Frontiers in Plant Science

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.

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

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

Hemp (Cannabis sativa L.) is an increasingly important crop with applications spanning fibre, seed oil and bioactive cannabinoid production, yet the development of reliable tissue culture systems for this species remains a significant challenge. The establishment of axenic seedling cultures is a prerequisite for hypocotyl-based regeneration and future genetic transformation pipelines, but hemp seeds harbour diverse endophytic microbial communities that frequently overwhelm standard surface sterilisation protocols. Here, we present a systematic comparative evaluation of seed sterilisation strategies across seven industrial hemp accessions, examining the effects of sterilisation chemistry, seed provenance and accession identity on both contamination outcome and the subsequent morphogenic competence of hypocotyl explants. Across all treatments and accessions, in-house glasshouse-harvested seeds achieved higher sterility rates than commercially sourced material regardless of protocol applied. This provenance effect, combined with considerable batch-to-batch variation within seed sources, indicates that contamination load is a primary determinant of successful hemp seed sterilisation. Among the sterilisation treatments evaluated, a baseline of 75% ethanol combined with sequential 1% hydrogen peroxide incubation performed consistently well for low-load seed batches, while supplementation with Plant Preservative Mixture (PPMTM) might be necessary to achieve acceptable rates of non-contaminated seedlings from high contamination load batches. Beyond their effect on contamination, sterilisation treatments influenced the morphogenic fate of hypocotyl explants independently of sterility outcomes. Notably, seedling treatment with the Prochloraz-based fungicide Octave promoted shoot and root co-regeneration in the absence of exogenous plant growth regulators in some cases. Hormone-free organogenesis from hypocotyl explants was achievable across multiple hemp accessions, demonstrating that this developmental capacity is broadly distributed within hemp, though its frequency and consistency varied with accession identity and protocol conditions. Together, these findings provide a practical framework for axenic hemp seed culture that can be used as starting point requiring local adaptation based on seed source, batch history and the intended downstream application.

Applications of tissue culture are critical for Cannabis sativa L. (cannabis), supporting clonal propagation, germplasm preservation, pathogen elimination, among other biotechnological applications. However, extensive genetic diversity associated with cannabis results in highly variable responses to in vitro conditioning, and no consensus basal media formulation exists to support reproducible micropropagation across genotypes. To address these limitations, a hybridized ensemble-NSGA-II approach was employed for concurrent optimization of individual media components to create a species specific, cultivar inclusive basal salt formulation for cannabis micropropagation. The resulting PHJ media represents a unique formulation that overcomes recalcitrance across a wide array of cannabis cultivars, facilitating improved growth and uniformity for the nine cultivars used in its development and validation. These results remain consistent from explant initiation through multiple rounds of subculture. The ability of PHJ to overcome genotypic recalcitrance is telling of its potential applicability with an array of plant species beyond cannabis. Additionally, robust performance both with and without plant growth regulators underscores the plausible use of PHJ for diverse applications beyond standard micropropagation. Ultimately, this cultivar-inclusive basal medium demonstrates utility for both scientific research and industrial-scale operations.

Lima bean (Phaseolus lunatus) is a broadly adapted, economically important leguminous crop and a susceptible host of root-knot nematodes (Meloidogyne spp.; RKN), which are a devastating plant pathogen in agricultural systems worldwide. To date, there have been few studies to elucidate the genetic determinants of RKN resistance in lima beans. Understanding the genetic mechanisms underlying resistance is essential for improving resistance traits and incorporating them into lima bean breeding programs. To assist in marker-assisted selection, we aimed to identify and map quantitative trait loci (QTLs) conferring RKN resistance-related traits. Three recombinant inbred line (RIL) populations were used in this study. Three populations were derived by crossing two RKN-resistant parents with the same RKN-susceptible parent and with each other. All populations were genotyped using genome-wide single-nucleotide polymorphism (SNP) markers. Each population was screened for root galling (RG) and RKN egg reproduction (ER) in response to M. incognita and M. javanica in greenhouse experiments. Three major QTLs were detected and mapped on chromosome Pl04 (QRk-pl04.1), Pl05 (QRk-pl05.1) and Pl10 (QRk-pl10.1) across populations. Among them, QRk-pl05.1 and QRk-pl10.1 affected levels of RG and ER of both RKN species, while QRk-pl04.1 suppressed root galling and reproduction responses of M. incognita but not of M. javanica. These chromosomal regions defined by flanking markers will help guide marker-assisted breeding and gene discovery for broad-based RKN resistance in lima beans.

