Journal of Experimental Botany

O_LICereal grafting using embryonic tissues has recently become technically feasible; however, the physiological consequences of cereal grafting remain uncharacterized. C_LIO_LIWe systematically evaluate photosynthetic performance and stomatal dynamics across different graft combinations in two photosynthetically distinct species, rice (C3) and pearl millet (C4). We first assessed steady-state photosynthetic performance and dynamic stomatal responses in five-week-old rice and pearl millet grafts grown under a saturated water regime, to establish whether cereal grafting alters physiology at early stages. Next, we assessed same traits at the onset of optimal water regime, and after five days to determine whether any graft-induced effects on photosynthesis or growth persisted over time. C_LIO_LIWe observed that across contrasting water regimes and at different plant developmental stages, cereal grafting did not alter growth, photosynthesis or stomatal kinetics in either species, while revealing modest early stage C4-specific adjustments in stomatal dynamics without affecting photosynthetic capacity or biochemical parameters. C_LIO_LIWe demonstrate that cereal grafting does not alter core physiological traits in rice or pearl millet and can be deployed without long-term impact on photosynthesis. These findings establish cereal grafting as a tractable platform for mechanistic dissection of root-shoot signaling and trait combination across different C3 and C4 cereals. C_LI

Drought is a major abiotic stress threatening plant productivity and agricultural sustainability, yet the molecular mechanisms underlying adaptive root responses to water deficit in the water use strategies continuum remain insufficiently understood, particularly in perennial crops. In this study, we explored drought responses in nine accessions belonging to three wild Vitis species (V. acerifolia, V. candicans, and V. doaniana) displaying varying drought-response strategies. Plants were subjected to moderate drought stress (40% soil water content) for three weeks under greenhouse conditions. By integrating physiological, metabolic, and transcriptomic analyses, we aimed to identify both conserved and species-specific mechanisms associated with drought adaptation. Differential expression analyses revealed a conserved core set of drought-responsive genes shared among species, including genes involved in abscisic acid signaling, reactive oxygen species detoxification, solute transport, and plant defense. In parallel, each species exhibited distinct transcriptional and metabolic signatures reflecting alternative adaptive strategies related to osmoregulation, and oxidative stress mitigation. Weighted gene co-expression network analysis (WGCNA) further revealed significant associations between constitutive, non-plastic gene expression and root phenotypic traits. Overall, our findings demonstrate that wild Vitis species rely on both conserved stress-responsive pathways and species-specific constitutive regulation to cope with drought stress. These results highlight the importance of root-associated traits and intrinsic regulatory networks in shaping drought adaptation and provide new targets for the development of drought-resilient grapevine rootstocks.

The sequence of leaf physiological thresholds underlying plant responses to water deficit is thought to be functionally coordinated; yet, to what extent this coordination is maintained across genotypes and environments remains poorly documented at the intraspecific level. We characterized the sequence of stomatal closure, turgor loss and xylem embolism in the leaves of two genotypes of the riparian species Populus nigra (DRA-038 vs. PG-31) subjected to control, additional nitrogen or additional potassium treatments. Under control conditions, embolism measurements using the optical vulnerability method showed that DRA-038 was more vulnerable than PG-31, in agreement with measurements performed on stems with the reference Cavitron method. Stomatal closure consistently preceded xylem embolism, while bulk leaf turgor loss was typically observed once xylem embolism had already reached 50%. Hydraulic thresholds responded to treatments in a genotype-dependent manner, the intrinsically more vulnerable genotype DRA-038 being typically more plastic. However, despite variations across genotypes and treatments, the trait sequence remained tightly coordinated such that stomatal safety margins (SSMs) remained virtually null. These findings support a strong mechanistic integration of leaf hydraulic thresholds in poplar across genetic units and varying environments, questioning whether to favour intrinsic tolerance or plastic capacities in breeding future drought-tolerant genotypes.

