The Plant Journal

Fagus sylvatica L. (European beech) is a dominant hardwood forest tree species across Central Europe, supporting diverse ecosystem services and forming the basis of a significant market for high-value timber. However, climate change increasingly threatens beech vitality and productivity, making molecular insights into its stress resilience and functional validation of underlying genes urgently needed. Here, we report an improved protocol for protoplast isolation from seedling leaves and demonstrate, for the first time, transient genetic transformation and CRISPR/Cas-mediated genome editing in F. sylvatica. PEG-mediated transformation was sequentially optimized, achieving efficiencies of 59 {+/-} 6.19%. Transformation efficiency was strongly influenced by season, which also affected protoplast yield. Both, a basic molecular toolkit for functional genomics and future biotechnological applications were established by testing a set of promoters and reporters. For proof-of-concept genome editing, we achieved 4.75 to 32.69% editing efficiencies in the PHYTOENE DESATURASE gene (FsPDS) using temperature-tolerant LbCas12a (ttLbCas12a). The establishment of a reliable protoplast transformation and editing system provides a crucial foundation for future genetic improvement and functional studies in this non-model tree species.

Lignin plays a central role in the formation and function of secondary cell walls in vascular plants. However, the structural consequences of lignin modification for cell wall properties and cellular function in grasses remain poorly understood. Here, we investigated how cinnamyl alcohol dehydrogenase (CAD) deficiency alters vascular cell architecture in Sorghum bicolor, using the brown midrib-6 (bmr6) mutant as a model system. Biochemical and histochemical analyses confirmed altered lignin chemistry in bmr6, including increased incorporation of hydroxycinnamaldehyde residues and reduced tricin levels. We applied ptychographic X-ray computed tomography (PXCT) to quantify the cell wall geometry, in three dimensions, at nanometer-scale resolution. PXCT enabled measurements of wall thickness distribution and lumen shape along tracheary elements. Analyses revealed no significant differences in wall thickness between wild-type and bmr6 plants. However, three-dimensional morphometric descriptors indicated reduced lumen convexity in bmr6, suggesting localized modifications not detectable by conventional two-dimensional imaging. Water flow numerical simulations through PXCT-derived images indicated reduced vessel permeability and simulated hydraulic conductivity in bmr6, suggesting that subtle geometric changes may influence performance. These findings highlight the value of three-dimensional imaging for resolving cell wall organization and provide new insight into the architectural resilience of grass xylem in response to targeted lignin modification. HighlightThree-dimensional X-ray nano-imaging reveals alterations in the cell wall architecture that affect simulated hydraulic performance under reduced CAD activity in sorghum.

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.

Sunflower broomrape (Orobanche cumana Wallr.) is a holoparasitic weed that parasitizes sunflower (Helianthus annuus L.) and severely constrains yield. The OrDeb2 gene confers resistance to broomrape race G in the sunflower line DEB2. It has been previously located within a 1.38-Mbp physical region in the H. annuus XRQ reference genome that contains a cluster of receptor-like proteins and receptor-like kinases (RLKs). In this study, an OrDeb2 candidate has been proposed though high-resolution genetic and physical mapping, multi-allelic comparisons across resistant and susceptible lines, and exclusion of other potential candidates based on their function and genetic features. OrDeb2 was delimited to a 219.4-kbp region in the DEB2 genome assembly that contained six annotated protein-coding genes belonging to two groups: three small heat shock proteins (HSP20) and three RLKs. Given that the only sunflower broomrape resistance gene cloned to date encodes an RLK, the key role of RLKs in resistance to biotic stresses in plants, and the similar genetic features between RLK-type resistance genes and OrDeb2 as major dominant genes in gene-for-gene interactions, RLKs in the interval emerged as the most plausible candidates. Within them, the RLK-2 gene encoding a root-expressed full-length kinase-pseudokinase protein belonging to the tandem kinase proteins (TKPs) family was prioritized, because the other two RLKs corresponded to a truncated TKP with a stop codon within the kinase domain. RLK-2 also carried sequence variants unique to DEB2 relative to multiple susceptible genotypes. The results of this research reveal the potential role of TKP genes as new players in resistance to parasitic plants and provide a framework to dissect the genetic dialog between O. cumana and sunflower in gene-for-gene interactions.

