Journal of Integrative Plant Biology

Higher plants possess two classes of myosin molecular motors, class XI and class VIII, both unique to the plant lineage. The diverse cellular functions of class XI myosins, including organelle transport and nuclear positioning, have been elucidated largely through systematic identification of cargo adaptor proteins that bind to their globular tail domains (GTDs). In contrast, no proteome-wide screen for class VIII myosin tail-binding proteins has been reported; the few known interacting proteins were each discovered through studies focused on the binding partner rather than on the myosin itself, leaving the full repertoire of class VIII myosin-associated proteins largely unknown. Here, we employed TurboID-based proximity labeling to systematically identify proteins associated with the GTD of the class VIII myosin ATM1 in Arabidopsis thaliana, as this approach covalently biotinylates neighboring proteins in vivo, enabling their identification even after proteolytic degradation during cell lysis. We identified 233 non-redundant candidate ATM1-proximal proteins. Candidates were prioritized by AlphaFold3-based protein complex structure prediction and validated by co-immunoprecipitation. We identified two ATM1-associated proteins: C3H61/AtTZF5, a tandem zinc finger protein involved in mRNA turnover at processing bodies and stress granules; and SFH7, a Sec14-nodulin domain protein that mediates phosphatidic acid transfer from the endoplasmic reticulum to chloroplasts. These findings provide initial evidence linking ATM1 to proteins involved in post-transcriptional gene regulation and interorganellar lipid transport, raising the possibility of previously unrecognized connections between class VIII myosins and these cellular processes.

Rapid Alkalinization Factor (RALF) peptides regulate plant growth and cell wall signaling, but their roles in fruit development remain unclear. Here, we characterized tomato (Solanum lycopersicum) fruit-associated RALF peptides and their interactions with leucine-rich repeat extensins (LRXs). Expression analyses identified SlRALF5, SlRALF7, and SlRALF10 as the main fruit-expressed RALFs. SlRALF10 was associated with early fruit development, whereas SlRALF5 and SlRALF7 remained expressed during ripening. Sequence analyses showed that SlRALF5/7 retain conserved motifs of canonical RALFs, while SlRALF10 displays divergent structural features and altered charge distribution. Synthetic SlRALF5 and SlRALF7 inhibited root growth and induced extracellular alkalinization, whereas SlRALF10 lacked both activities. Co-immunoprecipitation assays showed that all three peptides interact with the fruit-expressed proteins SlLRX2 and SlLRX5. Structural modeling predicted distinct electrostatic properties for the SlLRX5/SlRALF10 complex compared with SlRALF5. These results reveal structural and functional specialization among tomato fruit RALF peptides and suggest that distinct SlRALFs may differentially respond to cell wall remodeling during fruit development and ripening.

Survival in dynamic environments requires photosynthetic organisms to rapidly sense and respond to stress. The stringent response, mediated by the signaling molecule guanosine-3,5-bisdiphosphate (ppGpp), is crucial for acclimation to environmental changes such as darkness and nitrogen limitation. While it has been extensively characterized in heterotrophic bacteria such as Escherichia coli, the molecular mechanisms and regulatory targets of ppGpp in photosynthetic organisms remain less understood. Here, we report large-scale chemoproteomic identification of ppGpp-binding proteins across plant chloroplasts and cyanobacteria, revealing both conserved and novel targets compared to E. coli. In plants, we found that ppGpp regulates pyrimidine metabolism by inhibiting the chloroplastic enzyme aspartate transcarbamoylase (PyrB). In cyanobacteria, we found that ppGpp activates glucose-1-phosphate adenylyltransferase (GlgC) involved in glycogen synthesis, activates citrate synthase (GltA), and induces carboxysome aggregation. These findings expand the known ppGpp regulatory network in photosynthetic organisms and provide a foundation for understanding how ppGpp coordinates adaptation to nutrient and environmental stresses.

