The diversity of plant inflorescence architecture is specified by gene expression patterns. In wheat (Triticum aestivum), the lanceolate-shaped inflorescence (spike) is defined by rudimentary spikelets at the base, which form as a result of delayed spikelet and floral development compared with central spikelets. While previous studies identified gene expression differences between central and basal inflorescence sections, gene expression patterns along the apical-basal axis remain poorly resolved due to bulk tissue-level techniques. Here, we optimize Multiplexed Error Robust Fluorescence In Situ Hybridization, a spatial transcriptomics technique, in wheat inflorescence tissue, enabling transcript localization for 200 genes to cellular resolution across 4 stages of development. Cell segmentation and clustering of 50,000 cells identified 18 expression domains and their enriched genes, revealing the spatio-temporal organization of spikelet and floral development, and characterizing tissue-level gene markers. Using these domain- and cell-level maps, we characterize expression patterns of genes differentially expressed across the apical-basal axis. We identify distinct, spatially coordinated expression patterns distinguishing axillary meristems and their subtending leaf ridges across the apical-basal axis before visible spikelet formation, highlighting factors patterning meristem identity and transition. To support the broader research community, all raw and processed data are publicly available, including through an interactive WebAtlas interface (www.wheat-spatial.com).

Wheat is a staple crop critical for global food security, and its continuous genetic improvement is essential to meet the demands of a growing population. Efficient, genotype-independent transformation is a major bottleneck in wheat functional genomics and gene editing. The growth regulating factor (GRF)-GRF-interacting factor (GIF) fusion technology enhances regeneration efficiency and broadens the range of transformable cultivars, but constitutive expression can reduce fertility and spikelet number. Here, we present an optimised Agrobacterium-mediated wheat transformation protocol incorporating GRF4-GIF1, tested across multiple tetraploid and hexaploid cultivars. Transformation efficiency was improved through adjustments in selection pressure, zeatin concentration, and promoter choice, with GRF4-GIF1 consistently enabling successful transformation across genotypes. Tissue-specific promoters and heat-inducible excision strategies effectively minimised pleiotropic effects, such as reduced fertility, while maintaining high transformation rates. This refined system provides a robust and versatile platform for gene function studies and gene editing, advancing genotype-independent wheat transformation and supporting breeding efforts to improve crop productivity, resilience, and nutritional value.

Bread wheat ( Triticum aestivum L.) plays a vital role in global food security, and its continuous genetic improvement is essential to meet the demands of a rapidly growing world population. Advances in genome sequencing and assembly have positioned wheat as a model crop for functional genomics and have increased the demand for highly efficient, genotype-independent transformation systems. A fusion technology involving a GROWTH-REGULATING FACTOR (GRF) and a GRF-INTERACTING FACTOR (GIF) has emerged as a powerful tool to enhance regeneration efficiency and expand the range of transformable genotypes. In this study, we present an optimized and robust Agrobacterium -mediated wheat transformation protocol incorporating GRF4-GIF1 , tested across multiple wheat varieties. Across all tested wheat cultivars, GRF4-GIF1 containing constructs consistently enabled successful transformation, with varied efficiencies depending on the genotype and promotors used to drive the gene fusion. Our method significantly improves transformation efficiency while minimizing GRF4-GIF1 pleiotropic effects, providing a versatile platform for gene function analysis and gene editing. This work represents a critical step toward efficient, genotype-independent transformation in wheat, supporting both research and breeding applications aimed at improving crop resilience, nutritional value, and productivity. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, BB/X011003/1 Scientific and Technological Research Council of Turkey

The aim of this protocol is to micro-dissect young wheat plants to access developing meristems which are found enveloped within growing leaves. This protocol can be used to dissect spikes during early stages of development, for both wheat and barley.

Large differences exist in the number of grains per spikelet across an individual wheat (Triticum aestivum L.) spike. The central spikelets produce the highest number of grains, while apical and basal spikelets are less productive, and the most basal spikelets are commonly only developed in rudimentary form. Basal spikelets are delayed in initiation, yet they continue to develop and produce florets. The precise timing or the cause of their abortion remains largely unknown. Here, we investigated the underlying causes of basal spikelet abortion using shading applications in the field. We found that basal spikelet abortion is likely to be the consequence of complete floret abortion, as both occur concurrently and have the same response to shading treatments. We detected no differences in assimilate availability across the spike. Instead, we show that the reduced developmental age of basal florets pre-anthesis is strongly associated with their increased abortion. Using the developmental age pre-abortion, we were able to predict final grain set per spikelet across the spike, alongside the characteristic gradient in the number of grains from basal to central spikelets. Future efforts to improve spikelet homogeneity across the spike could thus focus on improving basal spikelet establishment and increasing floret development rates pre-abortion.

