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Cyanobacteria and chloroplasts of algae and plants harbor specialized thylakoid membranes (TMs) that convert sunlight into chemical energy. These membranes house PSII and I, the vital protein-pigment complexes that drive oxygenic photosynthesis. In the course of their evolution, TMs have diversified in structure. However, the core machinery for photosynthetic electron transport remained largely unchanged, with adaptations occurring primarily in the light-harvesting antenna systems. Whereas TMs in cyanobacteria are relatively simple, they become more complex in algae and plants. The chloroplasts of vascular plants contain intricate networks of stacked grana and unstacked stroma thylakoids. This review provides an in-depth view of TM architectures in phototrophs and the determinants that shape their forms, as well as presenting recent insights into the spatial organization of their biogenesis and maintenance. Its overall goal is to define the underlying principles that have guided the evolution of these bioenergetic membranes. [ABSTRACT FROM AUTHOR]

In the chloroplast, the 54 kDa subunit of the signal recognition particle (cpSRP54) is involved in the post-translational transport of the light-harvesting chlorophyll a / b -binding proteins (LHCPs) and the co-translational transport of plastid-encoded subunits of the photosynthetic complexes to the thylakoid membrane. It forms a high-affinity complex with plastid-specific cpSRP43 for post-translational transport, while a ribosome-associated pool coordinates its co-translational function. CpSRP54 constitutes a conserved multidomain protein, comprising a GTPase (NG) and a methionine-rich (M) domain linked by a flexible region. It is further characterized by a plastid-specific C-terminal tail region containing the cpSRP43-binding motif. To characterize the physiological role of the various regions of cpSRP54 in thylakoid membrane protein transport, we generated Arabidopsis cpSRP54 knockout (ffc1-2) lines producing truncated cpSRP54 variants or a GTPase point mutation variant. Phenotypic characterization of the complementation lines demonstrated that the C-terminal tail region of cpSRP54 plays an important role exclusively in post-translational LHCP transport. Furthermore, we show that the GTPase activity of cpSRP54 plays an essential role in the transport pathways for both nuclear as well as plastid-encoded proteins. In addition, our data revealed that plants expressing cpSRP54 without the C-terminal region exhibit a strongly increased accumulation of a photosystem I assembly intermediate. [ABSTRACT FROM AUTHOR]

Chloroplasts fix carbon by using light energy and have evolved a complex redox network that supports plastid functions by (i) protecting against reactive oxygen species and (ii) metabolic regulation in response to environmental conditions. In thioredoxin- and glutathione/glutaredoxin-dependent redox cascades, protein cysteinyl redox steady states are set by varying oxidation and reduction rates. The specificity and interplay of these different redox-active proteins are still under investigation, for example to understand how plants cope with adverse environmental conditions by acclimation. Genetically encoded biosensors with distinct specificity can be targeted to subcellular compartments such as the chloroplast stroma, enabling in vivo real-time measurements of physiological parameters at different scales. These data have provided unique insights into dynamic behaviours of physiological parameters and redox-responsive proteins at several levels of the known redox cascades. This review summarizes current applications of different biosensor types as well as the dynamics of distinct protein cysteinyl redox steady states, with an emphasis on light responses. [ABSTRACT FROM AUTHOR]

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana , but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis. [ABSTRACT FROM AUTHOR]

Plants, being sessile organisms, constantly need to respond to environmental stresses, often leading to the accumulation of reactive oxygen species (ROS). While ROS can be harmful, they also act as second messengers guiding plant growth and stress responses. Because chloroplasts are sensitive to environmental changes and are both a source and a target of ROS during stress conditions, they are important in conveying environmental changes to the nucleus, where acclimation responses are coordinated to maintain organellar and overall cellular homeostasis. ANAC102 has previously been established as a regulator of β-cyclocitral-mediated chloroplast-to-nucleus signaling, protecting plants against photooxidative stress. However, debates persist about where ANAC102 is located—in chloroplasts or in the nucleus. Our study, utilizing the genomic ANAC102 sequence driven by its native promoter, establishes ANAC102 primarily as a nuclear protein, lacking a complete N-terminal chloroplast-targeting peptide. Moreover, our research reveals the sensitivity of plants overexpressing ANAC102 to severe superoxide-induced chloroplast oxidative stress. Transcriptome analysis unraveled a dual role of ANAC102 in negatively and positively regulating genome-wide transcriptional responses to chloroplast oxidative stress. Through the integration of published data and our own study, we constructed a comprehensive transcriptional network, which suggests that ANAC102 exerts direct and indirect control over transcriptional responses through downstream transcription factor networks, providing deeper insights into the ANAC102-mediated regulatory landscape during oxidative stress. [ABSTRACT FROM AUTHOR]

The superoxide anion radical (O2·−) is a one-electron reduction product of molecular oxygen. Compared with other forms of reactive oxygen species (ROS), superoxide has limited reactivity. Nevertheless, superoxide reacts with nitric oxide, ascorbate, and the iron moieties of [Fe–S] cluster-containing proteins. Superoxide has largely been neglected as a signalling molecule in the plant literature in favour of the most stable ROS form, hydrogen peroxide. However, superoxide can accumulate in plant cells, particularly in meristems, where superoxide dismutase activity and ascorbate accumulation are limited (or absent), or when superoxide is generated within the lipid environment of membranes. Moreover, oxidation of the nucleus in response to environmental stresses is a widespread phenomenon. Superoxide is generated in many intracellular compartments including mitochondria, chloroplasts, and on the apoplastic/cell wall face of the plasma membrane. However, nuclear superoxide production and functions remain poorly documented in plants. Accumulating evidence suggests that the nuclear pools of antioxidants such as glutathione are discrete and separate from the cytosolic pools, allowing compartment-specific signalling in the nucleus. We consider the potential mechanisms of superoxide generation and targets in the nucleus, together with the importance of antioxidant processing in regulating superoxide signalling. [ABSTRACT FROM AUTHOR]

Mitochondria and plastids have both dramatically reduced their genomes since the endosymbiotic events that created them. The similarities and differences in the evolution of the two organelle genome types have been the target of discussion and investigation for decades. Ongoing work has suggested that similar mechanisms may modulate the reductive evolution of the two organelles in a given species, but quantitative data and statistical analyses exploring this picture remain limited outside of some specific cases like parasitism. Here, we use cross-eukaryote organelle genome data to explore evidence for coevolution of mitochondrial and plastid genome reduction. Controlling for differences between clades and pseudoreplication due to relatedness, we find that extents of mtDNA and ptDNA gene retention are related to each other across taxa, in a generally positive correlation that appears to differ quantitatively across eukaryotes, for example, between algal and nonalgal species. We find limited evidence for coevolution of specific mtDNA and ptDNA gene pairs, suggesting that the similarities between the two organelle types may be due mainly to independent responses to consistent evolutionary drivers. [ABSTRACT FROM AUTHOR]

