Photoprotective mechanisms in Elysia species hosting Acetabularia chloroplasts shed light on host-donor compatibility in photosynthetic sea slugs.

Sacoglossa sea slugs have garnered attention due to their ability to retain intracellular functional chloroplasts from algae, while degrading other algal cell components. While protective mechanisms that limit oxidative damage under excessive light are well documented in plants and algae, the photoprotective strategies employed by these photosynthetic sea slugs remain unresolved. Species within the genus Elysia are known to retain chloroplasts from various algal sources, but the extent to which the metabolic processes from the donor algae can be sustained by the sea slugs is unclear. By comparing responses to high-light conditions through kinetic analyses, molecular techniques, and biochemical assays, this study shows significant differences between two photosynthetic Elysia species with chloroplasts derived from the green alga Acetabularia acetabulum. Notably, Elysia timida displayed remarkable tolerance to high-light stress and sophisticated photoprotective mechanisms such as an active xanthophyll cycle, efficient D1 protein recycling, accumulation of heat-shock proteins and α-tocopherol. In contrast, Elysia crispata exhibited absence or limitations in these photoprotective strategies. Our findings emphasize the intricate relationship between the host animal and the stolen chloroplasts, highlighting different capacities to protect the photosynthetic organelle from oxidative damage.

Structure of the multi-subunit chloroplast RNA polymerase.

Chloroplasts contain a dedicated genome that encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase (PEP), a nearly 1 MDa complex composed of core subunits with homology to eubacterial RNA polymerases (RNAPs) and at least 12 additional chloroplast-specific PEP-associated proteins (PAPs). However, the architecture of this complex and the functions of the PAPs remain unknown. Here, we report the cryo-EM structure of a 19-subunit PEP complex from Sinapis alba (white mustard). The structure reveals that the PEP core resembles prokaryotic and nuclear RNAPs but contains chloroplast-specific features that mediate interactions with the PAPs. The PAPs are unrelated to known transcription factors and arrange around the core in a unique fashion. Their structures suggest potential functions during transcription in the chemical environment of chloroplasts. These results reveal structural insights into chloroplast transcription and provide a framework for understanding photosynthesis gene expression.

The newly assembled chloroplast genome of Aeluropus littoralis: molecular feature characterization and phylogenetic analysis with related species.

Aeluropus littoralis, a halophyte grass, is widely distributed from the Mediterranean to the Indian subcontinent through the Mongolian Gobi. This model halophyte has garnered increasing attention owing to its use as forage and its high tolerance to environmental stressors. The chloroplast genomes of many plants have been extensively examined for molecular, phylogenetic and transplastomic applications. However, no published research on the A. littoralis chloroplast (cp) genome was discovered. Here, the entire chloroplast genome of A. littoralis was assembled implementing accurate long-read sequences. The entire chloroplast genome, with an estimated length of 135,532 bp (GC content: 38.2%), has a quadripartite architecture and includes a pair of inverted repeat (IR) regions, IRa and IRb (21,012 bp each), separated by a large and a small single-copy regions (80,823 and 12,685 bp, respectively). The features of A. littoralis consist of 133 genes that synthesize 87 peptides, 38 transfer RNAs, and 8 ribosomal RNAs. Of these genes, 86 were unique, whereas 19 were duplicated in IR regions. Additionally, a total of forty-six simple sequence repeats, categorized into 32-mono, four-di, two-tri, and eight-tetranucleotides, were discovered. Furthermore, ten sets of repeats greater than 20 bp were located primarily in the LSC region. Evolutionary analysis based on chloroplast sequence data revealed that A. littoralis with A. lagopoides and A. sinensis belong to the Aeluropodinae subtribe, which is a sister to the Eleusininae in the tribe Cynodonteae and the subfamily Chloridoideae. This subfamily belongs to the PACMAD clade, which contains the majority of the C4 photosynthetic plants in the Poaceae. The newly constructed A. littoralis cp genome offers valuable knowledge for DNA barcoding, phylogenetic, transplastomic research, and other biological studies.

How Did Thylakoids Emerge in Cyanobacteria, and How Were the Primary Chloroplast and Chromatophore Acquired?