Spikelets are the fundamental building blocks of Poaceae inflorescences and their development and branching patterns determine the various inflorescence architectures and grain yield of grasses. In wheat, the central spikelets produce the most and largest grains, while spikelet size gradually decreases acro- and basipetally, giving rise to the characteristic lanceolate shape of wheat spikes. The acropetal gradient correlates with the developmental age of spikelets, however the basal spikelets are developed first and the cause of their small size and rudimentary development is unclear. Here, we adapted G&T-seq, a low-input transcriptomics approach, to characterise gene expression profiles within spatial sections of individual spikes before and after the establishment of the lanceolate shape. We observed larger differences in gene expression profiles between the apical, central and basal sections of a single spike than between any section belonging to consecutive developmental timepoints. We found that SVP MADS-box transcription factors, including VRT-A2, are expressed highest in the basal section of the wheat spike and display the opposite expression gradient to flowering E-class SEP1 genes. Based on multi-year field trials and transgenic lines, we show that higher expression of VRT-A2 in the basal sections of the spike is associated with increased numbers of rudimentary basal spikelets. Our results, supported by computational modelling, suggest that the delayed transition of basal spikelets from vegetative to floral developmental programmes results in the lanceolate shape of wheat spikes. This study highlights the value of spatially resolved transcriptomics to gain new insights into developmental genetics pathways of grass inflorescences. One sentence summary Large transcriptional gradients exist within a wheat spike and are associated with rudimentary basal spikelet development, resulting in the characteristic lanceolate shape of wheat spikes.

Spikelets are the fundamental building blocks of Poaceae inflorescences, and their development and branching patterns determine the various inflorescence architectures and grain yield of grasses. In wheat (Triticum aestivum), the central spikelets produce the most and largest grains, while spikelet size gradually decreases acropetally and basipetally, giving rise to the characteristic lanceolate shape of wheat spikes. The acropetal gradient corresponds with the developmental age of spikelets; however, the basal spikelets are developed first, and the cause of their small size and rudimentary development is unclear. Here, we adapted G&T-seq, a low-input transcriptomics approach, to characterize gene expression profiles within spatial sections of individual spikes before and after the establishment of the lanceolate shape. We observed larger differences in gene expression profiles between the apical, central, and basal sections of a single spike than between any section belonging to consecutive developmental time points. We found that SHORT VEGETATIVE PHASE MADS-box transcription factors, including VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT-A2), are expressed highest in the basal section of the wheat spike and display the opposite expression gradient to flowering E-class SEPALLATA1 genes. Based on multi-year field trials and transgenic lines, we show that higher expression of VRT-A2 in the basal sections of the spike is associated with increased numbers of rudimentary basal spikelets. Our results, supported by computational modeling, suggest that the delayed transition of basal spikelets from vegetative to floral developmental programs results in the lanceolate shape of wheat spikes. This study highlights the value of spatially resolved transcriptomics to gain insights into developmental genetics pathways of grass inflorescences.

Yellow rust, caused by Puccinia striiformis f. sp. tritici, is a common and serious fungal disease of wheat (Triticum aestivum L.) all over the world and particularly in the Central and West Asia and North Africa region. To identify effective yellow rust resistance loci, genome-wide association study (GWAS) was performed using 196 bread wheat genotypes based on 10,477 single nucleotide polymorphisms markers. Adult plants were evaluated under field conditions for resistance to yellow rust for 2 years (2014–2015) at ICARDA station, Marchouch, Morocco. Out of the 196 genotypes, 85 genotypes (43.37%) were resistant, 22 genotypes (11.22%) were moderately resistant, 13 genotypes (6.63%) were moderately susceptible, 48 genotypes (24.49%) were moderately susceptible to susceptible and 28 genotypes (14.29%) were susceptible to Puccinia striiformis f. sp. tritici. GWAS using mixed linear model identified 23 markers on chromosomes 2A, 2B, 2D and 7B significantly associated with adult plant resistance at false discovery rate (FDR-adjusted P ≤ 0.05). Of which, three markers were within 10 wheat functional genes involved in several plant disease resistance and defense mechanism. Five of the reported functional genes are annotated as disease resistance proteins with nucleotide-binding site leucine repeat domains. BLAST analysis confirmed that YrR61 and Yr17 were mostly the candidate genes linked to the marker Tdurum_contig29983_490 on chromosome 2A. Moreover, markers identified on chromosome 7B and Kukri_c12648_434 on 2D were not mapped within any of previously reported gene/QTL region hence, representing novel resistance loci for Pst and needs to be confirmed using an allelism test.