Polycomb Repressive Complex 2 (PRC2) is a histone H3K27 methyltransferase that represses gene expression. Arabidopsis thaliana (A. thaliana) has several different PRC2 isoforms that are functionally distinct during the life cycle of the plants. However, their biochemical and structural characteristics have not been investigated. Here, we biochemically characterized PRC2 isoforms having different catalytic subunits: SWNINGER (SWN; PRC2 SWN ) and CURLY LEAF (CLF; PRC2 CLF ). Interestingly, PRC2 SWN showed much lower activity than PRC2 CLF . In addition, PRC2 SWN methylates histone H3K27 in mono and di-methylation, while PRC2 CLF shows robust tri-methylase activity. We also determined the cryo-electron microscopy (cryo-EM) structures of PRC2 SWN and PRC2 CLF , revealing that the substrate binding pocket of the SWN SET domain is blocked by a loop in the pre-SET domain, functioning as an auto-inhibitory loop, while that of the CLF SET domain is freely accessible. Introduction of CLF-like mutations in the auto-inhibitory loop in SWN enhances PRC2 SWN activity. Furthermore, structure-guided in planta analysis shows that a CLF-mimetic SWN mutant rescues the CLF knockout phenotype. Our work provides structural and molecular insights into the isoform-specific regulatory mechanism of plant PRC2.

Heavy metal-associated isoprenylated plant proteins (HIPPs) are encoded by large gene families, which have diversified specifically in vascular plants. Their physiological functions and molecular mode of activity are currently largely unknown. In this study, we characterize a group of phylogenetically closely related genes HIPP32, HIPP33, and HIPP34 in Arabidopsis thaliana, revealing their essential roles in controlling diverse developmental pathways. Through comprehensive genetic analyses, we demonstrate that these genes exhibit partially overlapping pleiotropic functions, influencing multiple aspects of plant growth such as embryogenesis, maintenance of apical meristems, root architecture, shoot branching, leaf morphogenesis and floral organ formation. Transcriptomic profiling of hipp mutants identified significant deregulation in several regulatory pathways involved in plant hormone responses, with a specific impact on auxin signaling processes. Interestingly, we show that the analyzed HIPP proteins localize very specifically to plasmodesmata, suggesting their potential function in regulating intercellular communication in shaping plant development.

O_LIResearch on the functions and molecular mechanisms of long non-coding RNAs (lncRNAs) involved in regulating plant freezing tolerance is still in its infancy. Our previous research work identified that lncRNA DROUGHT INDUCED LNCRNA (DRIR) regulates gene expressions in Arabidopsis. However, the underlying molecular mechanism is still unknown. C_LIO_LIThis study demonstrates that lncRNA DRIR regulates plant freezing tolerance by affecting alternative splicing patterns of pre-mRNAs. C_LIO_LIThrough chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS), we identified two DRIR interacting proteins: CP29A and CP29B. We showed that the drirD mutant, which exhibits elevated DRIR expression and DRIR overexpression lines showed increased sensitivity to freezing stress, whereas DRIR RNAi lines were more tolerant to the stress. CP29A and CP29B bind to nuclear transcripts and, together with DRIR, regulate pre-mRNA alternative splicing under freezing stress. Notably, DRIR induces the relocalization of CP29A and CP29B to autophagosomes, leading to autophagy-mediated protein degradation. C_LIO_LICollectively, our findings elucidate the molecular mechanism by which DRIR influences the autophagy-based degradation of its binding proteins CP29A and CP29B, thereby regulating plant freezing tolerance by altering the alternative splicing patterns of pre-mRNAs, providing novel insights into the functions and mechanisms of lncRNAs in plants adapting to freezing environments. C_LI

