Lysine malonylation (K_mal), an evolutionarily conserved post-translational modification, serves as a critical regulator of cellular processes including transcriptional control, metabolic coordination, and enzyme activation. While K_mal sites have been mapped in rice (Oryza sativa L.) seeds, their dynamic regulation in rice responses to biotic stresses remains poorly characterized. Here, we reported a global profiling of lysine-malonylated proteins in rice leaf sheaths, and the changes in these proteins under herbivore (Nilaparvata lugens/Chilo suppressalis) or viral (rice stripe virus/rice black-streaked dwarf virus) stresses. Using affinity enrichment and proteomics, we identified 3,113 K_mal sites across 1,324 proteins in wild-type rice leaf sheaths, these data demonstrated that lysine-malonylated proteins are involved in diverse biological processes. K_mal levels were significantly upregulated following herbivore infestation or viral infection, with two herbivores inducing more pronounced changes than viruses infection, revealing stress-specific malonylation landscapes. K_mal preferentially targeted highly expressed proteins in energy metabolism (e.g., glycolysis and the tricarboxylic acid cycle) and photosynthesis, exhibiting an inverse correlation between hypermalonylation and protein abundance. Cleavage under targets and tagmentation analyses revealed K_mal-mediated chromatin remodeling through promoter occupancy at defense-related genes. In addition, the histone deacetylases OsHDA702-704, OsHDA711-713 were functionally characterized as key regulators mediating the erasure of specific malonylation marks in rice plants, with OsHDA711 knockout lines exhibiting enhanced resistance against both herbivore and virus infection. Our work establishes K_mal as a regulator in rice biotic immunity, uncovering novel insights into K_mal-mediated plant defense responses against herbivorous pests and viral pathogens. These findings identify potential genetic targets for developing rice varieties with broad-spectrum immunity to biotic stresses, enhancing crop resilience.

Drought stress represents a critical challenge to global agriculture, severely compromising plant growth and crop productivity through its disruption of intracellular signaling networks, with particular emphasis on protein kinase-mediated pathways and transcriptional regulation. In this study, we identified and characterized ZmDNL1 as a novel transcriptional regulator that serves as a negative modulator of drought tolerance in maize. Through comprehensive biochemical analyses, we demonstrated that ZmDNL1 physically interacts with ZmYAB15, a known negative regulator of drought tolerance, and potentiates its transcriptional regulatory activity. Most significantly, our investigation revealed that ZmSnRK2.10-mediated phosphorylation of three specific N-terminal residues in ZmDNL1 effectively attenuates ZmYAB15's transcriptional activity while maintaining the structural integrity of the ZmDNL1-ZmYAB15 protein complex, ultimately enhancing drought tolerance. These findings elucidate a previously unrecognized regulatory mechanism in which ZmSnRK2.10 orchestrates drought tolerance through phosphorylation-dependent fine tuning of the ZmDNL1–ZmYAB15 transcriptional regulatory module. Beyond advancing our fundamental understanding of drought response mechanisms in maize, this study provides valuable molecular targets for precision breeding strategies aimed at developing drought-resilient crop varieties.

Salt stress, especially the increasing secondary salt stress, severely compromises apple production worldwide. Mitigation of oxidative damage caused by salt stress is critical for salt tolerance in apple plants. However, it remains unclear how the salt signal triggers the excessive reactive oxygen species (ROS) mitigation system in apple. In this study, we identified a salt-induced gene MdGRF10 (encoding a 14-3-3 protein), whose overexpression conferred transgenic apple plants reduced oxidative damage and enhanced salt tolerance. Furthermore, a salt-activated receptor-like cytoplasmic kinase MdPBL34 was found to interact with and phosphorylate the C-terminal of MdGRF10. This phosphorylation promoted the interaction between MdGRF10 and a melatonin rate-limiting synthetase MdASMT1 (N-acetylserotonin methyltransferase). Its overexpression or knockdown by CRISPR/Cas9 in transgenic apple plants demonstrated that MdASMT1 is critical in melatonin-mediated ROS scavenging for salt tolerance. Their interaction stabilizes MdASMT1 by decreasing its ubiquitin-mediated degradation for increased melatonin level, decreased oxidative damage and therefore promoted salt tolerance. Our findings revealed that 14-3-3 protein could integrate the salt signal in a phosphorylation-dependent manner. Moreover, MdPBL34 was also identified for the first time to be involved in salt signaling. Our research uncovered a novel MdPBL34–MdGRF10–MdASMT1 regulatory module in response to salt stress in apple, which will contribute to the molecular breeding of melatonin-enriched salt-tolerant apple trees.

