John Innes Centre

Pulses are a valuable source of plant proteins for human and animal nutrition and have various industrial applications. Understanding the genetic basis for the relative abundance of different seed storage proteins is crucial for developing cultivars with improved protein quality and functional properties. In this study, we employed two complementary approaches, genome‐wide association study (GWAS) and quantitative trait locus (QTL) mapping, to identify genetic loci underlying seed protein composition in pea (Pisum sativum L.). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was used to separate the seed proteins, and their relative abundance was quantified using densitometric analysis. For GWAS, we analyzed a diverse panel of 209 accessions genotyped with an 84,691 single‐nucleotide polymorphism (SNP) array and identified genetic loci significantly associated with globulins, such as convicilin, vicilin, legumins, and non‐globulins, including lipoxygenase, late embryogenesis abundant protein, and annexin‐like protein. Additionally, using QTL mapping with 96 recombinant inbred lines, we mapped 11 QTL, including five that overlapped with regions identified by GWAS for the same proteins. Some of the significant SNPs were within or near the genes encoding seed proteins and other genes with predicted functions in protein biosynthesis, trafficking, and modification. This comprehensive genetic mapping study serves as a foundation for future breeding efforts to improve protein quality in pea and other legumes.

Cellular responses to abiotic stress involve multiple signals such as reactive oxygen species (ROS), Ca²⁺, abscisic acid (ABA), and chloroplast‐to‐nucleus retrograde signals such as 3′‐phosphoadenosine 5′‐phosphate (PAP). The mechanism(s) by which these messengers intersect for cell regulation remain enigmatic, as do the roles of retrograde signals in specialized cells. Here we demonstrate a mechanistic link enabling ABA and PAP to coordinate chloroplast and plasma membrane ROS production. Contrary to its role in upregulating processes leading to quenching of ROS in foliar tissue, we show that in guard cells, PAP induces chloroplast ROS accumulation via photosynthetic electron transport and apoplast ROS via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) proteins. Both subcellular ROS sources are necessary for stress hormone ABA‐mediated stomatal closure, as well as PAP‐mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD instead of RBOHF. Three calcium‐dependent protein kinases (CPKs) transcriptionally induced by PAP, namely CPK13, CPK32, and CPK34, concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two SLAC1 serine (S) residues, including S120, which is also targeted by the ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpression of the PAP‐induced CPKs rescues stomatal closure in ost1. Our data identify chloroplast retrograde signals as critical nodes in cellular stress response networks of guard cells.

An efficient nitrate uptake system contributes to the improvement of crop nitrogen use efficiency under low nitrogen availability. The High Affinity nitrate Transport System (HATS) in plants is active in low range of external nitrate and is mediated by a two‐component system (high affinity transporters NRT2 associated to a partner protein NRT3 (NAR2)). In Brachypodium, the model plant for C3 cereals, we investigated the role of BdNRT2A and BdNRT3.2 through various experimental approaches. Expression profile of BdNRT2.A and BdNRT3.2 genes in response to nitrate availability fits perfectly with the characteristics of the HATS components. ¹⁵Nitrate influx measurements decreased in bdnrt2a mutants (one NaN3 induced mutant with a truncated NRT2A protein and two amiRNA mutants). In addition, the N limited phenotype of the mutant with a truncated NRT2A protein confirmed that BdNRT2A is a major contributor of the HATS in Brachypodium. An effective nitrate transport in the heterologous expression system Xenopus oocytes required the coexpression of BdNRT2A and BdNRT3.2 that characterizes two‐component system of the HATS. Functional interaction between BdNRT2A‐GFP and BdNRT3.2‐RFP fusion proteins was observed at the plasma membrane in Arabidopsis protoplasts in transient expression experiments with BdNRT3.2 being necessary for the plasma membrane localization of BdNRT2A. The role of a conserved Ser residue in BdNRT2A (S461) specific to monocotyledons was evaluated in the BdNRT2A and BdNRT3.2 interaction leading to plasma membrane targeting. Assuming that S461 could be regulated by phosphorylation, a directed mutagenesis was performed to mimic a nonphosphorylated (S461A) or a constitutively phosphorylated (S461D), However, the mimicking the phosphorylation status of S461 by mutagenesis did not modify the BdNRT2A and BdNRT3.2 interaction, suggesting a more complex regulating mechanism. In conclusion, our data show that BdNRT2A and BdNRT3.2 are the main components of the nitrate HATS activity in Brachypodium (Bd21‐3) and allow an optimal growth in low N conditions.

