Journal of Integrative Plant Biology

Virus-induced genome editing (VIGE) using compact RNA-guided endonucleases is a transformational new approach in plant biotechnology, enabling tissue-culture-independent and transgene-free genome editing (Hu et al. 2025; Liu et al. 2025; Weiss et al. 2025). We recently established a VIGE approach for heritable editing at single loci in Arabidopsis by delivering the compact genome editor ISYmu1 TnpB (Ymu1) and its guide RNA (gRNA) via Tobacco Rattle Virus (TRV) (Weiss et al. 2025). Here, we greatly improved this approach by devising a multiple gRNA expression system and by utilizing an engineered high-activity Ymu1 variant (Ymu1-WFR) (Zhou et al. 2026) to develop an efficient multiplexed genome editing platform.

Plant immunity relies on the detection of microbes and the rapid activation of intracellular defense pathways. Catalyzed by protein kinases and E3 ubiquitin ligases, respectively, phosphorylation and ubiquitination are among the most abundant post-translational modifications that regulate immune pathways. It has been well established that members of the receptor-like cytoplasmic kinase (RLCK) and plant U-box E3 ligase (PUB) families are critical components of plant immune signaling. Interestingly, a group of proteins that contain both an RLCK domain and a PUB domain has been conserved throughout plant evolution, referred to as subgroups RLCK-IXb and PUB-VI within their respective families. While very little is known about these proteins, evidence from multiple independent studies indicates that orthologous PUB-VI/RLCK-IXb proteins in potato, tomato, Nicotiana benthamiana, and Arabidopsis thaliana associate with diverse pathogen effectors from the oomycete pathogen Phytophthora infestans, bacterial pathogen Ralstonia pseudosolanacearum, and the mirid bug Apolygus lucorum, suggesting that they may be critical virulence targets or components of the immune response. However, the biochemical activities of these proteins and how they contribute to plant health remain poorly defined. Here, we introduce the PUB-VI/RLCK-IXb clade in Arabidopsis, focusing on PUB32, PUB33, and PUB50. We show that PUB33 exhibits dual kinase and E3 ubiquitin ligase activities that are inversely regulated by autophosphorylation at Thr333. PUB33 forms homomers and heteromers with PUB32 which attenuate PUB33 catalytic activity. Although we did not observe clear defects in innate immune signaling in pub32, pub33, or pub50 mutants, we found that overexpression of PUB33 can suppress cell death triggered by the R. pseudosolanacearum effector RipV1 in N. benthamiana. Moreover, PUB33 directly ubiquitinates RipV1 in vitro and reduces RipV1 accumulation in planta, suggesting that it functions as part of the immune response against R. pseudosolanacearum.

The N-degron pathways of ubiquitin mediated proteolysis target proteins for degradation dependent on the amino terminal residue, often produced after endopeptidase activity. Very few substrates have been identified in plants even though enzymes of these pathways are highly conserved in eukaryotes. Here we identify ELONGATED HYPOCOTYL5 (HY5), a master transcriptional regulator involved in many aspects of plant development, as a target for the endopeptidase METACASPASE (MC)9, producing the carboxy-terminal protein fragment (proteoform) E59-HY5. E59-HY5 is shown to be a substrate of the arginyl transferase (ATE) N-degron pathway, and influences physiological processes known to be controlled by HY5, including photomorphogenesis and the unfolded protein response. Conditional stability of E59-HY5 was shown to result from environmentally controlled ATE function, which may highlight a general mechanism for N-degron pathway regulation of proteoform and proteome function during growth and development.

