Chloroplast Envelope Research Articles

LIPID RICH 1 Modulates Allocation of Carbon between Starch and Triacylglycerol in Arabidopsis Leaves.

Plants accumulate starch and triacylglycerols (TAGs) as carbon sources. Leaves primarily store starch in chloroplasts, with some TAGs stored in lipid droplets, but how carbon resource allocation is regulated in leaves during cellular metabolism is largely unknown. Using a forward genetics approach, we isolated an Arabidopsis thaliana mutant with more lipid droplets in its leaves than the wild type, named lipid rich 1 (liri1). The overaccumulation of lipid droplets was caused by the loss of function in the causal gene, encoding an uncharacterized protein. TAG levels were five-fold higher and starch levels two-fold lower in the leaves of liri1 than the wild type. LIRI1 localized to the chloroplasts, and the contents of chloroplast membrane lipids were 20% higher in liri1 leaves than in wild-type leaves. Co-immunoprecipitation assays revealed that LIRI1 interacts with acetyl-coenzyme A carboxylase carboxyltransferase alpha subunit (an enzyme for fatty acid biosynthesis) and starch synthase 4 (an enzyme for starch biosynthesis). In isotope tracer experiments using [1-13C]-sodium acetate, more 13C was incorporated into TAGs in liri1 leaves than in wild-type leaves. Moreover, liri1 plants showed growth defects and irregular chloroplasts. These results suggest that LIRI1 affects the carbon trade-off to inhibit lipid production in leaves.

Alternative transcriptional initiation of OsβCA1 produces three distinct subcellular localization isoforms involved in stomatal response regulation and photosynthesis in rice.

Plants adjust the size of their stomatal openings to balance CO2 intake and water loss. Carbonic anhydrases (CAs) facilitate the conversion between CO2 and HCO3 -, and theOsβCA1 mutant in rice (Oryza sativa) shows similar traits in carbon fixation and stomatal response to CO2 as the dual βCA mutants in Arabidopsis thaliana. However, the exact role of OsβCA1 in these processes was unclear. We used gene editing, molecular biology, and plant physiology to study how OsβCA1 contributes to carbon fixation, stomatal opening, and CO2 responses. OsβCA1 produces three isoforms (OsβCA1A, OsβCA1B, and OsβCA1C) through alternative transcriptional initiation, which localize to the chloroplast, cell membrane, and cytosol, respectively. Protein measurements revealed that OsβCA1A/C and OsβCA1B contribute 97 and 3% to OsβCA1, respectively. By creating specific mutants for each isoform, our results found that the chloroplast and cell membrane isoforms independently participate in carbon fixation and regulation of stomatal aperture. Furthermore, the complete knockout of OsβCA1 caused a delayed response to low CO2. Our findings provide new insights into the generation and function of different OsβCA1 isoforms, clarifying their roles in CO2 diffusion, CO2 fixation and stomatal regulation in rice.

Cryo-EM structure of the NDH-PSI-LHCI supercomplex from Spinacia oleracea.

The nicotinamide adenine dinucleotide phosphate (NADPH) dehydrogenase (NDH) complex is crucial for photosynthetic cyclic electron flow and respiration, transferring electrons from ferredoxin to plastoquinone while transporting H+ across the chloroplast membrane. This process boosts adenosine triphosphate production, regardless of NADPH levels. In flowering plants, NDH forms a supercomplex with photosystem I, enhancing its stability under high light. We report the cryo-electron microscopy structure of the NDH supercomplex in Spinacia oleracea at a resolution of 3.0-3.3 Å. The supercomplex consists of 41 protein subunits, 154 chlorophylls and 38 carotenoids. Subunit interactions are reinforced by 46 distinct lipids. The structure of NDH resembles that of mitochondrial complex I closely, including the quinol-binding site and an extensive internal aqueous passage for proton translocation. A well-resolved catalytic plastoquinone (PQ) occupies the PQ channel. The pronounced structural similarity to complex I sheds light on electron transfer and proton translocation within the NDH supercomplex.