Tar spot, caused by the obligate fungus Phyllachora maydis, significantly threatens maize (Zea mays L.) production across the Americas. Host genetic resistance offers the most viable long-term management strategy. Building on observed differential tar spot tolerance in parents B73 and Mo17, we utilized a strategic core subset of 94 intermated B73 x Mo17 (IBM-94) population to characterize the genetic architecture of resistance. Phenotypic analysis of a panel of 96 recombinant inbreds including the susceptible parent, Mo17, and the moderately resistant parent, B73 confirmed stable differences in susceptibility, with B73 consistently demonstrating moderate resistance compared to Mo17. Analysis of variance revealed highly significant genetic variation within the population (F = 12.96; p < 0.001). High Pearson correlation (r = 0.8706, p < 0.0001) and coefficient of determination (R2 = 0.7579) across environments indicated that 76% of the phenotypic variance is attributable to genetic factors. Linkage mapping identified a novel, consistent major QTL cluster on chromosome 1. This cluster comprises five regions (qTAR_1.1 through qTAR_1.5) exceeding the significance threshold (LOD 3.8) in both years. We identified 74 candidate genes including bZIP, and RING/U-box proteins at significant SNP peaks. Additionally, gene annotation revealed a high concentration of wall-associated kinases and S-locus lectin protein kinases within the qTAR_1.4 and qTAR_1.5 regions, alongside potential defense-related transcription factors (MYB, bZIP, and C2H2 zinc fingers).These findings provide a framework for high-resolution mapping and functional validation to accelerate the development of tar spot-resistant maize cultivars.

Pine needles contain two vascular cell types unique to gymnosperms: Transfusion parenchyma (tp) and tracheids (tt). Since they form the only connections between vascular bundles and bundle sheath, we hypothesised that they are involved in regulating the needles water import and photoassimilate export. Synchrotron-based tomography enabled us to quantify volume changes of tp and tt cells in Pinus pinea needles systematically along the needle and throughout a diurnal day cycle, as well as under rehydration. As a physiological indicator of tps carbohydrate status served their starch content. Segmentation of the comprehensive data uncovered dramatic volume changes during dehydration and showed a diurnal course of starch formation and degradation. These changes suggest a yet unknown osmotic water flux between tp and tt, balanced by the formers carbohydrate status. Confirming our hypothesis, excess of photoassimilates in tp cells went into starch synthesis during the day. Starch mobilisation during the night increased the osmotic potential in tp and led to water intake. According to the decreasing starch fraction from base to needle tip, this mechanism is predominant in the upper needle segments, particularly after rehydration of dehydrated needles. Mechanistically, osmolytes in tp cells maintain tension in tt for the needles water import. HighlightSynchrotron tomographic microscopy uncovers diurnal starch fluctuations and osmotic water pumping in inner tissues of pine needles that are utilised at night and when recovering from dehydration

Pathogen pressure threatens legume crop productivity worldwide. Nucleotide-binding leucine-rich repeat (NLR) immune receptors serve as crucial plant resistance genes, recognizing pathogens and triggering immunity. However, the extent and patterns of NLR expression in different tissues and organs, notably across evolutionary time, remain largely uncharacterized. To investigate tissue-specificity of NLR expression in the Fabaceae (legumes), we conducted comparative analyses integrating phylogenomics and transcriptomics in root and shoot tissues across different legume species. The NLR repertoires of 28 legumes were grouped into five monophyletic clades: coiled-coil NLR (CC-NLR), Toll/interleukin-1 receptor NLR (TIR-NLR), G10-subclade CC NLR (CCG10-NLR), RESISTANCE TO POWDERY MILDEW 8-like CC NLR (CCR-NLR), and TIR-NB-ARC-like {beta}-propeller WD40/tetratricopeptide repeats (TNPs). Most legume NLRs belonged to CC-NLR and TIR-NLR clades, followed by CCG10-NLR, CCR-NLR, and TNP clades. In seven of these species, comparative analysis of NLR expression in leaves versus roots revealed that over half ([~]57%) of expressed NLR genes showed predominant expression in one tissue: 34% in roots (451/1336), and 23% in leaves (311/1336). We identified 324 root-specific NLRs, 171 leaf-specific NLRs, and 841 non-specific NLRs, with an average tissue specificity per species of 32%. The closely related species grass pea (Lathyrus sativus) and pea (Pisum sativum) were an exception, showing higher levels of leaf-specific rather than root-specific NLR expression. We also identified conserved tissue expression patterns across legume species, resulting in a comprehensive resource describing tissue expression bias, enrichment, and specificity for 113 phylogenetic NLR subclasses. These legume NLR repertoires will support comparative studies between species and inform precision-breeding programs considering tissue expression patterns.