Histone variant H2A.X is a well-conserved histone that plays crucial roles in mediating DNA damage response across eukaryotes. Although H2A.X expresses even without any stress, and decorates gene bodies of actively expressed genes, it is not known if H2A.X has functions beyond DNA damage repair. Using genetic, high throughput genomics and molecular approaches, we identified a previously unappreciated role of H2A.X in regulating development-associated genes. Using custom-made antibodies specific to H2A.X variant, we show that it suppressed the deposition of active H3K4me3 marks over gene bodies and Transposable elements (TE)s, specifically regulating several root development, photosynthesis, and pigmentation-related genes as seen by the impairment of these processes in h2a.x ko (knockout) plants. H2A.X also suppressed global deposition of repressive mark H3K9me2 by restricting activity of H2A variant H2A.W. In agreement with this, there was a genome-wide re-localization of H2A.W to TEs and a few genes in h2a.x ko plants. H2A.X overexpressing plants exhibited stress phenotypes including increased anthocyanin levels, mimicking the transcriptome of DNA damaged wildtype plants. The transcriptome of kd lines of FACT complex, a known chaperone of H2A.X, was largely similar to that of h2a.x ko, suggesting that the development-associated functions of FACT are at least partially due to H2A.X. These results suggest a key role of H2A.X in regulating the competing histone marks and this function might be conserved across plants. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/707635v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1b2fe74org.highwire.dtl.DTLVardef@5fa3c8org.highwire.dtl.DTLVardef@f9b741org.highwire.dtl.DTLVardef@6e1101_HPS_FORMAT_FIGEXP M_FIG C_FIG

Wheat grain weight and flour quality largely depend on starch biosynthesis, yet the mechanisms by which transcription factors coordinate this process remain poorly understood. In this study, using an integrative strategy that combines genome-wide association analysis with yeast two-hybrid library screening, we identify TaNF-YC10, a Nuclear Factor Y transcription factor, as a positive regulator of starch accumulation in the wheat endosperm. Loss of TaNF-YC10 reduces starch content and alters starch granule size distribution, whereas overexpression enhances starch accumulation and increases grain weight. TaNF-YC10 binds and activates core starch biosynthetic-related genes, including AGPL1, GBSS1, YUC11, and NF-YB7, and forms higher-order transcriptional complexes with TaNF-YB1 and TabHLH95 to coordinate multiple regulatory pathways. TaNF-YC10-A1-Hap2 is associated with higher starch content and thousand grain weight and has been selected during wheat breeding in China. Collectively, our findings establish TaNF-YC10 as a pivotal transcriptional hub in starch regulation and highlight its potential as a target for genetic improvement of grain yield in wheat.

O_LIModerate leaf rolling in rice is crucial for plant architecture and stress adaptation, but its molecular regulation remains unclear. We investigated the role of RLC3/OsBLH4, a BELL-type homeobox transcription factor, in controlling leaf rolling and drought tolerance, addressing gaps in lignin biosynthesis and cell wall development mechanisms. C_LIO_LIWe used gene map-based cloning (rlc3-1, rlc3-2), CRISPR/Cas9 knockout lines (rlc3-ko#11, rlc3-ko#12), and allelic complementation to validate RLC3s function. Additionally, we employed biochemical assays, gene expression analysis, and protein interaction studies to explore its regulatory network. C_LIO_LIRLC3 mutations impaired lignin biosynthesis and secondary cell wall formation, reducing bulliform cells area and causing midrib defects. These structural abnormalities accelerated water loss, leading to excessive leaf rolling and compromised drought tolerance. Mechanistically, RLC3 directly activates lignin synthesis genes (OsPAL5, OsCOMT5, OsCCR4, OsCAld5H1) and interacts with KNOX transcription factors (OSH1, OSH45, OSH71) to form a KNOX-BELL complex, further regulating lignin content and cell wall development. C_LIO_LIRLC3 orchestrates lignin deposition and secondary cell wall development to control leaf rolling, water transport, and drought tolerance. This study reveals a novel KNOX-BELL-lignin regulatory module governing leaf morphology and stress adaptation, offering targets for crop improvement under drought conditions. C_LI

