Plant Cell Reports

Background and AimsPaspalum notatum reproduces through either sexuality or apomixis, two pathways that may coexist within the same individual and are regulated by interconnected molecular networks responsive to environmental cues. Here, we characterized the transcript structure and expression of BUD SITE SELECTION PROTEIN 13 (BUD13), a component of the RES spliceosomal complex previously reported as differentially expressed in florets of sexual and apomictic plants, as a first step toward testing its involvement in the molecular regulation of the apomixis-sexuality switch. MethodsPreviously generated floral and leaf transcriptomes from sexual and apomictic Paspalum notatum plants, including Oxford Nanopore long-read data, were mined to characterize BUD13 transcript structure and expression. Phylogenetic analyses and in silico mapping were conducted to infer evolutionary relationships and determine the origin of the transcripts. Differential expression was validated by RT-qPCR, while in situ hybridization was used to reveal cell-specific ovule expression patterns. Key resultsBUD13 is expressed in Paspalum notatum florets as a truncated isoform (SHORT) encoding a small protein lacking part of the herpes simplex virus regulatory protein (ICP4) domain. Two SHORT transcripts, SHORT1 and SHORT2, with different 5' untranslated region (UTR) regions, were identified in flowers. SHORT1 was consistently upregulated in apomictic ovules from premeiosis to anthesis. Both transcripts originated from a single genomic locus located in the subtelomeric region of the short arm of chromosome 6. SHORT isoforms with variable structures were detected in other monocots. In situ hybridization showed that, whereas BUD13 was expressed throughout sexual ovules, expression was absent from the female germline of apomictic ovules. A consistent expression was observed in somatic proembryos of aposporous embryo sacs. ConclusionsOur findings reveal structural, spatial and temporal divergence in BUD13 expression between sexual and apomictic reproductive programs, providing new insights into the molecular regulation of asexual seed formation.

Plant regeneration is a major determinant of transformation and genome-editing efficiency, yet the endogenous regulatory networks controlling regenerative competence in horticultural crops remain incompletely understood. The miR319-TCP module regulates multiple developmental processes in plants, but its function in lettuce regeneration has not been defined. Here, we performed a genome-wide analysis of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family in lettuce (Lactuca sativa). Thirty-three LsTCP genes were identified and classified into Class I/PCF, Class II/CIN, and Class II/CYC/TB1 groups. Five CIN-class genes, LsTCP2, LsTCP3, LsTCP4, LsTCP10, and LsTCP24, were predicted as high-confidence miR319 targets and supported by degradome-based cleavage evidence. MIR319-overexpression (OX319) explants showed enhanced de novo shoot regeneration, with 94.5% regeneration efficiency and 1.92 shoots per explant, whereas STTM-miR319 suppression (S319) explants showed reduced regeneration, with 28.5% regeneration efficiency and 0.36 shoots per explant. These phenotypes were associated with altered expression of several miR319-targeted CIN-TCP genes, particularly LsTCP4, LsTCP10, and LsTCP24. Disruption of LsTCP4 increased regeneration efficiency to 91.4% and shoot production to 2.05 shoots per explant, resembling the regeneration-enhancing effect of miR319 overexpression. In contrast, disruption of the non-target CIN gene LsTCP17 did not significantly affect regeneration under the tested conditions. Together, these results identify LsTCP4 as a key miR319-responsive negative regulator of de novo shoot regeneration and highlight miR319-mediated repression of LsTCP4 as a potential endogenous strategy for improving lettuce regeneration.

O_LIEngineering nitrogen fixing crops requires not only transferring the nitrogenase structural genes, but also the accessory genes to synthesize its iron-sulphur cofactors. Scaffold protein NifU is a critical element in this system as the starting point of nitrogenase cofactor assembly. NifU has been successfully produced in plants, however, its optimal production required high levels of iron in the medium. This is likely due to a faulty connection with the endogenous iron trafficking network C_LIO_LITo identify specific elements targeting iron to NifU, pull-down assays were performed to identify showing bacterioferritin A (BfrA) as a likely candidate. Co-immunopurification, mutant characterization, iron transfer assays, and co-expression in Nicotiana benthamiana assays were carried out. C_LIO_LIBfrA transfers iron to NifU through protein-protein interactions. When these two proteins were co-expressed in N. benthamiana leaves, there was an increase in NifU production. In turn, it led to doubling NifH synthesis, a nitrogenase structural protein that is also required for the synthesis of the more complex nitrogenase cofactors. C_LIO_LIOur results provide a new element towards engineering nitrogen-fixing crops. They also underscore the importance of transferring the metal delivery systems when expressing metalloproteins in heterologous systems. C_LI