Hypothetical chloroplast open reading frames (ycfs) are putative genes in the plastid genomes of photosynthetic eukaryotes. Many ycfs are also conserved in the genomes of cyanobacteria, the presumptive ancestors of present-day chloroplasts. The functions of many ycfs are still unknown. Here, we generated knock-out mutants for ycf51 (sll1702) in the cyanobacterium Synechocystis sp. PCC 6803. The mutants showed reduced photoautotrophic growth due to impaired electron transport between photosystem II (PSII) and PSI. This phenotype results from greatly reduced PSI content in the ycf51 mutant. The ycf51 disruption had little effect on the transcription of genes encoding photosynthetic complex components and the stabilization of the PSI complex. In vitro and in vivo analyses demonstrated that Ycf51 cooperates with PSI assembly factor Ycf3 to mediate PSI assembly. Furthermore, Ycf51 interacts with the PSI subunit PsaC. Together with its specific localization in the thylakoid membrane and the stromal exposure of its hydrophilic region, our data suggest that Ycf51 is involved in PSI complex assembly. Ycf51 is conserved in all sequenced cyanobacteria, including the earliest branching cyanobacteria of the Gloeobacter genus, and is also present in the plastid genomes of glaucophytes. However, Ycf51 has been lost from other photosynthetic eukaryotic lineages. Thus, Ycf51 is a PSI assembly factor that has been functionally replaced during the evolution of oxygenic photosynthetic eukaryotes. Chloroplast reading frame Ycf51 is a photosystem I assembly factor conserved in all sequenced cyanobacteria and glaucophyte plastids but lost from other photosynthetic eukaryotic lineages. [ABSTRACT FROM AUTHOR]

In heterotrophs, heme degradation produces bilirubin, a tetrapyrrole compound that has antioxidant activity. In plants, heme is degraded in plastids and is believed to be converted to phytochromobilin rather than bilirubin. Recently, we used the bilirubin-inducible fluorescent protein UnaG to reveal that plants produce bilirubin via a non-enzymatic reaction with NADPH. In the present study, we used an UnaG-based live imaging system to visualize bilirubin accumulation in Arabidopsis thaliana and Nicotiana benthamiana at the organelle and tissue levels. In chloroplasts, bilirubin preferentially accumulated in the stroma, and the stromal bilirubin level increased upon dark treatment. Investigation of intracellular bilirubin distribution in leaves and roots showed that it accumulated mostly in plastids, with low levels detected in the cytosol and other organelles, such as peroxisomes, mitochondria and the endoplasmic reticulum. A treatment that increased bilirubin production in chloroplasts decreased the bilirubin level in peroxisomes, implying that a bilirubin precursor is transported between the two organelles. At the cell and tissue levels, bilirubin showed substantial accumulation in the root elongation region but little or none in the root cap and guard cells. Intermediate bilirubin accumulation was observed in other shoot and root tissues, with lower levels in shoot tissues. Our data revealed the distribution of bilirubin in plants, which has implications for the transport and physiological function of tetrapyrroles. [ABSTRACT FROM AUTHOR]

Selective partitioning of amino acids among organelles, cells, tissues, and organs is essential for cellular metabolism and plant growth. Nitrogen assimilation into glutamine and glutamate and de novo biosynthesis of most protein amino acids occur in chloroplasts; therefore, various transport mechanisms must exist to accommodate their directional efflux from the stroma to the cytosol and feed the amino acids into the extraplastidial metabolic and long-distance transport pathways. Yet, Arabidopsis (Arabidopsis thaliana) transporters functioning in plastidial export of amino acids remained undiscovered. Here, USUALLY MULTIPLE ACIDS MOVE IN AND OUT TRANSPORTER 44 (UMAMIT44) was identified and shown to function in glutamate export from Arabidopsis chloroplasts. UMAMIT44 controls glutamate homeostasis within and outside of chloroplasts and influences nitrogen partitioning from leaves to sinks. Glutamate imbalances in chloroplasts and leaves of umamit44 mutants impact cellular redox state, nitrogen and carbon metabolism, and amino acid (AA) and sucrose supply of growing sinks, leading to negative effects on plant growth. Nonetheless, the mutant lines adjust to some extent by upregulating alternative pathways for glutamate synthesis outside the plastids and by mitigating oxidative stress through the production of other amino acids and antioxidants. Overall, this study establishes that the role of UMAMIT44 in glutamate export from chloroplasts is vital for controlling nitrogen availability within source leaf cells and for sink nutrition, with an impact on growth and seed yield. Plastidial glutamate exporter UMAMIT44 regulates amino acid availability within leaf cells and for sink nutrition, which impacts plant growth and seed yield. [ABSTRACT FROM AUTHOR]

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement. CHLOROPLAST UNUSUAL POSITIONING 1 is an actin polymerization factor that is unique to plants and essential for chloroplast movement in response to high-light stress. [ABSTRACT FROM AUTHOR]

Chlorophyll degradation causes the release of phytol, which is converted into phytyl diphosphate (phytyl-PP) by phytol kinase (VITAMIN E PATHWAY GENE5 [VTE5]) and phytyl phosphate (phytyl-P) kinase (VTE6). The kinase pathway is important for tocopherol synthesis, as the Arabidopsis (Arabidopsis thaliana) vte5 mutant contains reduced levels of tocopherol. Arabidopsis harbors one paralog of VTE5, farnesol kinase (FOLK) involved in farnesol phosphorylation. Here, we demonstrate that VTE5 and FOLK harbor kinase activities for phytol, geranylgeraniol, and farnesol with different specificities. While the tocopherol content of the folk mutant is unchanged, vte5-2 folk plants completely lack tocopherol. Tocopherol deficiency in vte5-2 plants can be complemented by overexpression of FOLK, indicating that FOLK is an authentic gene of tocopherol synthesis. The vte5-2 folk plants contain only ∼40% of wild-type amounts of phylloquinone, demonstrating that VTE5 and FOLK both contribute in part to phylloquinone synthesis. Tocotrienol and menaquinone-4 were produced in vte5-2 folk plants after supplementation with homogentisate or 1,4-dihydroxy-2-naphthoic acid, respectively, indicating that their synthesis is independent of the VTE5/FOLK pathway. These results show that phytyl moieties for tocopherol synthesis are completely but, for phylloquinone production, only partially derived from geranylgeranyl-chlorophyll and phytol phosphorylation by VTE5 and FOLK. The phytyl diphosphate and geranylgeranyl diphosphate pools in chloroplasts required for tocopherol and phylloquinone synthesis are derived from the isoprenoid alcohol kinases VITAMIN E PATHWAY GENE5 and farnesol kinase. [ABSTRACT FROM AUTHOR]

