Mitochondrial lipoamide dehydrogenase (mtLPD) is a crucial component of four NADH-generating multienzyme complexes essential to photorespiration, branched-chain amino acids (BCAAs) metabolism, and the tricarboxylic acid (TCA) cycle in plants. Despite its established biochemical function, the in vivo physiological significance of mtLPD remained largely unclear due to insufficient mutant analysis. Here, we addressed this critical knowledge gap by characterizing the loss-of-function mutants for the two Arabidopsis mtLPDs. While the two mtLPDs both contribute to photorespiration, mtLPD1 plays a more predominant role, especially under high-light stress. In addition, the two mtLPDs are largely functionally redundant in BCAA degradation during carbon starvation. Finally, we uncovered a critical and dose-dependent role for both mtLPDs in male and female gametophytic development. Our findings established the physiological importance of a critical and conserved plant mitochondrial enzyme in plant resilience and reproduction, which has significantly advanced the understanding of mitochondrial metabolism and may have important implications in crop improvement.

Pre-harvest sprouting (PHS), defined as the premature germination of grains on the panicle before harvest, significantly compromises grain yield and seed quality. Optimizing seed dormancy is therefore crucial for mitigating PHS. Here, we report that loss of function in Stress-associated Protein 5 (OsSAP5) enhances resistance to PHS without grain yield reduction. Mechanistically, OsSAP5 participates in the GA-mediated modulation of the DELLA protein OsSLR1 abundance to orchestrate downstream responses; it accelerates GA signaling transduction to induce amylase gene expression and elevate soluble sugar content, while concurrently suppressing the expression of ABA signaling regulators, thereby collaboratively governing seed dormancy and germination. In ossap5 mutants, this bipartite control-manifested as intensified dormancy alongside delayed germination-acts in concert to repress PHS. We further confirm an interaction between OsSAP5 and OsSLR1, which likely plays a role in this regulatory process. Correspondingly, we also established that OsSLR1 positively regulates seed dormancy and negatively regulates seed germination, thereby further elaborating its biological functions. In conclusion, our findings identify OsSAP5 as a pivotal modulator of GA signaling, offering a new theoretical basis and genetic target for breeding PHS-resistant varieties.

Protein structure bridges the sequence-function relationship, enabling deep exploration of biological processes across diverse organisms. Insects, the most diverse animal lineage, accounting for over 50% of all described animal species, provide an exceptional system for exploring sequence-structure-function relationships. Here, we reconstructed a comprehensive and well-resolved phylogeny of 4854 insects, spanning all orders. Leveraging this framework, we created an atlas of 13.29 million predicted protein structures from 824 representative species, including 11.63 million newly predicted structures. Structural clustering revealed that proteins with divergent sequences but similar structures could be effectively grouped together. Structural similarity searches against proteins with well-characterized functions yielded annotations for 7.61 million insect proteins, including up to 14% of previously unannotated proteins. We further identified 750 million remote homologs between insect proteins, many of which trace back to ancient branches of the insect phylogeny. Remarkably, despite extensive sequence divergence, cGAS-like receptors (cGLRs) were structurally conserved across all 824 insects. Experimental assays demonstrated that these structurally identified cGLRs play a crucial role in antiviral defense in the yellow fever mosquito. Our findings highlight the significance of structural genomics for understanding protein function and evolution across the tree of life.

Seed dormancy is a complex developmental and agronomic trait requiring a delicate balance: insufficient dormancy leads to pre-harvest sprouting (PHS), while excessive dormancy causes uneven germination. Understanding the intrinsic mechanisms resolving this trade-off is vital for crop improvement. In this study, our findings indicate that the heat shock transcription factor OsHsfA2a acts as a negative regulator of seed dormancy. The hsfa2a knockout mutants exhibited enhanced PHS resistance while maintaining yield potential and post-harvest germination rates, offering a potential strategy to uncouple PHS resistance from agronomic penalties. Mechanistically, our data suggest that OsHsfA2a self-associates and can activate the ABA catabolic gene OsABA8ox3, thereby promoting dormancy release. Furthermore, genetic analysis using the sd6 hsfa2a double mutant supports the hypothesis that the bHLH transcription factor OsSD6 is epistatic to OsHsfA2a, acting as an upstream initiator. Biochemically, OsSD6 transcriptionally activates OsHsfA2a. Once expressed, OsHsfA2a likely amplifies the signal via a self-activation loop. However, the accumulating OsSD6 protein physically interacts with OsHsfA2a, which we propose competitively attenuates its DNA-binding capacity. These results support a multi-tiered regulatory module operating across both transcriptional and post-translational levels. Collectively, our findings provide insights into an intrinsic physiological mechanism by which this module fine-tunes ABA catabolism to govern primary seed dormancy, providing precise genetic targets for mitigating PHS.