Plants employ cell surface receptors to recognize pathogen-associated molecular patterns (PAMPs) and activate pattern-triggered immunity, a crucial defense mechanism against invading pathogens. Pep-13 is a PAMP derived from a class of conserved cell wall transglutaminases present in Phytophthora species, and its receptor PERU was reported recently. In our parallel study, we observed distinct responses to Pep-13 between two diploid potato inbred lines: E454 recognizes Pep-13, whereas A018 does not. Genetic analysis demonstrated that Pep-13 recognition in E454 is controlled by a single genetic locus, tentatively designated TGER (Transglutaminase elicitor response). Through bulked segregant analysis sequencing, followed by complementation assays, we found that the TGERa gene in E454 is essential for Pep-13 recognition. Sequence alignment revealed that TGERa shares 99.91% amino acid sequence identity with PERU, indicating that TGERa and PERU are allelic variants of the same gene (PERU/TGERa). TGERb, a highly homologous gene of TGERa, was identified in the E454 genome; notably, TGERa, but not TGERb, can recognize Pep-13. We further demonstrated that TGERb exhibits defects in both ligand binding and association with the co-receptor StSERK3A. Additionally, we found that the TGERa allele in A018 is a weak allele with reduced expression levels, presumably resulting from a 3 kb DNA fragment insertion in its first intron. Heterologous introduction of TGERa into Nicotiana benthamiana and tomato significantly enhanced their resistance to Phytophthora infestans. Collectively, our findings confirm that PERU/TGERa functions as the Pep-13 receptor in potato and provide a valuable molecular target for improving Phytophthora resistance in plants.

Calcium-dependent protein kinases (CDPKs) decode cellular calcium transients and play diverse roles in plant growth and stress responses, including immunity. In Arabidopsis thaliana (At, Arabidopsis thereafter), AtCPK28 contributes to immune homeostasis by phosphorylating subgroup IV plant U-box proteins AtPUB22/24/25/26, which target the key immune receptor-like cytoplasmic kinase (RLCK) AtBIK1 for turnover. While this module is conserved in multiple angiosperms, it is unclear if the role of CPK28 in immune homeostasis is conserved more broadly across land plants. Here, we took an evolutionary comparative approach to understand the role of CPK28. We identified a single CPK28 ortholog in the liverwort Marchantia polymorpha, MpCPK28, which exhibits Ca2+-dependent kinase activity that is inhibited by calmodulin in vitro. We identified the subgroup IV plant U-box protein MpPUB20e as a substrate of MpCPK28. MpPUB20e is able to ubiquitinate MpPBLa, the functional ortholog of AtBIK1. We also provide preliminary evidence that MpPBLa undergoes proteasomal degradation in Marchantia, suggesting that optimization of MpPBLa protein accumulation is conserved across land plants. Interestingly, while loss of CPK28 function in multiple angiosperm species results in enhanced immune signaling, we find that Marchantia Mpcpk28 mutant alleles do not display enhanced immune-triggered production of reactive oxygen species or resistance to two pathogens. However, transgenic expression of MpCPK28 was able to restore function in Arabidopsis cpk28-1 mutants, suggesting latent functional conservation of MpCPK28. Furthermore, while AtCPK28-mediated phosphorylation of Thr95/94 on AtPUB25/26 is known to contribute to their activation, we could not observe a functional role for the equivalent residue Thr122 on MpPUB20e. Taken together, our results suggest that post-translational fine-tuning by CPK28 is likely to have refined the PUB-BIK1 module in the vascular plant lineages.

O-glycosylation of nucleocytosolic proteins by the Arabidopsis enzymes SPINDLY (SPY; O-fucosyltransferase) and SECRET AGENT (SEC; O-GlcNAc transferase) is essential for plant growth and development, yet the scope of their substrates and regulatory impact remains poorly defined. Here, we combined TurboID-based proximity labeling with quantitative proteomics to systematically map the SPY interactome and determine how SPY- and SEC-dependent modifications influence protein abundance. A functional SPY-TD enriched 221 proxiome proteins, including 80 known O-fucosylated substrates and 141 new interactors. The SPY-TD proxiome is enriched in nuclear pore components, chromatin regulators, transcription factors, and RNA-processing proteins. Integration with O-fucose and O-GlcNAc datasets yielded a comprehensive Arabidopsis SPY/SEC (At-S/S) protein list of 886 candidates. We quantified proteome-wide changes in spy single mutants and inducible spy sec double mutants. Loss of SPY alone caused selective stabilization or destabilization of targets, whereas combined SPY/SEC depletion triggered widespread, synergistic protein abundance changes, particularly affecting nucleoporins, transcriptional regulators, and RNA-binding proteins. Integration with ubiquitination datasets revealed extensive overlap, supporting potential crosstalk between O-fucosylation, O-GlcNAcylation, and ubiquitin-mediated protein turnover. Together, our study establishes proximity labeling as a powerful strategy to define plant O-glycosylation networks and reveals dual, context-dependent roles of SPY and SEC in controlling protein homeostasis and stress-responsive pathways.