RD21-like proteases are papain-like cysteine proteases with a C-terminal granulin domain that are abundant and ubiquitous in angiosperms and have often been implicated in immunity. We previously found that the activity of RD21 in Nicotiana benthamiana (NbRD21) is suppressed during infection with Pseudomonas syringae. Here, we studied the role of NbRD21 in immunity and proteome processing. NbRD21 was disrupted by genome editing and rd21 mutants were subjected to disease assays and shot-gun proteomics. Dipeptide substrate zLR-AMC was used in protease assays and agroinfiltration was used to transiently express NbRD21 and candidate substrates. Genome edited lines lacking NbRD21 develop normally but have drastically reduced zLRase activity and are significantly more susceptible to P. syringae. Shot gun proteomics revealed an increased accumulation of [~]20 diverse receptor-like kinases (RLKs) in untreated rd21 knockout lines, but their transcript levels are unaltered when compared to wild-type plants. 35S-driven GFP-tagged RLKs accumulate more upon transient expression in rd21 plants than in wild- type plants. These data indicate that NbRD21 post-translationally controls RLK homeostasis, either by directly degrading RLKs, or indirectly by regulating endocytic RLK recycling.

In tomato plants, the potassium (K) transporter SlHAK5 is integral to root K uptake and overall plant fertility. Under K deficiency, SlHAK5 expression is induced in roots and the encoded transporter is activated via the Ca{superscript 2}-sensing CIPK/CBL complex SlCIPK23/SlCBL1-9. In Arabidopsis, multiple CIPK/CBL complexes can activate AtHAK5, providing alternative regulatory pathways that enhance K uptake. However, the architecture of CIPK/CBL signaling networks has diverged among plant species, necessitating species-specific identification of novel regulatory components. Accordingly, we screened additional tomato CIPK proteins for their capacity to modulate SlHAK5 activity in yeast. SlCIPK15 and SlCIPK26 emerged as potent activators of SlHAK5, acting in concert with SlCBL9. Functional characterization of slcipk15 and slcipk26 mutants revealed that neither contributed significantly to SlHAK5-mediated K uptake in roots. Conversely, both mutants exhibited impaired pollen tube elongation, correlating with reduced K content in pollen relative to wild type. Notably, slcipk26 mutants displayed more severe pollen defects, phenocopying the slhak5 mutant. Further analyses demonstrated that slcipk26 plants suffered compromised seed set and pistil function, paralleling the reproductive deficiencies observed in slhak5 mutants. These findings implicate SlCIPK26 as the principal regulator of SlHAK5 in reproductive tissues. Collectively, our data underscore the role of CIPK paralogs in orchestrating tissue-specific regulation of target proteins, thereby enabling fine-tuned modulation of K transport essential for both vegetative and reproductive development.

Elongating rice internodes present a developmental gradient from dividing meristem to mature cells, providing an elegant pseudo-time course for study of plant vegetative development. We tested the hypothesis that DNA methylation regulates gene expression during rice internode development by integrating RNA-seq and bisulfite DNA sequencing across eight internode segments. Previously described topologically associated chromatin domain borders aligned with transcription start sites of constitutive expressed genes. CpG and CHG differential methylation was enriched in young segments, consistent with maintenance methylation; whereas CHH methylation showed similar differential abundance in young and old segments. CHH and CHG methylation in upstream regions, CpG methylation within gene bodies, and any methylation in 5' and 3' untranslated regions were permissive of moderate to high gene expression. Very low expression was associated with CpG methylation upstream, CHG and CHH methylation within gene bodies, and CpG and CHG methylation downstream. A nonrandom subset of genes, including cell wall-related glycoside hydrolases, lignin and tricin biosynthesis enzymes, and WD40 proteins, showed methylation-expression correlations, with expression changes enriched in triple-marked elements. These results suggest that internode phenotypes of DNA methylation machinery mutants relate to alteration of specific target genes, opening approaches for grass culm improvement for lodging resistance and biomass production.

