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.

In nature, plants are subjected to multiple environmental stress factors simultaneously or sequentially. Recent studies revealed that when three or more stress factors impact a plant simultaneously (termed multifactorial stress combination; MFSC), plant survival declines, even if the intensity of each individual stress involved in the MFSC is low. We previously identified RAP2.3 as a key transcription factor (TF) required for Arabidopsis thaliana survival, specifically under a MFSC of salt+excess light+heat stress (i.e., S+EL+HS). Here we report that RAP2.3 is required for the expression of SIGMA3, a nuclear-encoded factor that directs plastid RNA polymerase to specific plastid promoters, and MYB51, a key stress response TF involved in glucosinolate metabolism and oxidative stress responses, specifically during a MFSC of S+EL+HS. Like rap2.3 mutants, myb51 and sig3 mutants display significantly low survival rate specifically under the MFSC of S+EL+HS. Based on MYB51 gene regulatory network analysis and characterization of jasmonic acid (JA) mutants, we further reveal that suppression of JA signaling could play an important role in promoting plant survival under conditions of S+EL+HS. Our findings uncover an additional layer of the response of plants to MFSC, as well as identify potential targets for breeding crops with enhanced tolerance to climate change.

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.

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.

Iron (Fe) is an essential micronutrient for plant growth, and the hormone auxin is a key regulator of developmental processes, including root gravitropism. Here, we investigated the molecular mechanisms underlying the crosstalk between iron nutrition and auxin-mediated root growth in Arabidopsis thaliana. Phenotypic analysis revealed that iron deficiency strongly shaped root system architecture and root gravitropism, and these phenotypes were exacerbated in the iron uptake mutant irt1-1. Genetic analysis revealed that iron deficiency did not aggravate the gravitropic defect of the pin2 mutant, eir1-4, suggesting that iron availability modulates root gravitropism through a PIN2-dependent pathway. Further transcriptomic analysis confirmed that iron deficiency significantly altered the expression of numerous genes related to the auxin pathway, providing molecular evidence for the observed physiological connection. Collectively, this study revealed that iron availability regulates root gravitropic growth by modulating PIN-mediated auxin transport and distribution, providing insights into how plants integrate nutritional cues with developmental programs. Graphical abstract A brief descriptionIron modulates auxin transport and root tip distribution by regulating PIN2 protein, thereby mediating root gravitropism in Arabidopsis. Public summaryO_LIIron nutrition specifically regulates root gravitropism and architecture in Arabidopsis. C_LIO_LIIron deficiency disrupts local auxin homeostasis in root tips and impairs asymmetric distribution in response to gravity. C_LIO_LIIron deficiency stress significantly reduces the abundance of PIN2 protein in root tip cells and disrupts its polar localization pattern on the plasma membrane, thereby precisely modulating polar auxin transport by interfering with the vesicle trafficking and recycling efficiency of PIN2. C_LIO_LIRNA-seq results showed that iron deficiency induced differential expression of multiple auxin-related genes, indicating that iron nutrition affects root development through the auxin pathway. C_LI

Quantitative genetic approaches such as genome-wide association studies and genomic prediction are widely used to identify favourable genetic variation, but they have limited resolution due to linkage disequilibrium. Comparative genomics approaches, especially Protein Language Models (PLMs), have emerged as powerful alternatives, by detecting phylogenetic residue conservation (PRC) across evolutionary time scales. However, the extent to which these tools can guide the detection of impactful variants for field agronomic traits is still unclear. In this study, we used the pre-trained PLM ESM2 to predict PRC scores of nonsynonymous mutations segregating within a diverse panel of 387 accessions in sorghum (SAP). The distribution of fitness effects (DFE) of the same set of nonsynonymous mutations was inferred using unfolded site frequency spectra to assess whether the DFE distribution covaried with PRC scores. Furthermore, we estimated the load of putatively nonneutral mutations of SAP accessions and evaluated associations between this mutation load and phenotypic performance across multiple agronomic traits. Our results show that ESM2 can detect mutations associated with fitness-enhancing effects in SAP, as indicated by enrichments in positive selection signatures among the variants with positive PRC scores. Significant associations were also detected between phenotypic performance and mutation load for several agronomic traits, indicating that PLMs can identify functionally important genetic variation. However, these signals were not consistent across all traits in the SAP population. Altogether, our findings suggest that large language models may support breeding efforts, as PLM predictions covaried with fitness effects and captured agronomic performance for some traits in plant populations.