Copper (Cu) is an essential micronutrient whose bioavailability is strongly affected by soil physicochemical properties. During evolution, plants have developed mechanisms to flexibly adjust their metabolism to Cu status. Superoxide dismutases (SODs), including Cu/ZnSOD1 (CSD1) and FeSOD1 (FSD1), are key antioxidant enzymes regulated in Cu dependent manner in Arabidopsis thaliana. Examination of CSD1 cellular distribution and activity revealed that CSD1 is a nuclear and cytosolic SOD whose abundance and activity respond to Cu availability inversely to FSD1. Combined microscopic and biochemical analyses of Cu-dependent dynamics revealed that, unlike FSD1, CSD1 localization in guard cells (GCs) remains independent of Cu availability. CSD1 escapes miR398-mediated regulation in GCs through a cell type-specific splice variant (CSD1.2) that carries an altered miR398-binding site. In silico analyses indicate that this mechanism is also present in crop species. Functionally, the csd1 mutant showed reduced sensitivity to abscisic acid (ABA)-induced stomatal closure, a phenotype rescued by reintroducing CSD1. Biochemical and reactive oxygen species (ROS) level analyses indicate that CSD1.2 most likely acts independently of its canonical enzymatic activity in GCs and functions upstream of the ROS burst in the ABA signaling pathway. Together, we present a novel, cell-type-specific mechanism that safeguards ABA-driven stomatal closure under fluctuating Cu supply.

Leaf senescence directly affects lettuce quality and postharvest shelf life, but the regulatory roles of miR319-targeted and non-target CIN-TCP transcription factors remain unclear. Here, we examined whether the miR319-TCP module controls lettuce leaf senescence through separable genetic branches. MIR319 overexpression delayed dark-induced senescence, whereas STTM-mediated miR319 suppression accelerated chlorophyll loss, photosynthetic decline, and senescence-marker activation. Disruption of the miR319-targeted gene LsTCP4 phenocopied MIR319 overexpression, supporting LsTCP4 as a pro-senescence factor downstream of miR319. We further found that the miR319 non-target CIN gene LsTCP17 also promoted senescence, as tcp17 leaves retained more chlorophyll than wild type during dark treatment. Genetic combinations showed that tcp17 enhanced chlorophyll retention in the OX319 background and partially rescued the accelerated senescence phenotype of S319, indicating that LsTCP17 acts through a route separable from the miR319-targeted branch. Together, these results reveal a split CIN-TCP architecture in which miR319-targeted LsTCP4 and non-target LsTCP17 provide parallel pro-senescence inputs, offering a genetic framework for targeted improvement of lettuce quality.

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.

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.

Biuret, a nitrogen-rich by-product of urea and a common contaminant of urea-based fertilisers, has long been considered a passive phytotoxin, affecting plant performances. Yet its effects on root development and the existence of endogenous mechanisms of perception or tolerance remain largely uncharacterised. Here we combined physiological, developmental, genetic and transcriptomic approaches to investigate the response of Arabidopsis thaliana to biuret. Biuret inhibited primary root growth in a dose-dependent manner by reducing meristematic cell division rather than cell elongation, and concomitantly impaired shoot growth by limiting leaf expansion. This root inhibition was reversible upon biuret removal and was accompanied by increased auxin-responsive (DR5) and decreased cytokinin-responsive (TCS) outputs at the root apex, consistent with a regulated remodelling of meristem activity rather than purely cumulative damage. A forward genetic screen identified the biuret-resistant mutant bir29, which sustained root and inflorescence development under inhibitory concentrations. Using {superscript 1}N-labelled biuret, we showed that resistance occurred without any change in biuret influx or accumulation, uncoupling sensitivity from exposure. Whole-genome transcriptomics revealed that bir29 fails to execute the wild-type response, neither repressing the cell-cycle machinery nor deploying the stress-associated programme induced by biuret. Genetic characterisation linked resistance to multiple genomic loci required for full resistance. Together, the results indicate that biuret triggers an active, reversible and genetically tractable developmental response, suggesting that this xenobiotic compound is integrated into endogenous signalling networks. Significance StatementBiuret, a poorly metabolised contaminant of urea fertilisers, is generally regarded as a passive phytotoxin, yet we show that it inhibits Arabidopsis root growth through a reversible and genetically tractable developmental response, accompanied by reorganised auxin and cytokinin signalling, rather than through cumulative chemical injury. The isolation of the resistant mutant bir29 suggests that plants integrate this xenobiotic molecule into endogenous signalling networks, reframing biuret as an informative probe of root developmental regulation.