O_LIThe control of amount of nuclear proteins is fundamental in regulating plant gene expression, but the mechanisms of quantitative dynamics of the nuclear proteome are largely unstudied during adaptive responses to pathogens. C_LIO_LIHighly specific labeling, enrichment and measurement of the nuclear proteome was performed using TurboID LC-MS of Arabidopsis thaliana leaves treated with the pathogen-associated molecular pattern (PAMP), flg22, and/or cycloheximide. The chosen experimental approach allowed discrimination of the effects of translation, nuclear protein import, trafficking of preexisting proteins, derepression, and nuclear protein turn-over upon elicitation of basal immunity. C_LIO_LIThe highly specific, deep coverage of proteins in the nucleus makes this study a resource for anyone interested in plant nuclear proteome dynamics and defense. C_LIO_LIAround 2,000 nuclear proteins were repeatedly quantified, including more than 300 transcription factors or other proteins related to transcription. Several proteins with documented activity in endosomes were newly synthesized and imported into nuclei upon PAMP challenge, suggesting alternative nuclear functions in PAMP-triggered immunity (PTI). Circadian clock components, including the transcription factor, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)-HIKING EXPEDITION (CHE), were depleted upon PAMP challenge, suggesting a safeguard against untimely induction of systemic acquired resistance (SAR). C_LIO_LIBased on proteomic patterns, proteins moonlighting in the nucleus as well as trafficking and turn-over regulation of the proteome are common elements during plant immunity. C_LI

Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns (PAMPs) of Gram-negative pathogenic bacteria recognized by plants, triggering typical pattern-triggered immunity (PTI) responses. However, a LPS sensing receptor for the recognition of plants remains largely undefined. A plant receptor for lipopolysaccharide (LPS) has not yet been identified. Here, we identify a plant protein, OsML1, with homologies to animal MD-2, which is capable of binding LPS. Furthermore, it may act as a molecular chaperone to assist CK1 in perceiving LPS signals. Our results show that OsML1 functions as an LPS-binding protein recognizing LPS and participates in downstream rice immune response activation. Structural modeling and sequence analysis revealed that OsML1 contains both a typical ML domain and a conserved three-dimensional {beta}-barrel structure as mammalian MD-2 proteins. Microscale thermophoresis assays confirmed that OsML1 binds LPS with high affinity. Functional analyses further demonstrated that OsML1 knockout plants show reduced resistance to the rice bacterial blight pathogen, as well as attenuated ROS bursts upon LPS treatments, whereas overexpression plants show enhanced immune responses. Metabolomic profiling indicated significant metabolic changes in OsML1 knockout plants, particularly in immune-related pathways involving lipids, amino acids, and antimicrobial compounds. OsML1 is consequently a structurally conserved and functional LPS-binding protein linking lipid metabolism, LPS perception, immune activation, and metabolic regulation. Phylogenetic and structural analyses revealed that OsML1 likely arose from a duplication of OsML2, forming an independently functional subgroup within the PITP family. Our study identifies OsML1 as a LPS recognition factor involved in LPS sensing and downstream ROS bursts activation, callose deposition, and broad-spectrum gene expression of resistance. These findings expand our knowledge of bacterial LPS perception and immune regulation in plants, offering novel targets and strategies for disease-resistant breeding.

The red/far-red sensing photoreceptor phytochrome B (phyB) governs multifaceted plant development and responses to light and temperature stimuli. PhyB photoconversion between red-absorbing, inactive Pr and far red-absorbing, active Pfr states, imparted by its covalently bound bilin chromophore, enables rapid switching and plasticity of phyB signaling activities. The phyBY276H variant (YHB) is photochemically inert but adopts a constitutively active Pfr-like structure regardless of light conditions, which becomes a versatile model to dissect phyB signaling mechanisms. Here, we conducted a large-scale EMS mutagenesis screen on YHB-expressing transgenic lines, mining intragenic suppressor mutations that would unveil critical residues for phyB structure-function relationships. Comparative analyses of 26 nonsense variants suggested modular organization of phyB overall structure and dispensability of the C-terminal HKRD domain for phyB signaling. Amongst fourteen novel and nine known loss-of-function missense variants identified herein, G284E was of particular interest for its fully suppressed constitutive activity in darkness and its restored photochemistry and light responsiveness. The G284E mutation was further tested to also nullify another constitutively active phyBY303V allele by eliminating chromophore attachment. P309L was the sole variant identified which fully suppressed YHB in both dark and light conditions. C402Y profoundly elicited YHB protein instability. Three variants G118R, C402Y and G538D markedly reduced chromophorylation levels of YHB. Although the chromophore binding site variant C357Y was a strong loss-of-function allele, it retained residual signaling activity with respect to PIF3 protein turnover in dark-grown seedlings, presumably due to its ability to noncovalently bind chromophore. Two tandem prolines (P799, P800) proved critical to YHB structural integrity/stability as well as signaling activity. In summary, these diverse variants shed new insights into multiple levels by which the YHB (and thereby phyB) signaling is initiated, tuned, and disseminated.