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

In vitro regeneration of Citrus plants is a widely used method, however, induction of adventitious roots from regenerated shoots remains a major bottleneck, limiting the recovery of healthy plants for commercial production and genomic research for crop improvement. We established an in vitro regeneration system producing profuse, healthy roots for sweet orange (Citrus sinensis cv. Benyenda) by optimising combinations and concentrations of auxins. Prior to optimising the rooting media (RTMs), we obtained a shoot regeneration rate of 90.6% from sweet orange epicotyl explants using a cytokinin, 6-benzylaminopurine (BAP). Across twelve auxin-supplemented RTMs containing different concentrations of indole-3-butyric acid (IBA) and/or 1-naphthaleneacetic acid (NAA), rooting percentages ranged from 8 - 87.5%. The combination of IBA 1.0 mg L-1 and NAA 0.1 mg L-1 promoted the best overall performance, 75 {+/-} 7.2% rooting percentage with healthy, callus-free roots ([≥]5 cm in length), whereas other RTMs with other auxin combinations induced callus and limited root elongation. The best-performing SRM and RTM were subsequently used for selection and recovery of transgenic sweet orange lines carrying an empty CRISPR/Cas9 construct, resulting in an 4.8% transformation efficiency. Both transgenic and non-transgenic rooted plantlets were successfully acclimatised under glasshouse conditions with a survival rate of 90%. This enhanced regeneration system overcomes rooting bottleneck and improves plant survival,enabling faster recovery of transgenic citrus lines within four months. It supports accelerated development for commercial applications and advances in citrus genetic improvement.

Ovate Family Proteins (OFPs) are a class of plant-specific, negative nuclear transcriptional regulators characterized by conserved C-terminal OVATE domain. This study on comparative functional characterization of two head-to-head arranged OFPs - AtOFP2 (Ovate-OFP with full ovate domain) and AtOFP17 (Ovate-Like OFP with partial ovate domain) provides critical insight into how structural variations in ovate domain leads to functional divergence. Detailed phenotypic analysis of 28 physical and physiological traits of loss- and gain-of-function mutants revealed that both genes act as broad, pleotropic repressors of plant growth and development. Removal of repression in knock-down mutants of both genes exhibited reduced duration of seed dormancy, faster rate of germination and growth, bigger plants and significantly higher seed yield. In contrast, constitutive over-expression showed a generalized repressive nature of both genes, with nuanced differences for fine tuning of specific traits. For example, both genes showed antagonistic behaviours on root hair architecture. AtOFP2 act as a strong repressor of root hair development whereas AtOFP17 is a stronger repressor of hypocotyl and root cell architecture. AtOFP17 owing to partial ovate domain exerts a mild level of repression throughout life span as indicated by smaller plants and lesser yield in knock-down AtOFP17 mutants. On the contrary, AtOFP2 exerted a much stronger repressor effect in which > 90% over-expression mutants died at the juvenile stage ; the survival of remaining 10% is probably owing to activation of dosage-dependent feedback loop mechanism as indicated by normal growth of mature plants, and is also evident by transcriptome data. Transcriptome analysis of roots of 7-day old seedling of knock-down and over-expression mutants of AtOFP2 showed downregulation of OFP2 in over-expressed mutants. However, severely stunted phenotype indicated presence of stable OFP2 protein to exert effects. Analysis of DEGs in OFP2 mutants revealed that it acts as an important regulator working at intersection of hormonal signalling affecting critical genes required for auxin, cytokinin, GA, BR and ABA functioning. Perturbations across hormonal signalling pathways affects cell wall remodelling factors such as EXPANSINS, Xyloglucan hydrolases (XTHs) and cellulose synthases (CSLs) causing overall stunted growth; and epidermal patterning genes such as WER, GL1, EGL3, TTG1 leading to severely reduced root length and root hairs. Significantly, functional analysis of this master regulator highlighted a significant economic potential. Knockdown of both these genes relieves their natural repression on reproductive traits, leading to longer siliques, bigger and heavier seeds, and substantially increased overall seed yield, positioning AtOFP2 and AtOFP17 as highly valuable targets for agricultural crop improvement.