In higher plants, stomatal movements represent a critical physiological process that matains cellular water homestasis while enabling photosynthetic gas exchange. Open stomata 1 (OST1), a key protein kinase in the abscisic acid (ABA) signaling cascade, has been established as a central regulator of stomatal dynamics. This study reveals that two highly conserved mitogen-activated protein kinase 1 (MAP4K1) and MAP4K2 are positive regulators in ABA promoted stomatal closure, and ABA-activated OST1 potentiates MAP4K1/2 through phosphorylation at conserved serine and threonine residues (S166, T170, and S479/S488). The activated MAP4K1, in turn, phosphorylates two critical downstream targets: plasma membrane H⁺-ATPase 2 (AHA2) at residues T858, T881, and Y946, and slow anion channel-associated 1 (SLAC1) at T114 and S116. Functional analysis demonstrates that the phosphomimetic (3D: S166D/T170D/S479D) MAP4K1, but not non-phosphorylatable (3A: S166A/T170A/S479A) MAP4K1, could fully restore drought tolerance and reduced water loss in detached leaves of map4k1map4k2 double mutant. Our findings delineate a previously unrecognized signaling module comprising OST1–MAP4K1/2–AHA2/SLAC1, which crucially modulates ABA-mediated stomatal regulation. This work advances our mechanistic understanding of phosphorylation cascades governing plant water relations and stress responses.

Ubiquitination, a critical post-translational modification, plays a pivotal role in fine tuning the immune responses of plants. The tomato (Solanum lycopersicum) suffers significant yield and quality losses caused by the devastating pathogen Botrytis cinerea. We have discovered the role of SlRAE1, a gene encoding an E3 ubiquitin ligase, as a pivotal negative regulator of resistance to B. cinerea. SlRAE1 interacts with SlSKP1, a component of the SKP1–Cullin1–F-box (SCF) complex, to modulate the protein stability of the transcription factor SlWRKY1 through the 26S proteasome pathway. SlWRKY1 targets and inhibits the transcription of SlJAZ7, a suppressor of jasmonic acid (JA) signaling, thereby activating the JA-induced defense system and affecting tomato susceptibility to B. cinerea. The resistance enhancement observed with knock-out SlRAE1 was reduced when SlWRKY1 was also knocked out, highlighting SlWRKY1's role in SlRAE1's regulation of tomato defense against B. cinerea. Our findings elucidate the defense mechanism in tomato and suggest that targeting SlRAE1, by modulating SlWRKY1 stability, could help to develop resistant tomato varieties. These insights have broader implications for using gene-editing technologies to enhance crop defense against fungi.