Flower development is an important determinant of grain yield in crops. In wheat (Triticum spp.), natural variation for the size of spikelet and floral organs is particularly evident in Triticum turgidum ssp. polonicum (also termed Triticum polonicum), a tetraploid subspecies of wheat with long glumes, lemmas, and grains. Using map-based cloning, we identified VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2), which encodes a MADS-box transcription factor belonging to the SHORT VEGETATIVE PHASE family, as the gene underlying the T. polonicum long-glume (P1) locus. The causal P1 mutation is a sequence rearrangement in intron-1 that results in ectopic expression of the T. polonicum VRT-A2 allele. Based on allelic variation studies, we propose that the intron-1 mutation in VRT-A2 is the unique T. polonicum subspecies-defining polymorphism, which was later introduced into hexaploid wheat via natural hybridizations. Near-isogenic lines differing for the P1 locus revealed a gradient effect of P1 across spikelets and within florets. Transgenic lines of hexaploid wheat carrying the T. polonicum VRT-A2 allele show that expression levels of VRT-A2 are highly correlated with spike, glume, grain, and floral organ length. These results highlight how changes in expression profiles, through variation in cis-regulation, can affect agronomic traits in a dosage-dependent manner in polyploid crops.

Background Functional characterisation of genes using transgenic methods is increasingly common in cereal crops. Yet standard methods of gene over-expression can lead to undesirable developmental phenotypes, or even embryo lethality, due to ectopic gene expression. Inducible expression systems allow the study of such genes by preventing their expression until treatment with the specific inducer. When combined with the Cre-Lox recombination system, inducible promoters can be used to initiate constitutive expression of a gene of interest. Yet while these systems are well established in dicot model plants, like Arabidopsis thaliana , they have not yet been implemented in grasses. Results Here we present an irreversible heat-shock inducible system developed using Golden Gate-compatible components which utilises Cre recombinase to drive constitutive gene expression in barley and wheat. We show that a heat shock treatment of 38 °C is sufficient to activate the construct and drive expression of the gene of interest. Modulating the duration of heat shock controls the density of induced cells. Short durations of heat shock cause activation of the construct in isolated single cells, while longer durations lead to global construct activation. The system can be successfully activated in multiple tissues and at multiple developmental stages and shows no activation at standard growth temperatures (~ 20 °C). Conclusions This system provides an adaptable framework for use in gene functional characterisation in cereal crops. The developed vectors can be easily adapted for specific genes of interest within the Golden Gate cloning system. By using an environmental signal to induce activation of the construct, the system avoids pitfalls associated with consistent and complete application of chemical inducers. As with any inducible system, care must be taken to ensure that the expected construct activation has indeed taken place.

AbstractFlower development is a major determinant of yield in crops. In wheat, natural variation for the size of spikelet and floral organs is particularly evident inTriticum polonicum, a tetraploid subspecies of wheat with long glumes, lemmas, and grains. Using map-based cloning, we identifiedVRT2, a MADS-box transcription factor belonging to theSVPfamily, as the gene underlying theT. polonicumlong-glume (P1)locus. The causalP1mutation is a sequence re-arrangement in intron-1 that results in both increased and ectopic expression of theT. polonicum VRT-A2allele. Based on allelic variation studies, we propose that the intron-1 mutation inVRT-A2is the uniqueT. polonicumspecies defining polymorphism, which was later introduced into hexaploid wheat via natural hybridizations. Near-isogenic lines differing for theP1locus revealed a gradient effect ofP1across florets. Transgenic lines of hexaploid wheat carrying theT. polonicum VRT-A2allele show that expression levels ofVRT-A2are highly correlated with spike, glume, grain, and floral organ length. These results highlight how changes in expression profiles, through variation incis-regulation, can impact on agronomic traits in a dosage-dependent manner in polyploid crops.One-sentence summaryAn intron-1 rearrangement in the MADS-box transcription factorVRT-A2leads to its misexpression and defines the long-glume phenotype of Polish wheat (T. polonicum).