Bacterial blight of rice causes substantial crop losses in Asia and Africa. The recent introduction of Asian strains into African countries has led to two independent outbreaks detected in 2019, causing severe damage in Madagascar and Tanzania. The strains are highly virulent on local rice varieties and rapidly spread from Tanzania to neighboring countries. Multiplex genome editing of effector-binding elements in the promoter of SWEET rice promoters has successfully generated elite rice lines with broad-spectrum resistance against bacterial blight. While genome-edited crops can be released in countries with biosafety regulations, their use in countries that have yet to establish regulations, e.g., Tanzania and Madagascar, is hindered. To circumvent this, marker-assisted backcross breeding (MABB) of the African elite varieties Komboka, FARO-44, and NE-RICA-4 was adopted to introgress resistance genes to confer resistance to Xoo strains identified in Tanzania (iTz) and Madagascar (iMg). Resistance gene pyramiding, namely Xa1, Xa4, xa13, and Xa21, in the three elite rice varieties conferred resistance to iTz and iMg strains. Our study presents a solution to provide rice breeders in East Africa with bacterial blight resistance in three local rice varieties for field trials, line registration, and deployment.

Viroids are plant infectious agents that threaten agricultural production. Current viroid detection methods rely on RT-PCR-based assays, which require specialized laboratory equipment and can sometimes produce false-negative results or non-specific amplification due to the high sequence conservation among closely related viroid species. CRISPR-based diagnostics, particularly Cas12-based systems for DNA detection (DETECTR) and Cas13a-based systems (SHERLOCK) for RNA detection, have emerged as powerful tools for nucleic acid diagnostics. However, most existing workflows still rely on target amplification and, in the case of Cas13a systems, require additional in vitro transcription steps, limiting their simplicity and direct applicability for plant diagnostics. Here, we developed a direct amplification-free Cas13a-based detection platform for viroids using potato spindle tuber viroid (PSTVd) as a model. We optimized CRISPR RNA (crRNA) design, identified inhibitory effects of plant total RNA on readout signal, and employed simplified viroid RNA enrichment workflows enabling robust detection in plant samples. The system further supported both PSTVd-specific and broad-spectrum pospiviroid (genus Pospiviroid) detection and was successfully extended to avocado sunblotch viroid (family Avsunviroidae), demonstrating its adaptability across distinct viroid families. Together, these results establish a practical and modular Cas13a-based platform, not only for viroid diagnostics, but also for broader applications in RNA-derived plant pathogen detection. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/736049v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1d04170org.highwire.dtl.DTLVardef@1783aa3org.highwire.dtl.DTLVardef@51baa7org.highwire.dtl.DTLVardef@1b542b9_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance statementA simplified RNA enrichment workflow combined with CRISPR-Cas13a enables direct, amplification-free detection of plant viroids. The assay supports early and reliable diagnosis across different tomato varieties and provides a practical strategy for improving molecular detection of plant pathogens.

AbstractTomato production faces a persistent challenge from the tomato leaf miner, Phthorimaea absoluta, a pest that severely limits yields while effective resistance in cultivated varieties remains scarce. To address this gap, wild tomato relatives represent a promising reservoir of resistance traits. In this study, 24 tomato accessions, including both cultivated types and wild species, were evaluated under greenhouse (no-choice) and tunnel (choice) conditions. Resistance mechanisms were characterized through measures of antibiosis such as leaflet lesion type, proportion of attacked leaflets, and mine density. The results revealed substantial variation between and within species, allowing classification of accessions into resistant, intermediate, and susceptible groups through multivariate analysis. Notably, the wild accession Solanum habrochaites PI248707 exhibited strong resistance, in contrast to susceptible cultivated varieties such as Rose de Berne. Under choice conditions, PI248707 sustained limited damage and disrupted larval development, with early instar larvae present but few reaching advanced stages, indicating an inhibitory defense response. Untargeted metabolomic profiling further highlighted pronounced constitutive differences between wild and cultivated accessions, with S. pennellii and S. habrochaites displaying higher metabolic diversity. By integrating phenotypic and metabolic data, specific metabolite classes associated with resistance were identified. These findings underscore the potential of wild tomato germplasm in breeding programs, with PI248707 standing out as a strong candidate for resistance introgression.

