Research progress on differentiation and regulation of plant chromoplasts.

Carotenoids, natural tetraterpenoids found abundantly in plants, contribute to the diverse colors of plant non-photosynthetic tissues and provide fragrance through their cleavage products, which also play crucial roles in plant growth and development. Understanding the synthesis, degradation, and storage pathways of carotenoids and identifying regulatory factors represents a significant strategy for enhancing plant quality. Chromoplasts serve as the primary plastids responsible for carotenoid accumulation, and their differentiation is linked to the levels of carotenoids, rendering them a subject of substantial research interest. The differentiation of chromoplasts involves alterations in plastid structure and protein import machinery. Additionally, this process is influenced by factors such as the ORANGE (OR) gene, Clp proteases, xanthophyll esterification, and environmental factors. This review shows the relationship between chromoplast and carotenoid accumulation by presenting recent advances in chromoplast structure, the differentiation process, and key regulatory factors, which can also provide a reference for rational exploitation of chromoplasts to enhance plant quality.

Plastid dynamism integrates development and environment.

In land plants plastid type differentiation occurs concomitantly with cellular differentiation and the transition from one type to another is under developmental and environmental control. Plastid dynamism is based on a bilateral communication between plastids and nucleus through anterograde and retrograde signaling. Signaling occurs through the interaction with specific phytohormones (abscisic acid, strigolactones, jasmonates, gibberellins, brassinosteroids, ethylene, salicylic acid, cytokinin and auxin). The review is focused on the modulation of plastid capabilities at both transcriptional and post-translational levels at the crossroad between development and stress, with a particular attention to the chloroplast, because the most studied plastid type. The role of plastid-encoded and nuclear-encoded proteins for plastid development and stress responses, and the changes of plastid fate through the activity of stromules and plastoglobules, are discussed. Examples of plastid dynamism in response to soil stress agents (salinity, lead, cadmium, arsenic, and chromium) are described. Albinism and root greening are described based on the modulation activities of auxin and cytokinin. The physiological and functional responses of the sensory epidermal and vascular plastids to abiotic and biotic stresses along with their specific roles in stress sensing are described together with their potential modulation of retrograde signaling pathways. Future research perspectives include an in-depth study of sensory plastids to explore their potential for establishing a transgenerational memory to stress. Suggestions about anterograde and retrograde pathways acting at interspecific level and on the lipids of plastoglobules as a novel class of plastid morphogenic agents are provided.

Implicating the red body of Nannochloropsis in forming the recalcitrant cell wall polymer algaenan.

Stramenopile algae contribute significantly to global primary productivity, and one class, Eustigmatophyceae, is increasingly studied for applications in high-value lipid production. Yet much about their basic biology remains unknown, including the nature of an enigmatic, pigmented globule found in vegetative cells. Here, we present an in-depth examination of this "red body," focusing on Nannochloropsis oceanica. During the cell cycle, the red body forms adjacent to the plastid, but unexpectedly it is secreted and released with the autosporangial wall following cell division. Shed red bodies contain antioxidant ketocarotenoids, and overexpression of a beta-carotene ketolase results in enlarged red bodies. Infrared spectroscopy indicates long-chain, aliphatic lipids in shed red bodies and cell walls, and UHPLC-HRMS detects a C32 alkyl diol, a potential precursor of algaenan, a recalcitrant cell wall polymer. We propose that the red body transports algaenan precursors from plastid to apoplast to be incorporated into daughter cell walls.

A symbiont fungal effector relocalizes a plastidic oxidoreductase to nuclei to induce resistance to pathogens and salt stress.

The root endophytic fungus Serendipita indica establishes beneficial symbioses with a broad spectrum of plants and enhances host resilience against biotic and abiotic stresses. However, little is known about the mechanisms underlying S. indica-mediated plant protection. Here, we report S. indica effector (SIE) 141 and its host target CDSP32, a conserved thioredoxin-like protein, and underlying mechanisms for enhancing pathogen resistance and abiotic salt tolerance in Arabidopsis thaliana. SIE141 binding interfered with canonical targeting of CDSP32 to chloroplasts, leading to its re-location into the plant nucleus. This nuclear translocation is essential for both their interaction and resistance function. Furthermore, SIE141 enhanced oxidoreductase activity of CDSP32, leading to CDSP32-mediated monomerization and activation of NON-EXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1), a key regulator of systemic resistance. Our findings provide functional insights on how S. indica transfers well-known beneficial effects to host plants and indicate CDSP32 as a genetic resource to improve plant resilience to abiotic and biotic stresses.