Plants, as sessile organisms, must continually adapt to fluctuating temperatures to ensure survival. The plasma membrane-localized receptor-like kinase FERONIA (FER) coordinates diverse physiological processes and responses to various biotic and abiotic stresses. However, the role of FER in plant adaptation to elevated temperatures remains largely unexplored. Here, we report that FER is indispensable for plant thermotolerance. We found that fer loss-of-function mutants exhibit impaired thermomorphogenic growth and are hypersensitive to mild heat stress, displaying extensive oxidative stress-mediated cell death at elevated temperatures. Combined genetic and molecular analyses revealed that these temperature-sensitive defects in fer mutants are caused by an overaccumulation of jasmonic acid (JA), which subsequently triggers excessive production of reactive oxygen species. Furthermore, we show that this aberrant JA accumulation and oxidative stress are attributable to impaired FER-mediated regulation of turgor-dependent cell wall tensile stress. Taken together, our results suggest that FER-mediated cell wall tensile stress regulation serves as a critical mechanism to prevent aberrant JA accumulation and oxidative stress at elevated temperatures, thereby enabling plants to adapt to and survive under high-temperature conditions.

Plants continuously develop shoot branches derived from axillary meristems. In rice (Oryza sativa), TILLERS ABSENT1 (TAB1), an ortholog of Arabidopsis WUSCHEL, plays an essential role in axillary meristem formation by promoting stem cell proliferation. Although several genes associated with TAB1 function have been identified, the molecular mechanisms underlying stem cell proliferation during axillary meristem formation remain poorly understood. Here we identify ABERRANT SPIKELET AND PANICLE1 (ASP1), a TOPLESS-like transcriptional corepressor, as a novel regulator of axillary meristem formation, and investigate downstream mechanisms regulated by TAB1 and ASP1. In asp1, the stem cell region was expanded, indicating that ASP1 negatively regulates stem cell proliferation. Notably, WOX4, a paralog of TAB1, was precociously expressed in asp1, possibly in association with expansion of the stem cell region. Genetic analysis further revealed that asp1 mutation rescued the loss of axillary meristems in tab1. Transcriptome analysis showed that several type-A RESPONSE REGULATOR (OsRR) genes, encoding negative regulators of cytokinin signaling, were upregulated in tab1 relative to wild type, asp1, and the tab1 asp1 double mutant. Consistently, fluorescence of the synthetic cytokinin reporter was absent during axillary meristem formation in tab1 but was detected in wild type and tab1 asp1. Moreover, overexpression of OsRR10 inhibited axillary meristem formation, phenocopying tab1. Collectively, these findings suggest that TAB1 activates cytokinin signaling by repressing type-A OsRR expression, whereas ASP1 negatively regulates cytokinin signaling by promoting the expression of these genes. Thus, rescue of the tab1 phenotype by asp1 mutation probably reflects restoration of cytokinin signaling.