In Asteraceae species, the capitulum is a compact inflorescence, featuring a characteristic reproductive structure. Despite the identification of a few key regulatory factors, the transcriptome-level information on the developing capitulum remains limited. Here, we applied single-cell and spatial transcriptome sequencing to investigate the developing Gerbera hybridas capitulum during floret differentiation. We obtained a transcriptomics atlas encompassing different stages of the Gerbera capitulum and analyzed the cellular and spatial dynamics of gene expression. Using marker gene expression and GO enrichment of cluster-specific DEGs, we annotated putative cell types and described changes in gene expression across sampled stages, potentially associated with ongoing developmental processes. We detected activity of previously undescribed MADS-box genes and defined their spatial expression patterns. Notably, the MADS-box gene GAGL12 was found to be enriched in the putative capitulum phloem cells. The GAGL12 protein was shown in yeast two-hybrid assays to interact with several other MADS-domain proteins with hypothesized functions in vasculature development, and further detailed in silico analyses supported a candidate role in the development of capitulum vasculature. Altogether, we provide integrative and dynamic transcriptomic insight into capitulum and floret development and lay a basis for future functional studies of the control and development of this intriguing reproductive structure.

Transgenerational plasticity can shape plant phenotype and influence plant response to environmental changes in interaction with the current conditions. While how past stress interact with either current optimal or stress conditions is increasingly documented within a single plant, transgenerational plasticity remains particularly poorly understood especially at the metabolome level. In our study, we investigated whether heat stress induces transgenerational metabolic and phenotypic modifications along two successive clonal ramet generations of the sub-Antarctic aquatic plant Limosella australis. We performed untargeted metabolomic approaches and measured morphologic and performance traits, to assess both transgenerational plasticity of the metabolome and the phenotype. We found that heat stress remodelled the metabolic profile and influenced the foraging strategy of our clonal plant, and that some of these metabolic changes persisted into the first clonal generation. This one therefore adopted an intermediate growth strategy, even though culture conditions were optimal. By comparing differentially accumulated features between daughter ramets from heat stressed mother ramets and from unstressed mother ramets, we identified common and specific metabolites accumulation to heat stress response, belonging to diverse compound families. However, we did not observe any adaptative advantage and any metabolic imprint during another heat stress applied on the second clonal generation. This work provides especially new clues into how plant metabolome integrates and transfers previous stressed clonal generation's information.

Phenotyping stomatal traits and their developmental plasticity is time-consuming but holds potential to improve water use efficiency and photosynthesis for designing stress-tolerant crops under climate change. Here, we develop a robust, high-throughput pipeline for phenotyping 14 stomatal traits in winter wheat related to size, variation, maximum conductance, and spatial patterning. We (1) analyze over 25,000 images from 60 wheat cultivars grown in growth chamber, greenhouse, and field conditions; (2) investigate the impact of light, temperature, and reduced water and nitrogen supply on stomatal traits and their developmental plasticity across adaxial and abaxial surfaces; and (3) evaluate genetic diversity and breeding progress of stomatal traits. Stomatal traits were highly broad-sense heritable, were largely plastic in response to environmental conditions, and showed genotype-specific responses. Stomatal traits of third leaves under controlled environments with stable light and temperature conditions reliably captured the genetic variance of flag leaves under field conditions. Our data suggests that the upper leaf surface contributed more to transpiration and cooling through consistently higher stomatal density, area, and maximum conductance, while the lower surface facilitated CO2 diffusion via systematic proper patterning and spacing. Breeding maintains the genetic diversity of stomatal traits, and our pipeline facilitates breeders to target them to enhance water use efficiency in high-yielding modern cultivars.

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.

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.