Arabidopsis conditional photosynthesis mutants abc1k1 and var2 accumulate partially processed thylakoid preproteins and are defective in chloroplast biogenesis

Photosynthetic activity is established during chloroplast biogenesis. In this study we used 680 nm red light to overexcite Photosystem II and disrupt photosynthesis in two conditional mutants (var2 and abc1k1) which reversibly arrested chloroplast biogenesis. During biogenesis, chloroplasts import most proteins associated with photosynthesis. Some of these must be inserted in or transported across the thylakoid membrane into the thylakoid lumen. They are synthesized in the cytoplasm with cleavable targeting sequences and the lumenal ones have bi-partite targeting sequences (first for the chloroplast envelope, second for the thylakoid membrane). Cleavage of these peptides is required to establish photosynthesis and a critical step of chloroplast biogenesis. We employ a combination of Western blotting and mass spectrometry to analyze proteins in var2 and abc1k1. Under red light, var2 and abc1k1 accumulated incompletely cleaved processing intermediates of thylakoid proteins. These findings correlated with colorless cotyledons, and defects in both chloroplast morphology and photosynthesis. Together the results provide evidence for the requirement of active photosynthesis for processing of photosystem-associated thylakoid proteins and concomitantly progression of chloroplast biogenesis.

Get the Ball Rolling: Update and Perspective on the Role of Chloroplast Plastoglobule-associated Protein under Abiotic Stress.

Plastid-localized plastoglobules (PGs) are monolayer lipid droplets typically associated with the outer envelope of thylakoid membranes in chloroplasts. The size and number of PGs can vary significantly in response to different environmental stimuli. Since the early 21st century, a variety of proteins attached to the surface of PGs have been identified and experimentally characterized using advanced biotechnological techniques, revealing their biological functions. This article aims to review the latest discoveries regarding PG-associated proteins and explore their dynamics under both single and combined abiotic stress conditions, providing insights into the critical role of plastid lipid droplets in plant adaptation to global climate-related challenges.

Comprehensive Identification and Expression Profiling of Lipoxygenase Genes in Sweet Cherry During Fruit Development

Lipoxygenase (LOX) is involved in the oxidation of fatty acids in plants and is a ubiquitous oxygenase that plays an important role in the process of plant resistance to adversity. In this study, the LOX gene family in the sweet cherry genome was identified by bioinformatics methods, the chromosomal mapping of different LOX genes was carried out, and the homology alignment and functional domain analysis of the encoded proteins were performed. The results showed that there were nine LOX gene sequences in the sweet cherry LOX gene family, and the subcellular localization was mainly located in the cytoplasm, chloroplast, or plasma membrane, and was concentrated on chromosomes 1, 2, 3, 4, 6, and 8. During the ripening process of sweet cherry fruits, the LOX gene family showed five different expression patterns, the expression peak of different LOX genes reached the peak of expression at a specific development period, all LOX genes jointly promoted the growth and development of fruits, the enzyme activities of LOX in different varieties of early and late ripening cherries exhibited great differences during the development process, and the results of volatile content in the later stages also showed that different varieties of cherries had their specificity. The results of this study provide a theoretical basis for further revealing the specific functions of LOX gene family members in sweet cherry.

Antioxidant and Ultrastructural Alterations in Wheat During Drought-Induced Leaf Senescence