Far-red light (FR; 700-750 nm) is increasingly incorporated into controlled-environment lighting because it can improve photosynthetic efficiency when combined with comparatively shorter wavelengths. In long-day leafy crops such as spinach, however, FR may also promote the transition from vegetative to reproductive growth and thereby reduce marketable yield. Most studies have evaluated FR fraction, intensity or end-of-day exposure, whereas the developmental timing of FR has rarely been tested, particularly in spinach. Here, we evaluated six commercial spinach cultivars (Amador, Harp, Renegade, Responder, Rubino and Santa Cruz) in an indoor vertical farm under a common red-green-blue background (PPFD 260-264 {micro}mol m-{superscript 2} s-{superscript 1}, 12 h photoperiod, 24 {degrees}C) and four FR timing treatments: no FR (Control), FR throughout production (FullFR), FR during early development only (EarlyFR), and FR during late development only (LateFR). LateFR increased marketable fresh weight relative to Control (244 vs 224 g) and reduced flowering incidence, whereas far-red supplied during early development reduced fresh weight (158 g) and increased flowering. The magnitude of the timing response differed among cultivars: switching from EarlyFR to LateFR recovered 0 % fresh weight in Amador but 107 % in Renegade and Rubino, with the largest penalties occurring in otherwise bolt-resistant cultivars. EarlyFR also increased total chlorophyll and reduced the chlorophyll a:b ratio. These results show that FR response in spinach is strongly conditioned by developmental stage and cultivar. Although LateFR received more total far-red than EarlyFR, it behaved like the Control, indicating that the penalty was set by far-red timing rather than dose. Treatment differences in bolting and yield tracked an estimated phytochrome photostationary-state deficit during early development: a phytochrome-deficit model markedly outperformed a cumulative-dose model ({Delta}AIC = 441), and the deficit x cultivar interaction was strong (p < 0.001), with bolt-resistant cultivars losing most yield when far-red coincided with the early developmental window. We therefore propose that FR should be treated as a genotype-dependent management variable rather than as a fixed spectral input, with late application and bolt-resistant cultivars offering the most favourable combination for vertical-farm spinach production. Framed within the breeders equation, the close match between the trial and production environment and the scope for shorter breeding cycles indoors suggest that genotype and far-red timing can be optimised jointly to accelerate genetic gain.

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.

Bacterial leaf blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), causes yield losses exceeding 50% in affected areas, including the Bagre rice plain in Burkina Faso. Genome-edited (GEd) rice lines have been successful in tackling BB. Modifications in the Xoo virulence protein target site upstream of three SWEET susceptibility genes in two elite rice varieties, IR64 and Ciherang-Sub1, have been demonstrated to confer broad-spectrum resistance to Asian and East African Xoo strains. Here, we evaluate the potential of the GEd lines as a solution for BB management in Burkina Faso. We challenged the GEd lines against five locally collected Burkinabe Xoo strains under controlled green-house conditions and assessed their agro-morphological performance under field conditions representative of local agroecological conditions. Greenhouse pathogen assays demonstrated that GEd IR64 and Ciherang-Sub1 lines were resistant to all tested local Xoo strains across three successive generations. We identified TalC as the primary disease-causing effector in the local Xoo populations. Irrigated field trials conducted over two seasons in the Kou Valley, Burkina Faso, revealed absence of agro-morphological penalties in GEd lines compared to their parental wild-type lines. Observed trait variation was attributable to environmental fluctuations rather than genomic modifications. Collectively, our findings demonstrate that genome editing of the rice lines does not impose growth penalties, and support the suitability of GEd IR64 and Ciherang-Sub1 for large-scale adoption in Burkina Faso, pending multi-location validation and introgression into locally adapted varieties.

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.