Peroxisomes are single-membrane-bound organelles essential for diverse metabolic reactions and cellular redox homeostasis, yet the contribution of ubiquitin-proteasome system to peroxisomal biology remains unclear. Here, we demonstrate that the AAA-ATPase complex comprising Cell Division Cycle48 (CDC48), Nuclear Protein Localization4 (NPL4) and Ubiquitin Fusion Degradation1 (UFD1) is indispensable for peroxisomal biogenesis and physiological function in Arabidopsis. We identify the peroxisomal membrane peroxin PEX22 as a direct substrate of the CDC48 complex and show that this complex promotes ubiquitin-dependent PEX22 turnover. Genetic analyses place CDC48 complex upstream of PEX22 in controlling peroxisomal biogenesis and activity. Moreover, H₂O₂‑triggered Cys271 oxidation represses CDC48 ATPase activity, stabilizing PEX22 via slowed degradation; nucleoredoxin NRX1 reduces oxidized CDC48 to recover its function. Consistently, transgenic plants harboring the redox-insensitive CDC48-C271S variant display accelerated PEX22 turnover and enhanced susceptibility to oxidative stress. Collectively, our findings establish the CDC48 complex as a putative H₂O₂ sensor that governs ubiquitin-mediated peroxisome-associated protein degradation (PexAD), enabling fine-tuning of peroxisomal performance in plant development and upon environmental stress. Peroxisomes are essential for diverse biological functions. Here the authors show that H₂O₂‑triggered oxidation of the CDC48 AAA-ATPase complex in Arabidopsis regulates PEX22 abundance and fine-tunes peroxisomal activity.

Stress-associated proteins (SAPs), a class of zinc-finger proteins, play crucial roles in plant responses to abiotic stresses, including high temperature; however, their underlying mechanism is largely unknown. Here, we report that a heat-induced E3 ubiquitin ligase OsSAP3 negatively regulates thermotolerance in rice (Oryza sativa) by specifically interacting with the small heat shock protein OsHSP16. Knockout of OsSAP3 improves seedling survival and maintains grain-setting rates under heat stress. OsSAP3 ubiquitinates the lysine residues K128 and K145 in OsHSP16 to regulate its stability. Unlike OsSAP3, OsHSP16 functions as a positive regulator of thermotolerance in rice, and this enhanced heat resistance correlates with increased antioxidant enzyme activity, reduced reactive oxygen species (ROS) accumulation, decreased relative electrolyte leakage, and upregulation of heat stress defense genes. Genetic interaction analysis supports that OsSAP3 and OsHSP16 operate in the same pathway to modulate heat stress responses, albeit with opposing roles. Furthermore, OsHSP16 binds to and promotes the degradation of putatively misfolded or damaged OsAPX2 (ascorbate peroxidase 2) proteins, which may contribute to its role in enhancing thermotolerance in rice. Collectively, our findings reveal a key molecular framework controlling rice heat tolerance and provide a potential gene-editing-based strategy to enhance crop heat resilience.

Plants must continuously balance growth and stress resilience to survive in fluctuating environments. Selective autophagy and the ubiquitin-proteasome system serve as key post-translational regulatory mechanisms that enable this dynamic equilibrium. The ubiquitin receptor DOMINANT SUPPRESSOR OF KAR 2 (DSK2) is known to mediate stress-induced degradation of the brassinosteroid-activated transcription factor BES1, thereby suppressing growth under adverse conditions. In this perspective, we propose that DSK2 functions more broadly as a multi-client 'growth-switch' hub, coordinating the targeted transcription factors. Phenotypic evidence supports a role for DSK2-mediated TCP degradation in drought tolerance. We present a conceptual model in which DSK2 integrates diverse stress signals to reprioritize metabolic resources, maintain cellular homeostasis, and safeguard reproductive development. Elucidating the full target spectrum of DSK2 will deepen our understanding of post-translational regulation in plant stress adaptation and open new avenues for engineering climate-resilient crops, an urgent imperative for global food security in an era of accelerating climate change.

