe1 revealed increased sensitivity to HL stress as compared to wild type (WT), with oeGUN1 coe1 plants displaying the highest sensitivity. Accumulation of FTSH2 protein and degradation of D1 protein during the HL stress were shown to depend on both COE1 and GUN1. Overexpression of COE1 enhanced the induction of FTSH2 and the tolerance to HL stress. These results indicate that the COE1-GUN1 signaling pathway plays an important role in regulating the adaptation of plants to HL.

As a fascinating and complicated nanomotor, chloroplast ATP synthase comprises nine subunits encoded by both the nuclear and plastid genomes. Because of its uneven subunit stoichiometry, biogenesis of ATP synthase and expression of plastid-encoded ATP synthase genes requires assistance by nucleus-encoded factors involved in transcriptional, post-transcriptional, and translational steps. In this study, we report a P-class pentatricopeptide repeat (PPR) protein BFA2 (Biogenesis Factor required for ATP synthase 2) that is essential for accumulation of the dicistronic atpH/F transcript in Arabidopsis chloroplasts. A loss-of-function mutation in BFA2 results in a specific reduction of more than 3/4 of chloroplast ATP synthase, which is likely due to the absence of dicistronic atpH/F transcript. BFA2 protein contains 22 putative PPR motifs and exclusively localizes in the chloroplast. Bioinformatics and Electrophoretic Mobility Shift Assays (EMSA) analysis showed that BFA2 binds to the consensus sequence of the atpF-atpA intergenic region in a sequence-specific manner. However, translation initiation of the atpA was not affected in the bfa2 mutant. Thus, we propose that the chloroplast PPR protein BFA2 mainly acts as barrier to prevent the atpH/F transcript degradation by exoribonucleases by binding to the consensus sequence of the atpF-atpA intergenic region.

The reaction center (RC) of photosystem II (PSII), which is composed of D1, D2, PsbI, and cytochrome b559 subunits, forms at an early stage of PSII biogenesis. However, it is largely unclear how these components assemble to form a functional unit. In this work, we show that synthesis of the PSII core proteins D1/D2 and formation of the PSII RC is blocked specifically in the absence of ONE-HELIX PROTEIN1 (OHP1) and OHP2 proteins in Arabidopsis (Arabidopsis thaliana), indicating that OHP1 and OHP2 are essential for the formation of the PSII RC. Mutagenesis of the chlorophyll-binding residues in OHP proteins impairs their function and/or stability, suggesting that they may function in the binding of chlorophyll in vivo. We further show that OHP1, OHP2, and HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244), together with D1, D2, PsbI, and cytochrome b559, form a complex. We designated this complex the PSII RC-like complex to distinguish it from the RC subcomplex in the intact PSII complex. Our data imply that OHP1, OHP2, and HCF244 are present in this PSII RC-like complex for a limited time at an early stage of PSII de novo assembly and of PSII repair under high-light conditions. In a subsequent stage of PSII biogenesis, OHP1, OHP2, and HCF244 are released from the PSII RC-like complex and replaced by the other PSII subunits. Together with previous reports on the cyanobacterium Synechocystis, our results demonstrate that the process of PSII RC assembly is highly conserved among photosynthetic species.

In order to maintain optimal photosynthetic activity under a changing light environment, plants and algae need to balance the absorbed light excitation energy between photosystem I and photosystem II through processes called state transitions. Variable light conditions lead to changes in the redox state of the plastoquinone pool which are sensed by a protein kinase closely associated with the cytochrome b(6)f complex. Preferential excitation of photosystem II leads to the activation of the kinase which phosphorylates the light-harvesting system (LHCII), a process which is subsequently followed by the release of LHCII from photosystem II and its migration to photosystem I. The process is reversible as dephosphorylation of LHCII on preferential excitation of photosystem I is followed by the return of LHCII to photosystem II. State transitions involve a considerable remodelling of the thylakoid membranes, and in the case of Chlamydomonas, they allow the cells to switch between linear and cyclic electron flow. In this alga, a major function of state transitions is to adjust the ATP level to cellular demands. Recent studies have identified the thylakoid protein kinase Stt7/STN7 as a key component of the signalling pathways of state transitions and long-term acclimation of the photosynthetic apparatus. In this article, we present a review on recent developments in the area of state transitions.

Singlet oxygen (

International audience; Twelve cationic helicenes and one triangulene were tested for the specific labeling of mitochondria from algal cells. Octyl ester derivative 5 readily penetrates algal cells and gives rise to clear fluorescence patterns when it is used at concentrations in the μM range. Under these conditions, cell structures are well preserved and cell survival is not compromised. Cationic helicene compounds such as 5 provide new useful tools for examining the mitochondrial network and its dynamics including fission and fusion events.

We present here a Tribute to Klaus Apel (1942-2017), a photosynthesis pioneer-an authority on plant molecular genetics-in five parts. The first section is a prologue. The second section deals with a chronological discussion of Apel's research life, prepared by the editor Govindjee; it is based on a website article at the Boyce Thompson Institute (BTI) by Patricia Waldron ( https://btiscience.org/explore-bti/news/post/bti-says-goodbye-klaus-apel/ ), as approved for use here by Keith C. Hannon and David Stern of BTI. The third section, which focuses on Apel's pioneering work on singlet oxygen-mediated EXECUTER-dependent signaling in plants, is written by two of us (J-DR and CK). The fourth section includes three selected reminiscences, one from BTI and two from ETH (Eidgenossische Technische Hochschule). This tribute ends with section five, which is a very brief presentation of Klaus Apel's personal life, by Wiebke Apel.

Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria. © 2014 American Society of Plant Biologists. All rights reserved.

Photosynthetic organisms are continuously subjected to changes in light quantity and quality, and must adjust their photosynthetic machinery so that it maintains optimal performance under limiting light and minimizes photodamage under excess light. To achieve this goal, these organisms use two main strategies in which light-harvesting complex II (LHCII), the light-harvesting system of photosystem II (PSII), plays a key role both for the collection of light energy and for photoprotection. The first is energy-dependent nonphotochemical quenching, whereby the high-light-induced proton gradient across the thylakoid membrane triggers a process in which excess excitation energy is harmlessly dissipated as heat. The second involves a redistribution of the mobile LHCII between the two photosystems in response to changes in the redox poise of the electron transport chain sensed through a signaling chain. These two processes strongly diminish the production of damaging reactive oxygen species, but photodamage of PSII is unavoidable, and it is repaired efficiently.

Significance: Photosynthetic organisms are subjected to frequent changes in their environment that include fluctuations in light quality and quantity, temperature, CO2 concentration, and nutrient availability. They have evolved complex responses to these changes that allow them to protect themselves against photo-oxidative damage and to optimize their growth under these adverse conditions. In the case of light changes, these acclimatory processes can occur in either the short or the long term and are mainly mediated through the redox state of the plastoquinone pool and the ferredoxin/thioredoxin system. Recent Advances: Short-term responses involve a dynamic reorganization of photosynthetic complexes, and long-term responses (LTRs) modulate the chloroplast and nuclear gene expression in such a way that the levels of the photosystems and their antennae are rebalanced for an optimal photosynthetic performance. These changes are mediated through a complex signaling network with several protein kinases and phosphatases that are conserved in land plants and algae. The phosphorylation status of the light-harvesting proteins of photosystem II and its core proteins is mainly determined by two complementary kinase–phosphatase pairs corresponding to STN7/PPH1 and STN8/PBCP, respectively. Critical Issues: The activity of the Stt7 kinase is principally regulated by the redox state of the plastoquinone pool, which in turn depends on the light irradiance, ambient CO2 concentration, and cellular energy status. In addition, this kinase is also involved in the LTR. Future Directions: Other chloroplast kinases modulate the activity of the plastid transcriptional machinery, but the global signaling network that connects all of the identified kinases and phosphatases is still largely unknown. Antioxid. Redox Signal. 18, 2184–2201.

The pyrenoid is a membrane-less organelle that exists in various photosynthetic organisms, such as algae, and wherein most global CO2 fixation occurs. Two papers from the Jonikas lab in this issue of Cell provide new insights into the structure, protein composition, and dynamics of this important organelle.

Using a genetic approach, we have identified and characterized a novel protein, named Msf1 (Maintenance factor for photosystem I), that is required for the maintenance of specific components of the photosynthetic apparatus in the green alga Chlamydomonas reinhardtii Msf1 belongs to the superfamily of light-harvesting complex proteins with three transmembrane domains and consensus chlorophyll-binding sites. Loss of Msf1 leads to reduced accumulation of photosystem I and chlorophyll-binding proteins/complexes. Msf1is a component of a thylakoid complex containing key enzymes of the tetrapyrrole biosynthetic pathway, thus revealing a possible link between Msf1 and chlorophyll biosynthesis. Protein interaction assays and greening experiments demonstrate that Msf1 interacts with Copper target homolog1 (CHL27B) and accumulates concomitantly with chlorophyll in Chlamydomonas, implying that chlorophyll stabilizes Msf1. Contrary to other light-harvesting complex-like genes, the expression of Msf1 is not stimulated by high-light stress, but its protein level increases significantly under heat shock, iron and copper limitation, as well as in stationary cells. Based on these results, we propose that Msf1 is required for the maintenance of photosystem I and specific protein-chlorophyll complexes especially under certain stress conditions.

We have identified hpm91, a Chlamydomonas mutant lacking Proton Gradient Regulation5 (PGR5) capable of producing hydrogen (H

Chloroplast retrograde signals play important roles in coordinating the plastid and nuclear gene expression and are critical for proper chloroplast biogenesis and for maintaining optimal chloroplast functions in response to environmental changes in plants. Until now, the signals and the mechanisms for retrograde signalling remain poorly understood. Here we identify factors that allow the nucleus to perceive stress conditions in the chloroplast and to respond accordingly by inducing or repressing specific nuclear genes encoding plastid proteins. We show that ABI4, which is known to repress the LHCB genes during retrograde signalling, is activated through phosphorylation by the MAP kinases MPK3/MPK6 and the activity of these kinases is regulated through 14-3-3ω-mediated Ca2+-dependent scaffolding depending on the chloroplast calcium sensor protein CAS. These findings uncover an additional mechanism in which chloroplast-modulated Ca2+ signalling controls the MAPK pathway for the activation of critical components of the retrograde signalling chain., Retrograde signalling co-ordinates nuclear gene expression in response to stress perceived in the chloroplast. Here Guo et al. show that a MAPK cascade acting downstream of a plastid derived Ca2+ signal contributes to the regulation of nuclear gene expression by phosphorylation of ABI4 during retrograde signalling.