RNA-binding proteins play integral roles in the regulation of essential processes in cells and as such are attractive targets for engineering to manipulate gene expression at the RNA level. Expression of transcripts in chloroplasts and mitochondria is heavily regulated by pentatricopeptide repeat (PPR) proteins. The diverse roles of PPR proteins and their naturally modular architecture make them ideal candidates for engineering. Synthetic PPR proteins are showing great potential to become valuable tools for controlling the expression of plastid and mitochondrial transcripts. In this review, by 'synthetic', we mean both rationally modified natural PPR proteins and completely novel proteins designed using the principles learned from their natural counterparts. We focus on the many different applications of synthetic PPR proteins, covering both their use in basic research to learn more about protein–RNA interactions and their use to achieve specific outcomes in RNA processing and the control of gene expression. We describe the challenges associated with the design, construction and deployment of synthetic PPR proteins and provide perspectives on how they might be assembled and used in future biotechnology applications. [ABSTRACT FROM AUTHOR]

Plastids (including chloroplasts) and mitochondria are remnants of endosymbiotic bacteria, yet they maintain their own genomes, which encode vital components for photosynthesis and respiration, respectively. Organellar genomes have distinctive features, such as being present as multicopies, being mostly inherited maternally, having characteristic genomic structures and undergoing frequent homologous recombination. To date, it has proven to be challenging to modify these genomes. For example, while CRISPR/Cas9 is a widely used system for editing nuclear genes, it has not yet been successfully applied to organellar genomes. Recently, however, precise gene-editing technologies have been successfully applied to organellar genomes. Protein-based enzymes, especially transcription activator–like effector nucleases (TALENs) and artificial enzymes utilizing DNA-binding domains of TALENs (TALEs), have been successfully used to modify these genomes by harnessing organellar-targeting signals. This short review introduces and discusses the use of targeted nucleases and base editors in organellar genomes, their effects and their potential applications in plant science and breeding. [ABSTRACT FROM AUTHOR]

The proton motive force (pmf) generated across the thylakoid membrane rotates the Fo-ring of ATP synthase in chloroplasts. The pmf comprises two components: membrane potential (∆Ψ) and proton concentration gradient (∆pH). Acidification of the thylakoid lumen resulting from ∆pH downregulates electron transport in the cytochrome b 6 f complex. This process, known as photosynthetic control, is crucial for protecting photosystem I (PSI) from photodamage in response to fluctuating light. To optimize the balance between efficient photosynthesis and photoprotection, it is necessary to regulate pmf. Cyclic electron transport around PSI and pseudo-cyclic electron transport involving flavodiiron proteins contribute to the modulation of pmf magnitude. By manipulating the ratio between the two components of pmf, it is possible to modify the extent of photosynthetic control without affecting the pmf size. This adjustment can be achieved by regulating the movement of ions (such as K+ and Cl−) across the thylakoid membrane. Since ATP synthase is the primary consumer of pmf in chloroplasts, its activity must be precisely regulated to accommodate other mechanisms involved in pmf optimization. Although fragments of information about each regulatory process have been accumulated, a comprehensive understanding of their interactions is lacking. Here, I summarize current knowledge of the network for pmf regulation, mainly based on genetic studies. [ABSTRACT FROM AUTHOR]

Chloroplasts/plastids are unique organelles found in plant cells and some algae and are responsible for performing essential functions such as photosynthesis. The plastid genome, consisting of circular and linear DNA molecules, is packaged and organized into specialized structures called nucleoids. The composition and dynamics of these nucleoids have been the subject of intense research, as they are critical for proper plastid functions and development. In this mini-review, recent advances in understanding the organization and regulation of plastid nucleoids are overviewed, with a focus on the various proteins and factors that regulate the shape and dynamics of nucleoids, including DNA-binding proteins and membrane anchorage proteins. The dynamic nature of nucleoid organization, which is influenced by a variety of developmental cues and the cell cycle, is also examined. [ABSTRACT FROM AUTHOR]

The formation of chloroplasts can be traced back to an ancient event in which a eukaryotic host cell containing mitochondria ingested a cyanobacterium. Since then, chloroplasts have retained many characteristics of their bacterial ancestor, including their transcription and translation machinery. In this review, recent research on the maturation of rRNA and ribosome assembly in chloroplasts is explored, along with their crucial role in plant survival and their implications for plant acclimation to changing environments. A comparison is made between the ribosome composition and auxiliary factors of ancient and modern chloroplasts, providing insights into the evolution of ribosome assembly factors. Although the chloroplast contains ancient proteins with conserved functions in ribosome assembly, newly evolved factors have also emerged to help plants acclimate to changes in their environment and internal signals. Overall, this review offers a comprehensive analysis of the molecular mechanisms underlying chloroplast ribosome assembly and highlights the importance of this process in plant survival, acclimation and adaptation. [ABSTRACT FROM AUTHOR]

Background and Aims Extrafloral nectaries are nectar-secreting structures present on vegetative parts of plants which provide indirect defences against herbivore attack. Extrafloral nectaries in Clerodendrum chinense are patelliform-shaped specialized trichomatous structures. However, a complete understanding of patelliform extrafloral nectaries in general, and of C. chinense in particular, has not yet been established to provide fundamental insight into the cellular physiological machinery involved in nectar biosynthesis and secretory processes. Methods We studied temporal changes in the morphological, anatomical and ultrastructural features in the architectures of extrafloral nectaries. We also compared metabolite profiles of extrafloral nectar, nectary tissue, non-nectary tissue and phloem sap. Further, both in situ histolocalization and normal in vitro activities of enzymes related to sugar metabolism were examined. Key Results Four distinct tissue regions in the nectar gland were revealed from histochemical characterization, among which the middle nectariferous tissue was found to be the metabolically active region, while the intermediate layer was found to be lipid-rich. Ultrastructural study showed the presence of a large number of mitochondria along with starch-bearing chloroplasts in the nectariferous region. However, starch depletion was noted with progressive maturation of nectaries. Metabolite analysis revealed compositional differences among nectar, phloem sap, nectary and non-nectary tissue. Invertase activity was higher in secretory stages and localized in nectariferous tissue and adjacent region. Conclusions Our study suggests extrafloral nectar secretion in C. chinense to be both eccrine and merocrine in nature. A distinct intermediate lipid-rich layer that separates the epidermis from nectary parenchyma was revealed, which possibly acts as a barrier to water flow in nectar. This study also revealed a distinction between nectar and phloem sap, and starch could act as a nectar precursor, as evidenced from enzymatic and ultrastructural studies. Thus, our findings on changing architecture of extrafloral nectaries with temporal secretion revealed a cell physiological process involved in nectar biosynthesis and secretion. [ABSTRACT FROM AUTHOR]