The agricultural sector faces mounting challenges from climate change and crop losses caused by biotic and abiotic stresses, with traditional breeding and chemical controls offering limited solutions. Advances in artificial intelligence (AI) have enabled the de novo design of protein binders exhibiting high specificity and stability, presenting new opportunities for crop protection and stress tolerance. Here we review core AI-driven methodologies—including diffusion models and protein language models—that facilitate the rational design of mini-proteins targeting pathogen effectors and plant immune components, demonstrating potential for precise molecular interventions to enhance disease resistance and abiotic stress resilience. These approaches have the potential to simplify or navigate differently the regulatory complexities often associated with transgenic organisms. While traditional delivery systems still facing a critical challenge, emerging cell-penetrating peptide (CPP) systems offer a non-viral, targeted solution for protein translocation, potentially simplifying regulatory pathways by enabling transient expression without genomic integration. While promising, successful agricultural application requires expanding plant-specific structural data and developing compatible delivery systems. This emerging paradigm integrates computational protein design with plant biotechnology, offering a transformative strategy for sustainable crop improvement amid global food security challenges.

Salicylic acid (SA) is a central signaling molecule in systemic acquired resistance (SAR) in plants, yet long-term monitoring of its real-time dynamics in living tissues has remained technically challenging. Using the recently developed genetically encoded fluorescent sensor SalicS1, high-resolution spatiotemporal imaging of SA was successfully performed in intact plants for the first time, revealing an SA wave that propagates ahead of the invading pathogen. This breakthrough tool directly captures the dynamic spread of plant defense signals. In addition, SalicS1 provides a technological foundation for dissecting the temporal relationships between SA and upstream early signals such as calcium ions and reactive oxygen species and for identifying novel immune signaling components through genetic screening. The development of this sensor marks a transition in plant immunity research from static measurements to real-time dynamic imaging.

Protein structure serves as a critical bridge between sequence and functional annotation, particularly in establishing functional links among distantly homologous proteins with low sequence similarities. However, systematic protein structure-based functional annotations have been lacking in plants, where functions for a significant portion of the proteomes are still elusive. In this study, we leveraged protein structural data from 17 angiosperms to uncover previously unannotated protein functions in plants. After structural clustering, we used the plant clusters to query the UniProtKB/Swiss-Prot database (the expertly curated component of UniProtKB), a repository of expertly curated and reliably annotated proteins, and identified structural matches for thousands of plant clusters that were undetectable by sequence-based BLAST searches. We further selected 120 clusters, which are highly reliable in structural quality and alignment and are well-conserved across plant species, and uncovered various protein functions that are potentially widely important in plants. Finally, we experimentally analyzed one plant cluster structurally resembling the yeast peroxisomal peroxin 8 (PEX8) protein and verified that plant PEX8-like proteins can functionally complement yeast pex8 mutants. Our findings highlight the power of structural comparison in uncovering protein functions in plants.