Trichoderma species are widely used as biological control agents due to their ability to parasitize plant pathogens. However, substantial variability in mycoparasitic performance exists among strains, even within the same species, and the underlying molecular mechanisms remain poorly understood. Here, we performed comparative genomic and transcriptomic analyses of six Trichoderma atroviride strains exhibiting contrasting mycoparasitic performance (weakly or highly parasitic; WP or HP) against Alternaria brassicicola, Rhizoctonia solani, and Globisporangium ultimum. Comparative genomics revealed limited strain-specific differences, mainly restricted to NLR (NOD-like receptor) repertoires, with certain NLR-coding genes absent from WP strain genomes compared to HP strains, while overall genomic variation remained low. In contrast, transcriptomic analyses revealed strong differences in gene expression dynamics between HP and WP strains. Co-expression network analysis identified two modules associated with mycoparasitic performance. The first was specifically induced in response to pathogen contact and was enriched in genes encoding cell wall-degrading enzymes, with stronger expression in HP strains. The second module was more broadly overexpressed in HP strains across all conditions and included genes involved in detoxification and defense-related pathways. In addition, this module encompassed genes involved in specialized metabolite biosynthesis and effector-like protein secretion, with WP and HP strains differentially expressing distinct gene subsets within these categories. Together, these results provide a comprehensive framework for identifying the molecular drivers of mycoparasitic performance in T. atroviride. This study deepens our understanding of the functional diversity within the species and establishes a robust foundation for the future development of molecular markers to predict strain efficiency.

Potato leafroll virus (PLRV) is an economically important viral disease of potato (S. tuberosum). Genetic resistance to this phloem-limited virus is rare, and no cloned resistance (R) genes have been reported. Rladg confers resistance to PLRV in an Andean potato landrace, LOP-868 (Velasquez et al. 2007). We identified the functional Rladg gene as a homolog of the tomato TIR-NLR-encoding Bs4. Rladg interacts with the serine protease domain of the PLRV protein P1, which is essential for virus replication. This recognition is independent of the proteases enzymatic activity, and the Rladg immune receptor oligomerizes upon direct association with the protease. Like PLRV, many poleroviruses contain a serine protease. Despite their diverse amino acid sequences, these proteases are predicted to share similar structures. Rladg recognizes all ten tested polerovirus proteases, suggesting a conserved structural recognition mechanism. We propose that Rladgs broad recognition capacity could enable resistance to poleroviruses in many crop species. Rladg is the first R-gene reported to confer resistance to a phloem-limited pathogen and could provide enhanced resistance to many economically important poleroviruses.

Microalgal metabolism relies on their chloroplasts, and involves both nucleus and plastidial-encoded proteins of various evolutionary origins. The plastidial ATP synthase complex is a key player in photosynthesis, and has been extensively studied in plants. However, our knowledge in other photosynthetic eukaryotes remains limited, despite their importance in marine environments. Here, we report the characterisation of a novel homologue of the F-type ATP synthase alpha subunit, hereby named xATPA, widespread in microalgae but absent from other photosynthetic organisms. Comparisons of xATPA sequences and predicted structures revealed a specific feature, the bump domain, and highlighted the absence of an ATP-binding site. We assessed xATPA prevalence in microalgae in the global ocean using environmental data from Tara Oceans, with a particular focus on diatoms, and demonstrate that its expression is associated with polar summer conditions. Using a reverse genetic approach in the model diatom Phaeodactylum tricornutum, we show that xATPAP t has a plastidial localisation, and that xATPA KO mutants exhibit growth deficiencies in a combination of low temperature, low salinity and constant light, consistent with environmental analysis. Surprisingly, both RNAseq and physiological assays suggest that xATPA is not involved in ATP synthase functions. On the other hand, xATPA interacts with other F1 ATP synthase subunits in vitro, which we suggest forms transient unassembled complexes. This study hence represents a comprehensive analysis of a novel protein from the environment to the lab, and reveals a new player in the plastidial physiology of eukaryotic microalgae.