Rice leaf blades have intricate-shaped mesophyll cells (MCs) with a large volume of chloroplasts enhancing gas exchange between stroma and intercellular airspace (IAS). Since the rice MCs do not form palisade or spongy tissue cells and are considered monotypic cells, the structural analysis of MCs in the middle part of the leaf tissue has been done, neglecting the various shapes of MCs can be observed on the cross-section of rice leaves. Moreover, the middle MC layer is sandwiched between the upper and lower layers and is more restricted in its demand for light and CO2 entering from the outside. Therefore, the different layers of MCs may differ in their sensitivity to salt stress that causes structural changes in cells. This study aims to elucidate the intra- and extra-cellular structures of MC in different layers of leaf tissue and determine how salinity affects the MC structure in each layer. The mesophyll tissue was divided into adaxial, middle and abaxial layers, and eight MCs and chloroplast regions were selected from each layer and reconstructed into three-dimensional (3D) representations. The whole leaf anatomical and physiological parameters were measured to find the effects of salinity stress on the MC structures. As a result, the 3D analysis of rice leaf tissue revealed the different structures of MCs with greater diversity in the adaxial and abaxial layers than in the middle layer. Salinity stress reduced the size and height of the MCs and coverage of the chloroplast on the cytoplasm periphery of the adaxial and abaxial layers, as well as the chloroplast size of adaxial MCs. Overall, these results reveal the variation of rice MC in leaf tissue and suggest the higher sensitivity to salt stress in the adaxial mesophyll among the layers, which may partly account for the decrease in photosynthetic capacity. [ABSTRACT FROM AUTHOR]

In biomembranes, lipids form bilayer structures that serve as the fluid matrix for membrane proteins and other hydrophobic compounds. Additionally, lipid molecules associate with membrane proteins and impact their structures and functions. In both cyanobacteria and the chloroplasts of plants and algae, the lipid bilayer of the thylakoid membrane consists of four distinct glycerolipid classes: monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidylglycerol. These lipids are also integral components of photosynthetic complexes such as photosystem II and photosystem I. The lipid-binding sites within the photosystems, as well as the lipid composition in the thylakoid membrane, are highly conserved between cyanobacteria and photosynthetic eukaryotes, and each lipid class has specific roles in oxygenic photosynthesis. This review aims to shed light on the potential evolutionary implications of lipid utilization in membrane lipid bilayers and photosynthetic complexes in oxygenic photosynthetic organisms. [ABSTRACT FROM AUTHOR]

High-throughput sequencing methods have increased the accessibility of plastid genomes, which are crucial for clarifying phylogenetic relationships. Current large sequencing efforts require software tools for routine display of their distinctive quadripartite structure, which is denoted by four junction sites. By concentrating on these junctions and their close vicinity, IRscope has emerged as the standard tool for detection of this structure and creating simplified comparative graphical maps of plastid genomes. Here, we provide an augmented version (IRplus) that encompasses a novel set of functions such as integrated error detection, flexible color schemes, and an upgraded method to detect inverted repeats in genomic sequences. Spanning across the plant tree of life, IRplus allows the quick visualization of various sets of plastid genomes and features, next to smooth interoperability with other widely used annotation file formats and platforms. The IRplus can be accessed at https://irscope.shinyapps.io/IRplus/ , and source codes are freely available at https://github.com/AmiryousefiLab/IRplus. [ABSTRACT FROM AUTHOR]

Phase separation underpins many biologically important cellular events such as RNA metabolism, signaling, and CO2 fixation. However, determining the composition of a phase-separated organelle is often challenging due to its sensitivity to environmental conditions, which limits the application of traditional proteomic techniques like organellar purification or affinity purification mass spectrometry to understand their composition. In Chlamydomonas reinhardtii, Rubisco is condensed into a crucial phase-separated organelle called the pyrenoid that improves photosynthetic performance by supplying Rubisco with elevated concentrations of CO2. Here, we developed a TurboID-based proximity labeling technique in which proximal proteins in Chlamydomonas chloroplasts are labeled by biotin radicals generated from the TurboID-tagged protein. By fusing 2 core pyrenoid components with the TurboID tag, we generated a high-confidence pyrenoid proxiome that contains most known pyrenoid proteins, in addition to new pyrenoid candidates. Fluorescence protein tagging of 7 previously uncharacterized TurboID-identified proteins showed that 6 localized to a range of subpyrenoid regions. The resulting proxiome also suggests new secondary functions for the pyrenoid in RNA-associated processes and redox-sensitive iron–sulfur cluster metabolism. This developed pipeline can be used to investigate a broad range of biological processes in Chlamydomonas, especially at a temporally resolved suborganellar resolution. A high-confidence proteome of the CO2-fixing algal pyrenoid is identified by proximity labeling. [ABSTRACT FROM AUTHOR]

Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants. [ABSTRACT FROM AUTHOR]

Thiol/disulfide-based redox regulation is a ubiquitous post-translational protein modification. In plant chloroplasts, this regulatory mechanism is tightly associated with the light-dependent activation of photosynthetic enzymes (e.g. Calvin–Benson cycle enzymes). A thioredoxin (Trx)-mediated pathway was discovered to transmit light signals as a reducing power about half a century ago; since then, it has been accepted as the basic machinery of chloroplast redox regulation. However, during the past two decades, it has been increasingly apparent that plants have acquired multiple Trx isoforms and Trx-like proteins in chloroplasts. Furthermore, proteomics-based analyses have identified various chloroplast enzymes as potential targets of redox regulation. These facts highlight the necessity to revisit the molecular basis and physiological importance of the redox regulation system in chloroplasts. Recent studies have revealed novel aspects of this system, including unprecedented redox-regulated processes in chloroplasts and the functional diversity of Trx family proteins. Of particular significance is the identification of protein-oxidizing pathways that turn off photosynthetic metabolism during light-to-dark transitions. In this review, we summarize current insights into the redox regulation network in chloroplasts. [ABSTRACT FROM AUTHOR]

In the cytosol of plant cells, heat-induced protein aggregates are resolved by the CASEIN LYTIC PROTEINASE/HEAT SHOCK PROTEIN 100 (CLP/HSP100) chaperone family member HSP101, which is essential for thermotolerance. For the chloroplast family member CLPB3 this is less clear, with controversial reports on its role in conferring thermotolerance. To shed light on this issue, we have characterized two clpb3 mutants in Chlamydomonas reinhardtii. We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TRIGGER FACTOR (TIG1) and the small heat shock proteins 22E/F (HSP22E/F) in vivo , and for conferring thermotolerance under heat stress. Although CLPB3 accumulation is similar to that of stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the PLASTID RIBOSOMAL PROTEIN L1 (PRPL1) and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that a reduction in chloroplast protein synthesis capacity and an increase in proteolytic capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast, but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to HSP22E/F, which accumulated largely near the thylakoid membranes. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system. [ABSTRACT FROM AUTHOR]

Keywords: Heavy metal; metabolism; redox; signaling; sulfur; transport EN Heavy metal metabolism redox signaling sulfur transport 3271 3275 5 06/08/23 20230606 NES 230606 B Sulfur homeostasis is of vital importance for plant health and human nutrition. The redox-sensitive module of cyclophilin 20-3, 2-cysteine peroxiredoxin and cysteine synthase integrates sulfur metabolism and oxylipin signaling in the high light acclimation response. Boxes highlight responses to changes in sulfur conditions: under sulfur-replete conditions (left), plants accumulate SO SB 4 sb SP 2- sp and synthesize Cys, GSH, and sulfur-containing specialized metabolites. Cys desulfurase is the enzyme which is central to the extraction of sulfur from Cys, facilitating subsequent sulfur transfer or "sulfur trafficking" in the Fe-S cluster and Moco biogenesis pathways. [Extracted from the article]