Lipids are essential for building cells and are used as important seed reserves. Fatty acids (FAs) are the key structural units of lipids, forming their hydrophobic tails in triglycerides and phospholipids. FA synthesis starts in plastids and is completed in the endoplasmic reticulum (ER). SYNTAXIN OF PLANTS 81 (SYP81), a Qa-soluble N-ETHYLMALEIMIDE SENSITIVE FACTOR attachment protein receptor (Qa-SNARE), regulates vesicle trafficking between the ER and Golgi apparatus, yet its role in FA synthesis is unknown. Here, we examined the expression of SYP81 during Arabidopsis thaliana seed development and found that the syp81 mutation reduced Acyl-Acyl Desaturase 5 (AAD5) accumulation in plastids of Arabidopsis embryonic cells and thus significantly decreased unsaturated FA production. Pull-down experiments identified possible interactors with SYP81, including various Stearoyl-Acyl carrier protein Desaturases (SADs), notably AAD5, and Translocons at Outer envelope membranes of Chloroplast (TOCs), especially TOC33. To validate these interactions, CoIP, yeast-two-hybridization assays, and bimolecular-fluorescence-complementation experiments were performed. The results of these experiments supported the interaction between SYP81 and AAD5, as well as SYP81 and TOC33. Based on these findings, a model was proposed, suggesting that pre-AAD5, recruited by SYP81, translocates from the ER to the plastids through the TIC-TOC complex mediations. Within the plastids, pre-AAD5 then matures into its catalytically active form, enabling subsequent FA desaturation.

According to the endosymbiotic theory, both mitochondria and plastids arose from bacteria. As semi-autonomous organelles, they play key roles in aerobic respiration and photosynthesis as well as many other physiological and metabolic processes in plants. Although nuclear gene editing technology has become relatively mature, the editing technology for organelle genomes still faces huge challenges. Gene editing technology based on the CRISPR-Cas system has not yet achieved substantial breakthroughs in organelle genomes, but transcription activator-like effector nucleases (TALENs) technology has been successfully applied to organelle gene knockout, which demonstrates broad application prospects, particularly in the field of cytoplasmic male sterility. Additionally, TALE-dependent base editors, such as DddA-derived cytosine base editors (DdCBEs)and TALE-linked deaminases (TALEDs), have enabled single-base editing in plant organellar genomes, offering new opportunities for precise mitochondrial and plastid genome editing. This review discusses the structural characteristics of organellar genomes, the challenges in developing organellar gene editing technologies, and the progress made in TALEN-mediated gene knockout and TALE-dependent base editing in plants. Two key factors complicate organelle gene editing: the difficulty of RNA delivery and the unique genomic structures and DNA repair mechanisms. TALENs, which use TALE proteins for DNA recognition, have circumvented the limitations of RNA delivery, enabling targeted gene knockout in organelles. MitoTALENs and cpTALENs, targeting mitochondria and plastids, respectively, have been used to study CMS and other mitochondrial functions. For instance, mitoTALENs have been employed to knockout CMS-related genes in rice, rapeseed, and tomato, restoring male fertility and demonstrating the potential for crop breeding. Single-base editing in organelles has been achieved using DdCBEs and TALEDs. DdCBEs, derived from the bacterial toxin DddA, enable C-to-T editing in mitochondrial and plastid genomes. TALEDs, combining DddA with adenine deaminases, facilitate A-to-G editing. These tools have been successfully applied in various plants, including lettuce, rapeseed, and Arabidopsis, to introduce precise mutations in organellar genes. Despite these advancements, TALEN-based editing in organelles can lead to unpredictable outcomes due to the complex repair mechanisms of organellar genomes. CRISPR-Cas9, while widely used for nuclear genome editing, faces challenges in organellar genome editing due to difficulties in delivering guide RNAs (sgRNAs) into organelles. However, recent studies have shown the potential for CRISPR-Cas9 in mitochondrial and plastid editing by optimizing RNA delivery systems. For example, engineered Cas9 proteins with mitochondrial targeting sequences have been used to edit mitochondrial DNA in human cells and zebrafish. In conclusion, while organellar genome editing tools are still limited compared to nuclear genome editing, significant progress has been made with TALENs and TALE-dependent base editors. These tools have enabled precise gene knockout and single-base editing in mitochondria and plastids, offering new possibilities for studying organellar gene functions and improving crop breeding. Future research should focus on developing more efficient and precise editing tools and exploring the potential of CRISPR-based systems for organellar genome editing. These advancements will not only enhance our understanding of organellar biology but also provide powerful tools for precision agriculture and medical applications.