High grain quality is a key target in wheat breeding and is influenced by genetic and environmental factors. This study evaluated 94 recombinant inbred lines (RILs) from a Pamyati Azieva × Paragon (PA × P) mapping population grown in two regions in Kazakhstan to assess the genetic basis of six grain quality traits: the test weight per liter (TWL, g/L), grain protein content (GPC, %), gluten content (GC, %), gluten deformation index in flour (GDI, unit), sedimentation value in a 2% acetic acid solution (SV, mL), and grain starch content (GSC, %). A correlation analysis revealed a trade-off between protein and starch accumulation and an inverse relationship between grain quality and yield components. Additionally, GPC exhibited a negative correlation with yield per square meter (YM2), underscoring the challenge of simultaneously improving grain quality and yield. With the use of the QTL Cartographer statistical package, 71 quantitative trait loci (QTLs) were identified for the six grain quality traits, including 20 QTLs showing stability across multiple environments. Notable stable QTLs were detected for GPC on chromosomes 4A, 5B, 6A, and 7B and for GC on chromosomes 1D and 6A, highlighting their potential for marker-assisted selection (MAS). A major QTL found on chromosome 1D (QGDI-PA × P.ipbb-1D.1, LOD 19.4) showed a strong association with gluten deformation index, emphasizing its importance in improving flour quality. A survey of published studies on QTL identification in common wheat suggested the likely novelty of 12 QTLs identified for GDI (five QTLs), TWL (three QTLs), SV, and GSC (two QTLs each). These findings underscore the need for balanced breeding strategies that optimize grain composition while maintaining high productivity. With the use of SNP markers associated with the identified QTLs for grain quality traits, the MAS approach can be implemented in wheat breeding programs.

Hybrids offer a promising approach to improve crop performance because the progeny are often superior to their parent lines and they outyield inbred varieties. A major challenge in producing hybrid progeny in wheat, however, lies in the inefficient fertilization of maternal parent ovaries by airborne pollen from male donor lines. This is often attributed to suboptimal synchronisation of male and female flowering as delayed pollination can result in reproductive failure due to female stigma deterioration. To test this accepted dogma, we examined the seed set capacity of six male sterile (MS) cultivars, each varying in the onset of stigma deterioration. To mimic a hybrid seed production scenario, MS cultivars were grown during two consecutive field seasons, and open pollination was allowed up to 15 days after flowering of the female parent using a blend of seven male fertile cultivars with varying flowering times. Detailed analysis of the temporal and spatial distribution of hybrid seed set along the spike across the six MS cultivars showed that seed production remained remarkably stable during the pollination window tested. These findings suggest sustained receptivity of stigma to pollen across all tested MS cultivars throughout the entire time course. We therefore conclude that stigma longevity does not represent a limiting factor in hybrid wheat seed production, and that breeding efforts should prioritise the study of other female traits, such as enhanced access to airborne pollen.