Whether Photosystem I (PSI) core subunits accumulate prior to light exposure in developing angiosperm seedlings remains unresolved, with conflicting reports across species. Here, we investigated the presence and membrane colocalization of the PSI core subunit PsaA in etioplasts of dark-grown angiosperms representing dicot and monocot species. Immunoblotting showed that PsaA accumulates in etioplasts of all three dicot species examined (pea, Arabidopsis, and runner bean), whereas in the monocot oat it was detected only after prolonged etiolation, at substantially lower levels and with an anomalously high apparent molecular weight. Blue-native PAGE analysis reveals that a fraction of PsaA co-migrates with LPOR, PsaB, FNR, and chlorophyll synthase, suggesting co-localization within a shared membrane microdomain rather than stable complex formation. The thylakoid insertase Alb3 was more abundant in dicot etioplasts, consistent with a potential role in the early integration of PsaA into the membrane. Upon illumination, pea reached PSI functionality faster than oat, with P700 oxidation detectable 30 min earlier, linking the dark accumulation of PsaA to an accelerated photosynthetic onset. These findings demonstrate light-independent accumulation of a PSI core subunit in a species-dependent manner and point to early steps in PSI biogenesis that precede full photosynthetic complex assembly. Highlight Contrary to prevailing models, a Photosystem I core subunit PsaA accumulates in dark-grown angiosperm seedlings before light exposure, revealing light-independent early steps in photosynthetic complex biogenesis.

RationaleDeveloping climate-resilient crops requires understanding the developmental and physiological mechanisms governing responses to unpredictable environmental stresses. While agricultural selection historically favored phenotypic uniformity, highly volatile conditions can favor risk-spreading "bet-hedging". However, because standard genetic mapping relies on shifting plot-level averages, loci controlling population-level variance remain cryptic. MethodsWe investigated the pleiotropic effects of the Dry2.2 quantitative trait locus (candidate gene HvCEN) using an allelic series of wild barley (Hordeum vulgare ssp. spontaneum) introgressions housed in distinct cultivated backgrounds, evaluated via high-resolution single-plant physiological phenotyping and mini-plot field trials. ResultsSpecific wild alleles confer robust developmental canalization under water limitation, maintain harvest traits, stabilize vascular lignification, and drive a uniform senescence escape strategy. Conversely, carriers of cultivated alleles deploy a bet-hedging strategy under stress, more than doubling inter-plant developmental variation (volume, maturity timing). This variance-driven strategy relies on epistatic interactions, rendering Dry2.2 invisible to traditional mean-centric GWAS plots. ConclusionImproving crop resilience in volatile climates requires expanding selection focus beyond static, plot-level averages to include the active genetic design of population-level variance strategies.

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.

Bacterial canker remains a major constraint affecting apricot production in South-East of France. It is primarily caused by Pseudomonas syringae, a Gram-negative bacterium, many strains of which exhibit ice nucleation activity. By promoting ice formation at relatively high subzero temperatures, ice nucleation-active bacteria may facilitate tissue disruption and pathogen entry. Concurrently, climate-driven shifts toward warmer winter-spring periods have advanced flowering phenology, increasing exposure to late frost events. Despite breeders having developed less susceptible varieties to bacterial canker and early flowering varieties, the link between these traits and frost sensitivity, an emerging risk in this location, remains unresolved. Here, we have evaluated the links between canker susceptibility and frost sensitivity using three cultivar pairs contrasting in disease response and flowering time. Ice nucleation temperature was measured in excised buds under controlled conditions throughout the frost-risk period, alongside field-based diameter variation monitoring over two years. Disease susceptibility (P < 0.001), phenology (P = 0.003), varieties (P < 0.001), locations (P < 0.001), and sampling date (P < 0.001) significantly affected nucleation temperature, whereas epiphytic bacterial abundance and xylem vessel diameter did not. Trees froze at higher temperatures in situ than in laboratory assays (1 to -2{degrees}C versus -3 to 4{degrees}C, respectively), indicating strong environmental modulation of freezing processes beyond Psy-like bacterial activity, which is reflected in contrasting disease susceptibilities (P < 0.001) and precocities (P < 0.001). These results shed light on the complexity of the freezing process in trees under natural conditions. We discuss the potential roles of microclimatic conditions and alternative ice nucleation sources beyond Psy-like bacteria in driving these physiological processes.