Fruit shape is a key horticultural trait shaped by conserved genetic pathways and hormonal interactions, yet the mechanisms underlying shape diversity in fleshy fruits remain incompletely understood. Studies in model species such as Arabidopsis thaliana, tomato, and rice have established Ovate Family Proteins (OFPs) as central regulators of organ morphology through their interactions with brassinosteroid (BR) and gibberellin (GA) pathways and cytoskeleton dynamics. Here, we combine phylogenetic, transcriptomic, and co-expression network analysis to investigate fruit shape regulation in peach and apple, two major Rosaceae crops. We show that flat and oblong phenotypes are associated with distinct OFP expression patterns and with coordinated changes in hormone-related modules, revealing conserved OFP-hormone-cytoskeleton regulatory circuits. Flat shapes were linked to the activation of flat-associated OFPs in the absence of brassinosteroid signalling, whereas oblong shapes were associated with the activation of elongation-related OFPs under brassinosteroid-responsive conditions. Our findings extend current models of fruit morphology by providing species-specific mechanistic insight into OFP-mediated regulation in Rosaceae, offering a refined framework for breeding fruit shape.

The establishment of aphid-plant interaction involves the secretion of a salivary MIF protein. Morphological analyses revealed that aphid MpMIF1 prevents plant cell death, protects organelles from stress, and may promote plant cellular recovery. Co-expression of aphid MpMIF1 and the cell death inducer Npp1 revealed that MpMIF1 modulates autophagy-related genes ATG7/BECLIN1, impair plant senescence regulator ATAF1 and regulate apoptosis-like via Caspase-3-like activity. This effect on multiple-cell death pathways helps to maintain cellular homeostasis during aphid infection. Investigations on DNA Damage Response (DDR) signaling pathways demonstrated that aphid MpMIF1 reduces {gamma}H2A.X phosphorylation, maintains activity of the DNA repair protein RAD51 and stabilizes cell cycle checkpoint expression WEE1 under genotoxic stress. Therefore, MpMIF1 actively participates to the maintenance of a functional DDR. Finally, we showed that aphid MpMIF1 physically interacts with SOG1, a functional analog of animal p53 and central regulator of DDR, cell cycle arrest and programmed cell death in plants. These findings establish MpMIF1 as a key regulator of plant cell death during aphid-plant interactions and highlight its potential as a biotechnological tool for protecting major crops against aphid infection.

Salt stress alters plant development, including the floral transition, but regulation of timing of flowering by salt is poorly understood at the molecular level. To identify genetic loci regulating the floral transition under high soil salinity, we performed a genome-wide association study (GWAS) in Arabidopsis thaliana and identified natural variation at the UGT74E1-UGT74E2-BT3 (UUB) locus that correlates with bolting time specifically in response to salt stress. Genetic analysis revealed BT3 as a novel repressor of the floral transition in control conditions. Similarly, the putative IBA glycosylases UGT74E1 & UGT74E2 delay the floral transition in control conditions. Furthermore, we identified that IBA homeostasis regulators TOB1 and ECH2/IBR10 play a key role in the floral transition, and that ECH2/IBR10 are required for the early flowering phenotype of the ugt74e1/ugt74e2 double mutant, indicating that UGT74E1 & UGT74E2 delay flowering by altering IBA homeostasis. A pangenome analysis of the UUB locus revealed variation in the occurrence of the DNA transposon SAUERKRAUT (SKRT). CRISPR-mediated SKRT deletion in Col-0 affected gene expression both within and outside the UUB locus and caused a salt-dependent delayed floral transition. The delayed bolting phenotype of the skrt-2 mutant also depends on ECH2/IBR10 function, indicating that SKRT accelerates the floral transition by altering IBA homeostasis. Finally, targeted demethylation of SKRT resulted in delayed floral transition under salt stress. Taken together, our data show a role for SKRT and its DNA methylation levels in the salt-dependent bolting time response in Arabidopsis, revealing a novel molecular mechanism to control flowering in adverse conditions.