Peroxisomes, as essential eukaryotic organelles, are known to be involved in many oxidative metabolic processes including β-oxidative biosynthesis and/or metabolism of plant hormones and their substrates that are less or not known. The small thioesterase (ST) gene family encodes enzymes, called thioesterases that are notably involved in β-oxidative benzoic acid metabolism, as well as the biosynthesis of aromatic compounds and phylloquinone. To delve deeper into the role of these proteins in plant peroxisomes, we conducted an in-silico analysis to identify peroxisomal ST genes in Arabidopsis, focusing on identifying peroxisome-targeting signal peptide. In Arabidopsis, out of seven ST genes, three of them were experimentally verified as peroxisomal for the first time by confirming their sub-cellular localization. Phylogenetic studies revealed that peroxisomal ST genes are present in various plant species, underlining their functional conservation in peroxisomal metabolism. Insights into known peroxisomal STs in other species also aided in predicting the functions of Arabidopsis ST proteins within the same subgroup. Structural modeling indicates that Arabidopsis ST proteins possess a hotdog fold domain, a structural motif essential for thioesterase activity. In silico gene expression profiling showed that several ST genes are strongly expressed during the development of siliques, pods, and seeds, suggesting a role in these processes. This evidence supports the identification of a novel peroxisomal ST subfamily, prompting further investigation into their functions in Arabidopsis peroxisomes. This study provides a basis for further exploration of STs' roles in plant peroxisomal biology and their contributions to the redox regulation of peroxisome functions.

Submergence poses a deleterious threat to plant development and the agricultural production of Brassica napus (B. napus), an important worldwide oil crop. However, the molecular mechanisms underlying submergence tolerance in B. napus remain elusive. Here, through a genome-wide association study, we identify a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE, BKK1/BAK7, which is crucial for plant survival after submergence stress. Submergence rapidly activates the expression of BAK7 and induces the accumulation of its kinase domain in the nucleus. BAK7 interacts with and phosphorylates class I TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTOR 1 and 2 (TCP) transcription factor TCP21, thereby stabilizing it and modulating transcriptomic reprogramming after submergence. Moreover, overexpression of TCP21 rescues the submergence-intolerant phenotype of the bak1-3 bkk1-1 mutant. Interestingly, TCP21 directly binds to the promoter regions of BAK7 to activate its expression after submergence. This study provides insights into the molecular mechanism of plant adaptation to submergence stress and offers promising solutions for breeding submergence-resistant B. napus accessions.

Salicylic acid (SA) is a ubiquitous plant hormone with a long history in human civilization1,2. Because of the central role of SA in orchestrating plant pathogen defence, understanding SA biosynthesis is fundamental to plant immunity research and crop improvement. Isochorismate-derived SA biosynthesis has been well defined in Arabidopsis. However, increasing evidence suggests a crucial function for phenylalanine-derived SA biosynthesis in many other plant species1. Here we reveal the phenylalanine-derived SA biosynthetic pathway in rice by identifying three dedicated enzymes - peroxisomal benzoyl-CoA:benzyl alcohol benzoyltransferase (BEBT), the endoplasmic reticulum-associated cytochrome P450 enzyme benzylbenzoate hydroxylase (BBH), and cytosolic benzylsalicylate esterase (BSE) that sequentially convert benzoyl-CoA to benzylbenzoate, benzylsalicylate and SA. The pathogen-induced gene expression pattern and SA biosynthetic functions of this triple-enzyme module are conserved in diverse plants. This work fills a major knowledge gap in the biosynthesis of a key plant defence hormone, establishing a foundation for new strategies to create disease-resistant crops.

In plant chassis, peroxisomes, as key hubs of metabolism, play important roles in fatty acid β-oxidation, reactive oxygen species (ROS) metabolism and photorespiration. Fatty acid β-oxidation breaks down fatty acids through a multi-step enzymatic reaction, providing precursors for energy metabolism and phytohormone synthesis; ROS metabolism maintains redox balance through the antioxidant system and enhances plant resilience; and photorespiration relies on the synergistic action of peroxisomes with other organelles to optimize the efficiency of carbon and nitrogen utilization. Notably, peroxisomes possess a clear and simple targeting signaling system and a flexible abundance regulation mechanism, which provide unique advantages for their metabolic engineering. Recent studies have achieved effective control of fatty acid accumulation and promoted crop lipid yield by modulating the activity and transport mechanism of specific enzymes within the peroxisome. In addition, by reconfiguring the metabolic pathways in peroxisomes, the researchers successfully optimized the photorespiratory pathway, which significantly improved the energy utilization efficiency and enhanced the stress tolerance of crops. Meanwhile, the modification of ROS metabolism also showed the potential to enhance the antioxidant capacity and environmental adaptability of plants. These advances not only deepen our understanding of the peroxisome function, but also provide new strategies and technical means for crop breeding improvement and efficient secondary metabolite synthesis, which are expected to have broader application prospects in agricultural biotechnology and natural product biosynthesis in the future.