Heteroplasmy Is Rare in Plant Mitochondria Compared with Plastids despite Similar Mutation Rates.

Plant cells harbor two membrane-bound organelles containing their own genetic material-plastids and mitochondria. Although the two organelles coexist and coevolve within the same plant cells, they differ in genome copy number, intracellular organization, and mode of segregation. How these attributes affect the time to fixation or, conversely, loss of neutral alleles is currently unresolved. Here, we show that mitochondria and plastids share the same mutation rate, yet plastid alleles remain in a heteroplasmic state significantly longer compared with mitochondrial alleles. By analyzing genetic variants across populations of the marine flowering plant Zostera marina and simulating organelle allele dynamics, we examine the determinants of allele segregation and allele fixation. Our results suggest that the bottlenecks on the cell population, e.g. during branching or seeding, and stratification of the meristematic tissue are important determinants of mitochondrial allele dynamics. Furthermore, we suggest that the prolonged plastid allele dynamics are due to a yet unknown active plastid partition mechanism. The dissimilarity between plastid and mitochondrial novel allele fixation at different levels of organization may manifest in differences in adaptation processes. Our study uncovers fundamental principles of organelle population genetics that are essential for further investigations of long-term evolution and molecular dating of divergence events.

Properties and Functional Analysis of Two Chorismate Mutases from Maritime Pine.

Through the shikimate pathway, a massive metabolic flux connects the central carbon metabolism with the synthesis of chorismate, the common precursor of the aromatic amino acids phenylalanine, tyrosine, and tryptophan, as well as other compounds, including salicylate or folate. The alternative metabolic channeling of chorismate involves a key branch-point, finely regulated by aromatic amino acid levels. Chorismate mutase catalyzes the conversion of chorismate to prephenate, a precursor of phenylalanine and tyrosine and thus a vast repertoire of fundamental derived compounds, such as flavonoids or lignin. The regulation of this enzyme has been addressed in several plant species, but no study has included conifers or other gymnosperms, despite the importance of the phenolic metabolism for these plants in processes such as lignification and wood formation. Here, we show that maritime pine (Pinus pinaster Aiton) has two genes that encode for chorismate mutase, PpCM1 and PpCM2. Our investigations reveal that these genes encode plastidial isoenzymes displaying activities enhanced by tryptophan and repressed by phenylalanine and tyrosine. Using phylogenetic studies, we have provided new insights into the possible evolutionary origin of the cytosolic chorismate mutases in angiosperms involved in the synthesis of phenylalanine outside the plastid. Studies based on different platforms of gene expression and co-expression analysis have allowed us to propose that PpCM2 plays a central role in the phenylalanine synthesis pathway associated with lignification.

Plastid-expressed AdDjSKI enhances photosystem II stability, delays leaf senescence, and increases fruit yield in tomato plants under heat stress.

Heat stress substantially reduces tomato (Solanum lycopersicum) growth and yield globally, thereby jeopardizing food security. DnaJ proteins, constituents of the heat shock protein system, protect cells from diverse environmental stresses as HSP-70 molecular co-chaperones. In this study, we demonstrated that AdDjSKI, a serine-rich DnaJ III protein induced by pathogens, plays an important role in stabilizing photosystem II (PSII) in response to heat stress. Our results revealed that transplastomic tomato plants expressing the AdDjSKI gene exhibited increased levels of total soluble proteins, improved growth and chlorophyll content, reduced malondialdehyde (MDA) accumulation, and diminished PSII photoinhibition under elevated temperatures when compared with wild-type (WT) plants. Intriguingly, these transplastomic plants maintained higher levels of D1 protein under elevated temperatures compared with the WT plants, suggesting that overexpression of AdDjSKI in plastids is crucial for PSII protection, likely due to its chaperone activity. Furthermore, the transplastomic plants displayed lower accumulation of superoxide radical (O2 •─) and H2O2, in comparison with the WT plants, plausibly attributed to higher superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities. This also coincides with an enhanced expression of corresponding genes, including SlCuZnSOD, SlFeSOD, SlAPX2, and SltAPX, under heat stress. Taken together, our findings reveal that chloroplastic expression of AdDjSKI in tomatoes plays a critical role in fruit yield, primarily through a combination of delayed senescence and stabilizing PSII under heat stress.

Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number.

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.

Connecting Species-Specific Extents of Genome Reduction in Mitochondria and Plastids.

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.

Plastome structure, phylogeny and evolution of plastid genes in Reevesia (Helicteroideae, Malvaceae).

Reevesia is an eastern Asian-eastern North American disjunction genus in the family Malvaceae s.l. and comprises approximately 25 species. The relationships within the genus are not well understood. Here, 15 plastomes representing 12 Reevesia species were compared, with the aim of better understanding the species circumscription and phylogenetic relationships within the genus and among genera in the family Malvaceae s.l. The 11 newly sequenced plastomes range between 161,532 and 161, 945 bp in length. The genomes contain 114 unique genes, 18 of which are duplicated in the inverted repeats (IRs). Gene content of these plastomes is nearly identical. All the protein-coding genes are under purifying selection in the Reevesia plastomes compared. The top ten hypervariable regions, SSRs, and the long repeats identified are potential molecular markers for future population genetic and phylogenetic studies. Phylogenetic analysis based on the whole plastomes confirmed the monophyly of Reevesia and a close relationship with Durio (traditional Bombacaceae) in subfamily Helicteroideae, but not with the morphologically similar genera Pterospermum and Sterculia (both of traditional Sterculiaceae). Phylogenetic relationships within Reevesia suggested that two species, R. pubescens and R. thyrsoidea, as newly defined, are not monophyletic. Six taxa, R. membranacea, R. xuefengensis, R. botingensis, R. lofouensis, R. longipetiolata and R. pycnantha, are suggested to be recognized.

Cysteine oxidation as a regulatory mechanism of Arabidopsis plastidial NAD-dependent malate dehydrogenase.

Malate dehydrogenases (MDHs) catalyze a reversible NAD(P)-dependent-oxidoreductase reaction that plays an important role in central metabolism and redox homeostasis of plant cells. Recent studies suggest a moonlighting function of plastidial NAD-dependent MDH (plNAD-MDH; EC 1.1.1.37) in plastid biogenesis, independent of its enzyme activity. In this study, redox effects on activity and conformation of recombinant plNAD-MDH from Arabidopsis thaliana were investigated. We show that reduced plNAD-MDH is active while it is inhibited upon oxidation. Interestingly, the presence of its cofactors NAD+ and NADH could prevent oxidative inhibition of plNAD-MDH. In addition, a conformational change upon oxidation could be observed via non-reducing SDS-PAGE. Both effects, its inhibition and conformational change, were reversible by re-reduction. Further investigation of single cysteine substitutions and mass spectrometry revealed that oxidation of plNAD-MDH leads to oxidation of all four cysteine residues. However, cysteine oxidation of C129 leads to inhibition of plNAD-MDH activity and oxidation of C147 induces its conformational change. In contrast, oxidation of C190 and C333 does not affect plNAD-MDH activity or structure. Our results demonstrate that plNAD-MDH activity can be reversibly inhibited, but not inactivated, by cysteine oxidation and might be co-regulated by the availability of its cofactors in vivo.

Cutinsomes of Malus Mill. (Rosaceae) leaf and pericarp: genesis, localization, and transport.

New data were obtained on specific bionanostructures, cutinsomes, which are involved in the formation of cuticles on the surface of leaf blades and pericarp of Malus domestica Borkh (Malus Mill., Rosaceae)introduced to the mountains at the altitudes of 1200 and 1700 m above sea level. Cutinsomes, which are electron-dense structures of spherical shape, have been identified by transmission electron microscopy. It was demonstrated that plastids can be involved in the synthesis of their constituent nanocomponents. The greatest number of nanoparticles was observed in the granal thylakoid lumen of the chloroplasts in palisade mesophyll cells and pericarp hypodermal cells. The transmembrane transport of cutinsomes into the cell wall cuticle proper by exocytosis has been visualized for the first time. The plasma membrane is directly involved in the excretion of nanostructures from the cell. Nanoparticles of cutinsomes in the form of necklace-like formations line up in a chain near cell walls, merge into larger conglomerates and are loaded into plasmalemma invaginations, and then, in membrane packing, they move into the cuticle, which covers both outer and inner cell walls of external tissues. The original materials obtained by us supplement the ideas about the non-enzymatic synthesis of cuticle components available in the literature and expand the cell compartment geography involved in this process.