The emergence of thylakoid membranes in cyanobacteria is a key event in the evolution of all oxygenic photosynthetic cells, from prokaryotes to eukaryotes. Recent analyses show that they could originate from a unique lipid phase transition rather than from a supposed vesicular budding mechanism. Emergence of thylakoids coincided with the great oxygenation event, more than two billion years ago. The acquisition of semi-autonomous organelles, such as the mitochondrion, the chloroplast, and, more recently, the chromatophore, is a critical step in the evolution of eukaryotes. They resulted from primary endosymbiotic events that seem to share general features, i.e., an acquisition of a bacterium/cyanobacteria likely via a phagocytic membrane, a genome reduction coinciding with an escape of genes from the organelle to the nucleus, and, finally, the appearance of an active system translocating nuclear-encoded proteins back to the organelles. An intense mobilization of foreign genes of bacterial origin, via horizontal gene transfers, plays a critical role. Some third partners, like Chlamydia, might have facilitated the transition from cyanobacteria to the early chloroplast. This chapter further details our current understanding of primary endosymbiosis, focusing on primary chloroplasts, thought to have appeared over a billion years ago, and the chromatophore, which appeared around a hundred years ago.

Diversification of Plastid Structure and Function in Land Plants.

Plastids represent a largely diverse group of organelles in plant and algal cells that have several common features but also a broad spectrum of morphological, ultrastructural, biochemical, and physiological differences. Plastids and their structural and metabolic diversity significantly contribute to the functionality and developmental flexibility of the plant body throughout its lifetime. In addition to the multiple roles of given plastid types, this diversity is accomplished in some cases by interconversions between different plastids as a consequence of developmental and environmental signals that regulate plastid differentiation and specialization. In addition to basic plastid structural features, the most important plastid types, the newly characterized peculiar plastids, and future perspectives in plastid biology are also provided in this chapter.

Purification of Chloroplast Envelope, Thylakoids, and Stroma from Angiosperm Leaves.

Plant cell chloroplasts are bounded by a two-membrane envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are distinct from the chloroplast envelope, and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three compartments. A protocol is detailed for leaves of spinach, Arabidopsis or pea.

Isolation of High-Quality Plastids from the Diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum, a model pennate diatom, carries a secondary plastid surrounded by four membranes. Its biological function remains mysterious, supposed to combine features of the primary chloroplast and the endomembrane system. Isolation of high-quality plastid from the diatom enables a more conclusive understanding of the special structure and metabolic pathways in the plastid. Due to the direct continuity between the chloroplast endoplasmic reticulum membrane (cERM) and the outer nuclear envelope together with the integration of cERM into the cellular endoplasmic reticulum (ER) system, the plastid isolation is still challenging. In this study, highly purified P. tricornutum plastids with the four-layered membrane are obtained by Percoll density gradient centrifugation. The isolated plastids are unlikely to contain any residue of nuclear and coatomer compartments, and they might contain a relatively small contamination of mitochondrion and ER debris.

Isolation of the Inner and Outer Membranes of the Chloroplast Envelope from Angiosperms.

The outer and the inner membranes of the chloroplast envelope, also called OEM and IEM, have distinct lipid and protein compositions. They host molecular systems involved in the biogenesis of the organelle, its cellular function, and its communication with other compartments. Here we describe a method for the isolation of these two membranes starting from intact chloroplast preparations, with two alternative procedures based on the starting material. One was developed from spinach leaves, the other from pea leaves. The two procedures differ in the method used to isolate and rupture chloroplasts and separate each membrane.

The Use of Nanopore Sequencing to Analyze the Chloroplast Transcriptome Part II: Bioinformatic Analyzes and Virtual RNA Blots.

Nanopore sequencing of full-length cDNAs offers unprecedented details of the plastid RNA metabolism. After the generation of the nanopore reads, several bioinformatic steps are required to analyze the data. In this chapter, we describe in a few simple command lines the processing and mapping of the reads as well as the generation of virtual Northern blots as a simple and familiar way to visualize Nanopore sequencing data.

Isolation of Cytosolic Ribosomes Associated with Plant Mitochondria and Chloroplasts.

Excluding the few dozen proteins encoded by the chloroplast and mitochondrial genomes, the majority of plant cell proteins are synthesized by cytosolic ribosomes. Most of these nuclear-encoded proteins are then targeted to specific cell compartments thanks to localization signals present in their amino acid sequence. These signals can be specific amino acid sequences known as transit peptides, or post-translational modifications, ability to interact with specific proteins or other more complex regulatory processes. Furthermore, in eukaryotic cells, protein synthesis can be regulated so that certain proteins are synthesized close to their destination site, thus enabling local protein synthesis in specific compartments of the cell. Previous studies have revealed that such locally translating cytosolic ribosomes are present in the vicinity of mitochondria and emerging views suggest that localized translation near chloroplasts could also occur. However, in higher plants, very little information is available on molecular mechanisms controlling these processes and there is a need to characterize cytosolic ribosomes associated with organelles membranes. To this goal, this protocol describes the purification of higher plant chloroplast and mitochondria and the organelle-associated cytosolic ribosomes.

Quantitative Assessment of the Chloroplast Lipidome.