Wheat is one of the most important crops to ensure food production globally. Understanding the mechanism of leaf senescence in wheat plays a crucial role in improving its productivity and resilience under various stress scenarios. In this study, we investigated biochemical, functional, and ultrastructural changes during leaf senescence in wheat genotypes with contrasting drought tolerance. For this, key parameters such as chlorophyll and total protein content, membrane stability, malondialdehyde level, and the activity of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, benzidine peroxidase, and catalase) were comparatively analyzed during both natural and drought-induced senescence. Additionally, the expression of superoxide dismutase isoform genes functioning in different cellular compartments was studied, alongside ultrastructural changes in flag leaves. The experiments involved genotypes of bread wheat (Triticum aestivum L.) and durum (Triticum durum Desf.) wheat. The plants were grown in controlled environment chambers under control and drought conditions using a completely randomized design. After the booting stage, irrigation was discontinued for drought-treated plants. Flag leaves were sampled at 7, 14, 21, 28, and 35 days after anthesis. Drought-tolerant genotypes exhibited slower chlorophyll degradation, lower lipid peroxidation, enhanced membrane stability, and stronger antioxidant responses, allowing them to maintain cellular function longer, whereas sensitive genotypes showed accelerated leaf senescence. Transcript levels of FeSOD increased significantly post-flowering but declined as senescence progressed, while MnSOD expression exhibited a rise towards the later stages of ontogenesis across all studied genotypes. Ultrastructural analysis revealed progressive damage to chloroplast membranes, thylakoid structures, and mesophyll cell walls under stress conditions, particularly in sensitive genotypes. These findings contribute to a deeper understanding of the physiological and molecular responses of wheat to drought stress, offering potential targets for improving crop performance in water-limited environments.

A Bifunctional Nuclease Promotes the Infection of Zucchini Yellow Mosaic Virus in Watermelon by Targeting P3.

Potyviral P3 is involved in viral replication, movement, and pathogenicity; however, its biochemical function is unknown. In this study, the P3 of the zucchini yellow mosaic virus (ZYMV) interacted with ClBBD, a protein with high ortholog bifunctional nuclease activity, in watermelon. The binding site was shown via yeast two-hybrid screening and BiFC assay to be located at the N-terminus of P3 rather than P3N-PIPO. ClBBD localized predominantly to the chloroplast and plasma membrane. ZYMV P3 was also present in the nucleus and cytoplasm as aggregates. When co-expressed with P3 in tobacco, ClBBD formed aggregates with P3 in the cytoplasm. The knockdown of ClBBD using the VIGS vector pV190 and challenge with ZYMV revealed a positive correlation between viral accumulation and ClBBD expression, indicating that ClBBD reduces the resistance of watermelon to ZYMV. Furtherly, we found that when P3 and ClBBD were transiently co-expressed in tobacco, the level of P3 was significantly higher than that when it was expressed alone or co-expressed with GUS. It inferred that ClBBD may be able to stabilize the expression of P3. Overall, the results suggest that the interaction of P3 with ClBBD promotes virus infection, and ClBBD may be involved in stabilizing the expression level of P3.

Genome-Wide Transcriptional Profiling and Functional Analysis Reveal That OsPHT4;4 Is Critical for the Growth and Development of Rice.

Phosphorus (P) is an essential macronutrient required for various vital processes in crop growth and development, including signal transduction, CO2 fixation, and photosynthetic phosphorylation. Phosphate transporters (PHTs) in plants play critical roles in the uptake, distribution, and internal transport of Phosphate (Pi). Among these transporters, the PHT4 family is widely distributed across plant species; however, the specific functions of many members within this family remain to be fully elucidated. This study focuses on unraveling the function of OsPHT4;4 in Pi utilization and photoprotection. The findings demonstrate that OsPHT4;4 acts as a low-affinity Pi transporter localized to the chloroplast membrane and reveal predominant expression of OsPHT4;4 in leaves, with peak expression during tillering and clear induction by light, exhibiting circadian rhythmicity. The ospht4;4 mutants display stunted growth. Transcriptomic analysis comparing ospht4;4 mutants and wild-types (WT) identified 1482 differentially expressed genes (DEGs), including 729 upregulated genes and 753 downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis reveals enrichment DEGs related to photosynthesis-antenna proteins, carbohydrate metabolism, and phenylpropanoid biosynthesis. These findings suggest that OsPHT4;4 plays crucial roles not only in photosynthesis but also in plant defense as an integral component involved in Pi metabolism.

Localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus to thylakoid subdomains in Arabidopsis.

Thylakoid membranes in chloroplasts and cyanobacteria harbor the multisubunit protein complexes that catalyze the light reactions of photosynthesis. In plant chloroplasts, the thylakoid membrane system comprises a highly organized network with several subcompartments that differ in composition and morphology: grana stacks, unstacked stromal lamellae, and grana margins at the interface between stacked and unstacked regions. The localization of components of the photosynthetic apparatus among these subcompartments has been well characterized. However, less is known about the localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus, the partitioning of proteins between two recently resolved components of the traditional margin fraction (refined margins and curvature), and the effects of light on these features. In this study, we analyzed the partitioning of numerous thylakoid biogenesis and repair factors among grana, curvature, refined margin, and stromal lamellae fractions of Arabidopsis thylakoid membranes, comparing the results from illuminated and dark-adapted plants. Several proteins previously shown to localize to a margin fraction partitioned in varying ways among the resolved curvature and refined margin fractions. For example, the ALB3 insertase and FtsH protease involved in photosystem II (PSII) repair were concentrated in the refined margin fraction, whereas TAT translocon subunits and proteins involved in early steps in photosystem assembly were concentrated in the curvature fraction. By contrast, two photosystem assembly factors that facilitate late assembly steps were depleted from the curvature fraction. The enrichment of the PSII subunit OE23/PsbP in the curvature fraction set it apart from other PSII subunits, supporting the previous conjecture that OE23/PsbP assists in PSII biogenesis and/or repair. The PSII assembly factor PAM68 partitioned differently among thylakoid fractions from dark-adapted plants and illuminated plants and was the only analyzed protein to convincingly do so. These results demonstrate an unanticipated spatial heterogeneity of photosystem biogenesis and repair functions in thylakoid membranes and reveal the curvature fraction to be a focal point of early photosystem biogenesis.

Monomer unfolding of a bacterial ESCRT-III superfamily member is coupled to oligomer disassembly.

The inner membrane associated protein of 30 kDa (IM30), a member of the endosomal sorting complex required for transport (ESCRT-III) superfamily, is crucially involved in the biogenesis and maintenance of thylakoid membranes in cyanobacteria and chloroplasts. In solution, IM30 assembles into various large oligomeric barrel- or tube-like structures, whereas upon membrane binding it forms large, flat carpet structures. Dynamic localization of the protein in solution, to membranes and changes of the oligomeric states are crucial for its in vivo function. ESCRT-III proteins are known to form oligomeric structures that are dynamically assembled from monomeric/smaller oligomeric proteins, and thus these smaller building blocks must be assembled sequentially in a highly orchestrated manner, a still poorly understood process. The impact of IM30 oligomerization on function remains difficult to study due to its high intrinsic tendency to homo-oligomerize. Here, we used molecular dynamics simulations to investigate the stability of individual helices in IM30 and identified unstable regions that may provide structural flexibility. Urea-mediated disassembly of the IM30 barrel structures was spectroscopically monitored, as well as changes in the protein's tertiary and secondary structure. The experimental data were finally compared to a three-state model that describes oligomer disassembly and monomer unfolding. In this study, we identified a highly stable conserved structural core of ESCRT-III proteins and discuss the advantages of having flexible intermediate structures and their putative relevance for ESCRT-III proteins.

Two critical membranes: how does the chloroplast envelope affect plant acclimation properties?

Chloroplasts play a pivotal role in the metabolism of leaf mesophyll cells, functioning as a cellular hub that orchestrates molecular reactions in response to environmental stimuli. These organelles contain complex protein machinery for energy conversion and are indispensable for essential metabolic pathways. Proteins located within the chloroplast envelope membranes facilitate bidirectional communication with the cell and connect essential pathways, thereby influencing acclimation processes to challenging environmental conditions such as temperature fluctuations and light intensity changes. Despite their importance, a comprehensive overview of the impact of envelope-located proteins during acclimation to environmental changes is lacking. Understanding the role of these proteins in acclimation processes could provide insights into enhancing stress tolerance under increasingly challenging environments. This review highlights the significance of envelope-located proteins in plant acclimation.