Crop photoperiodism models assume that flowering time is primarily controlled by daylength, yet many field observations contradict this view. We previously proposed an alternative framework integrating daily changes in sunrise and sunset times (dSR and dSS). Variety trials in Madagascar and in Argentina supported this concept: mid-late sorghum varieties from the northern hemisphere flowered late or very late when sown in November and December, consistent with the higher dSR/dSS values of the southern hemisphere summer. One Malian variety, sown monthly over six years in West Africa, exhibited high interannual variability in flowering time when sown between November and February. This revealed that up to four photoperiodic responses -- two quantitative and two qualitative, occurring at different times of the year -- may coexist within a single late photoperiod sensitive variety. All responses use only dSR and dSS cues. The qualitative responses are triggered by an internal phasic coincidence, which is set by a linear relationship between dSR and dSS at the onset of plant photoperiod sensitivity, and between dSR+dSS at panicle initiation. The research model fitted data from 28 varieties grown in Mali well. It also accurately fitted the duration to PI observed in three varieties sown at tropical and temperate latitudes. HighlightThe seasonal photoperiodic adaptation of flowering time in sorghum plants may rely on several signal transduction pathways regulated by sunrise and sunset times rather than day length.

Globe artichoke (Cynara cardunculus var. scolymus L.) comprises a broad range of local ecotypes and varietal groups whose genetic diversity has been investigated through different molecular markers. However, recent advances in next-generation sequencing and pangenomics approaches provide new opportunities to capture genome-wide variation at higher resolution and to develop practical tools for varietal discrimination, traceability, and germplasm conservation. In this study, we developed the first pangenomic framework for cultivated artichoke and evaluated pangenome-informed SNP markers for varietal fingerprinting. Whole-genome resequencing data from the Italian local ecotype Asti Sori were integrated with publicly available genomic data from representative globe artichoke and cultivated cardoon accessions to construct and annotate a pangenome. Genome-wide SNP and presence/absence variation (PAV) analyses were combined with pangenome-anchored genotyping-by-sequencing (GBS) data from 45 accessions representing the main cultivated varietal groups. The pangenome revealed a largely conserved core gene repertoire alongside a smaller accessory component, with gene accumulation curves suggesting a tendency toward saturation within the sampled cultivated germplasm. SNP- and PAV-based analyses provided complementary views of accession relationships and consistently resolved the principal cultivated groups. Across the broader germplasm panel, pangenome-anchored GBS-derived SNPs identified well-supported phylogenetic clusters corresponding to recognized varietal types. A reduced panel of 50 SNPs, selected through iterative random subsampling, retained at least 90% of the genetic diversity captured by the full dataset and reproduced its main population structure. This compact pangenome-anchored marker set provides a practical foundation for varietal fingerprinting, DUS-oriented applications, traceability, and conservation of traditional globe artichoke germplasm. Validation across independent collections will be required before routine deployment.

Metribuzin is a versatile herbicide effective against various annual grasses and broadleaf weeds found in wheat fields. However, it can cause foliar damage to wheat, impacting plant health and yield. A clearer understanding of the genetic architecture associated with metribuzin tolerance is necessary to guide marker-based breeding strategies. This study evaluated 351 historic Gulf Atlantic Wheat Nursery (GAWN) wheat breeding lines representative of southern US soft red winter wheat (SRWW) germplasm. Field trials were conducted at Winnsboro (WN) and Baton Rouge (BR), Louisiana, in 2016 and 2017. Metribuzin was applied at specific growth stages[DN1.1], and tolerance was assessed based on visual foliar damage. Genomic data from 6,252 filtered single nucleotide polymorphism (SNP) markers were used to estimate narrow-sense heritability, conduct genome-wide association (GWAS), and assess genomic prediction accuracy using genomic best linear unbiased prediction (GBLUP). Broad-sense heritability ranged from 0.54 to 0.69 within environments and reached 0.77 across environments, while narrow-sense heritability ranged from 0.35 to 0.47, indicating moderate additive genetic control. No SNP surpassed the significance threshold, but genomic prediction (GP) showed moderate to strong predictive ability (PA) across environments, with the highest accuracy (r = 0.62) observed between BR17 and WN17. These results indicate that metribuzin tolerance in SRWW is primarily controlled by multiple small-effect loci and that GS provides a more effective breeding strategy than marker-assisted selection for improving tolerance in southern wheat germplasm.

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.