In plants and algae, photosynthetic membranes have a unique lipid composition. They differ from all other cellular membranes by their very low amount of phospholipids, besides some phosphatidylglycerol (PG), and high proportion of glycolipids. These glycolipids are the uncharged galactolipids, that is, mono- and digalactosyldiacylglycerol (MGDG and DGDG), and an anionic sulfolipid, that is, sulfoquinovosyldiacylglycerol (SQDG). In all photosynthetic membranes analyzed to date, from cyanobacteria to algae, protists, and plants, the lipid quartet constituted by MGDG, DGDG, SQDG, and PG has been highly conserved, but the composition in fatty acids of these lipids can vary a lot from an organism to another. To better understand the chloroplast biogenesis, it is therefore essential to know their lipid content. Establishing chloroplast lipidome requires first to purify chloroplast from plant or algae tissue. Here we describe the methods to extract the lipid, quantify the lipid amount of the chloroplast, and qualify and quantify the different lipid classes that might be present in these fractions.

The Use of Nanopore Sequencing to Analyze the Chloroplast Transcriptome Part I: Library Preparation.

Global understanding of plastid gene expression has always been impaired by its complexity. RNA splicing, editing, and intercistronic processing create multiple transcripts isoforms that can hardly be resolved using traditional molecular biology techniques. During the last decade, the wide adoption of RNA-seq-based techniques has, however, allowed an unprecedented understanding of all the different steps of chloroplast gene expression, from transcription to translation. Current strategies are nonetheless unable to identify and quantify full length transcripts isoforms, a limitation that can now be overcome using Nanopore Sequencing. We here provide a complete protocol to produce, from total leaf RNA, cDNA libraries ready for Nanopore sequencing. While most Nanopore protocols take advantage of the mRNA polyA tail we here first ligate an RNA adapter to the 3' ends of the RNAs and use it to initiate the template switching reverse transcription. The cDNA is then prepared and indexed for use with the regular Oxford Nanopore v14 chemistry. This protocol is of particular interest to researchers willing to simultaneously study the multiple post-transcriptional processes prevalent in the chloroplast.

Thylakoid ultrastructural variations in chlorophyll-deficient wheat: aberrations or structural acclimation?

MAIN CONCLUSION: A structural re-modeling of the thylakoid system, including granum size and regularity, occurs in chlorophyll-deficient wheat mutants affected by photosynthetic membrane over-reduction. In the chloroplast of land plants, the thylakoid system is defined by appressed grana stacks and unstacked stroma lamellae. This study focuses on the variations of the grana organization occurring in outdoor-grown wheat mutants characterized by low chlorophyll content and a tendency for photosynthetic membrane over-reduction. Triticum aestivum ANK-32A and Triticum durum ANDW-7B were compared to their corresponding WT lines, NS67 and LD222, respectively. Electron micrographs of chloroplasts were used to calculate grana ultrastructural parameters. Photosynthetic parameters were obtained by modulated chlorophyll fluorescence and applying Light Curves (LC) and Rapid Light Curves (RLC) protocols. For each photosynthetic parameter, the difference Δ(RLC-LC) was calculated to evaluate the flexible response to light in the examined lines. In the mutants, fewer and smaller disks formed grana stacks characterized by a marked increase in lateral and cross-sectional irregularity, both negatively correlated with the number of layers per granum. A relationship was found between membrane over-reduction and granum structural irregularity. The possible acclimative significance of a greater proportion of stroma-exposed grana domains in relieving the excess electron pressure on PSI is discussed.

AmCBF1 activates the expression of GhClpR1 to mediate dark-green leaves in cotton (Gossypium hirsutum).

KEY MESSAGE: The transcription factor AmCBF1 deepens the leaf colour of transgenic cotton by binding to the promoter of the chloroplast development-related protein GhClpR1 to promote photosynthesis. The ATP-dependent caseinolytic protease (Clp protease) family plays a crucial role within chloroplasts, comprising several Clp proteins to maintain chloroplast homeostasis. At present, research on Clp proteins mainly focuses on Arabidopsis, leaving its function in other plants, particularly in crops, less explored. In this study, we overexpressed AmCBF1 from Ammopiptanthus mongolicus (A. mongolicus) in wild type (R15), and found a significant darkening of leaf colour in transgenic plants (L28 and L30). RNA-seq analysis showed an enrichment of pathways associated with photosynthesis. Subsequent screening of differentially expressed genes revealed a significant up-regulation of GhClpR1, a gene linked to chloroplast development, in the transgenic strain. In addition, GhClpR1 was consistently expressed in upland cotton, with the highest expression observed in leaves. Subcellular localization analysis revealed that the protein encoded by GhClpR1 was located in chloroplasts. Yeast one hybrid and dual luciferase experiments showed that the AmCBF1 transcription factor positively regulates the expression of GhClpR1. VIGs-mediated silencing of GhClpR1 led to a significant yellowing phenotype in the leaves. This was accompanied by a reduction in chlorophyll content, and microscopic examination of chloroplast ultrastructure revealed severe developmental impairment. Finally, yeast two-hybrid assays showed that GhClpR1 interacts with the Clp protease complex accessory protein GhClpT2. Our study provides a foundation for studying the function of the Clp protease complex and a new strategy for cultivating high-light-efficiency cotton resources.