Rice bacterial blight, caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo), poses a significant threat to rice crops. Arginine methylation, a post-translational modification of proteins, plays a pivotal role in transcriptional regulation, RNA processing, and the biosynthesis of plant hormones. Previous research has established that protein arginine methyltransferases (PRMTs) significantly influence protein function through arginine methylation. Nonetheless, the specific role of PRMT5 in regulating salicylic acid (SA) biosynthesis and plant immunity has been relatively unexplored. In this study, we elucidate the role of a rice protein arginine methyltransferase, OsPRMT5, in enhancing rice resistance to Xoo infection by interacting with the SA biosynthesis enzyme phenylalanine ammonia lyase 1 in rice (OsPAL1). Our results indicate that OsPRMT5 methylates OsPAL1 at the arginine residue 75, which affects the interaction between OsPRMT5 and OsPAL1 and subsequently boosts phenylalanine ammonia lyase (PAL) enzyme activity, leading to heightened SA accumulation. Conversely, compared to OsPAL1 overexpression plants in wild-type TP309 background, OsPAL1 overexpression plants in osprmt5 knockout (KO) mutants background exhibited diminished PAL activity. Furthermore, osprmt5 ospal1 double mutants demonstrated reduced resistance to bacterial blight compared to the OsPAL1-KO group. Additionally, we discovered that the Xoo effector protein PXO_01039 undermines the interaction between OsPRMT5 and OsPAL1, thereby facilitating Xoo infection. PXO_01039 binds to OsPRMT5, preventing the formation of the OsPRMT5-OsPAL1 complex, which results in decreased PAL activity and lower SA accumulation. In conclusion, our findings unveil how OsPRMT5 modulates the methylation and enzymatic activity of OsPAL1, a crucial enzyme in SA biosynthesis, to bolster plant antibacterial defenses.

Reactive oxygen species (ROS) plays critical roles in modulating plant growth and stress response and its homeostasis is fine tuned using multiple peroxidases. H₂O₂, a major kind of ROS, is removed rapidly and directly using three catalases, CAT1, CAT2, and CAT3, in Arabidopsis. Although the activity regulations of catalases have been well studied, their degradation pathway is less clear. Here, we report that CAT2 and CAT3 protein abundance was partially controlled using the 26S proteasome. To further identify candidate proteins that modulate the stability of CAT2, we performed yeast-two-hybrid screening and recovered several clones encoding a protein with RING and vWA domains, CIRP1 (CAT2 Interacting RING Protein 1). Drought and oxidative stress downregulated CIRP1 transcripts. CIRP1 harbored E3 ubiquitination activity and accelerated the degradation of CAT2 and CAT3 by direct interaction and ubiquitination. The cirp1 mutants exhibited stronger drought and oxidative stress tolerance, which was opposite to the cat2 and cat3 mutants. Genetic analysis revealed that CIRP1 acts upstream of CAT2 and CAT3 to negatively regulate drought and oxidative stress tolerance. The increased drought and oxidative stress tolerance of the cirp1 mutants was due to enhanced catalase (CAT) activities and alleviated ROS levels. Our data revealed that the CIRP1–CAT2/CAT3 module plays a vital role in alleviating ROS levels and balancing growth and stress responses in Arabidopsis.

Glycosylation, a prevalent post-translational modification in eukaryotic secreted and membrane-associated proteins, plays a pivotal role in diverse physiological and pathological processes. Although UDP-N-acetylglucosamine (UDP-GlcNAc) is essential for this modification, the specific glycosylation mechanisms during plant leaf senescence and defense responses remain poorly understood. In our research, we identified a novel rice mutant named rbb1 (resistance to blast and bacterial blight1), exhibiting broad-spectrum disease resistance. This mutant phenotype results from a loss-of-function mutation in the gene encoding glucosamine-6-phosphate acetyltransferase, an important enzyme in D-glucosamine 6-phosphate acetylation. The rbb1 mutant demonstrates enhanced defense responses, evident in increased resistance to rice blast and bacterial blight, along with the upregulation of defense-response genes. Various biochemical markers indicate an activated defense mechanism in the rbb1 mutant, such as elevated levels of reactive oxygen species and malondialdehyde, reduced enzyme activity and UDP-GlcNAc content, and decreased expression of N-glycan and O-glycan modifying proteins. Moreover, proteome analysis of N-glycosylation modifications reveals alterations in the N-glycosylation of several disease-resistance-related proteins, with a significant reduction in Prx4 and Prx13 in rbb1-1. Additionally, the knockout of Prx4 or Prx13 also enhances resistance to Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae (M. oryzae). This study uncovers a novel mechanism of defense response in rice, suggesting potential targets for the development of disease-resistant varieties.