Cysteine biosynthesis is essential for translation and represents the entry point of reduced sulfur into plant metabolism. The two consecutively acting enzymes serine acetyltransferase (SAT) and O -acetylserine-thiol-lyase catalyse cysteine production and form the cysteine synthase complex, in which SAT is activated. Here we show that tobacco (Nicotiana tabacum) expressing active SAT in plastids (referred to as PSA lines) shows substantial cysteine accumulation in plastids. Remarkably, enhanced cysteine production in plastids entirely abolished granal stack formation, impaired photosynthesis capacity, and decreased the number of chloroplasts in mesophyll cells of the PSA lines. A transgenic tobacco line expressing active SAT in the cytosol accumulated comparable amounts of thiols but displayed no phenotype. To dissect the consequences of cysteine synthase complex formation from enhanced SAT activity in tobacco plastids, we expressed an enzymatically inactive SAT that can still form the cysteine synthase complex in tobacco plastids (PSI lines). The PSI lines were indistinguishable from the PSA lines, although the PSI lines displayed no increase in plastid-localized SAT activity. Neither PSA lines nor PSI lines suffered from an oxidized redox environment in plastids that could have been causative for the disturbed photosynthesis. From these findings, we infer that the association of the plastid cysteine synthase complex itself triggers a signaling cascade controlling sulfur assimilation and photosynthetic capacity in leaves. [ABSTRACT FROM AUTHOR]

Leaves are the most important photosynthetic organs in most woody plants, but chloroplasts are also found in organs optimized for other functions. However, the actual photosynthetic efficiency of these chloroplasts is still unclear. We analyzed bark and wood chloroplasts of Fraxinus ornus L. saplings. Optical and spectroscopic methods were applied to stem samples and compared with leaves. A sharp light gradient was detected along the stem radial direction, with blue light mainly absorbed by the outer bark, and far-red-enriched light reaching the underlying xylem and pith. Chlorophylls were evident in the xylem rays and the pith and showed an increasing concentration gradient toward the bark. The stem photosynthetic apparatus showed features typical of acclimation to a low-light environment, such as larger grana stacks, lower chlorophyll a/b and photosystem I/II ratios compared with leaves. Despite likely receiving very few photons, wood chloroplasts were photosynthetically active and fully capable of generating a light-dependent electron transport. Our data provide a comprehensive scenario of the functional features of bark and wood chloroplasts in a woody species and suggest that stem photosynthesis is coherently optimized to the prevailing micro-environmental conditions at the bark and wood level. [ABSTRACT FROM AUTHOR]

The chloroplast (plastid) arose via the endosymbiosis of a photosynthetic cyanobacterium by a nonphotosynthetic eukaryotic cell ∼1.5 billion years ago. Although the plastid underwent rapid evolution by genome reduction, its rate of molecular evolution is low and its genome organization is highly conserved. Here, we investigate the factors that have constrained the rate of molecular evolution of protein-coding genes in the plastid genome. Through phylogenomic analysis of 773 angiosperm plastid genomes, we show that there is substantial variation in the rate of molecular evolution between genes. We demonstrate that the distance of a plastid gene from the likely origin of replication influences the rate at which it has evolved, consistent with time and distance-dependent nucleotide mutation gradients. In addition, we show that the amino acid composition of a gene product constraints its substitution tolerance, limiting its mutation landscape and its corresponding rate of molecular evolution. Finally, we demonstrate that the mRNA abundance of a gene is a key factor in determining its rate of molecular evolution, suggesting an interaction between transcription and DNA repair in the plastid. Collectively, we show that the location, the composition, and the expression of a plastid gene can account for >50% of the variation in its rate of molecular evolution. Thus, these three factors have exerted a substantial limitation on the capacity for adaptive evolution in plastid-encoded genes and ultimately constrained the evolvability of the chloroplast. [ABSTRACT FROM AUTHOR]

Plant organellar RNA metabolism is run by a multitude of nucleus-encoded RNA-binding proteins (RBPs) that control RNA stability, processing, and degradation. In chloroplasts and mitochondria, these post-transcriptional processes are vital for the production of a small number of essential components of the photosynthetic and respiratory machinery—and consequently for organellar biogenesis and plant survival. Many organellar RBPs have been functionally assigned to individual steps in RNA maturation, often specific to selected transcripts. While the catalog of factors identified is ever-growing, our knowledge of how they achieve their functions mechanistically is far from complete. This review summarizes the current knowledge of plant organellar RNA metabolism taking an RBP-centric approach and focusing on mechanistic aspects of RBP functions and the kinetics of the processes they are involved in. [ABSTRACT FROM AUTHOR]

Chloroplast is a crucial site for photosynthesis in plants. Determining the location and distribution of proteins in subchloroplasts is significant for studying the energy conversion of chloroplasts and regulating the utilization of light energy in crop production. However, the prediction accuracy of the currently developed protein subcellular site predictors is still limited due to the complex protein sequence features and the scarcity of labeled samples. We propose DaDL-SChlo, a multi-location protein subchloroplast localization predictor, which addresses the above problems by fusing pre-trained protein language model deep learning features with traditional handcrafted features and using generative adversarial networks for data augmentation. The experimental results of cross-validation and independent testing show that DaDL-SChlo has greatly improved the prediction performance of protein subchloroplast compared with the state-of-the-art predictors. Specifically, the overall actual accuracy outperforms the state-of-the-art predictors by 10.7% on 10-fold cross-validation and 12.6% on independent testing. DaDL-SChlo is a promising and efficient predictor for protein subchloroplast localization. The datasets and codes of DaDL-SChlo are available at https://github.com/xwanggroup/DaDL-SChlo. [ABSTRACT FROM AUTHOR]

Inter-organelle communication is an integral subcellular process in cellular homeostasis. In plants, cellular membrane lipids are synthesized in the plastids and endoplasmic reticulum (ER). However, the crosstalk between these organelles in lipid biosynthesis remains largely unknown. Here, we show that a pair of lipid phosphate phosphatases (LPPs) with differential subcellular localizations is required for ER glycerolipid metabolism in Arabidopsis (Arabidopsis thaliana). LPPα2 and LPPε1, which function as phosphatidic acid phosphatases and thus catalyze the core reaction in glycerolipid metabolism, were differentially localized at ER and chloroplast outer envelopes despite their similar tissue expression pattern. No mutant phenotype was observed in single knockout mutants; however, genetic suppression of these LPP s affected pollen growth and ER phospholipid biosynthesis in mature siliques and seeds with compromised triacylglycerol biosynthesis. Although chloroplast-localized, LPPε1 was localized close to the ER and ER-localized LPPα2. This proximal localization is functionally relevant, because overexpression of chloroplastic LPPε1 enhanced ER phospholipid and triacylglycerol biosynthesis similar to the effect of LPPα2 overexpression in mature siliques and seeds. Thus, ER glycerolipid metabolism requires a chloroplast-localized enzyme in Arabidopsis, representing the importance of inter-organelle communication in membrane lipid homeostasis. [ABSTRACT FROM AUTHOR]