The rapid growth of moso bamboo is primarily attributed to the swift elongation of its internodes. While mitochondria are known to provide energy for various cellular processes, the specific mechanisms by which they facilitate rapid growth in bamboo remain elusive. In this study, we optimised the procedures for mitochondria isolation and performed a comprehensive analysis of mitochondrial dynamics and proteomics from internodes at various growth stages, including the initial growth (IG) stage, the starting of cell division (SD), and the rapid elongation (RE). Confocal observation demonstrated that cells in the RE stage have a higher mitochondrial density and increased mitochondrial motility compared to other stages. Proteomic analysis of isolated mitochondria revealed an upregulation of the tricarboxylic acid cycle, along with a synchronous increase in both mitochondrial- and nuclear-encoded components of oxidative phosphorylation in RE cells. Moreover, the upregulation of various mitochondrial membrane transporters in RE cells suggests an enhanced exchange of metabolic intermediates and inorganic ions with the cytosol. Intriguingly, ultrastructural analysis and pharmacological treatments revealed membrane interactions between the endoplasmic reticulum (ER) and mitochondria in RE cells. In conclusion, our study provides novel insights into mitochondrial function and the intracellular dynamics that regulate the rapid growth of moso bamboo.

Mitochondrial biogenesis requires the import of more than a thousand proteins encoded by nuclear DNA. The translocase of the outer mitochondrial membrane (TOM) complex serves as the primary gateway for specific recognition of precursor proteins, which are synthesized in the cytosol. Little is known about the regulation of the abundance of the TOM complex. Using forward genetics, we identified key 26S proteasome subunits, including REGULATORY PARTICLE NON-ATPASE1A (RPN1A), that affect the abundance of TOM-complex subunits through the ubiquitin-proteasome pathway. Loss of proteasome function through rpn1a mutation or MG132 treatment increased the abundance of TOM20 isoforms and induced mitochondrial stress marker genes. By contrast, overexpression of ANAC017, an endoplasmic reticulum-anchored transcription factor that activates mitochondrial retrograde signaling under stress, lowered TOM20 abundance and reduced mitochondrial protein import. The rates of mitochondrial protein import and respiratory activity were also altered. Genetic analyses placed the proteasome downstream of ANAC017, since the reduction in TOM20 required the RPN1a subunit. Transcriptome profiling after antimycin A treatment showed broad ANAC017-dependent reprogramming of ubiquitin-proteasome system genes. A second tier formed by ANAC053- and ANAC078-bound promoters of proteasome subunits, including RPN1a, is required to restrain TOM20 accumulation. These findings establish a two-step transcriptional circuit that engages the ubiquitin-proteasome system to tune TOM abundance and coordinate protein import with organelle function.

The mechanism underlying the ability of rice to germinate underwater is a largely enigmatic but key research question highly relevant to rice cultivation. Moreover, although rice is known to accumulate salicylic acid (SA), SA biosynthesis is poorly defined, and its role in underwater germination is unknown. It is also unclear whether peroxisomes, organelles essential to oilseed germination and rice SA accumulation, play a role in rice germination. Here, we show that submerged imbibition of rice seeds induces SA accumulation to promote germination in submergence. Two submergence-induced peroxisomal Oryza sativa cinnamate:CoA ligases (OsCNLs) are required for this SA accumulation. SA exerts this germination-promoting function by inducing indole-acetic acid (IAA) catabolism through the IAA-amino acid conjugating enzyme GH3. The metabolic cascade we identified may potentially be adopted in agriculture to improve the underwater germination of submergence-intolerant rice varieties. SA pretreatment is also a promising strategy to improve submerged rice germination in the field.