Protein and gene integration analysis through proteome and transcriptome brings new insight into salt stress tolerance in pigeonpea (Cajanus cajan L.).

Salt stress is a major constrain to the productivity of nutritionally rich pigeonpea, an important legume of SE Asia and other parts of the world. The present study provides a comprehensive insight on integrated proteomic and transcriptomic analysis of root and shoot tissues of contrasting pigeonpea varieties (ICP1071- salt-sensitive; ICP7- salt-tolerant) to unravel salt stress induced pathways. Proteome analysis revealed 82 differentially expressed proteins (DEPs) with ≥±1.5 fold expression on 2-Dimensional (2D) gel. Of these, 25 DEPs identified through MALDI-TOF/TOF were classified using Uniprot software into functional categories. Pathways analyses using KAAS server showed the highest abundance of functional genes regulating metabolisms of carbohydrate followed by protein folding/degradation, amino acids and lipids. Expression studies on six genes (triosephosphate isomerase, oxygen evolving enhancer protein 1, phosphoribulokinase, cysteine synthase, oxygen evolving enhancer protein 2 and early nodulin like protein 2) with ≥±3 fold change were performed, and five of these showed consistency in transcript and protein expressions. Transcript analysis of root and shoot led to positive identification of 25 differentially expressed salt-responsive genes, with seven genes having ≥±5 fold change have diverse biological functions. Our combinatorial analysis suggests important role of these genes/proteins in providing salt tolerance in pigeonpea.

Assessment of Pigeonpea (Cajanus cajan L.) transgenics expressing Bt ICPs, Cry2Aa and Cry1AcF under nethouse containment implicated an effective control against herbivory by Helicoverpa armigera (Hübner).

BACKGROUND: Pigeonpea is a source of quality proteins and the main constituent of a well-balanced diet for majority of Indian population. One of the major constraints in the production of pigeonpea is a polyphagous insect pest, Helicoverpa armigera. Non-availability of resistant sources in the germplasm and limitations in conventional breeding have been key factors for continued yield losses. Additionally, hazards of chemical fertilizers on the environment have prompted the scientific community to develop alternative strategies. Bacillus thuringiensis (Bt) insecticidal proteins (ICPs) have emerged as the most reliable source for the control of insect pests through transgenics. RESULTS: Transgenic pigeonpea plants harboring validated Bt ICPs, Cry2Aa and Cry1AcF were developed by a non-tissue culture based in planta transformation strategy and assessed for integration of Transfer-DNA (T-DNA) and efficacy against pod borer under in vitro conditions. For the first time this study demonstrates the successful evaluation of 19 transgenic pigeonpea events (11 with cry2Aa and 8 with cry1AcF) under soil and pot conditions in a nethouse containment. The stability in the performance was assessed stringently by deliberate H. armigera larval challenging. The trial identified ten promising events of both the genes that portrayed reduced damage to the herbivore. CONCLUSION: We present the first ever successful evaluation of pigeonpea transgenics with the ability to mitigate pod borer under nethouse conditions. The transgenics depicted molecular evidence for the stability of T-DNA integration, consistency in the expression of Cry proteins and resistance against H. armigera. These events can form a pool of useful transgenics to manage the devastating pod borer. © 2019 Society of Chemical Industry.

Comparative transcriptome analyses provide novel insights into the differential response of Pigeonpea (Cajanus cajan L.) and its wild relative (Cajanus platycarpus (Benth.) Maesen) to herbivory by Helicoverpa armigera (Hübner).

KEY MESSAGE: Deeper insights into the resistance response of Cajanus platycarpus were obtained based on comparative transcriptomics under Helicoverpa armigera infestation. Devastation by pod borer, Helicoverpa armigera is one of the major factors for stagnated productivity in Pigeonpea. Despite possessing a multitude of desirable traits including pod borer resistance, wild relatives of Cajanus spp. have remained under-utilized due to linkage drag and cross-incompatibility. Discovery and deployment of genes from them can provide means to tackle key pests like H. armigera. Transcriptomic differences between Cajanus platycarpus and Cajanus cajan during different time points (0, 18, 38, 96 h) of pod borer infestation were elucidated in this study. For the first ever time, we demonstrated captivating variations in their response; C. platycarpus apparently being reasonably agile with effectual transcriptomic reprogramming to deter the insect. Deeper insights into the differential response were obtained by identification of significant GO-terms related to herbivory followed by combined KEGG and ontology analyses. C. platycarpus portrayed a multilevel response with cardinal involvement of SAR, redox homeostasis and reconfiguration of primary metabolites leading to a comprehensive defense response. The credibility of RNA-seq analyses was ascertained by transient expression of selected putative insect resistance genes from C. platycarpus viz., chitinase (CHI4), Alpha-amylase/subtilisin inhibitor (IAAS) and Flavonoid 3_5 hydroxylase (C75A1) in Nicotiana benthamiana followed by efficacy analysis against H. armigera. qPCR validated results of the study provided innovative insights and useful leads for development of durable pod borer resistance.