Fruit taste quality is greatly influenced by the content of soluble sugars, which are predominantly stored in the vacuolar lumen. However, the accumulation and regulation mechanisms of sugars in most fruits remain unclear. Recently, we established the citrus fruit vacuole proteome and discovered the major transporters localized in the vacuole membrane. Here, we demonstrated that the expression of tonoplast sugar transporter 2 (CsTST2) is closely associated with sugar accumulation during sweet orange (Citrus sinensis) ripening. It was further demonstrated that CsTST2 had the function of transporting hexose and sucrose into the vacuole. Overexpression of CsTST2 resulted in an elevation of sugar content in citrus juice sac, calli, and tomato fruit, whereas the downregulation of its expression led to the reduction in sugar levels. CsTST2 was identified as interacting with CsCIPK23, which binds to the upstream calcium signal sensor protein CsCBL1. The phosphorylation of the three serine residues (Ser277, Ser337, and Ser354) in the loop region of CsTST2 by CsCIPK23 is crucial for maintaining the sugar transport activity of CsTST2. Additionally, the expression of CsCIPK23 is positively correlated with sugar content. Genetic evidence further confirmed that calcium and CsCIPK23-mediated increase in sugar accumulation depends on CsTST2 and its phosphorylation level. These findings not only unveil the functional mechanism of CsTST2 in sugar accumulation, but also explore a vital calcium signal regulation module of CsCBL1/CIPK23 for citrus sweetness quality.

Autophagy is a highly conserved cellular program in eukaryotic cells which mediates the degradation of cytoplasmic components through the lysosome, also named the vacuole in plants. However, the molecular mechanisms underlying the fusion of autophagosomes with the vacuole remain unclear. Here, we report the functional characterization of a rice (Oryza sativa) mutant with defects in storage protein transport in endosperm cells and accumulation of numerous autophagosomes in root cells. Cytological and immunocytochemical experiments showed that this mutant exhibits a defect in the fusion between autophagosomes and vacuoles. The mutant harbors a loss-of-function mutation in the rice homolog of Arabidopsis thaliana MONENSIN SENSITIVITY1 (MON1). Biochemical and genetic evidence revealed a synergistic interaction between rice MON1 and AUTOPHAGY-RELATED 8a in maintaining normal growth and development. In addition, the rice mon1 mutant disrupted storage protein sorting to protein storage vacuoles. Furthermore, quantitative proteomics verified that the loss of MON1 function influenced diverse biological pathways including autophagy and vacuolar transport, thus decreasing the transport of autophagic and vacuolar cargoes to vacuoles. Together, our findings establish a molecular link between autophagy and vacuolar protein transport, and offer insights into the dual functions of the MON1–CCZ1 (CAFFEINE ZINC SENSITIVITY1) complex in plants.

Soil salinity is a serious environmental threat to plant growth and flowering. Flowering in the right place, at the right time, ensures maximal reproductive success for plants. Salinity-delayed flowering is considered a stress coping/survival strategy and the molecular mechanisms underlying this process require further studies to enhance the crop's salt tolerance ability. A nuclear pore complex (NPC) component, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1), has been recognized as a negative regulator of plant cold responses and flowering. Here, we challenged the role of HOS1 in regulating flowering in response to salinity stress. Interestingly, we discovered that HOS1 can directly interact with and ubiquitinate transcription factor SPL9 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9) to promote its protein degradation in response to salinity stress. Moreover, we demonstrated that HOS1 and SPL9 antagonistically regulate plant flowering under both normal and salt stress conditions. HOS1 was further shown to negatively regulate the expression of SPLs and several key flowering genes in response to salinity stress. These results jointly revealed that HOS1 is an important integrator in the process of modulating salinity-delayed flowering, thus offering new perspectives on a salinity stress coping strategy of plants.