Plants adapt to recurrent drought through transcriptional memory, yet the upstream regulators remain largely unknown. This study integrated expression genome-wide association study (eGWAS) across 115 Arabidopsis thaliana ecotypes with differential methylome profiling to identify these regulators. Focusing on the memory genes LKR, HIS1-3, and DREB1A, eGWAS identified signaling and epigenetic loci involved in ABA/JA responses and DNA methylation. Methylome profiling by whole-genome bisulfite sequencing of ecotypes contrasting in drought tolerance, as well as in superinduction of memory genes, revealed significantly greater methylation variation during the second drought (D2) than during the first (D1), highlighting the role of epigenetic reprogramming in memory maintenance. Functional validation using T-DNA mutants demonstrated specific modulation of the D2/D1 induction ratio without affecting initial drought responses. Mutants of LKR eGWAS-delineated genes AT1G56660, AT2G19120, AT4G16490, and DEG3, those of HIS1-3 eGWAS genes AT1G14220, AT2G24960, AT3G10845, AT3G19340, CNGC10, EMB2770, GRF7, and RPP2A, and DREB1A eGWAS genes AT1G67000, AT3G61610, AT5G62110, HK2, JMJ12, and LUP5 abolished respective memory gene induction. The eGWAS and methylome approaches converged on DNA repair, chromatin modification, vesicular transport, and proteostasis as core memory hubs. These findings reveal a genetic-epigenetic interplay that coordinates transcriptional memory, priming plants for rapid reactivation of stress pathways during recurrent drought.

AbstractEarly sowing is a key strategy to improve maize productivity and resilience under climate change, but it exposes plants to prolonged chilling stress that can severely compromise seedling establishment. While previous genetic studies have focused on germination or very early stages, tolerance to long-term chilling during the autotrophic transition remains poorly characterized. Here, we combined genome-wide association studies (GWAS) and transcriptome analysis on QTL near-isogenic lines (NILs) to dissect the genetic architecture of early vigor under chilling in maize. We identified a major genomic region on chromosome 4 (LD_COL4), harboring two QTLs within a 2.7 Mb interval, that were consistently associated with early vigor under long-term chilling conditions. Transcriptomic analysis of contrasted NILs revealed a cluster of differentially expressed genes co-localizing with LD_COL4, pointing to two strong candidate genes, Zm00001d048582, an ortholog of the Arabidopsis OPS gene that regulates the brassinosteroid (BR) signaling pathway upstream of the key transcription factors BES1 and BZR1, and Zm00001d048612, a brassinosteroid-signaling kinase (BSK). Multiple orthologs of BES1/BZR1 modulators were differentially expressed between genotypes under chilling, supporting the involvment of brassinosteroid signaling in this response. These findings highlight both genes as promising targets for marker-assisted breeding and gene editing to improve maize adaptation to early sowing.

Understanding heat stress (HS) responses across wheat species with different ploidy is crucial for breeding climate-resilient varieties. We combined field experiments with RNA sequencing to compare diploid (T. monococcum), tetraploid (T. turgidum), and hexaploid (T. aestivum) wheat during early grain filling. Under severe HS, grain yield declined most drastically in the diploid (74%) and substantially in the hexaploid (37.8%), while the tetraploid showed the greatest resilience limiting loss to only 19%. Transcriptome profiling revealed ploidy-associated reprogramming, with the hexaploid exhibiting the largest set of differentially expressed genes (2,227 vs. 859 and 757 in diploid and tetraploid, respectively). Alternative splicing patterns also diverged; notably, we detected species-specific, heat-induced exon skipping of the NF-YB transcription factor exclusively in hexaploid wheat, potentially compromising the transcription factor complex stability. Gene co-expression analysis identified 12 modules linked to grain traits, underscoring the relationship between transcriptional control and phenotype. Together, these results reveal contrasting heat response strategies among the examined genotypes. While the tetraploid genotype displayed the greatest yield resilience coupled with a streamlined transcriptional response, the hexaploid genotype engaged more extensive regulatory networks. These patterns are consistent with ploidy-associated regulatory differences, though genotype-specific factors may also contribute. These insights provide candidates for breeding heat-tolerant wheat varieties and a framework for future multi-genotype studies.

