Einkorn genomics sheds light on history of the oldest domesticated wheat.

Einkorn (Triticum monococcum) was the first domesticated wheat species, and was central to the birth of agriculture and the Neolithic Revolution in the Fertile Crescent around 10,000 years ago1,2. Here we generate and analyse 5.2-Gb genome assemblies for wild and domesticated einkorn, including completely assembled centromeres. Einkorn centromeres are highly dynamic, showing evidence of ancient and recent centromere shifts caused by structural rearrangements. Whole-genome sequencing analysis of a diversity panel uncovered the population structure and evolutionary history of einkorn, revealing complex patterns of hybridizations and introgressions after the dispersal of domesticated einkorn from the Fertile Crescent. We also show that around 1% of the modern bread wheat (Triticum aestivum) A subgenome originates from einkorn. These resources and findings highlight the history of einkorn evolution and provide a basis to accelerate the genomics-assisted improvement of einkorn and bread wheat.

Physical mapping of the wheat genes in low-recombination regions: radiation hybrid mapping of the C-locus.

KEY MESSAGE: This work reports the physical mapping of an important gene affecting spike compactness located in a low-recombination region of hexaploid wheat. This work paves the way for the eventual isolation and characterization of the factor involved but also opens up possibilities to use this approach to precisely map other wheat genes located on proximal parts of wheat chromosomes that show highly reduced recombination. Mapping wheat genes, in the centromeric and pericentromeric regions (~ 2/3rd of a given chromosome), poses a formidable challenge due to highly suppressed recombination. Using an example of compact spike locus (C-locus), this study provides an approach to precisely map wheat genes in the pericentromeric and centromeric regions that house ~ 30% of wheat genes. In club-wheat, spike compactness is controlled by the dominant C-locus, but previous efforts have failed to localize it, on a particular arm of chromosome 2D. We integrated radiation hybrid (RH) and high-resolution genetic mapping to locate C-locus on the short arm of chromosome 2D. Flanking markers of the C-locus span a physical distance of 11.0 Mb (231.0-242 Mb interval) and contain only 11 high-confidence annotated genes. This work demonstrates the value of this integrated strategy in mapping dominant genes in the low-recombination regions of the wheat genome. A comparison of the mapping resolutions of the RH and genetic maps using common anchored markers indicated that the RH map provides ~ 9 times better resolution that the genetic map even with much smaller population size. This study provides a broadly applicable approach to fine map wheat genes in regions of suppressed recombination.

Comprehensive compositional assessment of bioactive compounds in diverse pea accessions.

Pea (Pisum sativum L.) is an important legume crop providing a good source of protein, vitamins, minerals and bioactive compounds with health benefits for humans. In this study, an improved method for simultaneous analysis of multiple phytoestrogens among 100 pea accessions was developed. Ipriflavone, (a synthetic isoflavone), was used as an internal standard for the semiquantitative analysis of 17 phytoestrogens including isoflavone aglycones and conjugates, allowing direct analysis of isoflavones in their naturally occurring forms. This comprehensive dataset demonstrated that the isoflavones varied greatly and some accessions tended to have high levels of multiple phytoestrogens among the 100 accessions analyzed. Isoliquiritigenin followed by glycitein were the predominant compounds detected in the accessions and showed the highest correlation with the total phytoestrogens content. Secoisolariciresinol content was consistently higher in yellow cotyledon peas than in green cotyledon peas, whereas the contents of coumestrol, genestein and secoisolariciresinol were significantly correlated with seed coat color. The total phenolics and saponins showed a wide range of variability among the accessions with higher concentrations of total phenolics observed in seeds with pigmented seed coat or yellow cotyledon seeds, suggesting the synthesis of saponins and phenolics are significantly affected by metabolic pathway genes controlling cotyledon color or seed coat color. This study profiled the variability of bioactive compounds of pea seed quality traits in diverse pea accessions and provides an immense resource for continued research, breeding and selection of genotypes for a wide range of applications.

Spatiotemporal transcriptomics and metabolic profiling provide insights into gene regulatory networks during lentil seed development.

Lentil (Lens culinaris Medik.) is a nutritious legume with seeds rich in protein, minerals and an array of diverse specialized metabolites. The formation of a seed requires regulation and tight coordination of developmental programs to form the embryo, endosperm and seed coat compartments, which determines the structure and composition of mature seed and thus its end-use quality. Understanding the molecular and cellular events and metabolic processes of seed development is essential for improving lentil yield and seed nutritional value. However, such information remains largely unknown, especially at the seed compartment level. In this study, we generated high-resolution spatiotemporal gene expression profiles in lentil embryo, seed coat and whole seeds from fertilization through maturation. Apart from anatomic differences between the embryo and seed coat, comparative transcriptomics and weighted gene co-expression network analysis revealed embryo- and seed coat-specific genes and gene modules predominant in specific tissues and stages, which highlights distinct genetic programming. Furthermore, we investigated the dynamic profiles of flavonoid, isoflavone, phytic acid and saponin in seed compartments across seed development. Coupled with transcriptome data, we identified sets of candidate genes involved in the biosynthesis of these metabolites. The global view of the transcriptional and metabolic changes of lentil seed tissues throughout development provides a valuable resource for dissecting the genetic control of secondary metabolism and development of molecular tools for improving seed nutritional quality.

Involvement of Target of Rapamycin (TOR) Signaling in the Regulation of Crosstalk between Ribosomal Protein Small Subunit 6 Kinase-1 (RPS6K-1) and Ribosomal Proteins.

The target of rapamycin (TOR) protein phosphorylates its downstream effector p70kDa ribosomal protein S6 kinases (S6K1) for ribosome biogenesis and translation initiation in eukaryotes. However, the molecular mechanism of TOR-S6K1-ribosomal protein (RP) signaling is not well understood in plants. In the present study, we report the transcriptional upregulation of ribosomal protein large and small subunit (RPL and RPS) genes in the previously established TOR overexpressing transgenic lines of rice (in Oryza sativa ssp. indica, variety BPT-5204, TR-2.24 and TR-15.1) and of Arabidopsis thaliana (in Col 0 ecotype, ATR-1.4.27 and ATR-3.7.32). The mRNA levels of RP genes from this study were compared with those previously available in transcriptomic datasets on the expression of RPs in relation to TOR inhibitor and in the TOR-RNAi lines of Arabidopsis thaliana. We further analyzed TOR activity, i.e., S6K1 phosphorylation in SALK lines of Arabidopsis with mutation in rpl6, rpl18, rpl23, rpl24 and rps28C, where the rpl18 mutant showed inactivation of S6K1 phosphorylation. We also predicted similar putative Ser/Thr phosphorylation sites for ribosomal S6 kinases (RSKs) in the RPs of Oryza sativa ssp. indica and Arabidopsis thaliana. The findings of this study indicate that the TOR pathway is possibly interlinked in a cyclic manner via the phosphorylation of S6K1 as a modulatory step for the regulation of RP function to switch 'on'/'off' the translational regulation for balanced plant growth.

Asymmetric gene expression in grain development of reciprocal crosses between tetraploid and hexaploid wheats.

Production of viable progeny from interploid crosses requires precise regulation of gene expression from maternal and paternal chromosomes, yet the transcripts contributed to hybrid seeds from polyploid parent species have rarely been explored. To investigate the genome-wide maternal and paternal contributions to polyploid grain development, we analyzed the transcriptomes of developing embryos, from zygote to maturity, alongside endosperm in two stages of development, using reciprocal crosses between tetraploid and hexaploid wheats. Reciprocal crosses between species with varied levels of ploidy displayed broad impacts on gene expression, including shifts in alternative splicing events in select crosses, as illustrated by active splicing events, enhanced protein synthesis and chromatin remodeling. Homoeologous gene expression was repressed on the univalent D genome in pentaploids, but this suppression was attenuated in crosses with a higher ploidy maternal parent. Imprinted genes were identified in endosperm and early embryo tissues, supporting predominant maternal effects on early embryogenesis. By systematically investigating the complex transcriptional networks in reciprocal-cross hybrids, this study presents a framework for understanding the genomic incompatibility and transcriptome shock that results from interspecific hybridization and uncovers the transcriptional impacts on hybrid seeds created from agriculturally-relevant polyploid species.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-generated diallelic mutants reveal Arabidopsis actin-related protein 2 function in the trafficking of syntaxin PEN1.

In plants, the actin cytoskeleton plays a critical role in defense against diverse pathogens. The formation of actin patches is essential for the intracellular transport of organelles and molecules toward pathogen penetration sites and the formation of papillae for an early cellular response to powdery mildew attack in Arabidopsis thaliana. This response process is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR complex (W/SRC). The ARP2/3 complex is also required for maintaining steady-state levels of the defense-associated protein, PENETRATION 1 (PEN1), at the plasma membrane and for its deposition into papillae. However, specific ARP2 functionalities in this context remain unresolved, as knockout mutants expressing GFP-PEN1 reporter constructs could not be obtained by conventional crossing approaches. In this study, employing a CRISPR/Cas9 multiplexing-mediated genome editing approach, we produced an ARP2 knockout expressing the GFP-PEN1 marker in Arabidopsis. This study successfully identified diallelic somatic mutations with both ARP2 alleles edited among the primary T1 transgenic plants, and also obtained independent lines with stable arp2/arp2 mutations in the T2 generation. Further analyses on these arp2/arp2 mutants showed similar biological functions of ARP2 to ARP3 in the accumulation of PEN1 against fungal invasion. Together, this CRISPR/Cas9-based approach offers highly efficient simultaneous disruption of the two ARP2 alleles in GFP-PEN1-expressing lines, and a rapid method for performing live-cell imaging to facilitate the investigation of important plant-pathogen interactions using a well-established and widely applied GFP marker system, thus gaining insights and elucidating the contributions of ARP2 upon fungal attack.

Spatiotemporal Transcriptomic Atlas of Developing Embryos and Vegetative Tissues in Flax.

Flax (Linum usitatissimum L.) is an important multipurpose crop widely grown for oil and fiber. Despite recent advances in genomics, detailed gene activities during the important reproductive phase of its development are not well defined. In this study, we employed high-throughput RNA-sequencing methods to generate in-depth transcriptome profiles of flax tissues with emphasis on the reproductive phases of five key stages of embryogenesis (globular embryo, heart embryo, torpedo embryo, cotyledon embryo, and mature embryo), mature seed, and vegetative tissues viz. ovary, anther, and root. These datasets were used to establish the co-expression networks covering 36 gene modules based on the expression patterns for each gene through weighted gene co-expression network analysis (WGCNA). Functional interrogation with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) of dominantly expressed genetic modules in tissues revealed pathways involved in the development of different tissues. Moreover, the essential genes in embryo development and synthesis of storage reserves were identified based on their dynamic expression patterns. Together, this comprehensive dataset for developing embryos, mature seeds and vegetative tissues provides new insights into molecular mechanisms of seed development with potential for flax crop improvement.

The Seed Coat's Impact on Crop Performance in Pea (Pisum sativum L.).

Seed development in angiosperms produces three genetically and developmentally distinct sub-compartments: the embryo, endosperm, and seed coat. The maternally derived seed coat protects the embryo and interacts closely with the external environment especially during germination and seedling establishment. Seed coat is a key contributor to seed composition and an important determinant of nutritional value for humans and livestock. In this review, we examined pea crop productivity through the lens of the seed coat, its contribution to several valued nutritional traits of the pea crop, and its potential as a breeding target. Key discoveries made in advancing the knowledge base for sensing and transmission of external signals, the architecture and chemistry of the pea seed coat, and relevant insights from other important legumes were discussed. Furthermore, for selected seed coat traits, known mechanisms of genetic regulation and efforts to modulate these mechanisms to facilitate composition and productivity improvements in pea were discussed, alongside opportunities to support the continued development and improvement of this underutilized crop. This review describes the most important features of seed coat development in legumes and highlights the key roles played by the seed coat in pea seed development, with a focus on advances made in the genetic and molecular characterization of pea and other legumes and the potential of this key seed tissue for targeted improvement and crop optimization.

PALE-GREEN LEAF 1, a rice cpSRP54 protein, is essential for the assembly of the PSI-LHCI supercomplex.

Although photosynthetic multiprotein complexes have received major attention, our knowledge about the assembly of these proteins into functional complexes in plants is still limited. In the present study, we have identified a chlorophyll-deficient mutant, pale-green leaf 1 (pgl1), in rice that displays abnormally developed chloroplasts. Map-based cloning of this gene revealed that OsPGL1 encodes a chloroplast targeted protein homologous to the 54-kDa subunit of the signal recognition particle (cpSRP54). Immunoblot analysis revealed that the accumulation of the PSI core proteins PsaA and PsaB, subunits from the ATP synthase, cytochrome, and light-harvesting complex (LHC) is dramatically reduced in pgl1. Blue native gel analysis of thylakoid membrane proteins showed the existence of an extra band in the pgl1 mutant, which located between the dimeric PSII/PSI-LHCI and the monomeric PSII. Immunodetection after 2D separation indicated that the extra band consists of the proteins from the PSI core complex. Measurements of chlorophyll fluorescence at 77 K further confirmed that PSI, rather than PSII, was primarily impaired in the pgl1 mutant. These results suggest that OsPGL1 might act as a molecular chaperone that is required for the efficient assembly and specific integration of the peripheral LHCI proteins into the PSI core complex in rice.

Salicylic acid fights against Fusarium wilt by inhibiting target of rapamycin signaling pathway in Fusarium oxysporum.

INTRODUCTION: Biofungicides with low toxicity and high efficiency are a global priority for sustainable agricultural development. Phytohormone salicylic acid (SA) is an ancient medicine against various diseases in humans and activates the immune system in plants, but little is known of its function as a biofungicide. OBJECTIVES: Here, Fusarium oxysporum, the causal agent of devastating Fusarium wilt and immunodepressed patients, was used as a model system to explore whether SA can enter the pathogen cells and suppress key targets of the pathogen. METHODS: Oxford Nanopore MinION sequencing and high-throughput chromosome conformation capture (Hi-C) sequencing were used to analyzed the genome of F. oxysporum. In addition, RNA-seq, qRT-PCR, and western blotting were conducted to detect gene and protein expression levels. RESULTS: We isolated and sequenced the genome of F. oxysporum from potato dry rot, and the F. oxysporum included 12 chromosomes and 52.3 Mb genomic length. Pharmacological assays showed that exogenous application of SA can efficiently arrest hyphal growth, spore production, and pathogenicity of F. oxysporum, whereas endogenous salicylate hydroxylases significantly detoxify SA. The synergistic growth inhibition of F. oxysporum was observed when SA was combined with rapamycin. Kinase assays showed that SA inhibits FoTOR complex 1 (FoTORC1) by activating FoSNF1 in vivo. Transgenic potato plants with the interference of FoTOR1 and FoSAH1 genes inhibited the invasive growth of hyphae and significantly prevented the occurrence of Fusarium wilt. CONCLUSION: This study revealed the underlying mechanisms of SA against F. oxysporum and provided insights into SA in controlling various fungal diseases by targeting the SNF1-TORC1 pathway of pathogens.

Evolutionary divergence in embryo and seed coat development of U's Triangle Brassica species illustrated by a spatiotemporal transcriptome atlas.

The economically valuable Brassica species include the six related members of U's Triangle. Despite the agronomic and economic importance of these Brassicas, the impacts of evolution and relatively recent domestication events on the genetic landscape of seed development have not been comprehensively examined in these species. Here we present a 3D transcriptome atlas for the six species of U's Triangle, producing a unique resource that captures gene expression data for the major subcompartments of the seed, from the unfertilized ovule to the mature embryo and seed coat. This comprehensive dataset for seed development in tetraploid and ancestral diploid Brassicas provides new insights into evolutionary divergence and expression bias at the gene and subgenome levels during the domestication of these valued crop species. Comparisons of gene expression associated with regulatory networks and metabolic pathways operating in the embryo and seed coat during seed development reveal differences in storage reserve accumulation and fatty acid metabolism among the six Brassica species. This study illustrates the genetic underpinnings of seed traits and the selective pressures placed on seed production, providing an immense resource for continued investigation of Brassica polyploid biology, genomics and evolution.

Auxin and Target of Rapamycin Spatiotemporally Regulate Root Organogenesis.

The programs associated with embryonic roots (ERs), primary roots (PRs), lateral roots (LRs), and adventitious roots (ARs) play crucial roles in the growth and development of roots in plants. The root functions are involved in diverse processes such as water and nutrient absorption and their utilization, the storage of photosynthetic products, and stress tolerance. Hormones and signaling pathways play regulatory roles during root development. Among these, auxin is the most important hormone regulating root development. The target of rapamycin (TOR) signaling pathway has also been shown to play a key role in root developmental programs. In this article, the milestones and influential progress of studying crosstalk between auxin and TOR during the development of ERs, PRs, LRs and ARs, as well as their functional implications in root morphogenesis, development, and architecture, are systematically summarized and discussed.

Target of rapamycin controls hyphal growth and pathogenicity through FoTIP4 in Fusarium oxysporum.

Fusarium oxysporum is the causal agent of the devastating Fusarium wilt by invading and colonizing the vascular system in various plants, resulting in substantial economic losses worldwide. Target of rapamycin (TOR) is a central regulator that controls intracellular metabolism, cell growth, and stress responses in eukaryotes, but little is known about TOR signalling in F. oxysporum. In this study, we identified conserved FoTOR signalling pathway components including FoTORC1 and FoTORC2. Pharmacological assays showed that F. oxysporum is hypersensitive to rapamycin in the presence of FoFKBP12 while the deletion mutant strain ΔFofkbp12 is insensitive to rapamycin. Transcriptomic data indicated that FoTOR signalling controls multiple metabolic processes including ribosome biogenesis and cell wall-degrading enzymes (CWDEs). Genetic analysis revealed that FoTOR1 interacting protein 4 (FoTIP4) acts as a new component of FoTOR signalling to regulate hyphal growth and pathogenicity of F. oxysporum. Importantly, transcript levels of genes associated with ribosome biogenesis and CWDEs were dramatically downregulated in the ΔFotip4 mutant strain. Electrophoretic mobility shift assays showed that FoTIP4 can bind to the promoters of ribosome biogenesis- and CWDE-related genes to positively regulate the expression of these genes. These results suggest that FoTOR signalling plays central roles in regulating hyphal growth and pathogenicity of F. oxysporum and provide new insights into FoTOR1 as a target for controlling and preventing Fusarium wilt in plants.

The ARP2/3 complex, acting cooperatively with Class I formins, modulates penetration resistance in Arabidopsis against powdery mildew invasion.

The actin cytoskeleton regulates an array of diverse cellular activities that support the establishment of plant-microbe interactions and plays a critical role in the execution of plant immunity. However, molecular and cellular mechanisms regulating the assembly and rearrangement of actin filaments (AFs) at plant-pathogen interaction sites remain largely elusive. Here, using live-cell imaging, we show that one of the earliest cellular responses in Arabidopsis thaliana upon powdery mildew attack is the formation of patch-like AF structures beneath fungal invasion sites. The AFs constituting actin patches undergo rapid turnover, which is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR regulatory complex (W/SRC). The focal accumulation of phosphatidylinositol-4,5-bisphosphate at fungal penetration sites appears to be a crucial upstream modulator of the W/SRC-ARP2/3 pathway-mediated actin patch formation. Knockout of W/SRC-ARP2/3 pathway subunits partially compromised penetration resistance with impaired endocytic recycling of the defense-associated t-SNARE protein PEN1 and its deposition into apoplastic papillae. Simultaneously knocking out ARP3 and knocking down the Class I formin (AtFH1) abolished actin patch formation, severely impaired the deposition of cell wall appositions, and promoted powdery mildew entry into host cells. Our results demonstrate that the ARP2/3 complex and formins, two actin-nucleating systems, act cooperatively and contribute to Arabidopsis penetration resistance to fungal invasion.

Conserved, divergent and heterochronic gene expression during Brachypodium and Arabidopsis embryo development.

KEY MESSAGE: Developmental and transcriptomic analysis of Brachypodium embryogenesis and comparison with Arabidopsis identifies conserved and divergent phases of embryogenesis and reveals widespread heterochrony of developmental gene expression. Embryogenesis, transforming the zygote into the mature embryo, represents a fundamental process for all flowering plants. Current knowledge of cell specification and differentiation during plant embryogenesis is largely based on studies of the dicot model plant Arabidopsis thaliana. However, the major crops are monocots and the transcriptional programs associated with the differentiation processes during embryogenesis in this clade were largely unknown. Here, we combined analysis of cell division patterns with development of a temporal transcriptomic resource during embryogenesis of the monocot model plant Brachypodium distachyon. We found that early divisions of the Brachypodium embryo were highly regular, while later stages were marked by less stereotypic patterns. Comparative transcriptomic analysis between Brachypodium and Arabidopsis revealed that early and late embryogenesis shared a common transcriptional program, whereas mid-embryogenesis was divergent between species. Analysis of orthology groups revealed widespread heterochronic expression of potential developmental regulators between the species. Interestingly, Brachypodium genes tend to be expressed at earlier stages than Arabidopsis counterparts, which suggests that embryo patterning may occur early during Brachypodium embryogenesis. Detailed investigation of auxin-related genes shows that the capacity to synthesize, transport and respond to auxin is established early in the embryo. However, while early PIN1 polarity could be confirmed, it is unclear if an active response is mounted. This study presents a resource for studying Brachypodium and grass embryogenesis and shows that divergent angiosperms share a conserved genetic program that is marked by heterochronic gene expression.

Integrative Modeling of Gene Expression and Metabolic Networks of Arabidopsis Embryos for Identification of Seed Oil Causal Genes.

Fatty acids in crop seeds are a major source for both vegetable oils and industrial applications. Genetic improvement of fatty acid composition and oil content is critical to meet the current and future demands of plant-based renewable seed oils. Addressing this challenge can be approached by network modeling to capture key contributors of seed metabolism and to identify underpinning genetic targets for engineering the traits associated with seed oil composition and content. Here, we present a dynamic model, using an Ordinary Differential Equations model and integrated time-course gene expression data, to describe metabolic networks during Arabidopsis thaliana seed development. Through in silico perturbation of genes, targets were predicted in seed oil traits. Validation and supporting evidence were obtained for several of these predictions using published reports in the scientific literature. Furthermore, we investigated two predicted targets using omics datasets for both gene expression and metabolites from the seed embryo, and demonstrated the applicability of this network-based model. This work highlights that integration of dynamic gene expression atlases generates informative models which can be explored to dissect metabolic pathways and lead to the identification of causal genes associated with seed oil traits.

High-resolution mapping of the Mov-1 locus in wheat by combining radiation hybrid (RH) and recombination-based mapping approaches.

KEY MESSAGE: This work reports a quick method that integrates RH mapping and genetic mapping to map the dominant Mov-1 locus to a 1.1-Mb physical interval with a small number of candidate genes. Bread wheat is an important crop for global human population. Identification of genes and alleles controlling agronomic traits is essential toward sustainably increasing crop production. The unique multi-ovary (MOV) trait in wheat holds potential for improving yields and is characterized by the formation of 2-3 grains per spikelet. The genetic basis of the multi-ovary trait is known to be monogenic and dominant in nature. Its precise mapping and functional characterization is critical to utilizing this trait in a feasible manner. Previous mapping efforts of the locus controlling multiple ovary/pistil formation in the hexaploid wheat have failed to produce a consensus for a particular chromosome. We describe a mapping strategy integrating radiation hybrid mapping and high-resolution genetic mapping to locate the chromosomal position of the Mov-1 locus in hexaploid wheat. We used RH mapping approach using a panel of 188 lines to map the Mov-1 locus in the terminal part of long arm of wheat chromosome 2D with a map resolution of 1.67 Mb/cR1500. Then using a genetic population of MOV × Synthetic wheat of F2 lines, we delineated the Mov-1 locus to a 1.1-Mb physical region with a small number of candidate genes. This demonstrates the value of this integrated strategy to mapping dominant genes in wheat.