Cryo-EM structures of the plant plastid-encoded RNA polymerase.

Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions. Our data show the PEP core adopts a typical fold as bacterial RNAP. Fifteen PAPs bind at the periphery of the PEP core, facilitate assembling the PEP-PAP supercomplex, protect the complex from oxidation damage, and likely couple gene transcription with RNA processing. Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts.

Structure of the plant plastid-encoded RNA polymerase.

Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined. Here, we present cryoelectron microscopy (cryo-EM) structures of native 21-subunit PEP and a PEP transcription elongation complex from white mustard (Sinapis alba). We identify that PAPs encase the core polymerase, forming extensive interactions that likely promote complex assembly and stability. During elongation, PAPs interact with DNA downstream of the transcription bubble and with the nascent mRNA. The models reveal details of the superoxide dismutase, lysine methyltransferase, thioredoxin, and amino acid ligase enzymes that are subunits of PEP. Collectively, these data provide a foundation for the mechanistic understanding of chloroplast transcription and its role in plant growth and adaptation.

The chloroplast and mitochondrial genomes of two Gomphonema parvulum (Bacillariophyta) environmental isolates from South Carolina (United States) and Virginia (United States).

Gomphonema parvulum is a cosmopolitan freshwater diatom that is used as an indicator in water quality biomonitoring. In this study, we report the culturing of two geographically separated isolates from southeastern North America, their morphology, and the sequencing and assembly of their mitochondrial and chloroplast genomes. Morphologically, both strains fit G. parvulum sensu lato, but the frustules from a protected habitat in South Carolina were smaller than those cited in the historic data of this species from the same location as well as a second culture from Virginia. Phylogenetic analyses using the rbcL gene placed both within a clade with G. parvulum. Genetic markers, including full chloroplast and mitochondrial genomes and the nuclear small subunit rRNA gene region were assembled from each isolate. The organellar genomes of the two strains varied slightly in size due to small differences in intergenic regions with chloroplast genomes of 121,035 bp and 121,482 bp and mitochondrial genomes of 34,639 bp and 34,654 bp. The intraspecific pairwise identities of the chloroplast and mitochondrial genomes of these two isolates were 97.9% and 95.4%, respectively. Multigene phylogenetic analysis demonstrated a close relationship between G. parvulum, Gomphoneis minuta, and Didymosphenia geminata.

The dicot homolog of maize PPR103 carries a C-terminal DYW domain and may have a role in C-to-U editing of some chloroplast RNA transcripts.

In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain. IPI1/emb175/PPR103 is a nuclear gene encoding a PLS-type PPR protein essential for survival in Arabidopsis thaliana and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and Nicotiana IPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing in N. benthamiana to gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in the Nicotiana and Arabidopsis IPI1 orthologs. Virus-induced gene silencing of NbIPI1 led to defects in chloroplast ribosomal RNA processing and changes to stability of rpl16 transcripts, revealing conserved function with its maize ortholog. NbIPI1-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing in N. benthamiana chloroplasts.

Adaptive evolution of chloroplast division mechanisms during plant terrestrialization.

Despite extensive research, the origin and evolution of the chloroplast division machinery remain unclear. Here, we employ recently sequenced genomes and transcriptomes of Archaeplastida clades to identify the core components of chloroplast division and reconstruct their evolutionary histories, respectively. Our findings show that complete division ring structures emerged in Charophytes. We find that Glaucophytes experienced strong selection pressure, generating diverse variants adapted to the changing terrestrial environments. By integrating the functions of chloroplast division genes (CDGs) annotated in a workflow developed using large-scale multi-omics data, we further show that dispersed duplications acquire more species-specific functions under stronger selection pressures. Notably, PARC6, a dispersed duplicate CDG, regulates leaf color and plant growth in Solanum lycopersicum, demonstrating neofunctionalization. Our findings provide an integrated perspective on the functional evolution of chloroplast division machinery and highlight the potential of dispersed duplicate genes as the primary source of adaptive evolution of chloroplast division.