Regulation of developmental gatekeeping and cell fate transition by the calpain protease DEK1 in Physcomitrium patens.

Calpains are cysteine proteases that control cell fate transitions whose loss of function causes severe, pleiotropic phenotypes in eukaryotes. Although mainly considered as modulatory proteases, human calpain targets are directed to the N-end rule degradation pathway. Several such targets are transcription factors, hinting at a gene-regulatory role. Here, we analyze the gene-regulatory networks of the moss Physcomitrium patens and characterize the regulons that are misregulated in mutants of the calpain DEFECTIVE KERNEL1 (DEK1). Predicted cleavage patterns of the regulatory hierarchies in five DEK1-controlled subnetworks are consistent with a pleiotropic and regulatory role during cell fate transitions targeting multiple functions. Network structure suggests DEK1-gated sequential transitions between cell fates in 2D-to-3D development. Our method combines comprehensive phenotyping, transcriptomics and data science to dissect phenotypic traits, and our model explains the protease function as a switch gatekeeping cell fate transitions potentially also beyond plant development.

Meiotic recombination dynamics in plants with repeat-based holocentromeres shed light on the primary drivers of crossover patterning.

Centromeres strongly affect (epi)genomic architecture and meiotic recombination dynamics, influencing the overall distribution and frequency of crossovers. Here we show how recombination is regulated and distributed in the holocentric plant Rhynchospora breviuscula, a species with diffused centromeres. Combining immunocytochemistry, chromatin analysis and high-throughput single-pollen sequencing, we discovered that crossover frequency is distally biased, in sharp contrast to the diffused distribution of hundreds of centromeric units and (epi)genomic features. Remarkably, we found that crossovers were abolished inside centromeric units but not in their proximity, indicating the absence of a canonical centromere effect. We further propose that telomere-led synapsis of homologues is the feature that best explains the observed recombination landscape. Our results hint at the primary influence of mechanistic features of meiotic pairing and synapsis rather than (epi)genomic features and centromere organization in determining the distally biased crossover distribution in R. breviuscula, whereas centromeres and (epi)genetic properties only affect crossover positioning locally.

Integrative multi-omics analyses of date palm (Phoenix dactylifera) roots and leaves reveal how the halophyte land plant copes with sea water.

Date palm (Phoenix dactylifera L.) is able to grow and complete its life cycle while being rooted in highly saline soils. Which of the many well-known salt-tolerance strategies are combined to fine-tune this remarkable resilience is unknown. The precise location, whether in the shoot or the root, where these strategies are employed remains uncertain, leaving us unaware of how the various known salt-tolerance mechanisms are integrated to fine-tune this remarkable resilience. To address this shortcoming, we exposed date palm to a salt stress dose equivalent to seawater for up to 4 weeks and applied integrative multi-omics analyses followed by targeted metabolomics, hormone, and ion analyses. Integration of proteomic into transcriptomic data allowed a view beyond simple correlation, revealing a remarkably high degree of convergence between gene expression and protein abundance. This sheds a clear light on the acclimatization mechanisms employed, which depend on reprogramming of protein biosynthesis. For growth in highly saline habitats, date palm effectively combines various salt-tolerance mechanisms found in both halophytes and glycophytes: "avoidance" by efficient sodium and chloride exclusion at the roots, and "acclimation" by osmotic adjustment, reactive oxygen species scavenging in leaves, and remodeling of the ribosome-associated proteome in salt-exposed root cells. Combined efficiently as in P. dactylifera L., these sets of mechanisms seem to explain the palm's excellent salt stress tolerance.

Barley shows reduced Fusarium head blight under drought and modular expression of differentially expressed genes under combined stress.