Acetylation is one of the most common chemical modifications found on a variety of molecules ranging from metabolites to proteins. Although numerous chloroplast proteins have been shown to be acetylated, the role of acetylation in the regulation of chloroplast functions has remained mainly enigmatic. The chloroplast acetylation machinery in Arabidopsis thaliana consists of eight General control non-repressible 5 (GCN5)-related N -acetyltransferase (GNAT)–family enzymes that catalyze both N-terminal and lysine acetylation of proteins. Additionally, two plastid GNATs have also been reported to be involved in the biosynthesis of melatonin. Here, we have characterized six plastid GNATs (GNAT1, GNAT2, GNAT4, GNAT6, GNAT7 and GNAT10) using a reverse genetics approach with an emphasis on the metabolomes and photosynthesis of the knock-out plants. Our results reveal the impact of GNAT enzymes on the accumulation of chloroplast-related compounds, such as oxylipins and ascorbate, and the GNAT enzymes also affect the accumulation of amino acids and their derivatives. Specifically, the amount of acetylated arginine and proline was significantly decreased in the gnat2 and gnat7 mutants, respectively, as compared to the wild-type Col-0 plants. Additionally, our results show that the loss of the GNAT enzymes results in increased accumulation of Rubisco and Rubisco activase (RCA) at the thylakoids. Nevertheless, the reallocation of Rubisco and RCA did not have consequent effects on carbon assimilation under the studied conditions. Taken together, our results show that chloroplast GNATs affect diverse aspects of plant metabolism and pave way for future research into the role of protein acetylation. [ABSTRACT FROM AUTHOR]

Lysine crotonylation (Kcr) is a novel post-translational modification and its function in plant salt-stress responses remains unclear. In this study, we performed the first comprehensive chloroplast crotonylome analysis of wheat seedling leaves to examine the potential functions of Kcr proteins in salt-stress responses. In a total of 471 chloroplast proteins, 1290 Kcr sites were identified as significantly regulated by salt stress, and the Kcr proteins were mainly involved in photosynthesis, protein folding, and ATP synthesis. The identified Kcr sites that responded to salt stress were concentrated within KcrK and KcrF motifs, with the conserved KcrF motif being identified in the Kcr proteins of wheat chloroplasts for the first time. Notably, 10 Kcr sites were identified in fructose-1,6-bisphosphate aldolase (TaFBA6), a key chloroplast metabolic enzyme involved in the Calvin–Benson cycle. Site-directed mutagenesis of TaFBA6 showed that the Kcr at K367 is critical in maintaining its enzymatic activity and in conferring salt tolerance in yeast. Further molecular dynamic simulations and analyses of surface electrostatic potential indicated that the Kcr at K367 could improve the structural stability of TaFBA6 by decreasing the distribution of positive charges on the protein surface to resist alkaline environments, thereby promoting both the activity of TaFBA6 and salt tolerance. [ABSTRACT FROM AUTHOR]

Background and Aims For a comprehensive understanding of the mechanisms of changing plant photosynthetic capacity during plant evolutionary history, knowledge of leaf gas exchange and optical properties are essential, both of which relate strongly to mesophyll anatomy. Although ferns are suitable for investigating the evolutionary history of photosynthetic capacity, comprehensive research of fern species has yet to be undertaken in this regard. Methods We investigated leaf optical properties, gas exchange and mesophyll anatomy of fern species with a wide range of divergence time, using 66 ferns from natural habitats and eight glasshouse-grown ferns. We used a spectroradiometer and an integrating sphere to measure light absorptance and reflectance by the leaves. Key Results The more newly divergent fern species had a thicker mesophyll, a larger surface area of chloroplasts facing the intercellular airspaces (S c), thicker cell walls and large light absorptance. Although no trend with divergence time was obtained in leaf photosynthetic capacity on a leaf-area basis, when the traits were expressed on a mesophyll-thickness basis, trends in leaf photosynthetic capacity became apparent. On a mesophyll-thickness basis, the more newly divergent species had a low maximum photosynthesis rate, accompanied by a low S c. Conclusions We found a strong link between light capture, mesophyll anatomy and photosynthesis rate in fern species for the first time. The thick mesophyll of the more newly divergent ferns does not necessarily relate to the high photosynthetic capacity on a leaf-area basis. Rather, the thick mesophyll accompanied by thick cell walls allowed the ferns to adapt to a wider range of environments through increasing leaf toughness, which would contribute to the diversification of fern species. [ABSTRACT FROM AUTHOR]

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth. Introducing a proteobacterial Rubisco with a greater carboxylation rate and a higher content of active sites into tobacco chloroplasts supports photosynthesis and growth at high CO2 concentrations. [ABSTRACT FROM AUTHOR]

Chloroplasts produce singlet oxygen (1O2), which causes changes in nuclear gene expression through plastid-to-nucleus retrograde signaling to increase plant fitness. However, the identity of this 1O2-triggered pathway remains unclear. Here, we identify mutations in GENOMES UNCOUPLED4 (GUN4) and GUN5 as suppressors of phytochrome-interacting factor1 (pif1) pif3 in regulating the photo-oxidative response in Arabidopsis thaliana. GUN4 and GUN5 specifically interact with EXECUTER1 (EX1) and EX2 in plastids, and this interaction is alleviated by treatment with Rose Bengal (RB) or white light. Impaired expression of GUN4, GUN5, EX1, or EX2 leads to insensitivity to excess light and overexpression of EX1 triggers photo-oxidative responses. Strikingly, upon light irradiation or RB treatment, EX1 transiently accumulates in the nucleus and the nuclear fraction of EX1 shows a similar molecular weight as the plastid-located protein. Point mutagenesis analysis indicated that nuclear localization of EX1 is required for its function. EX1 acts as a transcriptional co-activator and interacts with the transcription factors WRKY18 and WRKY40 to promote the expression of 1O2-responsive genes. This study suggests that EX1 may act in plastid-to-nucleus signaling and establishes a 1O2-triggered retrograde signaling pathway that allows plants adapt to changing light environments during chloroplast development. EX1 acts as a plastid-to-nucleus mobile protein that relays 1O2 signals, thus bridging the communication gap between plastids and the nucleus. [ABSTRACT FROM AUTHOR]

Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes' complex chloroplast biogenesis requirements. New Synbio approaches, however, now allow the production of some plant Rubisco isoforms in Escherichia coli. While this enhances opportunities for catalytic improvement, there remain limitations in the utility of the expression system. Here we generate, optimize, and test a robust Golden Gate cloning E. coli expression system incorporating the protein folding machinery of tobacco chloroplasts. By comparing the expression of different plant Rubiscos in both E. coli and plastome-transformed tobacco, we show that the E. coli expression system can accurately predict high level Rubisco production in chloroplasts but poorly forecasts the biogenesis potential of isoforms with impaired production in planta. We reveal that heterologous Rubisco production in E. coli and tobacco plastids poorly correlates with Rubisco large subunit phylogeny. Our findings highlight the need to fully understand the factors governing Rubisco biogenesis if we are to deliver an efficient, low-cost screening tool that can accurately emulate chloroplast expression. [ABSTRACT FROM AUTHOR]