Septoria nodorum blotch (SNB) is an economically important fungal disease of wheat caused by Parastagonospora nodorum. It is primarily controlled by the breeding of resistant wheat cultivars, but experience over the last 50 years shows that new pathogen populations soon evolve that are more virulent on the current popular cultivars. In this study, we assembled a panel of 360 P. nodorum isolates. The collection resolved into eight subpopulations. One core and seven transient populations were found possessing contrasting characters in term of spatial and temporal distribution, mating-type, effector haplotypes and patterns of intact and degraded copies of a Tc-1 mariner transposon, called Molly. Molly can proliferate and randomly insert throughout the fungal genome. Its multiplication in sexual population likely triggered RIP which partially explains the extensive genetic diversity and explains the ability to form new adapted lineages and the observed population structure of this important pathogen of wheat. When tested on wheat, the recently emerged groups exhibited greater pathogenicity on modern elite cultivars consistent with the low-amplitude boom-and-bust cycle observed previously. It is possible that active copies of Molly transpose and contribute to both the birth and death of the transient groups. This study identified and characterised a fungal specific transposable element (TE) which plays a vital role in shaping Australian P. nodorum population structure and creating extensive genetic diversity which potentially leads to better adaptation of the pathogen. The study suggests practical measures to improve the efficiency and longevity of resistance breeding for SNB.

Light leaf spot (LLS), caused by subcuticular hemibiotrophic ascomycete fungus Pyrenopeziza brassicae, is a major constraint on oilseed rape (Brassica napus) production, yet the genetic and biochemical mechanisms of quantitative disease resistance (QDR) remain poorly defined. Here, disease phenotyping, pathogen quantification, microscopy, gene expression profiling and glucosinolate (GSL) analysis were integrated to dissect resistance mechanisms in B. napus. Disease assays of 19 diverse lines revealed clear contrasts between susceptible and resistant genotypes, with the commercial cultivar Ambassador showing a phenotype inconsistent with the UK Recommended List rating. Microscopy demonstrated that resistance within doubled haploid line Cubs Root does not inhibit spore germination or penetration but restricts hyphal branching and subcuticular colonisation from 4 to 8 days post-inoculation. Expression profiling of seven candidate gene expression markers (GEMs) and pathogenesis-related PR1 showed that cinnamate-4-hydroxylase, phospholipase C4, {beta}-adaptin, universal stress protein and the 40S ribosomal subunit protein S24 were strongly pathogen-induced in resistant lines, whereas a BAHD acyltransferase, a putative susceptibility factor, was induced only in susceptible cultivars. GSL profiling identified negative correlations between disease severity and total GSLs, particularly aliphatic and aromatic GSLs, with 2{square}phenylethyl and 7-methylsulfinyl heptyl GSLs showing the strongest associations with resistance. Together, these results highlight coordinated transcriptional and metabolic responses that limit pathogen proliferation and provide targets for breeding durable LLS resistance in B. napus.

Most proteins targeted to the organelles of endosymbiotic origin are encoded in the nuclear genome, placing them under the regulatory dominance of the nucleus. For photosynthetic eukaryotes, nuclear-encoded chloroplast proteins arise via two routes: First, genes of cyanobacterial origin were relocated to the nucleus through endosymbiotic gene transfer (EGT). Second, proteins of eukaryotic origin emerged to support chloroplast function and structure. These proteins are reimported into the chloroplast via an import machinery. Reversing the transfer of such genes from the nucleus to the chloroplast genome may offer insights into chloroplast regulation and evolution. In this study, we established a highly efficient and accessible electroporation protocol for chloroplast transformation in the green alga Chlamydomonas reinhardtii, and used it to reverse-transfer two nuclear-encoded genes encoding proteins arising via the two routes described above: the cyanobacteria-derived chloroplast division protein FtsZ1 and the Rubisco-linker EPYC1 of eukaryotic origin. Regardless of origin, both chloroplast-encoded FtsZ1 and EPYC1 showed proper localization and functionality comparable to their nuclear-encoded counterparts. Together, our study provides a robust protocol for chloroplast transformation, a platform for investigating the evolutionary drivers of EGT, and a foundation for advancing chloroplast bioengineering. SIGNIFICANCE STATEMENTO_LIEndosymbiotic gene transfer has resulted in the mass migration of genes from the chloroplast genome to the nuclear genome. Reversing the gene transfer could reveal the evolutionary significance of genome partitioning. C_LIO_LIUsing the green alga Chlamydomonas reinhardtii, this study developed an efficient, electroporation-based protocol for chloroplast transformation. Relocating the genes encoding two chloroplast-targeted proteins, FTSZ1 and EPYC1, to the chloroplast genome showed that the proteins maintained normal localization and function. C_LIO_LIThe established transformation protocol facilitates systematic testing of reverse gene transfer to elucidate the potential evolutionary advantages of genome partitioning and opens new avenues for chloroplast bioengineering. C_LI

The TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are a plant-specific family proposed to function as peripheral membrane proteins that contribute to abiotic stress tolerance in Arabidopsis, likely by maintaining cell wall integrity through brassinosteroid signaling. Previously, we identified a TTL gene that was differentially regulated at the translational level in nitrogen-fixing soybean plants under water deficit (WD) conditions. This finding prompted the characterization of the soybean TTL gene family. Using the Glycine max v4.0 proteome, we identified ten TTL homologs (GmTTL1-GmTTL10), which are unevenly distributed across five chromosomes. Phylogenetic and structural analyses grouped these genes into three clades and revealed a highly conserved exon-intron organization. Likewise, GmTTL proteins display a conserved number and arrangement of TPR and TRXL motifs. To gain insights into their potential biological functions, we integrated co-expression and differential expression analyses. This approach identified a co-expression module enriched for translationally downregulated genes related to the Gene Ontology terms "cellular anatomical entity", "membrane", "cell periphery", "cell wall modification", "nitrate assimilation", and "cell wall organization or biogenesis". Protein-protein interaction network analysis of this specific subset of genes uncovered a novel GmTTL connection with two nitrate reductase enzymes in nitrogen-fixing plants subjected to WD, potentially linking the TTL gene family to new functions or roles. This study provides a framework for future functional studies of GmTTL proteins and their contribution to abiotic stress adaptation in soybean. Key MessageThis work presents the first functional characterization of TTLs proteins in legume species and highlights key processes that may link the TTL gene family to new functions or roles.

Exocyst was initially uncovered in yeast genetic sec-screen as a tethering complex for exocytotic vesicles and this function was later found to be evolutionarily conserved in other eukaryotes including plants. Later however, a surprising engagement of the exocyst complex in autophagy was observed in animals, plants and recently also in yeast. Using the genetic approach we observed EXO70E2 exocyst complex subunit engagement in the defence response to Pseudomonas syringae attack linked to the autophagy pathway. CRISPR/CAS LOF mutant of EXO70E2 is more sensitive to Pseudomonas infection (both virulent as well as T3SS mutant) and autophagy flux monitored by NBR1 antibody is compromised in comparison to WT. We conclude that the plant exocyst complex linked to the EXO70E2 subunit participates in defence against Pseudomonas bacteria in conjunction with the autophagy pathway. HighlightArabidopsis exocyst subunit EXO70E2 affects selective autophagic flux monitored by NBR1 and is participating in defense against Pseudomonas syringae infection.

Endophytic biocontrol agents may contribute to grapevine health, but their ability to establish, persist, and move within woody tissues remains poorly understood. In this study, a stem injection method was developed to introduce Pseudomonas chlororaphis M71 and Trichoderma atroviride SC1 into rooted and grafted grapevine plants, and their spatial and temporal colonization patterns were compared with the movement of a dye tracer. The dye tracer moved rapidly through xylem tissues, whereas both microorganisms showed more restricted early distribution. Over time, M71 and SC1 displayed distinct colonization patterns. M71 persisted after injection, but remained localized near the inoculation site, with limited movement toward roots or distal aerial tissues. In grafted plants, M71 recovery depended on the injection site and declined more markedly after rootstock injection than after scion injection. In contrast, SC1 showed broader and more persistent colonization. In rooted cuttings, SC1 was recovered from stem and root tissues up to 56 days post-injection, and in grafted plants it was recovered across the graft union, particularly after scion injection. Microscopy supported internal localization of both microorganisms. GFP-labelled M71 and SC1 hyphae were observed mainly within xylem vessels, and viable microorganisms were recovered from corresponding wood tissues. No contamination was observed in control plants. These results show that beneficial microorganisms can be introduced into grapevine tissues by stem injection and that bacterial and fungal biocontrol agents differ markedly in their internal movement and persistence. IMPORTANCEIntroducing beneficial microorganisms directly into plant tissues could help in establishing protective endophytic populations, but little is known about how such microorganisms move and persist inside grapevine. This study shows that stem injection can deliver Pseudomonas chlororaphis M71 and Trichoderma atroviride SC1 into grapevine tissues without visible phytotoxicity. The two microorganisms followed different colonization patterns. M71 remained mostly localized near the injection site, whereas SC1 spread more broadly and persisted in both rooted and grafted plants. These findings provide a basis for developing targeted endophytic biocontrol strategies in grapevine propagation and early plant establishment. This approach may be particularly relevant for grapevine trunk diseases and other vascular disorders, in which pathogens colonize internal woody tissues.