Plants often face simultaneous abiotic and biotic stress conditions; however, physiological and transcriptional responses under such combined stress conditions are still not fully understood. Spring barley (Hordeum vulgare) is susceptible to Fusarium head blight (FHB), which is strongly affected by weather conditions. We therefore studied the potential influence of drought on FHB severity and plant responses in three varieties of different susceptibility. We found strongly reduced FHB severity in susceptible varieties under drought. The number of differentially expressed genes (DEGs) and strength of transcriptomic regulation reflected the concentrations of physiological stress markers such as abscisic acid or fungal DNA contents. Infection-related gene expression was associated with susceptibility rather than resistance. Weighted gene co-expression network analysis revealed 18 modules of co-expressed genes that reflected the pathogen- or drought-response in the three varieties. A generally infection-related module contained co-expressed genes for defence, programmed cell death, and mycotoxin detoxification, indicating that the diverse genotypes used a similar defence strategy towards FHB, albeit with different degrees of success. Further, DEGs showed co-expression in drought- or genotype-associated modules that correlated with measured phytohormones or the osmolyte proline. The combination of drought stress with infection led to the highest numbers of DEGs and resulted in a modular composition of the single-stress responses rather than a specific transcriptional output.

Genome-wide association of dry (Tamar) date palm fruit color.

Date palm (Phoenix dactylifera) fruit (dates) are an economically and culturally significant crop in the Middle East and North Africa. There are hundreds of different commercial cultivars producing dates with distinctive shapes, colors, and sizes. Genetic studies of some date palm traits have been performed, including sex determination, sugar content, and fresh fruit color. In this study, we used genome sequences and image data of 199 dry dates (Tamar) collected from 14 countries to identify genetic loci associated with the color of this fruit stage. Here, we find loci across multiple linkage groups (LG) associated with dry fruit color phenotype. We recover both the previously identified VIRESCENS (VIR) genotype associated with fresh fruit yellow or red color and new associations with the lightness and darkness of dry fruit. This study will add resolution to our understanding of date color phenotype, especially at the most commercially important Tamar stage.

Genome analyses reveal population structure and a purple stigma color gene candidate in finger millet.

Finger millet is a key food security crop widely grown in eastern Africa, India and Nepal. Long considered a 'poor man's crop', finger millet has regained attention over the past decade for its climate resilience and the nutritional qualities of its grain. To bring finger millet breeding into the 21st century, here we present the assembly and annotation of a chromosome-scale reference genome. We show that this ~1.3 million years old allotetraploid has a high level of homoeologous gene retention and lacks subgenome dominance. Population structure is mainly driven by the differential presence of large wild segments in the pericentromeric regions of several chromosomes. Trait mapping, followed by variant analysis of gene candidates, reveals that loss of purple coloration of anthers and stigma is associated with loss-of-function mutations in the finger millet orthologs of the maize R1/B1 and Arabidopsis GL3/EGL3 anthocyanin regulatory genes. Proanthocyanidin production in seed is not affected by these gene knockouts.

Repeat-based holocentromeres influence genome architecture and karyotype evolution.

The centromere represents a single region in most eukaryotic chromosomes. However, several plant and animal lineages assemble holocentromeres along the entire chromosome length. Here, we compare genome organization and evolution as a function of centromere type by assembling chromosome-scale holocentric genomes with repeat-based holocentromeres from three beak-sedge (Rhynchospora pubera, R. breviuscula, and R. tenuis) and their closest monocentric relative, Juncus effusus. We demonstrate that transition to holocentricity affected 3D genome architecture by redefining genomic compartments, while distributing centromere function to thousands of repeat-based centromere units genome-wide. We uncover a complex genome organization in R. pubera that hides its unexpected octoploidy and describe a marked reduction in chromosome number for R. tenuis, which has only two chromosomes. We show that chromosome fusions, facilitated by repeat-based holocentromeres, promoted karyotype evolution and diploidization. Our study thus sheds light on several important aspects of genome architecture and evolution influenced by centromere organization.

Stalk cell polar ion transport provide for bladder-based salinity tolerance in Chenopodium quinoa.

Chenopodium quinoa uses epidermal bladder cells (EBCs) to sequester excess salt. Each EBC complex consists of a leaf epidermal cell, a stalk cell, and the bladder. Under salt stress, sodium (Na+ ), chloride (Cl- ), potassium (K+ ) and various metabolites are shuttled from the leaf lamina to the bladders. Stalk cells operate as both a selectivity filter and a flux controller. In line with the nature of a transfer cell, advanced transmission electron tomography, electrophysiology, and fluorescent tracer flux studies revealed the stalk cell's polar organization and bladder-directed solute flow. RNA sequencing and cluster analysis revealed the gene expression profiles of the stalk cells. Among the stalk cell enriched genes, ion channels and carriers as well as sugar transporters were most pronounced. Based on their electrophysiological fingerprint and thermodynamic considerations, a model for stalk cell transcellular transport was derived.

