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.

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.

Overexpression of the plastidial pseudo-protease AtFtsHi3 enhances drought tolerance while sustaining plant growth.

With climate change, droughts are expected to be more frequent and severe, severely impacting plant biomass and quality. Here, we show that overexpressing the Arabidopsis gene AtFtsHi3 (FtsHi3OE) enhances drought-tolerant phenotypes without compromising plant growth. AtFtsHi3 encodes a chloroplast envelope pseudo-protease; knock-down mutants (ftshi3-1) are found to be drought tolerant but exhibit stunted growth. Altered AtFtsHi3 expression therefore leads to drought tolerance, while only diminished expression of this gene leads to growth retardation. To understand the underlying mechanisms of the enhanced drought tolerance, we compared the proteomes of ftshi3-1 and pFtsHi3-FtsHi3OE (pFtsHi3-OE) to wild-type plants under well-watered and drought conditions. Drought-related processes like osmotic stress, water transport, and abscisic acid response were enriched in pFtsHi3-OE and ftshi3-1 mutants following their enhanced drought response compared to wild-type. The knock-down mutant ftshi3-1 showed an increased abundance of HSP90, HSP93, and TIC110 proteins, hinting at a potential downstream role of AtFtsHi3 in chloroplast pre-protein import. Mathematical modeling was performed to understand how variation in the transcript abundance of AtFtsHi3 can, on the one hand, lead to drought tolerance in both overexpression and knock-down lines, yet, on the other hand, affect plant growth so differently. The results led us to hypothesize that AtFtsHi3 may form complexes with at least two other protease subunits, either as homo- or heteromeric structures. Enriched amounts of AtFtsH7/9, AtFtsH11, AtFtsH12, and AtFtsHi4 in ftshi3-1 suggest a possible compensation mechanism for these proteases in the hexamer.

Superoxide Dismutase Premodulates Oxidative Stress in Plastids for Protection of Tobacco Plants from Cold Damage Ultrastructure Damage.

ROS-dependent induction of oxidative damage can be used as a trigger initiating genetically determined non-specific protection in plant cells and tissues. Plants are potentially able to withstand various specific (toxic, osmotic) factors of abiotic effects, but do not have sufficient or specific sensitivity to form an adequate effective response. In this work, we demonstrate one of the possible approaches for successful cold acclimation through the formation of effective protection of photosynthetic structures due to the insertion of the heterologous FeSOD gene into the tobacco genome under the control of the constitutive promoter and equipped with a signal sequence targeting the protein to plastid. The increased enzymatic activity of superoxide dismutase in the plastid compartment of transgenic tobacco plants enables them to tolerate the oxidative factor of environmental stresses scavenging ROS. On the other hand, the cost of such resistance is quite high and, when grown under normal conditions, disturbs the arrangement of the intrachloroplastic subdomains leading to the modification of stromal thylakoids, probably significantly affecting the photosynthesis processes that regulate the efficiency of photosystem II. This is partially compensated for by the fact that, at the same time, under normal conditions, the production of peroxide induces the activation of ROS detoxification enzymes. However, a violation of a number of processes, such as the metabolism of accumulation, and utilization and transportation of sugars and starch, is significantly altered, which leads to a shift in metabolic chains. The expected step for further improvement of the applied technology could be both the use of inducible promoters in the expression cassette, and the addition of other genes encoding for hydrogen peroxide-scavenging enzymes in the genetic construct that are downstream in the metabolic chain.

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.

The nematode effector Mj-NEROSs interacts with Rieske's iron-sulfur protein influencing plastid ROS production to suppress plant immunity.