The migratory endoparasitic pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, causing significant economic and ecological losses in conifer forest ecosystems in Europe and Asia. Understanding the molecular mechanisms regulating PWN parasitism-related genes may lead to new sustainable solutions for control. Based on previous PWN transcriptomic datasets from the pre-parasitic and parasitic stages and from the pharyngeal gland cells (GC), an in silico analysis was performed to identify transcription factors (TF) highly expressed in the GC. Seven candidates TF genes were selected, and their spatial expression validated by in situ hybridisation. From those, two GC-expressed TFs, BXY_079 and BXY_022, each encoding zinc finger domains, were successfully knocked down by RNA interference. Transcriptomic data from silenced BXY_079 and BXY_022 TFs, analysed with existing life cycle specific transcriptomic data, showed that both TFs control genes expressed at similar times, by repressing male-related genes while activating genes expressed during the J3 and D3 stages, yet each represents the extreme of the others minor function. In addition to these common roles, BXY_079 also activates parasitism-related genes in the J2 stage. These BXY_079-activated parasitism-related genes predominantly encode proteins with lytic functions, including secreted peptidases and glycoside hydrolases. Consistent with their proposed role in parasitism, these genes are highly expressed during the parasitic juvenile stages and are likely involved in nematode feeding, tissue penetration, and migration within the host. In contrast, BXY_022 also represses the expression of several genes related to the reproduction system, such as major sperm proteins and cytosolic motility proteins, particularly in the adult male stage. Taken together, both dual-functional TFs work together, non-redundantly, to regulate gene expression across the life cycle, while each is additionally specialised to regulate diverse and distinct gene sets: ranging from genes implicated in lytic parasitic functions to sexual dimorphism.

Heavy metals act as essential metalloprotein cofactors in numerous physiological processes but can become toxic when non-essential metals accumulate or when essential metals are in excess. As plants continuously encounter heavy metals through their roots, they have evolved complex homeostatic mechanisms to regulate metal uptake and distribution. The Heavy Metal ATPase (HMA) gene family encodes a group of heavy metal transporting P-type ATPases that have been linked to stress resistance and nutrient supply. Here, we used a bioinformatics approach to identify and characterise 13 HMA genes containing characteristic P1B-type ATPase domains and motifs in the barley Morex V3 reference genome. The genes are located on five of the seven barley chromosomes. Phylogenetic analysis revealed that they cluster into five sub-clades, including one clade unique to barley. Expression profiling across multiple datasets showed distinct temporal and tissue-specific expression patterns among HvHMAs, with several members exhibiting significant transcriptional responses to specific biotic and abiotic stresses. By utilising recently available pan-transcriptomic and pan-genomic resources, we have identified substantial allelic diversity and inter-accession variation in HvHMAs. Our findings suggest that HvHMAs have functions extending beyond canonical heavy metal homeostasis and warrant further investigation for their potential roles in broader physiological and stress-related processes.

Accessory genes are thought to contribute to fungal adaptation and pathogenicity by modulating host immunity, while core genes play crucial roles in maintaining fundamental biological processes. Rust fungi (order Pucciniales) are obligate biotrophic plant-pathogens and infect economically relevant crops. Here, we characterize core and accessory gene repertoires across rust fungi, with a particular focus on Phakopsora pachyrhizi, the causal agent of Asian soybean rust. Across Pucciniales genomes, accessory genes represented the largest fraction of gene content (~44.6% on average), whereas core genes accounted for a smaller proportion (~18-35%). Notably, variations in accessory gene content among rust fungi are perhaps attributed to lineage-specific gene expansions and losses. Core gene content was positively correlated with total gene number across Pucciniales genomes, suggesting retention after gene duplication events, consistent with their essential biological functions. Among P. pachyrhizi genes expressed during soybean infection, core effectors were associated with cysteine-rich proteins, pectin-degrading enzymes, and SPFH/Band 7 family, while accessory effectors included phosphatidylethanolamine-binding proteins, trehalose phosphatases, and CFEM domain-containing proteins. The in-plant induced core and accessory genes in P. pachyrhizi also comprised multiple families of CAZymes (GH5/GH7 cellulases, CE5 cutinases, CE8 pectinesterases, CE4/GH18 chitin-modifying enzymes); proteases (aspartyl proteases, serine carboxypeptidases, alpha/beta hydrolases); transporters (amino acid permeases, ferric reductase-like transmembrane proteins, and OPT oligopeptide transporter), and transcription factors (bZIP, GATA zinc finger, STE-like, and homeobox KN). Our study highlights that core and accessory gene families have shaped P. pachyrhizi-soybean interactions, identifying promising targets for functional studies aimed at elucidating host-adaptation mechanisms in rust fungi.

Intraspecific hybridization in allopolyploid plant genomes has the potential to induce non-additive changes in gene expression and DNA cytosine methylation, partly through interactions among divergent parental subgenomes. However, the extent to which intraspecific hybridization reshapes gene expression, coordinates homoeolog regulation, and remodels methylation in higher-order polyploids remains poorly quantified. To address this, we sequenced seedling leaf transcriptomes and methylomes from two parental cultivars of hexaploid bread wheat (Triticum aestivum L.) and their hybrids. More than 40% of genes were differentially expressed between hybrids and parents, although many were not differentially expressed between the parents themselves, consistent with complex trans-regulatory effects in the hybrid genome. This effect was more pronounced for homoeologs whose relative expression differed between the parents. These expression shifts often occurred simultaneously across all three homoeologs within triads, reducing homoeolog expression bias (HEB) in the hybrids. CG methylation levels were similar between the parents and hybrids in regions of low genetic divergence and in transposable element (TE)-rich regions, whereas CG sites in gene-rich regions showed more additive inheritance (hybrids intermediate between parents), particularly when parental haplotypes were themselves divergent. TE and gene body methylation (gbM) was strongly conserved in parents and hybrids. gbM was associated with more balanced homoeolog expression and fewer non-additive expression changes. CHH methylation showed overdominance, whereas non-conserved CHG methylation was enriched in TE-rich regions, suggesting that non-CG remodeling may reflect parental differences in TE and small-RNA content. Our results show that intraspecific hybridization within a hexaploid species can generate non-additive changes in gene expression and DNA methylation in seedling leaf tissue, while the presence of homoeologous genes, parental HEB, parental genetic and methylation divergence, and genomic location have varying levels of influence on expression or methylation remodeling.

O_LIHybridisation is frequently associated with plant invasions; however, its consequences for genome organisation and chromosome evolution remain poorly understood in invasive species. We investigated the extent of hybridisation in the invasive Carpobrotus edulis--acinaciformis hybrid complex and determined the cytogenomic contribution of parental species in hybrid accessions. C_LIO_LIWe combined whole-genome sequencing, population genomic analyses, genome size estimation, repeatome characterisation, chromosome counting and fluorescence in situ hybridisation to compare parental species and hybrid accessions from South Africa and the Mediterranean Basin. C_LIO_LIPopulation genomic analyses revealed widespread hybridisation and introgression, with most invasive accessions showing admixed ancestries. Pattersons D-statistic supported asymmetric allele sharing towards C. edulis. Hybrid accessions displayed genome sizes indistinguishable from C. edulis, whereas C. acinaciformis possessed significantly larger genomes. Repeatome analyses identified marked differences in repetitive DNA composition, particularly in satellite DNA abundance and chromosomal distribution. A newly identified satellite repeat (CarpoSat) showed contrasting chromosomal patterns between parental species, whereas hybrids resembled C. edulis satellite pattern. C_LIO_LIOur results demonstrate that Carpobrotus hybrid accessions are a swarm of later-generation hybrids and backcrosses showing a strong bias towards C. edulis, indicating asymmetric introgression. These findings highlight the value of integrating cytogenetic and genomic approaches to understand genome evolution in invasive hybrid complexes. C_LI