Soil acidity-associated toxicities of aluminum (Al), cadmium (Cd), and manganese (Mn) severely constrain rice productivity in upland ecosystems. To investigate the genomic basis of adaptation to acidic soil-related metal stress, we conducted an integrated meta-QTL (M-QTL) and functional genomics analysis in rice. Meta-analysis of 681 QTLs and MTAs from 53 QTL mapping and GWAS studies identified 79 robust M-QTLs, including ten overlapping regions associated with Al-, Cd-, and Mn-responsive traits. A multi-criteria prioritization framework identified 98 candidate genes supported by positional overlap, transcriptomic recurrence, and functional annotation, enriched for ion transport, detoxification, and redox regulation pathways. M-QTL10.9 emerged as a major hotspot enriched for glutathione-S-transferase genes, whereas M-QTL9.5 contained the highest density of prioritized candidates linked to Al and Cd responses. Comparative physiological & biochemical analyses of the contrasting rice genotypes Sahasarang and IR64 revealed genotype-dependent differences in antioxidant responses, metal partitioning, metabolic regulation, and cell wall remodeling under individual and combined metal stresses. Expression profiling of prioritized candidate genes, including OsACO family genes, OsZIP10, and OsGSTU10, further revealed genotype-dependent transcriptional divergence under combined stress. The identification of overlapping M-QTLs across Al, Cd, and Mn datasets suggests both shared and stress-specific adaptive responses to acidic soil-associated metal stress in rice.

High humidity greatly influences plant growth and development and triggers adaptive physiological responses such as leaf hyponasty (elongation of leaf petiole and upward leaf movement). A recent study identified Cyclic Nucleotide-Gated Ion Channels 2 and 4 (CNGC2/4)-mediated Ca2+ influx and Calmodulin Binding Transcription Activators 2 and 3 (CAMTA2/3)-mediated transcription as essential for high humidity response in Arabidopsis, but the upstream regulators that control these pathways remain unknown. Here, we show that the receptor-like kinase FERONIA and its co-receptor LORELEI-LIKE GPI-ANCHORED PROTEIN1 (LLG1) are required for a large portion of high humidity-associated Arabidopsis transcriptomic changes, including CNGC2, CAMTA-regulated genes, and cell wall remodeling genes, and for high humidity-induced leaf hyponasty. High humidity triggers a previously uncharacterized petiole-localized Ca2+ waves that precede hyponastic leaf movement. The petiole-localized Ca2+ signals were significantly altered in the fer-4 mutant. Thus, FERONIA is a key regulator of plant responses to extracellular high humidity. Highlights FERONIA plays a prominent role in transcriptomic responses to high humidity FERONIA is required for high humidity-induced leaf hyponasty High humidity induces petiole calcium waves FERONIA is required for normal petiole calcium waves in response to high humidity

Plants, as sessile organisms, have developed various mechanisms to respond to environmental stress conditions. The plant hormone abscisic acid (ABA) is necessary for the plant to adapt to osmotic stress conditions. However, the molecular mechanisms preceding ABA accumulation remain largely unknown. To isolate transcriptional complexes on the promoter region of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) encoding a rate-limiting enzyme in the ABA biosynthetic pathway in planta, we developed the insertional chromatin immunoprecipitation (iChIP) screen method. The identified ALBA proteins formed condensates through liquid-liquid phase separation (LLPS) in response to osmotic stress conditions. ALBA4 directly binds to stress-inducible genes, including NCED3, and suppresses their stress inducibility. Our results demonstrate how plants respond to osmotic stress at early timepoints before ABA biosynthesis through condensate formation as osmo-sensors.

The TOR kinase is an important and conserved signaling hub in plants, as in other eukaryotes. However, the identification of the TOR pathway components and regulators in plants is still fragmentary. Using a genetic screen based on altered sensitivity to TOR inhibitors, we have selected an Arabidopsis mutant that develops leaf ectopic cell clusters of enlarged cells in a TOR-inhibition dependent manner. We have named this mutant loki (Localized growth depending on TOR Kinase Inhibition) and identified the causal mutation in a gene coding for the Arabidopsis homolog of the yeast Rav1 protein. This protein serves as the scaffold for the RAVE complex (Regulator of the ATPase of Vacuolar and Endosomal membranes), which regulates the V-ATPase activity in yeasts and animals. The overall V-ATPase activity is decreased in loki mutants and consistently the endosomal pH is increased. However, the vacuolar pH was found to be unaffected by this mutation. Interestingly, the det3 mutant, which is affected in the C subunit of the V-ATPase, also develops similar cell clusters. Finally, transcriptomic and metabolic analyses revealed that many pathways are affected by both the loki and det3 mutations, including cell wall integrity. This study establishes a new connection between the V-ATPase and the central TOR kinase in plants.