Fitness costs of plant disease defence are often subtle and difficult to quantify. In this study, we therefore used comparative high-throughput phenotyping in two independent facilities to assess growth, morphology and physiology of potato (cv. Desiree) with high time-resolution monitoring different defence mechanisms under pathogen-free conditions. Plants were either treated weekly with the resistance inducers {beta}-aminobutyric acid (BABA; 10 mM) or potassium phosphite (KPhi; 36 mM) or comprised six transgenic lines expressing late blight resistance genes (single Rpi genes or a three-gene stack) or reduced jasmonate perception (StCOI1-RNAi). Over four weeks, image-derived traits revealed consistent cross-facility effects for plant height and colour: BABA treatment increased plant height but reduced canopy area and induced a paler greenness signature, whereas KPhi caused minimal and transient growth effects. Chlorophyll fluorescence at the NaPPI facility indicated reduced vitality (Rfd_Lss) in BABA-treated plants and increased Rfd_Lss following KPhi, while maximum PSII efficiency was largely unchanged. Several transgenic lines showed somewhat reduced above-ground biomass. Enzyme activity profiling produced distinct treatment and genotype signatures, but was strongly modulated by facility conditions that overrode these specificities. Overall, high-throughput phenotyping robustly detected subtle growth-defence trade-offs across platforms. HighlightHigh-throughput optical phenotyping validated across two independent research facilities reveals that stacked resistance genes and resistance inducers in potato trigger subtle growth trade-offs. Graphical abstracts O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/713143v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@89df47org.highwire.dtl.DTLVardef@1a1ce64org.highwire.dtl.DTLVardef@1f52f0dorg.highwire.dtl.DTLVardef@1e41c35_HPS_FORMAT_FIGEXP M_FIG C_FIG Experimental timeline for high-throughput plant phenotyping platforms. Created in BioRender. Poque, S. (2026) https://BioRender.com/nmkve7g

Chromatin organization regulates genome stability and gene expression by controlling DNA accessibility to transcription factors and regulatory complexes. DNA-protein interactions are commonly investigated using chromatin immunoprecipitation (ChIP), which relies on specific antibodies often involving technically demanding protocols. CRISPR-Cas technologies have enabled sequence-specific targeting of genomic loci using catalytically inactive Cas9 (dCas9), but most CRISPR-based chromatin capture approaches in plants require transient or stable transformation to express the CRISPR machinery, limiting their applicability across species, tissues and physiological contexts. Here, we present GRASP (Genomic Region Affinity Sequestration by CRISPR-Purification), a transformation-independent strategy for sequence-specific chromatin isolation operating directly on purified plant nuclei. In GRASP, dCas9-gRNA ribonucleoprotein complexes are used to capture predefined genomic regions from chromatin under native conditions, bypassing the need for transgene expression. Using grapevine and tomato as model systems, we demonstrate efficient and highly specific enrichment of target loci, including telomeric repeats as well as low-copy and single-copy genomic regions, with qPCR and NGS validation. These results establish GRASP as a robust and broadly applicable platform for locus-specific chromatin isolation in plants. Beyond sequence-specific DNA isolation, GRASP establishes a versatile platform for potential downstream analyses of locus-associated chromatin components, including protein complexes, distal DNA-DNA interactions and chromatin-associated RNAs, providing new opportunities to investigate regulatory architecture in plant genomes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/712347v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@13758e8org.highwire.dtl.DTLVardef@adfd82org.highwire.dtl.DTLVardef@de81f4org.highwire.dtl.DTLVardef@25c2d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

O_LIUnderstanding how plant populations respond to environmental variation through functional leaf traits remains challenging due to limitations of traditional phenotyping approaches. Hyperspectral reflectance offers a rapid, non-destructive and high-throughput method to capture functional trait variation and detect signatures of local adaptation across populations. C_LIO_LIWe combined hyperspectral data, inverse modeling, and network analysis to investigate population-level variation in Streptanthus tortuosus. Using a common garden experiment with four geographically distinct populations, we applied partial least square discriminant analysis (PLS-DA) and ridge regression for population discrimination, inverse PROSPECT modeling to estimate leaf biochemical traits, and canonical correlation analysis to examine trait-climate relationships across historical (1900-1994) and recent (1995-2024) periods. We developed a spectral network approach treating wavelength correlations as biologically meaningful trait networks. C_LIO_LIPopulations showed distinct, heritable spectral signatures with high classification accuracy. Significant population differences emerged in anthocyanins, carotenoids, chlorophyll, and water content. Trait-climate correlations shifted between time periods, consistent with historical climate adaptation. Network analysis revealed population-specific integration patterns, with more variable environments displaying greater spectral modularity. C_LIO_LIHyperspectral signatures provide a high-throughput tool for detecting population-level adaptation and trait coordination. Our findings provide a framework to investigate how plant populations respond to climate change through evolved shifts in trait networks rather than isolated traits alone. C_LI