Grain legumes such as pea (Pisum sativum L.) accumulate large amounts of seed storage proteins without nitrogen fertilization due to their symbiosis with nitrogen-fixing bacteria, making them a key source of plant-based proteins. Seed growth and the accumulation of seed storage proteins are tightly regulated by complex gene networks; however, the mechanisms governing these processes in pea remain poorly understood. In this study, we generated a comprehensive seed expression atlas covering six developmental stages in pea (cv Cameor), including the key transition stage from embryogenesis to early seed filling, providing a detailed temporal resolution of transcriptional dynamics throughout seed development in this species. Co-expression network analysis highlighted several candidate transcription factors potentially involved in the transition towards seed filling. Among them, we characterized the seed-specific NF-YB transcription factor PsLEC1-like (PsL1L), the major LEC1-type factor expressed during early pea seed development. Functional analyses using TILLING mutants demonstrated that loss of PsL1L function reduces seed size and seed nitrogen content and impairs early embryo growth from the end of embryogenesis. Finally, we show that the expression of the B3-domain transcription factor PsFUS3, but not that of PsLEC2 or PsABI3, is reduced in the loss-of-function l1l mutant, suggesting that PsL1L acts upstream of PsFUS3 to control seed size.

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.

The Nucleotide-Binding Domain Leucine-Rich Repeat (NLR) gene family is a central component of plant immune systems, yet its diversity and evolutionary dynamics remain poorly characterized in long-lived tree species. Here, we performed a genome-wide analysis of the NLR gene family in Quercus suber (cork oak) using InterNLR, a new annotation tool, and explored their expression regulation in response to biotic and abiotic stresses. A total of 918 NLR and NLR-like genes were identified, encompassing both canonical and non-canonical members. Phylogenetic analyses based on the NB-ARC domain highlighted the distinct evolutionary trajectory of RNL proteins, which function as helper NLRs and show evidence of clade-specific gene duplication. Transcriptomic analyses revealed pronounced tissue-specific expression patterns, with RNLs exhibiting significantly elevated expression in xylem, suggesting a specialized role in this tissue. Under drought stress, seven NLR genes were differentially expressed and shared orthology with known abiotic stress-responsive genes. Notably, a CNL gene (LOC111996439) responded to both biotic and abiotic stresses, indicating a potential role as an integrative regulator of early defence responses, while an ADR1 orthologue (LOC112022539) suggests molecular crosstalk between stress signaling pathways. Population genetic analyses further revealed signatures of both positive and balancing selection acting on NLR genes. Together, these results provide new insights into the evolution, expression, and functional diversification of NLRs in cork oak. This work advances our understanding of immune gene architecture in an ecologically and economically important forest tree species.

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.

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

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).

Climate change poses an increasing threat to the cultivation of deciduous fruit trees, placing greater demands on modern pear breeding. Using pear germplasm adapted to diverse environments, we assembled 11 chromosome-level genomes. In combination with 13 publicly accessible pear genomes, we analyzed presence-absence variations (PAVs) and constructed a graph-based pangenome for pear. By performing a PAV-eQTL analysis of the fruit of 123 pear accessions, we identified PAVs significantly associated with expression levels of genes that may be involved in regulating agronomic traits. Population analysis of 268 pear accessions revealed two stop-gained variants in DAM1 of independent origin, which may function in advancing the blooming date and reducing the chilling requirement. We detected complex PAVs at the NOR1 locus, including two copy-number variations and one deletion. These PAVs contributed to the rapid diversification of the NOR1 locus and the fruit development period through regulating ARF5 and other ripening-related genes. We revealed the selection history of the NOR1 locus and developed novel pear individuals that accumulated alleles for low chilling requirement, early blooming date, and short fruit development period. The results provide valuable resources for pear genomics research and offer a guideline for breeding modern pears with climate resilience.