Transcriptomic study of the role of MeFtsZ2-1 in pigment accumulation in cassava leaves.

MeFtsZ2-1 is a key gene for plant plastid division, but the mechanism by which MeFtsZ2-1 affects pigment accumulation in cassava (Manihot esculenta Crantz) through plastids remains unclear. We found that MeFtsZ2-1 overexpression in cassava (OE) exhibited darker colors of leaves, with increased levels of anthocyanins and carotenoids. Further observation via Transmission Electron Microscopy (TEM) revealed no apparent defects in chloroplast structure but an increase in the number of plastoglobule in OE leaves. RNA-seq results showed 1582 differentially expressed genes (DEGs) in leaves of OE. KEGG pathway analysis indicated that these DEGs were enriched in pathways related to flavonoid, anthocyanin, and carotenoid biosynthesis. This study reveals the role of MeFtsZ2-1 in cassava pigment accumulation from a physiological and transcriptomic perspective, providing a theoretical basis for improving cassava quality.

Towards a species-level phylogeny for Neotropical Myrtaceae: Notes on topology and resources for future studies.

PREMISE: Increasingly complete phylogenies underpin studies in systematics, ecology, and evolution. Myrteae (Myrtaceae), with ~2700 species, is a key component of the exceptionally diverse Neotropical flora, but given its complicated taxonomy, automated assembling of molecular supermatrices from public databases often lead to unreliable topologies due to poor species identification. METHODS: Here, we build a taxonomically verified molecular supermatrix of Neotropical Myrteae by assembling 3909 published and 1004 unpublished sequences from two nuclear and seven plastid molecular markers. We infer a time-calibrated phylogenetic tree that covers 712 species of Myrteae (~28% of the total diversity in the clade) and evaluate geographic and taxonomic gaps in sampling. RESULTS: The tree inferred from the fully concatenated matrix mostly reflects the topology of the plastid data set and there is a moderate to strong incongruence between trees inferred from nuclear and plastid partitions. Large, species-rich genera are still the poorest sampled within the group. Eastern South America is the best-represented area in proportion to its species diversity, while Western Amazon, Mesoamerica, and the Caribbean are the least represented. CONCLUSIONS: We provide a time-calibrated tree that can be more reliably used to address finer-scale eco-evolutionary questions that involve this group in the Neotropics. Gaps to be filled by future studies include improving representation of taxa and areas that remain poorly sampled, investigating causes of conflict between nuclear and plastid partitions, and the role of hybridization and incomplete lineage sorting in relationships that are poorly supported.

Plastid phylogenomics and cytonuclear discordance in Rubioideae, Rubiaceae.

In this study of evolutionary relationships in the subfamily Rubioideae (Rubiaceae), we take advantage of the off-target proportion of reads generated via previous target capture sequencing projects based on nuclear genomic data to build a plastome phylogeny and investigate cytonuclear discordance. The assembly of off-target reads resulted in a comprehensive plastome dataset and robust inference of phylogenetic relationships, where most intratribal and intertribal relationships are resolved with strong support. While the phylogenetic results were mostly in agreement with previous studies based on plastome data, novel relationships in the plastid perspective were also detected. For example, our analyses of plastome data provide strong support for the SCOUT clade and its sister relationship to the remaining members of the subfamily, which differs from previous results based on plastid data but agrees with recent results based on nuclear genomic data. However, several instances of highly supported cytonuclear discordance were identified across the Rubioideae phylogeny. Coalescent simulation analysis indicates that while ILS could, by itself, explain the majority of the discordant relationships, plastome introgression may be the better explanation in some cases. Our study further indicates that plastomes across the Rubioideae are, with few exceptions, highly conserved and mainly conform to the structure, gene content, and gene order present in the majority of the flowering plants.

Plastid DNA is a major source of nuclear genome complexity and of RNA genes in the orphan crop moringa.