Genome-wide identification and characterization of InDels and SNPs in Glycine max and Glycine soja for contrasting seed permeability traits.

BACKGROUND: Water permeability governed by seed coat is a major facet of seed crops, especially soybean, whose seeds lack physiological dormancy and experience rapid deterioration in seed viability under prolonged storage. Moreover, the physiological and chemical characteristics of soybean seeds are known to vary with seed coat color. Thus, to underpin the genes controlling water permeability in soybean seeds, we carried out an in-depth characterization of the associated genomic variation. RESULTS: In the present study, we have analyzed genomic variation between cultivated soybean and its wild progenitor with implications on seed permeability, a trait related to seed storability. Whole genome resequencing of G.max and G. soja, identified SNPs and InDels which were further characterized on the basis of their genomic location and impact on gene expression. Chromosomal density distribution of the variation was assessed across the genome and genes carrying SNPs and InDels were characterized into different metabolic pathways. Seed hardiness is a complex trait that is affected by the allelic constitution of a genetic locus as well as by a tricky web of plant hormone interactions. Seven genes that hold a probable role in the determination of seed permeability were selected and their expression differences at different stages of water imbibition were analyzed. Variant interaction network derived 205 downstream interacting partners of 7 genes confirmed their role in seed related traits. Interestingly, genes encoding for Type I- Inositol polyphosphate 5 phosphatase1 and E3 Ubiquitin ligase could differentiate parental genotypes, revealed protein conformational deformations and were found to segregate among RILs in coherence with their permeability scores. The 2 identified genes, thus showed a preliminary association with the desirable permeability characteristics. CONCLUSION: In the light of above outcomes, 2 genes were identified that revealed preliminary, but a relevant association with soybean seed permeability trait and hence could serve as a primary material for understanding the molecular pathways controlling seed permeability traits in soybean.

Elucidation of salt-tolerance metabolic pathways in contrasting rice genotypes and their segregating progenies.

KEY MESSAGE: Differentially expressed antioxidant enzymes, amino acids and proteins in contrasting rice genotypes, and co-location of their genes in the QTLs mapped using bi-parental population, indicated their role in salt tolerance. Soil salinity is a major environmental constraint limiting rice productivity. Salt-tolerant 'CSR27', salt-sensitive 'MI48'and their extreme tolerant and sensitive recombinant inbred line (RIL) progenies were used for the elucidation of salt stress tolerance metabolic pathways. Salt stress-mediated biochemical and molecular changes were analyzed in the two parents along with bulked-tolerant (BT) and bulked-sensitive (BS) extreme RILs. The tolerant parent and BT RILs suffered much lower reduction in the chlorophyll as compared to their sensitive counterparts. Activities of antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD) and non-enzymatic antioxidant ascorbic acid were much higher in salt-stressed CSR27 and BT RILs than MI48 and BS RILs. Further, the tolerant lines showed significant enhancement in the levels of amino acids methionine and proline in response to salt stress in comparison to the sensitive lines. Similarly, the tolerant genotypes showed minimal reduction in cysteine content whereas sensitive genotypes showed a sharp reduction. Real time PCR analysis confirmed the induction of methionine biosynthetic pathway (MBP) enzymes cystathionine-ß synthase (CbS), S-adenosyl methionine synthase (SAMS), S-adenosyl methionine decarboxylase (SAMDC) and serine hydroxymethyl transferase (SHMT) genes in tolerant lines, suggesting potential role of the MBP in conferring salt tolerance in rice variety CSR27. Proteome profiling also confirmed higher expression of SOD, POD and plastidic CbS and other proteins in the tolerant lines, whose genes were co-located in the QTL intervals for salt tolerance mapped in the RIL population. The study signifies integrated biochemical-molecular approach for identifying salt tolerance genes for genetic improvement for stress tolerant rice varieties.