Gene regulatory networks are powerful tools which facilitate hypothesis generation and candidate gene discovery. However, the extent to which the network predictions are biologically relevant is often unclear. Recently a GENIE3 network which predicted targets of wheat transcription factors was produced. Here we used an independent RNA-Seq dataset to test the predictions of the wheat GENIE3 network for the senescence-regulating transcription factor NAM-A1 (TraesCS6A02G108300). We re-analyzed the RNA-Seq data against the RefSeqv1.0 genome and identified a set of differentially expressed genes (DEGs) between the wild-type and nam-a1 mutant which recapitulated the known role of NAM-A1 in senescence and nutrient remobilisation. We found that the GENIE3-predicted target genes of NAM-A1 overlap significantly with the DEGs, more than would be expected by chance. Based on high levels of overlap between GENIE3-predicted target genes and the DEGs, we identified candidate senescence regulators. We then explored genome-wide trends in the network related to polyploidy and found that only homeologous transcription factors are likely to share predicted targets in common. However, homeologs which vary in expression levels across tissues are less likely to share predicted targets than those that do not, suggesting that they may be more likely to act in distinct pathways. This work demonstrates that the wheat GENIE3 network can provide biologically-relevant predictions of transcription factor targets, which can be used for candidate gene prediction and for global analyses of transcription factor function. The GENIE3 network has now been integrated into the KnetMiner web application, facilitating its use in future studies.

Gene regulatory networks are powerful tools which facilitate hypothesis generation and candidate gene discovery. However, the extent to which the network predictions are biologically relevant is often unclear. Recently, as part of an analysis of the RefSeqv1.0 wheat transcriptome, a GENIE3 network which predicted targets of wheat transcription factors was produced. Here we have used an independent and publicly-available RNA-Seq dataset to validate the predictions of the wheat GENIE3 network for the senescence-regulating transcription factor NAM-A1 (TraesCS6A02G108300). We re-analysed the RNA-Seq data against the RefSeqv1.0 genome and identified a de novo set of differentially expressed genes (DEGs) between the wild-type and nam-a1 mutant which recapitulated the known role of NAM-A1 in senescence and nutrient remobilisation. We found that the GENIE3-predicted target genes of NAM-A1 overlap significantly with the de novo DEGs, more than would be expected for a random transcription factor. Based on high levels of overlap between GENIE3-predicted target genes and the de novo DEGs, we also identified a set of candidate senescence regulators. We then explored genome-wide trends in the network related to polyploidy and homoeolog expression levels and found that only homoeologous transcription factors are likely to share predicted targets in common. However, homoeologs in dynamic triads, i.e. with higher variation in homoeolog expression levels across tissues, are less likely to share predicted targets than stable triads. This suggests that homoeologs in dynamic triads are more likely to act on distinct pathways. This work demonstrates that the wheat GENIE3 network can provide biologically-relevant predictions of transcription factor targets, which can be used for candidate gene prediction and for global analyses of transcription factor function. The GENIE3 network has now been integrated into the KnetMiner web application, facilitating its use in future studies.

Manipulation of the soil microbiota associated with crop plants has huge promise for the control of crop pathogens. However, to fully realize this potential we need a better understanding of the relationship between the soil environment and the genes and phenotypes that enable microbes to colonize plants and contribute to biocontrol. A recent 2 years of investigation into the effect of wheat variety on second year crop yield in the context of take-all fungal infection presented the opportunity to examine soil microbiomes under closely defined field conditions. Amplicon sequencing of second year soil samples showed that Pseudomonas spp. were particularly affected by the wheat cultivar grown in year one. Consequently, 318 rhizosphere-associated Pseudomonas fluorescens strains were isolated and characterized across a variety of genetic and phenotypic traits. Again, the wheat variety grown in the first year of the study was shown to exert considerable selective pressure on both the extent and nature of Pseudomonas genomic diversity. Furthermore, multiple significant correlations were identified within the phenotypic/genetic structure of the Pseudomonas population, and between individual genotypes and the external wheat field environment. The approach outlined here has considerable future potential for our understanding of plant-microbe interactions, and for the broader analysis of complex microbial communities.