Phtheirospermum japonicum is a genetic model for parasitic Orobanchaceae, a plant family that includes noxious parasitic weeds from the genera Striga, Orobanche, and Phelipanche (Ishida et al., 2011). Striga species alone cause billions of dollars in annual losses by reducing yields of major crops (Pennisi, 2010). The lack of stable transgenesis protocols often hinders heritable CRISPR/Cas9 genome editing for gene function analysis in crops and species beyond standard model plants, including parasitic Orobanchaceae (Steinberger and Voytas, 2025). Here, we adapted a virus-mediated delivery system for ultracompact TnpB nucleases, enabling genome editing independently of tissue regeneration or floral dip transformation in the parasitic plant P. japonicum (Nagalakshmi et al., 2025; Weiss et al., 2025).

Neck blast (NB), caused by Magnaporthe oryzae, damages rice panicles and reduces yield. Knowledge of NB resistance remains limited due to the lack of reliable resistance evaluation methods. Here, we applied a newly established neck injection method and performed a GWAS on 335 diverse accessions from the 3K Rice Genomes Project to identify loci associated with NB resistance. We detected a significant association on chromosome 12, explaining 15-18% of the symptom variations caused by a highly virulent Philippine blast isolate (M64-1-3-9-1). Linkage disequilibrium analysis refined this region to a 42.3-kb interval containing Pita2, a known leaf blast resistance gene. We found that two Pita2 allelic variants, Pita2a and Pita2c, both harboring the variant A/G (Lys879) in the last exon (Chr12:10,833,400), are associated with NB resistance. IR64 and a CO39 near-isogenic line (NIL) IRBLta2-Pi[CO] harboring Pita2a were resistant, whereas CRISPR-Cas9 knock-out of Pita2a in IR64 caused susceptibility to M64-1-3-9-1 and IK81-25. These results indicate that Pita2a is required for NB resistance. Furthermore, the CO39 NIL, IRBLta2-Pi[CO], and Lijiangxintuanheigu monogenic line (IRBLta2-Pi) harboring the Pita2a allele exhibited broad-spectrum resistance to 75% and 80% of Philippine differential blast isolates, respectively. The superior haplotype of Pita2 contains two major SNPs (A/G and A/C at Chr12:10,833,400 and Chr12:10,845,095) occurs in 83% of IRRI elite breeding lines and can be used to select NB-resistant genotypes with an accuracy of 86%. Our findings identify Pita2a as a major gene for NB resistance and provide a valuable genetic resource for developing blast-resistant rice. PLAIN LANGUAGE SUMMARYRice blast, caused by the fungus Magnaporthe oryzae, is a major threat to global rice production. Neck blast (NB) is the most severe type of blast, however, the genetic basis of NB resistance remains poorly understood. In this study, we analyzed 335 rice accessions to identify genes underlying the resistance against a Philippine blast isolate. We found that allelic variants Pita2a and Pita2c are strongly-associated with NB resistance. Knock-out of Pita2a allele made resistant rice plants susceptible while introgression into susceptible rice lines enhanced resistance to multiple blast isolates, confirming its role in NB resistance. Importantly, the superior alleles of Pita2 are already present in 83% of elite breeding lines and can be used to select NB-resistant genotypes with an accuracy of 86%. Our findings clarify the genetic control of NB resistance and offer new tools for protecting rice yields in blast-endemic regions.