BACKGROUND: Unlike Transposable Elements (TEs) and gene/genome duplication, the role of the so-called nuclear plastid DNA sequences (NUPTs) in shaping the evolution of genome architecture and function remains poorly studied. We investigate here the functional and evolutionary fate of NUPTs in the orphan crop Moringa oleifera (moringa), featured by the highest fraction of plastid DNA found so far in any plant genome, focusing on (i) any potential biases in their distribution in relation to specific nuclear genomic features, (ii) their contribution to the emergence of new genes and gene regions, and (iii) their impact on the expression of target nuclear genes. RESULTS: In agreement with their potential mutagenic effect, NUPTs are underrepresented among structural genes, although their overall transcription levels and broadness were only lower when involved exonic regions; the occurrence of plastid DNA generally did not result in a broader expression, except among those affected in introns by older NUPTs. In contrast, we found a strong enrichment of NUPTs among specific superfamilies of retrotransposons and several classes of RNA genes, including those participating in the protein biosynthetic machinery (i.e., rRNA and tRNA genes) and a specific class of regulatory RNAs. A significant fraction of NUPT RNA genes was found to be functionally expressed, thus potentially contributing to the nuclear pool. CONCLUSIONS: Our results complete our view of the molecular factors driving the evolution of nuclear genome architecture and function, and support plastid DNA in moringa as a major source of (i) genome complexity and (ii) the nuclear pool of RNA genes.

Testing plastomes and nuclear ribosomal DNA sequences as the next-generation DNA barcodes for species identification and phylogenetic analysis in Acer.

BACKGROUND: Acer is a taxonomically intractable and speciose genus that contains over 150 species. It is challenging to distinguish Acer species only by morphological method due to their abundant variations. Plastome and nuclear ribosomal DNA (nrDNA) sequences are recommended as powerful next-generation DNA barcodes for species discrimination. However, their efficacies were still poorly studied. The current study will evaluate the application of plastome and nrDNA in species identification and perform phylogenetic analyses for Acer. RESULT: Based on a collection of 83 individuals representing 55 species (c. 55% of Chinese species) from 13 sections, our barcoding analyses demonstrated that plastomes exhibited the highest (90.47%) species discriminatory power among all plastid DNA markers, such as the standard plastid barcodes matK + rbcL + trnH-psbA (61.90%) and ycf1 (76.19%). And the nrDNA (80.95%) revealed higher species resolution than ITS (71.43%). Acer plastomes show abundant interspecific variations, however, species identification failure may be due to the incomplete lineage sorting (ILS) and chloroplast capture resulting from hybridization. We found that the usage of nrDNA contributed to identifying those species that were unidentified by plastomes, implying its capability to some extent to mitigate the impact of hybridization and ILS on species discrimination. However, combining plastome and nrDNA is not recommended given the cytonuclear conflict caused by potential hybridization. Our phylogenetic analysis covering 19 sections (95% sections of Acer) and 128 species (over 80% species of this genus) revealed pervasive inter- and intra-section cytonuclear discordances, hinting that hybridization has played an important role in the evolution of Acer. CONCLUSION: Plastomes and nrDNA can significantly improve the species resolution in Acer. Our phylogenetic analysis uncovered the scope and depth of cytonuclear conflict in Acer, providing important insights into its evolution.

A molecular atlas of plastid and mitochondrial proteins reveals organellar remodeling during plant evolutionary transitions from algae to angiosperms.

Algae and plants carry 2 organelles of endosymbiotic origin that have been co-evolving in their host cells for more than a billion years. The biology of plastids and mitochondria can differ significantly across major lineages and organelle changes likely accompanied the adaptation to new ecological niches such as the terrestrial habitat. Based on organelle proteome data and the genomes of 168 phototrophic (Archaeplastida) versus a broad range of 518 non-phototrophic eukaryotes, we screened for changes in plastid and mitochondrial biology across 1 billion years of evolution. Taking into account 331,571 protein families (or orthogroups), we identify 31,625 protein families that are unique to primary plastid-bearing eukaryotes. The 1,906 and 825 protein families are predicted to operate in plastids and mitochondria, respectively. Tracing the evolutionary history of these protein families through evolutionary time uncovers the significant remodeling the organelles experienced from algae to land plants. The analyses of gained orthogroups identifies molecular changes of organelle biology that connect to the diversification of major lineages and facilitated major transitions from chlorophytes en route to the global greening and origin of angiosperms.