Oxygenating respiratoid biosystem for therapeutic cell transplantation

In this study, we address the persistent challenge of providing adequate oxygen to transplanted cells by introducing a respiratoid biosystem. Central to our strategy is the chloroplast-transit-peptide (CTP), crucial for optimal oxygenation. Through conjugation of CTP with alginate, we achieve stabilization of chloroplast structure. Strategically anchored to the outer chloroplast membrane, CTP not only ensures structural integrity but also upregulates key photosynthesis-associated genes. This biosystem demonstrates exceptional efficacy in spontaneously generating oxygen, particularly under hypoxic conditions (~1% pO2). In an application, pancreatic islets encapsulated within the respiratoid biosystem and intraperitoneally implanted in diabetic mice maintain normal glucose levels effectively. Insulin secretion persists for 100 days post-xenotransplantation without the need for immunosuppressant administration, highlighting the reliance on the respiratoid biosystem’s oxygen supply and structural stability. Our study demonstrates the respiratoid biosystem as a platform in tissue engineering, offering a nature-inspired solution to the critical challenge of spontaneous oxygen supply.

Vitis vinifera Lipoxygenase LoxA is an Allosteric Dimer Activated by Lipidic Surfaces

Lipoxygenases catalyze the peroxidation of poly-unsaturated fatty acid chains either free or esterified in membrane lipids. Vitis vinifera LoxA is transcriptionally induced at ripening onset and localizes at the inner chloroplast membrane where it is responsible for galactolipid regiospecific mono- and di-peroxidation. Here we present a kinetic and structural characterization of LoxA. Our X-ray structures reveal a constitutive dimer with detergent induced conformational changes affecting substrate binding and catalysis. In a closed conformation, a LID domain prevents substrate access to the catalytic site by steric hindrance. Detergent addition above the CMC destabilizes the LID and opens the dimer with both catalytic sites accessible from the same surface framed by the PLAT domains. As a consequence, detergent molecules occupy allosteric sites in the PLAT/catalytic domain interface. These structural changes are mirrored by increased enzymatic activity and positive cooperativity when the substrate is provided in micelles. The ability to interact with micelles is lost upon dimer destabilization by site-directed mutagenesis as assessed by tryptophan fluorescence. Our data allow to propose a model for protein activation at the membrane, classifying LoxA as an interfacial enzyme acting on fatty acid chains directly from the membrane similar to mammalian 15-LOX and 5-LOX.

Monogalactosyldiacylglycerol synthase isoforms play diverse roles inside and outside the diatom plastid.

Diatoms derive from a secondary endosymbiosis event, which occurred when a eukaryotic host cell engulfed a red alga. This led to the formation of a complex plastid enclosed by four membranes: two innermost membranes originating from the red alga chloroplast envelope, and two additional peri- and epiplastidial membranes (PPM, EpM). The EpM is linked to the endoplasmic reticulum (ER). The most abundant membrane lipid in diatoms is monogalactosyldiacylglycerol (MGDG), synthesized by galactosyltransferases called MGDG synthases (MGDs), conserved in photosynthetic eukaryotes and considered to be specific to chloroplast membranes. Similar to angiosperms, a multigenic family of MGDs has evolved in diatoms, but through an independent process. We characterized MGDα, MGDβ and MGDγ in Phaeodactylum tricornutum, combining molecular analyses, heterologous expression in Saccharomyces cerevisiae, and studying overexpressing and CRISPR-Cas9-edited lines. MGDα localizes mainly to thylakoids, MGDβ to the PPM, and MGDγ to the ER and EpM. MGDs have distinct specificities for diacylglycerol, consistent with their localization. Results suggest that MGDα is required for thylakoid expansion under optimal conditions, while MGDβ and MGDγ play roles in plastid and non-plastid membranes and in response to environmental stress. Functional compensation among MGDs likely contributes to diatom resilience under adverse conditions and to their ecological success.