Early sowing of maize (Zea mays L.) is increasingly required to mitigate summer drought under climate change, making the acquisition of chilling tolerance a major agronomic challenge. Here, we investigated the molecular and physiological bases of cold tolerance using two maize near-isogenic lines (NILs) differing at two major chilling tolerance quantitative trait loci (QTLs) located on chromosome 4. Plants were exposed to low temperature (14{degrees}C day/10{degrees}C night) for 20 days and analyzed using an integrated multi-omics approach combining transcriptomics, soluble and cell wall proteomics, and metabolomics (primary and specialized metabolites), together with physiological measurements. Univariate and multivariate analyses revealed significant chilling-induced variability across all molecular layers, affecting [~]0.2% of genes, [~]6% of proteins, and a subset of specialized metabolites, while primary metabolites were largely stable. Integrative statistical analyses demonstrated that the soluble and cell wall proteomes contributed most strongly to the genotype effect, highlighting protein-level regulation as a major determinant of chilling tolerance. A restricted 5.15 Mb divergence region on chromosome 4 was sufficient to drive contrasting physiological responses, including differences in photosynthetic charge separation efficiency and leaf development, favoring the chilling-tolerant NIL. Notably, several components of the benzoxazinoid pathway located within the divergence region, including BX1 and associated specialized metabolites (BZX-like glucoside, DIBOA-glucoside-2, HBOA-glucoside-2), were specifically associated with chilling tolerance, suggesting a role in stress signaling and hormonal crosstalk. Overall, this study demonstrates that integrative multi-omics analyses provide a powerful framework to resolve genotype-specific regulatory mechanisms underlying chilling tolerance in maize and to identify candidate molecular targets for breeding. HighlightsO_LIFirst organ-resolved multi-omics dissection of chilling responses in maize NILs. C_LIO_LIA 5.1Mb divergence on chromosome 4 drives major physiological and molecular differences. C_LIO_LIChilling tolerance is linked to more robust photochemical homeostasis and sustained leaf development. C_LIO_LISoluble and cell-wall proteomes dominate the genotype-discriminating -omics signal. C_LIO_LIBenzoxazinoids and defense-related transcriptional modules are differentially activated. C_LIO_LICell wall remodeling enzymes and apoplastic peroxidases emerge as key tolerance players. C_LI

Common bean (Phaseolus vulgaris L.) is one of the most important grain legumes for direct human consumption. Currently, 60% of its production is estimated to be at risk due to drought. However, the genetic basis of common beans drought resistance is poorly understood. To this end, we assessed the genetic architecture of drought-responsive changes in a whole genome-sequenced population of 218 common bean accessions. Using multi-omics-based trait evaluation, including lipidomics, photosynthetic and agronomic traits, followed by multi-omics genome-wide association studies (moGWAS), yielded in the detection of a myriad of moQTL for photosynthesis and yield, as well as the levels of various lipids. QTL associated with glycolipids, which are integral to photosynthesis, since they constitute the major membrane components of chloroplasts, were identified. In addition, we molecularly validated several lipid-related candidate genes via P. vulgaris hairy root transformation as well as transient expression in tobacco. In particular, a lipoxygenase and an allene oxide synthase were identified as explaining the variation in triacylglycerol by oxylipin production. These data provide a blueprint for multi-omics-assisted improvement of crop water stress resilience.

The transition from hulled to free-threshing grain was a pivotal event in wheat domestication, enabling efficient harvesting and processing. Threshability in tetraploid wheat is controlled primarily by the Q locus and two Tenacious glume (Tg) loci on chromosomes 2A and 2B, yet the molecular basis of the major Tg1-B locus remains incompletely characterized. Here, we phenotyped a durum wheat x wild emmer wheat (WEW) recombinant inbred line (RIL) population across two field environments and performed QTL analysis for glume tenacity (TG), threshability ratio (THRR), and seed number per spike (SDNPS). A total of 19 significant QTLs were detected across six chromosomes. The largest-effect loci for both TG and THRR co-localized on chromosome 2B, with LOD scores up to 14.22 and phenotypic variance explained up to 31.2%, corresponding to the previously described Tg1-B locus. To validate this QTL, the donor RIL was backcrossed three times to Svevo to generate a near-isogenic line, NIL-65 (BC3F5), confirmed by whole-genome skim sequencing to carry a homozygous WEW introgression at Tg1-B. A segregating BC4F2 population derived from NIL-65 confirmed that plants homozygous for the dominant Tg1-B allele displayed significantly higher glume tenacity and intact glume morphology compared to tg1-B sister lines, which exhibited basal glume cracking characteristic of the free-threshing phenotype. Genotyping-by-sequencing delimited the causal interval to an approximately 11 Mb introgression on chromosome 2B. These results confirm the major role of Tg1-B in determining glume tenacity in tetraploid wheat, provide a validated near-isogenic germplasm resource, and lay the foundation for fine-mapping and functional characterization of the underlying gene(s).