The mosaic oat genome gives insights into a uniquely healthy cereal crop.

Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.

The Barley and Wheat Pan-Genomes.

To unlock the genetic potential in crops, multi-genome comparisons are an essential tool. Decreasing costs and improved sequencing technologies have democratized plant genome sequencing and led to a vast increase in the amount of available reference sequences on the one hand and enabled the assembly of even the largest and most complex and repetitive crops genomes such as wheat and barley. These developments have led to the era of pan-genomics in recent years. Pan-genome projects enable the definition of the core and dispensable genome for various crop species as well as the analysis of structural and functional variation and hence offer unprecedented opportunities for exploring and utilizing the genetic basis of natural variation in crops. Comparing, analyzing, and visualizing these multiple reference genomes and their diversity requires powerful and specialized computational strategies and tools.

Genome sequences of three Aegilops species of the section Sitopsis reveal phylogenetic relationships and provide resources for wheat improvement.

Aegilops is a close relative of wheat (Triticum spp.), and Aegilops species in the section Sitopsis represent a rich reservoir of genetic diversity for the improvement of wheat. To understand their diversity and advance their utilization, we produced whole-genome assemblies of Aegilops longissima and Aegilops speltoides. Whole-genome comparative analysis, along with the recently sequenced Aegilops sharonensis genome, showed that the Ae. longissima and Ae. sharonensis genomes are highly similar and are most closely related to the wheat D subgenome. By contrast, the Ae. speltoides genome is more closely related to the B subgenome. Haplotype block analysis supported the idea that Ae. speltoides genome is closest to the wheat B subgenome, and highlighted variable and similar genomic regions between the three Aegilops species and wheat. Genome-wide analysis of nucleotide-binding leucine-rich repeat (NLR) genes revealed species-specific and lineage-specific NLR genes and variants, demonstrating the potential of Aegilops genomes for wheat improvement.

Aegilops tauschii genome assembly Aet v5.0 features greater sequence contiguity and improved annotation.

Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and an important genetic resource. The reference-quality genome sequence Aet v4.0 for Ae. tauschii acc. AL8/78 was therefore an important milestone for wheat biology and breeding. Further advances in sequencing acc. AL8/78 and release of the Aet v5.0 sequence assembly are reported here. Two new optical maps were constructed and used in the revision of pseudomolecules. Gaps were closed with Pacific Biosciences long-read contigs, decreasing the gap number by 38,899. Transposable elements and protein-coding genes were reannotated. The number of annotated high-confidence genes was reduced from 39,635 in Aet v4.0 to 32,885 in Aet v5.0. A total of 2245 biologically important genes, including those affecting plant phenology, grain quality, and tolerance of abiotic stresses in wheat, was manually annotated and disease-resistance genes were annotated by a dedicated pipeline. Disease-resistance genes encoding nucleotide-binding site domains, receptor-like protein kinases, and receptor-like proteins were preferentially located in distal chromosome regions, whereas those encoding transmembrane coiled-coil proteins were dispersed more evenly along the chromosomes. Discovery, annotation, and expression analyses of microRNA (miRNA) precursors, mature miRNAs, and phasiRNAs are reported, including miRNA target genes. Other small RNAs, such as hc-siRNAs and tRFs, were characterized. These advances enhance the utility of the Ae. tauschii genome sequence for wheat genetics, biotechnology, and breeding.

Fusarium head blight resistance in European winter wheat: insights from genome-wide transcriptome analysis.