Reconstitution of functional nitrogenase in plants requires the coordinated expression of the [Fe-S] cluster assembly proteins NifU and NifS. However, the extent to which these proteins interact with endogenous Fe-S metabolism and affect plant physiology remains unclear. Here, we compared NifU and NifS homologs from diverse diazotrophs to identify variants compatible with the plant chloroplast environment. Selected variants of Azotobacter vinelandii, Fischerella thermalis, and Marinobacter lutimaris were characterized by transient expression in Nicotiana benthamiana and stable transformation in rice. Plant-produced NifU was largely devoid of [Fe-S] clusters when isolated but retained strong capacity for in vitro [Fe-S] cluster reconstitution and apo-NifH activation in a Ft > Av >Ml gradient, indicating correct folding and function but limited cluster loading or stability in vivo. NifU and NifS expression in transgenic rice resulted in variant-dependent proteome and phenotype effects, with A. vinelandii-expressing lines exhibiting severe defects, F. thermalis lines showing intermediate phenotype, and M. lutimaris lines being indistinguishable from wild type. These results reveal a trade-off between the biochemical activity of NifU and NifS and their compatibility with host metabolism, which must be considered for successful nitrogenase engineering in plants. HighlightNifU/NifS homolog selection determines trade-offs between [Fe-S] cluster assembly activity and plant compatibility, identifying variants that minimize physiological disruption while supporting nitrogenase cofactor assembly in chloroplasts.

Adaptation of plant photosynthesis to dynamic light conditions experienced in natural environments is achieved through specific protective mechanisms. Energy-dependent non-photochemical quenching (qE), regulated by Photosystem II Subunit S (PsbS), is a key process facilitating acclimation to fluctuating light in C3 plants, which operate conventional photosynthesis. C4 plants, which include some of the world's most productive and agriculturally important crops, have evolved a distinct high-efficiency photosynthetic pathway. Little is known about the role of specific processes, like qE, in acclimation of C4 plants to dynamic light environments. We generated gene-edited lines of the model C4 grass Setaria viridis lacking PsbS, which were found to be deficient in qE. This deficiency resulted in a modest increase in PSII photoinhibition and a CO2 assimilation penalty under light stress in short-term experiments, but photosynthesis and growth under fluctuating light were unaffected. Instead, keeping Photosystem I oxidised through photosynthetic control, negative feedback regulation of the Cytochrome b6f complex, was critical. Therefore, unlike in C3 plants, qE does not provide a significant adaptive advantage to C4 plants under dynamic light conditions. These findings provide important insights into the biology of C4 plants and help prioritise future strategies for improving the productivity and resilience of C4 crops.

The availability of pangenome and resequencing of wheat collections have facilitated the discovery of gene-trait associations in wheat. Yellow stripe-like (YSL) proteins play a key role in the uptake and translocation of metals and yet have not been fully identified and analyzed at the genome-wide level in wheat. In this study, 26 TaYSL genes were identified and divided into four distinct clades, each clade sharing similar domains and motif compositions. Most genes were upregulated under iron deficiency, whereas homoeologs of TaYSL1 were downregulated. Both SNP-based and haplotype-based association studies were used to dissect the role of TaYSLs underpinning grain iron contents (GFeC) and zinc contents (GZnC) in wheat. TaYSL6-2B and TaYSL16-1A haplotypes showed strong association with GFeC, and TaYSL14-6A showed strong association with GZnC in multiple field trials. The distribution of favorable haplotypes in global wheat collection of [~]3000 accessions showed that majority of haplotypes were more prevalent in landraces and winter wheat compared to modern cultivars and spring types, indicating their potential for use in breeding. The combination of favorable haplotypes of three YSL genes associated with GFeC and GZnC were very rare, and most of the wheat accessions has single or double favorable haplotypes. These findings provide the first comprehensive characterization of the TaYSL gene family in wheat and identify significant SNPs and elite haplotypes that can be utilized for genetic improvement and biofortification.