Nature's vital, but notoriously inefficient, CO2-fixing enzyme Rubisco often limits the growth of photosynthetic organisms including crop species. Form I Rubiscos comprise eight catalytic large subunits and eight auxiliary small subunits and can be classified into two distinct lineages—'red' and 'green'. While red-type Rubiscos (Form IC and ID) are found in rhodophytes, their secondary symbionts, and certain proteobacteria, green-type Rubiscos (Form IA and IB) exist in terrestrial plants, chlorophytes, cyanobacteria, and other proteobacteria. Eukaryotic red-type Rubiscos exhibit desirable kinetic properties, namely high specificity and high catalytic efficiency, with certain isoforms outperforming green-type Rubiscos. However, it is not yet possible to functionally express a high-performing red-type Rubisco in chloroplasts to boost photosynthetic carbon assimilation in green plants. Understanding the molecular and evolutionary basis for divergence between red- and green-type Rubiscos could help us to harness the superior CO2-fixing power of red-type Rubiscos. Here we review our current understanding about red-type Rubisco distribution, biogenesis, and sequence–structure, and present opportunities and challenges for utilizing red-type Rubisco kinetics towards crop improvements. [ABSTRACT FROM AUTHOR]

Chloroplast ATP synthases consist of a membrane-spanning coupling factor (CFO) and a soluble coupling factor (CF1). It was previously demonstrated that CONSERVED ONLY IN THE GREEN LINEAGE160 (CGL160) promotes the formation of plant CFO and performs a similar function in the assembly of its c-ring to that of the distantly related bacterial Atp1/UncI protein. Here, we show that in Arabidopsis (Arabidopsis thaliana) the N-terminal portion of CGL160 (AtCGL160N) is required for late steps in CF1-CFO assembly. In plants that lacked AtCGL160N, CF1-CFO content, photosynthesis, and chloroplast development were impaired. Loss of AtCGL160N did not perturb c-ring formation, but led to a 10-fold increase in the numbers of stromal CF1 subcomplexes relative to that in the wild type. Co-immunoprecipitation and protein crosslinking assays revealed an association of AtCGL160 with CF1 subunits. Yeast two-hybrid assays localized the interaction to a stretch of AtCGL160N that binds to the DELSEED-containing CF1-β subdomain. Since Atp1 of Synechocystis (Synechocystis sp. PCC 6803) could functionally replace the membrane domain of AtCGL160 in Arabidopsis, we propose that CGL160 evolved from a cyanobacterial ancestor and acquired an additional function in the recruitment of a soluble CF1 subcomplex, which is critical for the modulation of CF1-CFO activity and photosynthesis. The green-lineage specific N-terminal domain of CGL160 recruits coupling factor 1 and its lack affects chloroplast development, photosynthesis and thylakoid ATP synthase assembly in Arabidopsis. [ABSTRACT FROM AUTHOR]

Background and Aims The surface area of mesophyll cells (S mes) and chloroplasts (S c) facing the intercellular airspace (IAS) are important parameters for estimating photosynthetic activity from leaf anatomy. Although S mes and S c are estimated based on the shape assumption of mesophyll cells (MCs), it is questionable if the assumption is correct for rice MCs with concave–convex surfaces. Therefore, in this study, we establish a reconstruction method for the 3-D representation of the IAS in rice leaf tissue to calculate the actual S mes and S c with 3-D images and to determine the correct shape assumption for the estimation of S mes and S c based on 2-D section images. Methods We used serial section light microscopy to reconstruct 3-D representations of the IAS, MCs and chloroplasts in rice leaf tissue. Actual S mes and S c values obtained from the 3-D representation were compared with those estimated from the 2-D images to find the correct shape-specific assumption (oblate or prolate spheroid) in different orientations (longitudinal and transverse sections) using the same leaf sample. Key Results The 3-D representation method revealed that volumes of the IAS and MCs accounted for 30 and 70 % of rice leaf tissue excluding epidermis, respectively, and the volume of chloroplasts accounted for 44 % of MCs. The shape-specific assumption on the sectioning orientation affected the estimation of S mes and S c using 2-D section images with discrepancies of 10–38 %. Conclusions The 3-D representation of rice leaf tissue was successfully reconstructed using serial section light microscopy and suggested that estimation of S mes and S c of the rice leaf is more accurate using longitudinal sections with MCs assumed as oblate spheroids than using transverse sections with MCs as prolate spheroids. [ABSTRACT FROM AUTHOR]

Signals emanating from chloroplasts influence nuclear gene expression, but roles of retrograde signals during chloroplast development are unclear. To address this gap, we analyzed transcriptomes of non-photosynthetic maize mutants and compared them to transcriptomes of stages of normal leaf development. The transcriptomes of two albino mutants lacking plastid ribosomes resembled transcriptomes at very early stages of normal leaf development, whereas the transcriptomes of two chlorotic mutants with thylakoid targeting or plastid transcription defects resembled those at a slightly later stage. We identified ∼2,700 differentially expressed genes, which fall into six major categories based on the polarity and mutant-specificity of the change. Downregulated genes were generally expressed late in normal development and were enriched in photosynthesis genes, whereas upregulated genes act early and were enriched for functions in chloroplast biogenesis and cytosolic translation. We showed further that target-of-rapamycin (TOR) signaling was elevated in mutants lacking plastid ribosomes and declined in concert with plastid ribosome buildup during normal leaf development. Our results implicate three plastid signals as coordinators of photosynthetic differentiation. One signal requires plastid ribosomes and activates photosynthesis genes. A second signal reflects attainment of chloroplast maturity and represses chloroplast biogenesis genes. A third signal, the consumption of nutrients by developing chloroplasts, represses TOR, promoting termination of cell proliferation during leaf development. [ABSTRACT FROM AUTHOR]

RNA editing converts cytidines to uridines in plant organellar transcripts. Editing typically restores codons for conserved amino acids. During evolution, specific C-to-U editing sites can be lost from some plant lineages by genomic C-to-T mutations. By contrast, the emergence of novel editing sites is less well documented. Editing sites are recognized by pentatricopeptide repeat (PPR) proteins with high specificity. RNA recognition by PPR proteins is partially predictable, but prediction is often inadequate for PPRs involved in RNA editing. Here we have characterized evolution and recognition of a recently gained editing site. We demonstrate that changes in the RNA recognition motifs that are not explainable with the current PPR code allow an ancient PPR protein, QED1, to uniquely target the ndhB -291 site in Brassicaceae. When expressed in tobacco, the Arabidopsis QED1 edits 33 high-confident off-target sites in chloroplasts and mitochondria causing a spectrum of mutant phenotypes. By manipulating the relative expression levels of QED1 and ndhB -291, we show that the target specificity of the PPR protein depends on the RNA:protein ratio. Finally, our data suggest that the low expression levels of PPR proteins are necessary to ensure the specificity of editing site selection and prevent deleterious off-target editing. [ABSTRACT FROM AUTHOR]