Protein S-acylation or palmitoylation is a reversible post-translational modification that influences many proteins encoded in plant genomes. Exciting progress in the past 3 years demonstrates that S-acylation modulates subcellular localization, interacting profiles, activity, or turnover of substrate proteins in plants, participating in developmental processes and responses to abiotic or biotic stresses. In this review, we summarize and discuss the role of S-acylation in the targeting of substrate proteins. We highlight complex roles of S-acylation in receptor signaling. We also point out that feedbacks of protein S-acyl transferase by signaling initiated from their substrate proteins may be a recurring theme. Finally, the reversibility of S-acylation makes it a rapid and efficient way to respond to environmental cues. Future efforts on exploring these important aspects of S-acylation will give a better understanding of how plants enhance their fitness under ever changing and often harsh environments.

Lysine acetylation, an evolutionarily conserved post-translational protein modification, is reversibly catalyzed by lysine acetyltransferases and lysine deacetylases. Lysine acetylation, which was first discovered on histones, mainly functions to configure the structure of chromatin and regulate gene transcriptional activity. Over the past decade, with advances in high-resolution mass spectrometry, a vast and growing number of non-histone proteins modified by acetylation in various plant species have been identified. Lysine acetylation of non-histone proteins is widely involved in regulating biological processes in plants such as photosynthesis, energy metabolism, hormone signal transduction and stress responses. Moreover, in plants, lysine acetylation plays crucial roles in regulating enzyme activity, protein stability, protein interaction and subcellular localization. This review summarizes recent progress in our understanding of the biological functions and mechanisms of non-histone protein acetylation in plants. Research prospects in this field are also noted.

Ubiquitination-mediated protein degradation is integral to plant immunity, with E3 ubiquitin ligases acting as key factors in this process. Here, we report the functions of OsATL32, a plasma membrane-localized Arabidopsis Tóxicos En Levadura (ATL)-type E3 ubiquitin ligase, in rice (Oryza sativa) immunity and its associated regulatory network. We found that the expression of OsATL32 is downregulated in both compatible and incompatible interactions between rice and the rice blast fungus Magnaporthe oryzae. The OsATL32 protein level declines in response to infection by a compatible M. oryzae strain or to chitin treatment. OsATL32 negatively regulates rice resistance to blast and bacterial leaf blight diseases, as well as chitin-triggered immunity. Biochemical and genetic studies revealed that OsATL32 suppresses pathogen-induced reactive oxygen species (ROS) accumulation by mediating ubiquitination and degradation of the ROS- producing OsRac5–OsRbohB module, which enhances rice immunity against M. oryzae. The protein phosphatase PHOSPHATASE AND TENSIN HOMOLOG enhances rice blast resistance by dephosphorylating OsATL32 and promoting its degradation, preventing its negative effect on rice immunity. This study provides insights into the molecular mechanism by which the E3 ligase OsATL32 targets a ROS-producing module to undermine rice immunity.

Drought stress has negative effects on crop growth and production. Characterization of transcription factors that regulate the expression of drought-responsive genes is critical for understanding the transcriptional regulatory networks in response to drought, which facilitates the improvement of crop drought tolerance. Here, we identified an Alfin-like (AL) family gene ZmAL14 that negatively regulates drought resistance. Overexpression of ZmAL14 exhibits susceptibility to drought while mutation of ZmAL14 enhances drought resistance. An abscisic acid (ABA)-activated protein kinase ZmSnRK2.2 interacts and phosphorylates ZmAL14 at T38 residue. Knockout of ZmSnRK2.2 gene decreases drought resistance of maize. A dehydration-induced Rho-like small guanosine triphosphatase gene ZmROP8 is directly targeted and repressed by ZmAL14. Phosphorylation of ZmAL14 by ZmSnRK2.2 prevents its binding to the ZmROP8 promoter, thereby releasing the repression of ZmROP8 transcription. Overexpression of ZmROP8 stimulates peroxidase activity and reduces hydrogen peroxide accumulation after drought treatment. Collectively, our study indicates that ZmAL14 is a negative regulator of drought resistance, which can be phosphorylated by ZmSnRK2.2 through the ABA signaling pathway, thus preventing its suppression on ZmROP8 transcription during drought stress response.