Proteases regulate important biological functions. Here, we present the structural and functional characterization of three previously uncharacterized aspartic proteases in Pseudomonas aeruginosa . We show that these proteases have structural hallmarks of retropepsin peptidases and play redundant roles for cell survival under hypoosmotic stress conditions. Consequently, we named them retropepsin-like osmotic stress tolerance peptidases (Rlo). Our research shows that while Rlo proteases are homologous to RimB, an aspartic peptidase involved in rhizosphere colonization and plant infection, they contain N-terminal signal peptides and perform distinct biological functions. Mutants lacking all three secreted Rlo peptidases show defects in antibiotic resistance, biofilm formation, and cell morphology. These defects are rescued by mutations in the inactive transglutaminase transmembrane protein RloB and the cytoplasmic ATP-grasp protein RloC, two previously uncharacterized genes in the same operon as one of the Rlo proteases. These studies identify Rlo proteases and rlo operon products as critical factors in clinically relevant processes, making them appealing targets for therapeutic strategies against Pseudomonas infections. IMPORTANCE Bacterial infections have become harder to treat due to the ability of pathogens to adapt to different environments and the rise of antimicrobial resistance. This has led to longer illnesses, increased medical costs, and higher mortality rates. The opportunistic pathogen Pseudomonas aeruginosa is particularly problematic because of its inherent resistance to many antibiotics and its capacity to form biofilms, structures that allow bacteria to withstand hostile conditions. Our study uncovers a new class of retropepsin-like proteases in P. aeruginosa that are required for biofilm formation and bacterial survival under stress conditions, including antibiotic exposure. By identifying critical factors that determine bacterial fitness and adaptability, our research lays the foundation for developing new therapeutic strategies against bacterial infections.

We assembled a European bread wheat (Triticum aestivum L.) association mapping panel (n = 480) genotyped using a 90,000 feature single nucleotide polymorphism array, with the aim of identifying genetic loci controlling resistance to four fungal diseases: yellow (stripe) rust (YR), brown (leaf) rust (BR), Septoria tritici blotch (ST) and powdery mildew (PM). Simulations showed our panel to have good power to detect genetic loci, with > 50% probability of identifying loci controlling as little as 5% of the variance when heritability was 0.6 or more. Using disease infection data collected across 31 trials undertaken in five European countries, genome-wide association studies (GWAS) identified 34 replicated genetic loci (20 for YR, 12 for BR, two for PM, 0 for ST), with seven loci associated with resistance to two or more diseases. Construction and analysis of eight bi-parental populations enabled two selected genetic loci, yellow rust resistance locus YR_2A010 (chromosome 2A) and YR_6A610 (6A), to be independently cross-validated, along with the development of genetic markers to track resistance alleles at these loci. Notably, the chromosome 2A yellow and brown rust resistance locus corresponds to the 2NvS introgression from the wild wheat species, Aegilops ventricosa. We found evidence of strong selection for 2NvS over recent breeding history, being present in 48% of the most recent cultivars in our panel. Collectively, we define the genetic architectures controlling resistance to major fungal diseases of wheat under European field environments, and provide resources to exploit these for the development of new wheat cultivars with improved disease resistance.

The formation of new species often involves the correlated divergence of multiple traits and genetic regions. However, the mechanisms by which such trait covariation builds up remain poorly understood. In this context, we consider two non‐exclusive hypotheses. First, genetic covariance between traits can cause divergent selection on one trait to promote population divergence in correlated traits (a genetic covariation hypothesis). Second, correlated environmental pressures can generate selection on multiple traits, facilitating the evolution of trait complexes (an environmental covariation hypothesis). Here, we test these hypotheses using cryptic colouration (controlled by a likely incipient supergene) and chemical traits (i.e., cuticular hydrocarbons, CHCs) involved in desiccation resistance and mate choice in Timema cristinae stick insects. We first demonstrate that population divergence in colour‐pattern is correlated with divergence in some (but not all) CHC traits. We show that correlated population divergence is unlikely to be explained by genetic covariation, as our analyses using genotyping‐by‐sequencing data reveal weak within‐population genetic covariance between colour‐pattern and CHC traits. In contrast, we find that correlated geographic variation in climate and host plant likely generates selection jointly on colour‐pattern and some CHC traits. This supports the environmental covariation hypothesis, likely via the effects of two correlated environmental axes selecting on different traits. Finally, we provide evidence that misalignment between natural and sexual selection also contributes to patterns of correlated trait divergence. Our results shed light on transitions between phases of speciation by showing that environmental factors can promote population divergence in trait complexes, even without strong genetic covariance.