BACKGROUND: Fusarium head blight (FHB) is a devastating disease of wheat worldwide. Resistance to FHB is quantitatively controlled by the combined effects of many small to medium effect QTL. Flowering traits, especially the extent of extruded anthers, are strongly associated with FHB resistance. RESULTS: To characterize the genetic basis of FHB resistance, we generated and analyzed phenotypic and gene expression data on the response to Fusarium graminearum (Fg) infection in 96 European winter wheat genotypes, including several lines containing introgressions from the highly resistant Asian cultivar Sumai3. The 96 lines represented a broad range in FHB resistance and were assigned to sub-groups based on their phenotypic FHB severity score. Comparative analyses were conducted to connect sub-group-specific expression profiles in response to Fg infection with FHB resistance level. Collectively, over 12,300 wheat genes were Fusarium responsive. The core set of genes induced in response to Fg was common across different resistance groups, indicating that the activation of basal defense response mechanisms was largely independent of the resistance level of the wheat line. Fg-induced genes tended to have higher expression levels in more susceptible genotypes. Compared to the more susceptible non-Sumai3 lines, the Sumai3-derivatives demonstrated higher constitutive expression of genes associated with cell wall and plant-type secondary cell wall biogenesis and higher constitutive and Fg-induced expression of genes involved in terpene metabolism. Gene expression analysis of the FHB QTL Qfhs.ifa-5A identified a constitutively expressed gene encoding a stress response NST1-like protein (TraesCS5A01G211300LC) as a candidate gene for FHB resistance. NST1 genes are key regulators of secondary cell wall biosynthesis in anther endothecium cells. Whether the stress response NST1-like gene affects anther extrusion, thereby affecting FHB resistance, needs further investigation. CONCLUSION: Induced and preexisting cell wall components and terpene metabolites contribute to resistance and limit fungal colonization early on. In contrast, excessive gene expression directs plant defense response towards programmed cell death which favors necrotrophic growth of the Fg pathogen and could thus lead to increased fungal colonization.

Protein expression plasticity contributes to heat and drought tolerance of date palm.

Climate change is increasing the frequency and intensity of warming and drought periods around the globe, currently representing a threat to many plant species. Understanding the resistance and resilience of plants to climate change is, therefore, urgently needed. As date palm (Phoenix dactylifera) evolved adaptation mechanisms to a xeric environment and can tolerate large diurnal and seasonal temperature fluctuations, we studied the protein expression changes in leaves, volatile organic compound emissions, and photosynthesis in response to variable growth temperatures and soil water deprivation. Plants were grown under controlled environmental conditions of simulated Saudi Arabian summer and winter climates challenged with drought stress. We show that date palm is able to counteract the harsh conditions of the Arabian Peninsula by adjusting the abundances of proteins related to the photosynthetic machinery, abiotic stress and secondary metabolism. Under summer climate and water deprivation, these adjustments included efficient protein expression response mediated by heat shock proteins and the antioxidant system to counteract reactive oxygen species formation. Proteins related to secondary metabolism were downregulated, except for the P. dactylifera isoprene synthase (PdIspS), which was strongly upregulated in response to summer climate and drought. This study reports, for the first time, the identification and functional characterization of the gene encoding for PdIspS, allowing future analysis of isoprene functions in date palm under extreme environments. Overall, the current study shows that reprogramming of the leaf protein profiles confers the date palm heat- and drought tolerance. We conclude that the protein plasticity of date palm is an important mechanism of molecular adaptation to environmental fluctuations.

Long-read sequence assembly: a technical evaluation in barley.

Sequence assembly of large and repeat-rich plant genomes has been challenging, requiring substantial computational resources and often several complementary sequence assembly and genome mapping approaches. The recent development of fast and accurate long-read sequencing by circular consensus sequencing (CCS) on the PacBio platform may greatly increase the scope of plant pan-genome projects. Here, we compare current long-read sequencing platforms regarding their ability to rapidly generate contiguous sequence assemblies in pan-genome studies of barley (Hordeum vulgare). Most long-read assemblies are clearly superior to the current barley reference sequence based on short-reads. Assemblies derived from accurate long reads excel in most metrics, but the CCS approach was the most cost-effective strategy for assembling tens of barley genomes. A downsampling analysis indicated that 20-fold CCS coverage can yield very good sequence assemblies, while even five-fold CCS data may capture the complete sequence of most genes. We present an updated reference genome assembly for barley with near-complete representation of the repeat-rich intergenic space. Long-read assembly can underpin the construction of accurate and complete sequences of multiple genomes of a species to build pan-genome infrastructures in Triticeae crops and their wild relatives.

Chromosome-scale genome assembly provides insights into rye biology, evolution and agronomic potential.