Most retrograde signalling research in plants was performed using Arabidopsis, so an evolutionary perspective on mitochondrial retrograde regulation (MRR) is largely missing. Here, we used phylogenetics to track the evolutionary origins of factors involved in plant MRR. In all cases, the gene families can be traced to ancestral green algae or earlier. However, the specific subfamilies containing factors involved in plant MRR in many cases arose during the transition to land. NAC transcription factors with C-terminal transmembrane domains, as observed in the key regulator ANAC017, can first be observed in non-vascular mosses, and close homologs to ANAC017 can be found in seed plants. Cyclin-dependent kinases (CDKs) are common to eukaryotes, but E-type CDKs that control MRR also diverged in conjunction with plant colonization of land. AtWRKY15 can be traced to the earliest land plants, while AtWRKY40 only arose in angiosperms and AtWRKY63 even more recently in Brassicaceae. Apetala 2 (AP2) transcription factors are traceable to algae, but the ABI4 type again only appeared in seed plants. This strongly suggests that the transition to land was a major driver for developing plant MRR pathways, while additional fine-tuning events have appeared in seed plants or later. Finally, we discuss how MRR may have contributed to meeting the specific challenges that early land plants faced during terrestrialization. [ABSTRACT FROM AUTHOR]

Plant seeds do not contain differentiated chloroplasts. Upon germination, the seedlings thus need to gain photoautotrophy before storage energies are depleted. This requires the coordinated expression of photosynthesis genes encoded in nuclear and plastid genomes. Chloroplast biogenesis needs to be additionally coordinated with the light regulation network that controls seedling development. This coordination is achieved by nucleus to plastid signals called anterograde and plastid to nucleus signals termed retrograde. Retrograde signals sent from plastids during initial chloroplast biogenesis are also called biogenic signals. They have been recognized as highly important for proper chloroplast biogenesis and for seedling development. The molecular nature, transport, targets, and signalling function of biogenic signals are, however, under debate. Several studies disproved the involvement of a number of key components that were at the base of initial models of retrograde signalling. New models now propose major roles for a functional feedback between plastid and cytosolic protein homeostasis in signalling plastid dysfunction as well as the action of dually localized nucleo-plastidic proteins that coordinate chloroplast biogenesis with light-dependent control of seedling development. This review provides a survey of the developments in this research field, summarizes the unsolved questions, highlights several recent advances, and discusses potential new working modes. [ABSTRACT FROM AUTHOR]

Communication from plastids to the nucleus via retrograde signal cascades is essential to modulate nuclear gene expression, impacting plant development and environmental responses. Recently, a new class of plastid retrograde signals has emerged, consisting of acyclic and cyclic carotenoids and/or their degradation products, apocarotenoids. Although the biochemical identity of many of the apocarotenoid signals is still under current investigation, the examples described herein demonstrate the central roles that these carotenoid-derived signals play in ensuring plant development and survival. We present recent advances in the discovery of apocarotenoid signals and their role in various plant developmental transitions and environmental stress responses. Moreover, we highlight the emerging data exposing the highly complex signal transduction pathways underlying plastid to nucleus apocarotenoid retrograde signaling cascades. Altogether, this review summarizes the central role of the carotenoid pathway as a major source of retrograde signals in plants. [ABSTRACT FROM AUTHOR]

The complexes translocon at the outer envelope membrane of chloroplasts and translocon at the inner envelope membrane of chloroplasts (TIC) mediate preprotein translocation across the chloroplast outer and inner envelope membranes, respectively. Tic20, Tic56, Tic100, and Tic214 form a stable one-megadalton TIC whose function is essential for Arabidopsis thaliana and Chlamydomonas reinhardtii. Tic20 plays a central role in preprotein translocation by forming a protein-conducting channel. Tic56, Tic100, and Tic214 are also indispensable for TIC function, but whether other components are required for this process remain unclear. Here, we demonstrate that a 12-kDa protein named Tic12 is part of the TIC in A. thaliana and participates in preprotein translocation across the inner envelope membrane. Tic12 was tightly associated with the TIC but disassociated under high-salt conditions in combination with Triton X-100. Site-specific UV crosslinking experiments revealed that Tic12 and Tic20 directly interact with the transit peptide of a translocating preprotein. The tic12 null mutants are albino and seedling lethal, similar to the other tic null mutants. Tic12 and Tic20 were also involved in preprotein translocation in (Pisum sativum) pea chloroplasts. Thus, Tic12 is an essential constituent that forms the functional core together with Tic20 in the one-megadalton TIC. [ABSTRACT FROM AUTHOR]

Chloroplast movements are controlled by ultraviolet/blue light through phototropins. In Arabidopsis thaliana , chloroplast accumulation at low light intensities and chloroplast avoidance at high light intensities are observed. These responses are controlled by two homologous photoreceptors, the phototropins phot1 and phot2. Whereas chloroplast accumulation is triggered by both phototropins in a partially redundant manner, sustained chloroplast avoidance is elicited only by phot2. Phot1 is able to trigger only a small, transient chloroplast avoidance, followed by the accumulation phase. The source of this functional difference is not fully understood at either the photoreceptor or the signalling pathway levels. In this article, we review current understanding of phototropin functioning and try to dissect the differences that result in signalling to elicit two distinct chloroplast responses. First, we focus on phototropin structure and photochemical and biochemical activity. Next, we analyse phototropin expression and localization patterns. We also summarize known photoreceptor systems controlling chloroplast movements. Finally, we focus on the role of environmental stimuli in controlling phototropin activity. All these aspects impact the signalling to trigger chloroplast movements and raise outstanding questions about the mechanism involved. [ABSTRACT FROM AUTHOR]

Identification of autophagic protein cargo in plants in autophagy-related genes (ATG) mutants is complicated by changes in protein synthesis and protein degradation. To detect autophagic cargo, we measured protein degradation rate in shoots and roots of Arabidopsis (Arabidopsis thaliana) atg5 and atg11 mutants. These data show that less than a quarter of proteins changing in abundance are probable cargo and revealed roles of ATG11 and ATG5 in degradation of specific glycolytic enzymes and of other cytosol, chloroplast, and ER-resident proteins, and a specialized role for ATG11 in degradation of proteins from mitochondria and chloroplasts. Protein localization in transformed protoplasts and degradation assays in the presence of inhibitors confirm a role for autophagy in degrading glycolytic enzymes. Autophagy induction by phosphate (Pi) limitation changed metabolic profiles and the protein synthesis and degradation rates of atg5 and atg11 plants. A general decrease in the abundance of amino acids and increase in secondary metabolites in autophagy mutants was consistent with altered catabolism and changes in energy conversion caused by reduced degradation rate of specific proteins. Combining measures of changes in protein abundance and degradation rates, we also identify ATG11 and ATG5-associated protein cargo of low Pi-induced autophagy in chloroplasts and ER-resident proteins involved in secondary metabolism. [ABSTRACT FROM AUTHOR]