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.

Plants are continuously exposed to a myriad of DNA-damaging agents, including environmental cues such as sunlight. At the cellular level, plants respond to DNA damage by activating DNA damage response (DDR) pathways, in which chromatin remodelers play an important role. Among them, the evolutionary conserved INO80 complex (INO80c) has been shown in Arabidopsis to play a key role in DDR, notably by positively regulating Homologous Recombination (HR). Arabidopsis EIN6 ENHANCER (EEN) is the homolog of Yeast INO EIGHTY SUBUNIT 6 and interacts with the N-terminal region of INO80 in the INO80c. Using plant phenotyping, cellular and molecular biology, and third-generation sequencing technology we investigated how INO80 and EEN regulate plant development and genome integrity. We uncovered new roles for INO80 and EEN in plant growth and for INO80 in fine tuning endoreduplication. In addition, linear genome analysis revealed an important and unexpected function for the INO80-EEN complex in preventing Protein Coding Genes (PCGs) from structural rearrangements in somatic tissue and upon exposure to UV-B. Therefore, our results shed new light on the previously overlooked roles of INO80 and EEN in protecting genome integrity at PCGs.

Durable resistance to soil-borne pathogens remains elusive in cereals, partly because susceptibility (S) genes that facilitate root infection have not been identified in monocots. In the model legume Medicago truncatula, the SCAR/WAVE complex member MtAPI functions as a root S-gene for microbial invasion. Whether SCAR gene associated susceptibility function is conserved in monocots, and whether SCAR gene inactivation can enhance root resistance in cereals, remains unknown. Here, we identify and characterize three SCAR genes in barley: HvSCAR-A, HvSCAR-B, and HvSCAR-C. Cross-species complementation assays indicate that HvSCAR-B and HvSCAR-C are functionally similar to MtAPI. While hscar-b and hvscar-c single mutants exhibited no major growth defects, hvscar-a mutants showed strongly reduced seed production, and a hvscar-b/c double mutant displayed shorter root hairs. Notably, the hvscar-b/c double mutant exhibited increased resistance to the hemibiotrophic pathogen Phytophthora palmivora but greater colonization by the symbiotic arbuscular mycorrhizal fungus Funneliformis mosseae, underscoring a complex role in plant root - microbe interactions. Our findings reveal a conserved susceptibility function of SCAR genes in monocots and identify api monocot homologs as promising targets for engineering disease resistance in cereals. This study offers new insights into SCAR protein functional diversification and its potential for improving root health in crop plants.

Translation initiation factors of the eIF4E family play a crucial role in regulating translation and the cellular metabolism of mRNAs. Research has addressed the role of canonical 4E family isoforms in development, stress response, and during viral infection. Nevertheless, the class-II eIF4E family member cap-binding protein 4EHP (nCBP), has remained poorly characterized in plant stress responses. In this study, we show that loss of 4EHP confers enhanced basal and acquired thermotolerance and causes a mild flowering delay without major root defects. Under heat stress, 4EHP-GFP re-localizes from a diffuse cytosolic pattern to cytoplasmatic foci, co-localizing with canonical stress granule (SG) markers. Transcriptomic analysis under control, acclimation and heat stress conditions reveals that 4EHP limits the accumulation of heat-responsive mRNAs, especially those encoding heat shock proteins (HSPs), which remain constitutively expressed in 4ehp-1 mutant under control and heat stress conditions. Proteomic analysis also indicates that the absence of 4EHP alters the repertoire of HSPs compared with wild type (Col-0), especially upon heat stress, without significantly impairing the recruitment of corresponding mRNAs to translationally active polysomes. Together, our results indicate that 4EHP negatively modulates the accumulation of a specific subset of heat-responsive mRNAs fine-tuning chaperone production, via heat-responsive SG regulatory pathways.