An Overview of Multifaceted Applications and the Future Prospects of Glyceroglycolipids in Plants.

Glyceroglycolipids (GGLs) are a class of lipid molecules that contain a glycerol backbone and one or more carbohydrate moieties, giving them amphipathic properties with both hydrophilic and hydrophobic regions. This amphipathic nature is fundamental for composing cell membrane lipid bilayers. These compounds are primarily distributed on the inner chloroplast membranes of plants and exhibit a unique structure with numerous biological activities. Moreover, GGLs play a pivotal role in photosynthesis and energy conversion in plants and effectively respond to environmental stressors. This Review discusses the distribution, synthesis pathways, and functions of GGLs in plants and describes the recent updates on various methods for extracting, isolating, and identifying GGLs. Finally, this Review discusses the biological activities of plant GGLs, including their anti-inflammatory, antiviral, and anticancer properties, and highlights their potential applications in the fields of pharmaceuticals, food, and cosmetics. This Review provides insights into GGLs, offering research support for the application of these natural molecules in the realm of holistic health.

Dehydration priming remodels protein abundance and phosphorylation level regulating tolerance to subsequent dehydration or salt stress in creeping bentgrass

Dehydration priming (DP) induces stress memory which plays a positive role in plant adaptability, but it is not well understood how DP differentially regulates subsequent dehydration (cis priming) or salt (trans priming) tolerance at the post-translational level. Purpose of this study was to identify proteins, phosphorylation levels and sites, and relevant metabolic pathways for DP-induced dehydration or salt tolerance in Agrostis stolonifera. DP-induced differentially regulated proteins (DRPs) were mostly located in the cytoplasm, chloroplast, and cell membrane, and differentially regulated phosphoproteins (DRPPs) were mostly nuclear proteins and cytoplasmic proteins. DP regulated common phosphorylation sites ([SP] and [RxxS]) under dehydration and salt conditions and also individually affected 8 or 11 phosphorylation sites under dehydration or salt stress. DP-regulated DRPPs were mainly rich in glycolysis and glutathione metabolism pathways, RNA splicing, and dynamin family proteins under dehydration stress, whereas DP-regulated salt tolerance was mainly related to chlorophyll metabolism, photosynthesis, MAPK signaling cascade, and ABC transporter I family at the phosphorylation level. In addition, the DP also significantly up-regulated phosphorylation of histones (ATXR3 and SETD1A) in response to subsequent dehydration and salt stress as well as abundances of antioxidant enzymes, dynamin family protein, and KCS6 under dehydration stress or abundances of PETE, HMGA, XTH, and ABCI6 under salt stress, respectively. Transcriptomics analysis further indicated that DP-regulated dehydration or salt tolerance was also related to transcriptional regulation in the early stage. Current results provided better understanding of the role of stress memory in plant adaptability to repeated or crossed stress via post-translational modifications (PTMs). SignificanceRecurrent moderate drought may buffer drought legacies in many plant species. When plants were exposed to repeated drought stress, their adaptability to subsequent stress could be enhanced, which is known as “stress memory”. Dehydration priming has been found to be an important approach to induce stress memory. Current results provided better understanding of the role of stress memory in plant adaptability to repeated or crossed stress via post-translational modifications.

Functional Analysis of the PoSERK-Interacting Protein PorbcL in the Embryogenic Callus Formation of Tree Peony (Paeonia ostii T. Hong et J. X. Zhang).