Background Differences in the relative level of expression of homoeologs, known as homoeolog expression bias, are widely observed in allopolyploids. While the evolution of homoeolog expression bias through hybridization has been characterized, on shorter timescales such as those found in crop breeding programs, the extent to which homoeolog expression bias is preserved or altered between generations remains elusive. Results Here we use biparental mapping populations of hexaploid wheat (Triticum aestivum) with a common “Paragon” parent to explore the inheritance of homoeolog expression bias in the F5 generation. We found that homoeolog expression bias is inherited for 26–27% of triads in both populations. Most triads conserved a similar homoeolog expression bias pattern as one or both parents. Inherited patterns were largely driven by changes in the expression of one homoeolog, allowing homoeolog expression bias in subsequent generations to match parental expression. Novel patterns of homoeolog expression bias occurred more frequently in the biparental population from a landrace × elite cross, than in the population with two elite parents. Conclusions These results demonstrate that there is significant reprogramming and stabilization of homoeolog expression bias within a small number of generations that differs significantly based on the parental lines used in the crossing.

Tef (Eragrostis tef) is an indigenous African cereal that is gaining global attention as a gluten-free “superfood” with high protein, mineral, and fibre contents. However, tef yields are limited by lodging and by losses during harvest owing to its small grain size (150× lighter than wheat). Breeders must also consider a strong cultural preference for white-grained over brown-grained varieties. Tef is relatively understudied with limited “omics” resources. Here, we resequence 220 tef accessions from an Ethiopian diversity collection and also perform multi-locational phenotyping for 25 agronomic and grain traits. Grain metabolome profiling reveals differential accumulation of fatty acids and flavonoids between white and brown grains. k-mer and SNP-based genome-wide association uncover important marker-trait associations, including a significant 70 kb peak for panicle morphology containing the tef orthologue of rice qSH1—a transcription factor regulating inflorescence morphology in cereals. We also observe a previously unknown relationship between grain size, colour, and fatty acids. These traits are highly associated with retrotransposon insertions in homoeologues of TRANSPARENT TESTA 2, a known regulator of grain colour. Our study provides valuable resources for tef research and breeding, facilitating the development of improved cultivars with desirable agronomic and nutritional properties.

Reaction–diffusion equations are commonly used to model a diverse array of complex systems, including biological, chemical, and physical processes. Typically, these models are phenomenological, requiring the fitting of parameters to experimental data. In the present work, we introduce a novel formalism to construct reaction–diffusion models that is grounded in the principle of maximum entropy. This new formalism aims to incorporate various types of experimental data, including ensemble currents, distributions at different points in time, or moments of such. To this end, we expand the framework of Schrödinger bridges and maximum caliber problems to nonlinear interacting systems. We illustrate the usefulness of the proposed approach by modeling the evolution of (i) a morphogen across the fin of a zebrafish and (ii) the population of two varieties of toads in Poland, so as to match the experimental data.

Ferritins are ubiquitous proteins that function in iron storage/detoxification by catalyzing the oxidation of Fe²⁺ ions and solubilizing the resulting Fe³⁺-oxo mineral. Mammalian tissues that are metabolically highly active contain, in addition to the widespread cytosolic ferritin, a ferritin that is localized to mitochondria. Mitochondrial ferritin (FtMt) protects against oxidative stress and is found at higher levels in diseases associated with abnormal iron accumulation, including Alzheimer’s and Parkinson’s. Here we demonstrate that, despite 80% sequence identity with cytosolic human H-chain ferritin, Fe²⁺ oxidation at the catalytic diiron ferroxidase center of FtMt proceeds via a distinct mechanism. This involves a mixed-valent ferroxidase center (MVFC) that is readily detected under the O2-limiting conditions typical of mitochondria, and formation of a radical on a strictly conserved Tyr residue (Tyr34) that is key for the activation of O2 and stability of the MVFC. The possible origin of the mechanistic differences exhibited by the highly-related human mitochondrial and cytosolic H-chain ferritins is explored.

The natural products actinonin and matlystatin feature an N -hydroxy-2-pentyl-succinamyl (HPS) chemophore that facilitates metal chelation and confers their metalloproteinase inhibitory activity. Actinonin is the most potent natural inhibitor of peptide deformylase (PDF) and exerts antimicrobial and herbicidal bioactivity by disrupting protein synthesis. Here, we used a genomics-led approach to identify candidate biosynthetic gene clusters (BGCs) hypothesized to produce HPS-containing natural products. We show that one of these BGCs is on the pathogenicity megaplasmid of the plant pathogen Rhodococcus fascians and produces lydiamycin A, a macrocyclic pentapeptide. The presence of genes predicted to make an HPS-like chemophore informed the structural recharacterization of lydiamycin via NMR and crystallography to show that it features a rare 2-pentyl-succinyl chemophore. We demonstrate that lydiamycin A inhibits bacterial PDF in vitro and show that a cluster-situated PDF gene confers resistance to lydiamycin A, representing an uncommon self-immunity mechanism associated with the production of a PDF inhibitor . In planta competition assays showed that lydiamycin enhances the fitness of R. fascians during plant colonization. This study highlights how a BGC can inform the structure, biochemical target, and ecological function of a natural product.

In oilseed crops, e.g. oilseed rape (OSR; Brassica napus), a key developmental process is seed maturation, during which the embryo transitions from the early, globular state to the mature state. Seed development involves cell division, differentiation, and oil accumulation in specific tissue types (embryo, endosperm, and seed coat). These developmental processes impact seed quality and oil yield. High quality RNA from Brassica spp. seed tissues, from heart to mature developmental stages, was obtained using previously reported methods for five Brassica genotypes comprising winter, semi-winter and spring OSR varieties, a B. napus heritage kale and a rapid-cycling double-haploid Brassica oleracea line. RNA-seq was performed on 240 sets of samples. The resulting dataset contains detailed spatio-temporal expression profiles during seed development. In addition to the repository data, we provide easy access to this through the Seed Oilseed Rape Developmental Expression Resource (SeedORDER), which enables users to search for genes of interest and visualise expression patterns. Knowledge of where and when genes are expressed during seed development will inform future breeding efforts.

The rice blast fungus Magnaporthe oryzae secretes a battery of effector proteins to facilitate host infection. Among these effectors, Pathogenicity toward Weeping Lovegrass 2 (Pwl2) was originally identified as a host specificity determinant for the infection of weeping lovegrass (Eragrostis curvula) and is also recognized by the barley (Hordeum vulgare) Mla3 resistance protein. However, the biological activity of Pwl2 remains unknown. Here, we showed that the Pmk1 MAP kinase regulates PWL2 expression during the cell-to-cell movement of M. oryzae at plasmodesmata-containing pit fields. Consistent with this finding, we provided evidence that Pwl2 binds to the barley heavy metal-binding isoprenylated protein HIPP43, which results in HIPP43 displacement from plasmodesmata. Transgenic barley lines overexpressing PWL2 or HIPP43 exhibit attenuated immune responses and increased disease susceptibility. In contrast, a Pwl2SNDEYWY variant which does not interact with HIPP43 fails to alter the plasmodesmata localization of HIPP43. Targeted deletion of three PWL2 copies in M. oryzae resulted in a Δpwl2 mutant showing gain of virulence toward weeping lovegrass and barley Mla3 lines, but reduced blast disease severity on susceptible host plants. Taken together, our results provide evidence that Pwl2 is a virulence factor that suppresses host immunity by perturbing the plasmodesmatal deployment of HIPP43.

Background and Objectives The size distribution of starch granules is an important factor in determining the functional and nutritional properties of starch. However, a simple, standardized method for their analysis is lacking. Here, we developed an approach for estimating granule size parameters using a Python script that fits curves to volumetric granule size distributions generated using a Coulter counter. Findings The bimodal size distribution of starch from most wheat and barley cultivars could be best described with a mixed distribution curve. A log‐normal distribution was fitted to the small B‐type granules, and a normal distribution was fitted to the large A‐type granules, allowing estimation of their relative abundance and size parameters despite their overlapping size distributions. However, the optimal fitting is altered in wheat mutants with large perturbations in B‐type granule content. In maize and rice, which have unimodal granule size distributions, size parameters were calculated by fitting a single normal distribution. Conclusions Curve fitting is an effective approach for estimating starch granule size parameters in diverse cereals, particularly the Triticeae with A‐ and B‐type granules. Significance and Novelty We provide new tools and guidelines for the quantitative analysis of granule size in cereals.