In Arabidopsis thaliana, epigenetic changes in the DNA methylome can prime transcriptional responses to biotic and abiotic stress, resulting in enhanced resistance. Epigenetic recombinant inbred lines have further enabled the identification of trans-generationally stable epialleles controlling stress resistance without adverse effects on plant growth, highlighting their potential for crop improvement. Unfortunately, extending these approaches to crops has remained largely unsuccessful due to differences in genome architecture. The rice (Oryza sativa) genome consists for [~]40% of transposable elements and other epigenetically regulated repeat sequences. Therefore, perturbation of DNA methylation typically leads to severe developmental defects, sterility or lethality, which precludes the use of methylome engineering strategies for epiallele mapping and crop improvement. Here, we exploit an inducible system to introduce for the first time widespread epigenetic variation in rice without detrimental phenotypic consequences. We combined the A. thaliana DNA demethylase AtROS1 with the {beta}-estradiol-activated XVE cassette (XVE:AtROS1-YFP) to enable transient DNA demethylation during early development. Induction of the construct in transgenic Nipponbare seedlings yielded genome-wide changes in the DNA methylome which persisted for at least one generation. Strikingly, these methylome changes did not cause developmental defects or reduced seed yield, but instead correlated with enhanced resistance against Xanthomonas oryzae, the causal agent of bacterial leaf blight in rice. Our study demonstrates that controlled, transient DNA demethylation can uncouple epigenetic variation from deleterious phenotypes in rice. This approach provides a practical framework for generating epigenetic mapping populations and opens new avenues for harnessing epigenetic variation in crop improvement.

Elevated temperatures compromise plant immunity and increase susceptibility to bacterial pathogens through extensive reprogramming of hormone signaling pathways. Although autophagy contributes to both stress adaptation and pathogen defense, its role in hormone-dependent immune regulation under warm conditions remains unclear. Here, we investigated the contribution of NBR1 (NEIGHBOR OF BRCA1 GENE 1)-mediated selective autophagy to Arabidopsis immunity against Pseudomonas cannabina pv. alisalensis at elevated temperature. Bacterial infection under warming enhanced autophagic flux and promoted NBR1 turnover, indicating increased autophagic activity. Analysis of atg5 and nbr1 mutants, and NBR1-overexpressing lines, demonstrated that both core autophagy and NBR1-mediated selective autophagy contribute to bacterial immunity under warm conditions. Hormone and gene expression analyses indicated that NBR1 negatively regulates abscisic acid (ABA)-associated transcriptional responses during infection, while salicylic acid signaling was largely unaffected. Mechanistically, NBR1 physically associated with the ABA-responsive transcription factor ABI5 (ABA INSENSITIVE 5) and promoted its autophagy-dependent turnover in planta. ABI5 turnover was strongly reduced under warm conditions, leading to its accumulation in nbr1 and atg5 plants. Consistent with a functional role for ABI5 in this phenotype, genetic disruption of ABI5 largely reversed the increased susceptibility of nbr1 mutants at elevated temperature, whereas ABI5 overexpression increased susceptibility to bacterial infection. Together, our results identify NBR1-mediated selective autophagy as a regulatory mechanism that restrains ABA-associated susceptibility through the autophagy-dependent turnover of ABI5. These findings reveal a previously unrecognized connection between selective autophagy and ABA-dependent immune regulation and identify NBR1-mediated ABI5 turnover as a temperature-dependent mechanism that prevents stronger bacterial susceptibility under warm conditions.