Long-read sequencing has shown a rapid technological development during the last years. It has been established as the standard method for the sequencing of plant genomes and has also gained importance for full plasmid sequencing. As Sanger sequencing has a limited read length of about 1 kb, long read sequencing offers a great advantage, as the full plasmid can be sequenced in one read. Here, we present a cost-effective workflow to sequence full plasmids and compare the results against an expectation. The per plasmid cost of this workflow is determined by the number of plasmids investigated simultaneously, but can be lower than the price of a single Sanger sequencing reaction. We developed a workflow for automatic data processing, which allows us to complete sequencing and data analysis within a day.

The Multiple Synthetic Derivatives (MSD) population is a unique hexaploid wheat resource that captures extensive genetic diversity from Aegilops tauschii and exhibits wide variation in agronomic traits. However, root system architecture (RSA), a key determinant of resource acquisition and stress adaptation, remains poorly characterized in this population. Here, we established a practical phenotyping framework for RSA analysis and evaluated MSD417 as a representative genotype. A two-dimensional cultivation platform enabling continuous imaging of seedling root growth under controlled conditions was established to quantify RSA traits and their responses to high temperatures. MSD417 was compared with its recurrent parent, Norin 61 (N61). Under controlled conditions, MSD417 displayed greater total root length, root system width, and convex hull area than N61, indicating enhanced early root vigor. This genotype also exhibited a wider seminal root angle, suggesting improved horizontal soil exploration while maintaining root depth. High-temperature treatment reduced overall root growth and minimized genotypic differences, indicating that temperature stress constrains RSA expression. Microscopic observations further revealed a lower height-to-width ratio of coleorhiza tissue of MSD417, suggesting restricted downward expansion. Collectively, this study establishes a practical framework for RSA phenotyping and demonstrates the potential of Aegilops tauschii-derived germplasm to enhance wheat root-related adaptive traits.

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.

Phenylpropanoids are L-phenylalanine (Phe)-derived specialized metabolites with crucial roles in plant stress adaptation. Among various abiotic stresses, high-light (HL) is a major threat that leads to oxidative damage and therefore upregulates the biosynthesis of antioxidant phenylpropanoids, including anthocyanins, in plants. Phenylpropanoid production is initiated by the conversion of Phe by phenylalanine ammonia-lyase (PAL) enzyme, which acts as the key gatekeeper controlling carbon influx from the Phe pool into phenylpropanoid biosynthesis. Despite the importance of Phe as a precursor for phenylpropanoid production, we have limited knowledge of how Phe precursor availability influences downstream pathways, particularly in the context of stress adaptation. To tackle this question, Arabidopsis thaliana wild-type and production of anthocyanin pigment 1 dominant (pap1D) mutant, a line genetically enhanced for anthocyanin production, were subjected to HL treatment, followed by proteome and metabolome analyses. Our multi-omics data indicated that Phe biosynthesis was insufficiently responsive to meet the increased precursor demand from the downstream phenylpropanoid pathway, likely making Phe availability a rate-limiting factor for HL-induced phenylpropanoid production. We then generated pap1D double mutants with Phe-overaccumulating mutants. Untargeted metabolomics revealed that enhanced Phe availability had little impact on phenylpropanoid accumulation under standard growth conditions but additively promoted the accumulation of specific phenylpropanoid intermediates and anthocyanin species under HL stress. This metabolic difference between light treatment and genotypes was correlated with the activation of PAL enzymatic activity. This study demonstrates that enhanced Phe biosynthesis amplifies HL-induced phenylpropanoid biosynthesis.