Root-knot nematodes (RKN; Meloidogyne species) are plant pathogens that introduce several effectors in their hosts to facilitate infection. The actual targets and functioning mechanism of these effectors largely remain unexplored. This study illuminates the role and interplay of the Meloidogyne javanica nematode effector ROS suppressor (Mj-NEROSs) within the host plant environment. Mj-NEROSs suppresses INF1-induced cell death as well as flg22-induced callose deposition and reactive oxygen species (ROS) production. A transcriptome analysis highlighted the downregulation of ROS-related genes upon Mj-NEROSs expression. NEROSs interacts with the plant Rieske's iron-sulfur protein (ISP) as shown by yeast-two-hybrid and bimolecular fluorescence complementation. Secreted from the subventral pharyngeal glands into giant cells, Mj-NEROSs localizes in the plastids where it interacts with ISP, subsequently altering electron transport rates and ROS production. Moreover, our results demonstrate that isp Arabidopsis thaliana mutants exhibit increased susceptibility to M. javanica, indicating ISP importance for plant immunity. The interaction of a nematode effector with a plastid protein highlights the possible role of root plastids in plant defense, prompting many questions on the details of 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.

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.

Transcriptome analysis reveals molecular mechanisms underlying chloroplast biogenesis in albino Agave angustifolia plantlets.

Albino plants display partial or complete loss of photosynthetic pigments and defective thylakoid membrane development, consequently impairing plastid function and development. These distinctive attributes render albino plants excellent models for investigating chloroplast biogenesis. Despite their potential, limited exploration has been conducted regarding the molecular alterations underlying these phenotypes, extending beyond photosynthetic metabolism. In this study, we present a novel de novo transcriptome assembly of an albino somaclonal variant of Agave angustifolia Haw., which spontaneously emerged during the micropropagation of green plantlets. Additionally, RT-qPCR analysis was employed to validate the expression of genes associated with chloroplast biogenesis, and plastome copy numbers were quantified. This research aims to gain insight into the molecular disruptions affecting chloroplast development and ascertain whether the expression of critical genes involved in plastid development and differentiation is compromised in albino tissues of A. angustifolia. Our transcriptomic findings suggest that albino Agave plastids exhibit high proliferation, activation of the protein import machinery, altered transcription directed by PEP and NEP, dysregulation of plastome expression genes, reduced expression of photosynthesis-associated nuclear genes, disruption in the tetrapyrrole and carotenoid biosynthesis pathway, alterations in the plastid ribosome, and an increased number of plastome copies, among other alterations.

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.

Fatty acid de novo biosynthesis in plastids: Key enzymes and their critical roles for male reproduction and other processes in plants.

Fatty acid de novo biosynthesis in plant plastids is initiated from acetyl-CoA and catalyzed by a series of enzymes, which is required for the vegetative growth, reproductive growth, seed development, stress response, chloroplast development and other biological processes. In this review, we systematically summarized the fatty acid de novo biosynthesis-related genes/enzymes and their critical roles in various plant developmental processes. Based on bioinformatic analysis, we identified fatty acid synthase encoding genes and predicted their potential functions in maize growth and development, especially in anther and pollen development. Finally, we highlighted the potential applications of these fatty acid synthases in male-sterility hybrid breeding, seed oil content improvement, herbicide and abiotic stress resistance, which provides new insights into future molecular crop breeding.

FLOURY ENDOSPERM24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice.

Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure. Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map-based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant. The flo24 mutant develops more severe floury endosperm when grown under high-temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp-2 to regulate starch biosynthesis and endosperm development in rice. Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.

The plastid-localized lipoamide dehydrogenase 1 is crucial for redox homeostasis, tolerance to arsenic stress and fatty acid biosynthesis in rice.

Soil contamination with arsenic (As) can cause phytotoxicity and reduce crop yield. The mechanisms of As toxicity and tolerance are not fully understood. In this study, we used a forward genetics approach to isolate a rice mutant, ahs1, that exhibits hypersensitivity to both arsenate and arsenite. Through genomic resequencing and complementation tests, we identified OsLPD1 as the causal gene, which encodes a putative lipoamide dehydrogenase. OsLPD1 was expressed in the outer cell layer of roots, root meristem cells, and in the mesophyll and vascular tissues of leaves. Subcellular localization and immunoblot analysis demonstrated that OsLPD1 is localized in the stroma of plastids. In vitro assays showed that OsLPD1 exhibited lipoamide dehydrogenase (LPD) activity, which was strongly inhibited by arsenite, but not by arsenate. The ahs1 and OsLPD1 knockout mutants exhibited significantly reduced NADH/NAD+ and GSH/GSSG ratios, along with increased levels of reactive oxygen species and greater oxidative stress in the roots compared with wild-type (WT) plants under As treatment. Additionally, loss-of-function of OsLPD1 also resulted in decreased fatty acid concentrations in rice grain. Taken together, our finding reveals that OsLPD1 plays an important role for maintaining redox homeostasis, conferring tolerance to arsenic stress, and regulating fatty acid biosynthesis in rice.

Complex Endosymbioses I: From Primary to Complex Plastids, Serial Endosymbiotic Events.

A considerable part of the diversity of eukaryotic phototrophs consists of algae with plastids that evolved from endosymbioses between two eukaryotes. These complex plastids are characterized by a high number of envelope membranes (more than two) and some of them contain a residual nucleus of the endosymbiotic alga called a nucleomorph. Complex plastid-bearing algae are thus chimeric cell assemblies, eukaryotic symbionts living in a eukaryotic host. In contrast, the primary plastids of the Archaeplastida (plants, green algae, red algae, and glaucophytes) possibly evolved from a single endosymbiosis with a cyanobacterium and are surrounded by two membranes. Complex plastids have been acquired several times by unrelated groups of eukaryotic heterotrophic hosts, suggesting that complex plastids are somewhat easier to obtain than primary plastids. Evidence suggests that complex plastids arose twice independently in the green lineage (euglenophytes and chlorarachniophytes) through secondary endosymbiosis, and four times in the red lineage, first through secondary endosymbiosis in cryptophytes, then by higher-order events in stramenopiles, alveolates, and haptophytes. Engulfment of primary and complex plastid-containing algae by eukaryotic hosts (secondary, tertiary, and higher-order endosymbioses) is also responsible for numerous plastid replacements in dinoflagellates. Plastid endosymbiosis is accompanied by massive gene transfer from the endosymbiont to the host nucleus and cell adaptation of both endosymbiotic partners, which is related to the trophic switch to phototrophy and loss of autonomy of the endosymbiont. Such a process is essential for the metabolic integration and division control of the endosymbiont in the host. Although photosynthesis is the main advantage of acquiring plastids, loss of photosynthesis often occurs in algae with complex plastids. This chapter summarizes the essential knowledge of the acquisition, evolution, and function of complex plastids.

The Main Functions of Plastids.

Plastids are semi-autonomous organelles like mitochondria and derive from a cyanobacterial ancestor that was engulfed by a host cell. During evolution, they have recruited proteins originating from the nuclear genome, and only parts of their ancestral metabolic properties were conserved and optimized to limit functional redundancy with other cell compartments. Furthermore, large disparities in metabolic functions exist among various types of plastids, and the characterization of their various metabolic properties is far from being accomplished. In this review, we provide an overview of the main functions, known to be achieved by plastids or shared by plastids and other compartments of the cell. In short, plastids appear at the heart of all main plant functions.

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.

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.

Determining the Subcellular Localization of Proteins in the Different Membranes of Diatom Secondary Plastid.

Diatoms such as Phaeodactylum tricornutum arose through a process termed secondary endosymbiosis, in which red alga-derived plastids are surrounded by a complicated membrane system. Subcellular marker proteins provide defined localizations on the compartmental and even sub-compartmental levels in the complex plastids of diatoms. Here we introduce how to use subcellular marker proteins and in vivo co-localization in the diatom P. tricornutum by presenting a step-by-step method allowing the determination of subcellular localization of proteins in different membranes of the secondary plastid. This chapter describes the materials required and the procedures of transformation and microscopic observation.