It is generally accepted that jasmonate-ZIM domain (JAZ) repressors act to mediate jasmonate (JA) signaling via CORONATINE-INSENSITIVE1 (COI1)-mediated degradation. Here, we report a cryptic signaling cascade where a JAZ repressor, FvJAZ12, mediates multiple signaling inputs via phosphorylation-modulated subcellular translocation rather than the COI1-mediated degradation mechanism in strawberry (Fragaria vesca). FvJAZ12 acts to regulate flavor metabolism and defense response, and was found to be the target of FvMPK6, a mitogen-activated protein kinase that is capable of responding to multiple signal stimuli. FvMPK6 phosphorylates FvJAZ12 at the amino acid residues S179 and T183 adjacent to the PY residues, thereby attenuating its nuclear accumulation and relieving its repression for FvMYC2, which acts to control the expression of lipoxygenase 3 (FvLOX3), an important gene involved in JA biosynthesis and a diverse array of cellular metabolisms. Our data reveal a previously unreported mechanism for JA signaling and decipher a signaling cascade that links multiple signaling inputs with fruit trait development.

In plants, thousands of nucleus-encoded proteins translated in the cytosol are sorted to chloroplasts and mitochondria by binding to specific receptors of the TOC (translocon on the outer chloroplast membrane) and the TOM (translocon on the outer mitochondrial membrane) complexes for import into those organelles. The degradation pathways for these receptors are unclear. Here, we discovered a converged ubiquitin-proteasome pathway for the degradation of Arabidopsis thaliana TOC and TOM tail-anchored receptors. The receptors are ubiquitinated by E3 ligase(s) and pulled from the outer membranes by the AAA⁺ adenosine triphosphatase CDC48, after which a previously uncharacterized cytosolic protein, transmembrane domain (TMD)-binding protein for tail-anchored outer membrane proteins (TTOP), binds to the exposed TMDs at the C termini of the receptors and CDC48, and delivers these complexes to the 26S proteasome.

Roots are fundamental for plants to adapt to variable environmental conditions. The development of a robust root system is orchestrated by numerous genetic determinants and, among them, the MADS-box gene ANR1 has garnered substantial attention. Prior research has demonstrated that, in chrysanthemum, CmANR1 positively regulates root system development. Nevertheless, the upstream regulators involved in the CmANR1-mediated regulation of root development remain unidentified. In this study, we successfully identified bric-a-brac, tramtrack and broad (BTB) and transcription adapter putative zinc finger (TAZ) domain protein CmBT1 as the interacting partner of CmANR1 through a yeast-two-hybrid (Y2H) screening library. Furthermore, we validated this physical interaction through bimolecular fluorescence complementation and pull-down assays. Functional assays revealed that CmBT1 exerted a negative influence on root development in chrysanthemum. In both in vitro and in vivo assays, it was evident that CmBT1 mediated the ubiquitination of CmANR1 through the ubiquitin/26S proteasome pathway. This ubiquitination subsequently led to the degradation of the CmANR1 protein and a reduction in the transcription of CmANR1-targeted gene CmPIN2, which was crucial for root development in chrysanthemum. Genetic analysis suggested that CmBT1 modulated root development, at least in part, by regulating the level of CmANR1 protein. Collectively, these findings shed new light on the regulatory role of CmBT1 in degrading CmANR1 through ubiquitination, thereby repressing the expression of its targeted gene and inhibiting root development in chrysanthemum.