T-DNA insertion mutants are widely used to disrupt genes and infer their functions, yet the insertions can also trigger unintended genomic changes that confound phenotypic interpretation. Here, we used T-DNA insertion mutants affecting the major mitochondrial malate dehydrogenase (MDH1) and the heterodimeric NAD-dependent malic enzymes (ME1 and ME2) to examine their functional coordination across photoperiods and irradiance regimes. Under short days, especially at low light intensity, mdh1xme2 mutants were markedly smaller than wild type and, unexpectedly, than the mdh1xme1xme2 triple mutant, and they showed a more pronounced reduction in photosynthetic capacity. ME1 was undetectable in mdh1xme2, implying that the double and triple mutants effectively lack heterodimeric ME and should therefore behave similarly, contrary to what we observed. Whole-genome analysis resolved this discrepancy by revealing that the MDH1 T-DNA insertion in mdh1xme2 is accompanied by a major rearrangement, a 137-kbp duplication downstream of the insertion site, which was absent in the mdh1xme1xme2 triple mutant. This duplication increased gene dosage and elevated transcript abundance across the duplicated interval, while proteomics detected 5 of the 38 encoded proteins, including PEPC1. mdh1xme2 accumulated oxaloacetate-derived amino acids and displayed an altered carbon/nitrogen balance, making PEPC1 a plausible contributor to the exacerbated mdh1xme2 phenotype. Together, our data indicate that a T-DNA-linked structural variant can amplify expression of dozens of genes and intensify phenotypes at specific conditions, thereby affecting the interpretation of genotype-phenotype relationships. Because Agrobacterium-mediated DNA transfer also underpins many genome-editing workflows, our findings argue that structural validation around insertion/editing loci should be considered essential when interpreting T-DNA-derived plant lines.

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

Fusion of gametes possessing meiotically reduced (haploid) chromosome complements is the main pathway of propagation among eukaryotes. However, duckweeds, the smallest angiosperms, propagate mainly vegetatively, and meiosis has not yet been documented in detail for this plant family. The more surprising was the recent evidence of rather frequent interspecific hybrids and triploid clonal accessions which became obvious by genome size measurements, genomic in situ hybridization (GISH) and combined plastid and nuclear DNA markers. These observations indicated sexual propagation involving reduced as well as unreduced male and female gametes in Lemna minor and L. turionifera leading to allodiploid and allotriploid hybrids (MT, MMT, MTT) and autotriploid L. minor (MMM) accessions. Here, we i) documented the meiotic stages of Lemna species for the first time; ii) provided evidence of unreduced male gametes through fluorescent in situ hybridization (FISH) with single locus probes; iii) determined their abundance in different individuals and iv) hypothesized about the reasons of unreduced male gamete formation. These findings open new insights into the modes of sexual reproduction and evolution of duckweeds which may be useful for future breeding efforts in this emerging crop.

Pears (genus Pyrus) are among the most extensively cultivated tree fruits with a wide-reaching economic impact. Despite this, the genetic basis of most pear traits of interest, including abiotic stress tolerance, tree architecture, precocity, parthenocarpy, disease resistance, and fruit ripening, remains poorly understood. Although extensive efforts have been made to identify quantitative trait loci (QTLs) that explain the genetic basis of pear traits, many are poorly transferable, limiting their utility for informing genetic improvement or management of pears across most genetic backgrounds. To provide a whole-genome context and enable the exploration of functional variation in Pyrus, we developed a pangenome graph using 31 accessions representing 23 Pyrus species from the National Clonal Germplasm Repository. Whole-genome sequencing was performed solely with Oxford Nanopore, generating highly contiguous assemblies for pangenome construction, demonstrating the viability of a single-platform approach to pangenomic analysis in Viridiplantae. Exploration of the pangenome graph reveals genes present in some lineages, with potential functional implications. A group of arid-adapted Pyrus species exhibits signs of selective sweeps in regions associated with transcription factors, likely impacting abiotic stress tolerance. With the development of this pangenomic resource, resequencing analysis in Pyrus is now possible without the limitations imposed by single-reference genome assemblies.

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

Self-incompatibility (SI), a reproductive mechanism that prevents self-pollen from fertilizing the ovule, is widespread in flowering plants, including the Brassicaceae family, where it promotes outcrossing, genetic diversity, and hybrid vigor. Although prevalent in Brassica rapa, an economically vital crop, it remains poorly characterized in widely grown varieties, such as toria and yellow sarson, with prior studies primarily focused on Brassica napus. Given its potential for hybrid breeding and crop improvement in rapeseed (B. rapa), we characterized key SI-regulatory genes, analyzing their phylogenetic relationships, structure-function dynamics, and expression patterns. Our results indicate sequence, structural, and functional homology as well as conservation with previously known candidates. This study identifies SRK, FER, and ARC1 as essential, while MLPK plays a minor role in SI for the varieties under study. Furthermore, we identified that SRK, FER, and MLPK activate ROS during the SI response, while ARC1 does not. Our findings establish a foundation for harnessing this natural system to integrate agriculturally important traits and sustain them across generations via outcrossing.