SERK is a marker gene for early somatic embryogenesis. We screened and functionally verified a SERK-interacting protein to gain insights into tree-peony somatic embryogenesis. Using PoSERK as bait, we identified PorbcL (i.e., the large subunit of Rubisco) as a SERK-interacting protein from a yeast two-hybrid (Y2H) library of cDNA from developing tree-peony somatic embryos. The interaction between PorbcL and PoSERK was verified by Y2H and bimolecular fluorescence complementation analyses. PorbcL encodes a 586-amino-acid acidic non-secreted hydrophobic non-transmembrane protein that is mainly localized in the chloroplast and plasma membrane. PorbcL was highly expressed in tree-peony roots and flowers and was up-regulated during zygotic embryo development. PorbcL overexpression caused the up-regulation of PoSERK (encoding somatic embryogenesis receptor-like kinase), PoAGL15 (encoding agamous-like 15), and PoGPT1 (encoding glucose-6-phosphate translocator), while it caused the down-regulation of PoLEC1 (encoding leafy cotyledon 1) in tree-peony callus. PorbcL overexpression led to increased indole-3-acetic acid (IAA) content but decreasing contents of abscisic acid (ABA) and 6-benzyladenosine (BAPR). The changes in gene expression, high IAA levels, and increased ratio of IAA to ABA, BAPR, 1-Aminocyclopropanecarboxylic acid (ACC), 5-Deoxystrigol (5DS), and brassinolide (BL) promoted embryogenesis. These results provide a foundation for establishing a tree-peony embryogenic callus system.

Architecture of the ATP-driven motor for protein import into chloroplasts

Thousands of nuclear-encoded proteins are transported into chloroplasts through the TOC–TIC translocon that spans the chloroplast envelope membranes. A motor complex pulls the translocated proteins out of the TOC–TIC complex into the chloroplast stroma by hydrolyzing ATP. The Orf2971–FtsHi complex has been suggested to serve as the ATP-hydrolyzing motor in Chlamydomonas reinhardtii, but little is known about its architecture and assembly. Here, we report the 3.2-Å resolution structure of the Chlamydomonas Orf2971–FtsHi complex. The 20-subunit complex spans the chloroplast inner envelope, with two bulky modules protruding into the intermembrane space and stromal matrix. Six subunits form a hetero-hexamer that potentially provides the pulling force through ATP hydrolysis. The remaining subunits, including potential enzymes/chaperones, likely facilitate the complex assembly and regulate its proper function. Taken together, our results provide the structural foundation for a mechanistic understanding of chloroplast protein translocation.

Overexpression of SIMK in menadione-treated alfalfa enhances antioxidant machinery and leads to oxidative stress resistance

Mitogen-activated protein kinases (MAPKs) transduce stress and developmental signals related to the production of reactive oxygen species (ROS). Alfalfa (Medicago sativa L.) is a valuable forage and human nutrition crop, however, the involvement of MAPKs in plant resistance to oxidative stress is poorly understood in this species. Therefore, we elucidated the role of STRESS-INDUCED MAPK (SIMK) in alfalfa response to menadione, a compound inducing ROS generation, exploiting transgenic alfalfa lines with contrasting SIMK abundance. SIMK was activated by short-term menadione treatment and relocated from the nucleus to the cytoplasm. Proteomic analysis revealed that menadione caused changes in the abundance of proteins involved in metabolism, oxidative stress, biotic stress response, detoxification of carbonyl species, glutathione homeostasis, chloroplast protein turnover, photosynthesis, and membrane trafficking. Genetic manipulations of SIMK altered the abundance of proteins involved in mitochondrial and chloroplast protein import and processing, as well as GLUTATHIONE S-TRANSFERASES (GSTs). Increased GST abundance and activity in roots, and modifications in mitochondrial and chloroplast protein turnover might be responsible for the elevated oxidative stress resistance of alfalfa line overexpressing SIMK. This was supported by the reduced ROS levels in this line. These results reveal a complex nature of plant stress response and suggest a new role of SIMK in the alfalfa resistance to menadione-induced oxidative stress.