Rye (Secale cereale L.) is an exceptionally climate-resilient cereal crop, used extensively to produce improved wheat varieties via introgressive hybridization and possessing the entire repertoire of genes necessary to enable hybrid breeding. Rye is allogamous and only recently domesticated, thus giving cultivated ryes access to a diverse and exploitable wild gene pool. To further enhance the agronomic potential of rye, we produced a chromosome-scale annotated assembly of the 7.9-gigabase rye genome and extensively validated its quality by using a suite of molecular genetic resources. We demonstrate applications of this resource with a broad range of investigations. We present findings on cultivated rye's incomplete genetic isolation from wild relatives, mechanisms of genome structural evolution, pathogen resistance, low-temperature tolerance, fertility control systems for hybrid breeding and the yield benefits of rye-wheat introgressions.

PYL8 ABA receptors of Phoenix dactylifera play a crucial role in response to abiotic stress and are stabilized by ABA.

The identification of those prevalent abscisic acid (ABA) receptors and molecular mechanisms that trigger drought adaptation in crops well adapted to harsh conditions such as date palm (Phoenix dactylifera, Pd) sheds light on plant-environment interactions. We reveal that PdPYL8-like receptors are predominantly expressed under abiotic stress, with Pd27 being the most expressed receptor in date palm. Therefore, subfamily I PdPYL8-like receptors have been selected for ABA signaling during abiotic stress response in this crop. Biochemical characterization of PdPYL8-like and PdPYL1-like receptors revealed receptor- and ABA-dependent inhibition of PP2Cs, which triggers activation of the pRD29B-LUC reporter in response to ABA. PdPYLs efficiently abolish PP2C-mediated repression of ABA signaling, but loss of the Trp lock in the seed-specific AHG1-like phosphatase PdPP2C79 markedly impairs its inhibition by ABA receptors. Characterization of Arabidopsis transgenic plants that express PdPYLs shows enhanced ABA signaling in seed, root, and guard cells. Specifically, Pd27-overexpressing plants showed lower ABA content and were more efficient than the wild type in lowering transpiration at negative soil water potential, leading to enhanced drought tolerance. Finally, PdPYL8-like receptors accumulate after ABA treatment, which suggests that ABA-induced stabilization of these receptors operates in date palm for efficient boosting of ABA signaling in response to abiotic stress.

De Novo Genome Assembly of the Japanese Wheat Cultivar Norin 61 Highlights Functional Variation in Flowering Time and Fusarium-Resistant Genes in East Asian Genotypes.

Bread wheat is a major crop that has long been the focus of basic and breeding research. Assembly of its genome has been difficult because of its large size and allohexaploid nature (AABBDD genome). Following the first reported assembly of the genome of the experimental strain Chinese Spring (CS), the 10+ Wheat Genomes Project was launched to produce multiple assemblies of worldwide modern cultivars. The only Asian cultivar in the project is Norin 61, a representative Japanese cultivar adapted to grow across a broad latitudinal range, mostly characterized by a wet climate and a short growing season. Here, we characterize the key aspects of its chromosome-scale genome assembly spanning 15 Gb with a raw scaffold N50 of 22 Mb. Analysis of the repetitive elements identified chromosomal regions unique to Norin 61 that encompass a tandem array of the pathogenesis-related 13 family. We report novel copy-number variations in the B homeolog of the florigen gene FT1/VRN3, pseudogenization of its D homeolog and the association of its A homeologous alleles with the spring/winter growth habit. Furthermore, the Norin 61 genome carries typical East Asian functional variants different from CS, ranging from a single nucleotide to multi-Mb scale. Examples of such variation are the Fhb1 locus, which confers Fusarium head-blight resistance, Ppd-D1a, which confers early flowering, Glu-D1f for Asian noodle quality and Rht-D1b, which introduced semi-dwarfism during the green revolution. The adoption of Norin 61 as a reference assembly for functional and evolutionary studies will enable comprehensive characterization of the underexploited Asian bread wheat diversity.

The barley pan-genome reveals the hidden legacy of mutation breeding.

Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop species (known as the 'pan-genome'1). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley (Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions2. Here we report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley-comprising landraces, cultivars and a wild barley-that were selected as representatives of global barley diversity. We catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. We discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding.