Genome sequencing and assembly of Lathyrus sativus - a nutrient-rich hardy legume crop.

Grass pea (Lathyrus sativus) is a cool-season legume crop tolerant to drought, salinity, waterlogging, insects, and other biotic stresses. Despite these beneficial traits, this crop is not cultivated widely due to the accumulation of a neurotoxin - ß-N-oxalyl-L-α, ß-diaminopropionic acid (ß-ODAP) in the seeds and its association with neurolathyrism. In this study, we sequenced and assembled the genome of Lathyrus sativus cultivar Pusa-24, an elite Indian cultivar extensively used in breeding programs. The assembled genome of Lathyrus was 3.80 Gb in length, with a scaffold N50 of 421.39 Mb. BUSCO assessment indicated that 98.3% of highly conserved Viridiplantae genes were present in the assembly. A total of 3.17 Gb (83.31%) of repetitive sequences and 50,106 protein-coding genes were identified in the Lathyrus assembly. The Lathyrus genome assembly reported here thus provides a much-needed and robust foundation for various genetic and genomic studies in this vital legume crop.

Contrasting ß-ODAP content correlates with stress gene expression in Lathyrus cultivars.

Lathyrus sativus, commonly known as grass pea, is a nutrient-rich pulse crop with remarkable climate-resilient attributes. However, wide use of this nutritious crop is not adopted owing to the presence of a non-protein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), which is neurotoxic if consumed in large quantities. We conducted a de novo transcriptomic profiling of two ODAP contrasting cultivars, Pusa-24 and its somaclonal variant Ratan, to understand the genetic changes leading to and associated with ß-ODAP levels. Differential gene expression analysis showed that a variety of genes are downregulated in low ß-ODAP cultivar Ratan and include genes involved in biotic/abiotic stress tolerance, redox metabolism, hormonal metabolism, and sucrose, and starch metabolism. Several genes related to chromatin remodeling are differentially expressed in cultivar Ratan. ß-ODAP biosynthetic genes in these cultivars showed differential upregulation upon stress. ODAP content of these cultivars varied differentially upon stress and development. Physiological experiments indicate reduced relative water content and perturbed abscisic acid levels in the low ODAP cultivar. Altogether, our results suggest that the low ODAP cultivar may have a reduced stress tolerance. The dataset provides insight into the biological role of ODAP and will be helpful for hypothesis-driven experiments to understand ODAP biosynthesis and regulation.

Physiological and molecular responses to combinatorial iron and phosphate deficiencies in hexaploid wheat seedlings.

Iron (Fe) and phosphorus (P) are the essential mineral nutrients for plant growth and development. However, the molecular interaction of the Fe and P pathways in crops remained largely obscure. In this study, we provide a comprehensive physiological and molecular analysis of hexaploid wheat response to single (Fe, P) and its combinatorial deficiencies. Our data showed that inhibition of the primary root growth occurs in response to Fe deficiency; however, growth was rescued when combinatorial deficiencies occurred. Analysis of RNAseq revealed that distinct molecular rearrangements during combined deficiencies with predominance for genes related to metabolic pathways and secondary metabolite biosynthesis primarily include genes for UDP-glycosyltransferase, cytochrome-P450s, and glutathione metabolism. Interestingly, the Fe-responsive cis-regulatory elements in the roots in Fe stress conditions were enriched compared to the combined stress. Our metabolome data also revealed the accumulation of distinct metabolites such as amino-isobutyric acid, arabinonic acid, and aconitic acid in the combined stress environment. Overall, these results are essential in developing new strategies to improve the resilience of crops in limited nutrients.

Decoding the genome of superior chapatti quality Indian wheat variety 'C 306' unravelled novel genomic variants for chapatti and nutrition quality related genes.

An Indian wheat variety, 'C 306' has good chapatti quality, which is controlled by multiple genes that have not been explored. We report the high quality de novo assembled genome of 'C 306' by combining short and long read sequencing data. The hybrid assembly covered 93% of gene space and identified about 142 K coding genes, 34% repetitive DNA and ~ 501 K SSR motifs. The phylogenetic analysis of about 83 K orthologous protein groups suggested the closest relationship with T. turgidum, T. aestivum and Ae. tauschii. Genome wide analysis annotated 69,217,536 genomic variants. Out of them, 1423 missense and 117 deleterious variants identified in processing, nutrition, and chapatti quality related genes such as alpha- and beta-gliadin, SSI, SSIII, SUT1, SBEI, CHS, YSL, DMAS, and NAS encoded proteins. These variants may affect quality genes. The genomic data will be potential genomic resources in wheat breeding programs for quality improvement.

Temporal and spatial Bean pod mottle virus-induced gene silencing in soybean.

Virus-induced gene silencing (VIGS) is a powerful reverse genetics tool in plant science. In this study, we investigated the temporal and spatial silencing patterns achieved by Bean pod mottle virus (BPMV)-based VIGS in soybean using virus constructs targeting green fluorescence protein (GFP). Silencing GFP enabled an in-depth analysis of silencing in soybean tissues over time in a transgenic line constitutively expressing GFP. We discovered evidence for variable GFP silencing based on insert orientation and targeted region in the coding sequence. A 3' sequence in reverse orientation produced the strongest silencing phenotypes. Furthermore, we documented that BPMV VIGS can achieve widespread silencing in a broad range of tissues, including leaves, stems, flowers and roots. Near-complete silencing was attained in leaves and flowers. Although weaker than in shoots, the observed gene silencing in soybean roots will also allow reverse genetics studies in this tissue. When GFP fluorescence was assayed in cross-sections of stems and leaf petioles, near-complete and uniform silencing was observed in all cell types. Silencing was observed from as early as 2 weeks post-virus inoculation in leaves to 7 weeks post-virus inoculation in flowers, suggesting that this system can induce and maintain silencing for significant durations.

The Soybean Rhg1 locus for resistance to the soybean cyst nematode Heterodera glycines regulates the expression of a large number of stress- and defense-related genes in degenerating feeding cells.

To gain new insights into the mechanism of soybean (Glycine max) resistance to the soybean cyst nematode (Heterodera glycines), we compared gene expression profiles of developing syncytia in soybean near-isogenic lines differing at Rhg1 (for resistance to Heterodera glycines), a major quantitative trait locus for resistance, by coupling laser capture microdissection with microarray analysis. Gene expression profiling revealed that 1,447 genes were differentially expressed between the two lines. Of these, 241 (16.8%) were stress- and defense-related genes. Several stress-related genes were up-regulated in the resistant line, including those encoding homologs of enzymes that lead to increased levels of reactive oxygen species and proteins associated with the unfolded protein response. These results indicate that syncytia induced in the resistant line are undergoing severe oxidative stress and imbalanced endoplasmic reticulum homeostasis, both of which likely contribute to the resistance reaction. Defense-related genes up-regulated within syncytia of the resistant line included those predominantly involved in apoptotic cell death, the plant hypersensitive response, and salicylic acid-mediated defense signaling; many of these genes were either partially suppressed or not induced to the same level by a virulent soybean cyst nematode population for successful nematode reproduction and development on the resistant line. Our study demonstrates that a network of molecular events take place during Rhg1-mediated resistance, leading to a highly complex defense response against a root pathogen.

Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3 function in the systemin-mediated defense response against herbivorous insects.

Systemin is a wound-signaling peptide that mediates defenses of tomato plants against herbivorous insects. Perception of systemin by the membrane-bound receptor SR160 results in activation of MAPKs, synthesis of jasmonic acid (JA), and expression of defense genes. To test the function of MAPKs in the response to systemin, we used virus-induced gene silencing (VIGS) in plants that overexpress the systemin precursor prosystemin (35S::prosys plants). These transgenic plants accumulate high levels of defense proteins and exhibit increased resistance to herbivorous insects. Cosilencing of the MAPKs MPK1 and MPK2 reduced MPK1/2 kinase activity, JA biosynthesis, and expression of JA-dependent defense genes. Application of methyl-JA restored the full defense response. These data show that MPK1 and MPK2 are essential components of the systemin signaling pathway and most likely function upstream of JA biosynthesis. MPK1 and MPK2 are 95% identical at the amino acid level. Specific VIGS of only MPK1 or MPK2 resulted in the same reduction of defense gene expression as cosilencing of MPK1 and MPK2, indicating that gene dosage effects may be important for MPK signaling. In addition, VIGS of the closely related MPK3 also reduced systemin-induced defense responses. The function of MPK1/2 and orthologs in pathogen-induced defenses is well established. Here we show that cosilencing of MPK1 and MPK2 compromised prosystemin-mediated resistance to Manduca sexta (Lepidoptera) herbivory, demonstrating that MPK1 and MPK2 are also required for successful defenses against herbivorous insects.