BackgroundThe commercialization of Nigerias single-line pod borer-resistant (PBR) cowpea (IT97KT), the first transgenic cowpea variety in the world expressing Cry1Ab gene, has raised questions about potential unintended molecular changes and their ecological implications. This study employed integrated proteomic and metabolomic profiling to compare the transgenic line with its non-transgenic isoline (IT97KN) and assess molecular indicators associated with ecosystem resilience. MethodsProteomic analyses were conducted using LC-MS/MS following filter-assisted sample preparation, while metabolomic profiling employed GC-MS and UHPLC-MS/MS platforms. Differential protein and metabolite abundance were assessed using label-free quantification, volcano plot analysis, principal component analysis (PCA), hierarchical clustering, and Gene Ontology (GO) enrichment analyses. ResultsProteomic profiling revealed substantial overlap between IT97KT and IT97KN, with only a limited subset of proteins exhibiting significant differential abundance. Upregulated proteins in IT97KT were primarily associated with seed storage, redox regulation, oxidative stress mitigation, and defense-related functions, including Late Embryogenesis Abundant Protein 1 (LEA1), vicilins, thioredoxin, and iron superoxide dismutase. Among 37 proteins linked to ecological adaptation, only LEA1, CPRD22, and Bg7S showed significant differences. Similarly, only carbonic anhydrase II displayed differential abundance among proteins associated with potential ecological risk. PCA and clustering analyses demonstrated high proteomic similarity between genotypes. Metabolomic analyses identified sixteen major metabolites, predominantly fatty acids, with no statistically significant differences in abundance or composition between transgenic and non-transgenic lines ConclusionsThe transgenic PBR cowpea exhibited minimal unintended proteomic and metabolomic alterations relative to its non-transgenic isoline. These findings indicate that Cry1Ab insertion did not substantially disrupt molecular pathways associated with ecological adaptation, environmental risk, or metabolic homeostasis, providing molecular evidence supporting the environmental and biosafety equivalence of PBR cowpea.

As extreme heat events increase in frequency and intensity worldwide, understanding how woody perennial crops respond to higher maximum temperatures is critical. Perennials face distinct challenges, persisting across many seasons under increasingly variable and extreme conditions, and heat waves threaten the viability of wine grape cultivars through impacts on yield, wine quality, and long-term vine health. To test whether irrigation practices before and during heat waves affect grapevine carbon (C) storage and health, we experimentally manipulated irrigation regimes surrounding heat waves from 2019-2021 in a commercial Cabernet Sauvignon vineyard in the Lodi AVA of Californias Central Valley. Vine physiological traits and yield were measured throughout, and whole-vine nonstructural carbohydrate (NSC) concentrations were quantified after three growing seasons. Although lower supplemental irrigation reduced photosynthesis, stomatal conductance, and fruit yield, whole-vine NSCs did not differ significantly in any perennial organ by the experiments end, indicating that reproductive output and final NSC status responded to irrigation on different timescales. These results suggest that moderate supplemental irrigation during heat events is sufficient to mitigate negative impacts on yield and quality while supporting recovery of NSC reserves, though longer-term monitoring is needed to confirm that this short-term resilience persists.

Rising winter temperatures threaten deciduous fruit tree productivity by depleting carbohydrate reserves during dormancy. This study investigated Stem Photosynthetic Capacity (SPC), a rare adaptive trait from wild Prunus arabica, as a mechanism to enhance almond carbon economy. Using extreme segregating groups from the F1 population (P. dulcis X P. arabica), we evaluated physiological performance through high-resolution lysimetric and multi-year orchard monitoring. High-SPC [SPC(+)] genotypes maintained significantly greater stem CO2 assimilation and transpiration during leafless periods compared to low-SPC [SPC(-)] progenies. Over five successive seasons, SPC(+) trees exhibited a 33.3% increase in trunk secondary growth and reached 10% bloom approximately 8 days earlier. Most importantly, the SPC(+) group achieved a 4.6-fold increase in mean kernel yield when compared to SPC(-) group. These findings demonstrate that SPC provides a flexible, supplementary winter carbon source that directly supports both vegetative and reproductive development. Integrating SPC into commercial almond breeding programs may offer a valuable strategy to improve climate resilience and help sustain yields under warming conditions. HighlightIntegrating stem photosynthesis into commercial almond hybrids provides a winter carbon source that advances blooming, expands trunk growth by [~]33%, and increases kernel yields by more than 4.5-fold.