Concerns over climate change have intensified the demand for stress resistant crops like hybrid wheatgrass (HWG; Elymus hoffmannii, StStStStHH), a perennial forage species known for its exceptional salt and drought tolerance. However, hexaploidy and high heterozygosity have complicated efforts to resolve its genomic structure and evolutionary history. Here, we present high-quality, haplotype-resolved, chromosome-level genome assemblies for HWG (CDC Saltking) and its putative progenitor, bluebunch wheatgrass (Pseudoroegneria spicata). By integrating PacBio HiFi and ultra-long Oxford Nanopore sequencing with Hi-C scaffolding, we assembled the 10.7 Gb HWG genome into 21 pseudochromosomes per haplotype. Our phylogenomic analysis redefines the origin of the H subgenome, positioning it as an intermediate between Old-World Hordeum marinum (sea barley) and Hordeum brevisubulatum. Notably, we identified significant chromosomal rearrangements, including a unique duplication on St chromosome 4. Transcriptome analysis across multiple tissues revealed a pronounced expression dominance of the H subgenome. This dominance was not associated with reduced LTR density, suggesting that selective pressures for rapid adaptation of the latest subgenome entrant may drive its dominance. Finally, using the f-branch statistic, population genomic analysis of 189 accessions representing eight Elymus and Pseudoroegneria species revealed extensive reticulate evolutionary relationships and identified P. spicata as a major, asymmetric genetic donor within the wheatgrass complex. These resources provide a foundational framework for future genomic research and genetic improvement in grasses and for the introgression of stress-tolerance traits into cereal crops such as wheat. Key MessagesDevelopment of world-first high-quality chromosomal-level haplotype-resolved genome assemblies of hexaploid HWG and diploid progenitor, Pseudoroegneria spicata, enabled the identification of the subgenome origins. This study resolved the evolutionary placement of the St genome and clarified the history of polyploidization and hybridization in HWG. Homeolog expression bias in the H subgenome likely reflects selective pressure favoring greater gene retention and upregulation of functionally important genes, thereby enhancing hybrid fitness. Population structure analysis distinctly differentiates P. spicata, E. repens, E. hoffmannii from other European Pseudoroegneria species. The findings reveal the complex patterns of interspecific gene flow and population dynamics within the Elymus and Pseudoroegneria species.

Fruit development is a typical angiosperm feature that greatly facilitates seed dispersal. Despite extensive studies on the gene regulatory network underlying pod shattering in the dry Arabidopsis fruit and the ripening process in the fleshy tomato fruit, it is yet unclear if a conserved regulatory network acts in early fruit development. Here, we investigated the roles of Petunia x hybrida (petunia) FRUITFULL (FUL), SHATTERPROOF (SHP) and APETALA 2 (AP2) homologs, three types of transcription factors repeatedly associated with fruit development and/or ripening. Petunia is closely related to tomato but produces dry dehiscent fruits like Arabidopsis. Our functional analysis revealed that the three petunia FUL-like genes, PETUNIA FLOWERING GENE (PFG), FLORAL BINDING PROTEIN 26 (FBP26) and FBP29, redundantly regulate endocarp development. They promote the formation of regularly shaped inner endocarp cells, probably via auxin/brassinosteroid signalling and cell wall modification. Furthermore, we discovered that the SHP-like gene FLORAL BINDING PROTEIN 6 (FBP6) has an opposite role, promoting more mesocarp-shaped endocarp cells, indicating that the FUL-like and SHP-like genes act antagonistically in early pericarp development. Finally, we show that the AP2-like genes REPRESSOR OF B-FUNCTION 1 (ROB1), ROB2 and ROB3 are crucial factors in petunia fruit development. rob1 rob2 rob3 mutants completely fail to dehisce and show major defects in pericarp patterning. The ROB transcription factors repress the activity of the FUL-like genes, and have, together with FBP6, an opposite effect on auxin and brassinosteroid signalling genes. Our study suggests that a module consisting of antagonistically acting TFs, including co-orthologs of AP2, FUL and SHP, regulates early pericarp patterning, at least partially via auxin and brassinosteroids.