Novel insights into drought-induced regulation of ribosomal genes through DNA methylation in chickpea.

Modifications within the epigenome of an organism in response to external environmental conditions allow it to withstand the hostile stress factors. Drought in chickpea is a severely limiting abiotic stress factor which is known to cause huge yield loss. To analyse the methylome of chickpea in response to drought stress conditions and how it affects gene expression, we performed whole-genome bisulfite sequencing (WGBS) and RNA-seq of two chickpea genotypes which contrast for drought tolerance. It was observed that the mCHH was most variable under drought stress and the drought tolerant (DT) genotype exhibited substantial genome-wide hypomethylation as compared to the drought sensitive (DS) genotype. Specifically, there was substantial difference in gene expression and methylation for the ribosomal genes for the tolerant and sensitive genotypes. The differential expression of these genes was in complete agreement with earlier reported transcriptomes in chickpea. Many of these genes were hypomethylated (q < 0.01) and downregulated under drought stress (p < 0.01) in the sensitive genotype. The gene RPS6 (ribosomal protein small subunit) was found to be downregulated and hypomethylated in the drought sensitive genotype which could possibly lead to reduced ribosomal biosynthesis. This study provides novel insights into regulation of drought-responsive genes in chickpea.

Development of modified CTAB and Trizol protocols to isolate high molecular weight (HMW) RNA from polyphenol and polysaccharides rich pigeonpea (Cajanuscajan (L.) Millsp.

Pigeonpea (Cajanuscajan L.) is a legume crop that contains high levels of polyphenolic compounds and polysaccharides that become a hindrance in extracting good-quality and enough amount of RNA from its tissues. With the existing methods of RNA isolation, the phenolic compounds may co-precipitate or bind to the RNA giving false results. Therefore, in the present study, we have modified conventional CTAB and Trizol-based methods which resulted in good quality with the absorbance A260/A280 ratios in the range of 1.83 to 1.98 and A260/230 ratios in the range of 2.0-2.23, revealed RNA to be of high purity and free of contaminants. Both of the proposed protocols yielded a good quantity of RNA ranging from 289 to 422µg per gram of tissue. Distinctly visible bands of 28S and 18S rRNA were observed without degradation or smear, which indicated the presence of intact RNA. RT-PCR analysis showed that isolated RNA was quantitatively sufficient and compliant for the subsequent gene expression analysis.

The SPL transcription factor genes are potential targets for epigenetic regulation in response to drought stress in chickpea (C. arietinum L.).

BACKGROUND: Crop improvement for tolerance to various biotic and abiotic stress factors necessitates understanding the key gene regulatory mechanisms. One such mechanism of gene regulation involves changes in cytosine methylation at the gene body and flanking regulatory sequences. The present study was undertaken to identify genes which might be potential targets of drought-induced DNA methylation in chickpea. METHODS AND RESULTS: Two chickpea genotypes, which contrast for drought tolerance, were subjected to drought stress conditions and their differential response was studied by analysing different morpho-physiological traits. Utilizing the in-house, high throughput sequencing data, the SQUAMOSA promoter-binding (SBP) protein-like (SPL) transcription factor genes were identified to be differentially methylated and expressed amongst the two genotypes, in response to drought stress. The methylation status of one of these genes was examined and validated through bisulfite PCR (BS-PCR). The identified genes could be possible homologs to known epialleles and can therefore serve as potential epialleles which can be utilized for crop improvement in chickpea. CONCLUSION: The SPL TF genes are potential targets of epigenetic regulation in response to drought stress in chickpea. Since these are TFs, they might play important roles in controlling the expression of other genes, thus contributing to differential drought response of the two genotypes.

Interactome of millet-based food matrices: A review.

Millets are recently being recognized as emerging food ingredients with multifaceted applications. Whole grain flours made from millets, exhibit diverse chemical compositions, starch digestibility and physicochemical properties. A food matrix can be viewed as a section of food microstructure, commonly coinciding with a physical spatial domain that interacts or imparts specific functionalities to a particular food constituent. The complex millet-based food matrices can help individuals to attain nutritional benefits due to the intricate and unique digestive properties of these foods. This review helps to fundamentally understand the binary and ternary interactions of millet-based foods. Nutritional bioavailability and bioaccessibility are also discussed based on additive, synergistic, masking, the antagonistic or neutralizing effect of different food matrix components on each other and the surrounding medium. The molecular basis of these interactions and their effect on important functional attributes like starch retrogradation, gelling, pasting, water, and oil holding capacity is also discussed.

Characterization of STP4 promoter in Indian mustard Brassica juncea for use as an aphid responsive promoter.

OBJECTIVE: Brassica juncea, a major oilseed crop, suffers substantial yield losses due to infestation by mustard aphids (Lipaphis erysimi). Unavailability of resistance genes within the accessible gene pool underpins significance of the transgenic strategy in developing aphid resistance. In this study, we aimed for the identification of an aphid-responsive promoter from B. juncea, based on the available genomic resources. RESULTS: A monosaccharide transporter gene, STP4 in B. juncea was activated by aphids and sustained increased expression as the aphids colonized the plants. We cloned the upstream intergenic region of STP4 and validated its stand-alone aphid-responsive promoter activity. Further, deletion analysis identified the putative cis-elements important for the aphid responsive promoter activity. CONCLUSION: The identified STP4 promoter can potentially be used for driving high level aphid-inducible expression of transgenes in plants. Use of aphid-responsive promoter instead of constitutive promoters can potentially reduce the metabolic burden of transgene-expression on the host plant.

Purification of a protease inhibitor from Dolichos biflorus using immobilized metal affinity chromatography.

Plant protease inhibitors (PIs) are generally small proteins which play key roles in regulation of endogenous proteases and may exhibit antifeedant, antifungal, antitumor and cytokine inducing activities. Dolichos biflorus (horse gram) is an unexploited legume, which is rich in nutrients and also has therapeutic importance. It contains a double-headed PI, which is an anti-nutritional factor. As there is no report available on its simultaneous removal and purification in single step, in this study, a double-headed PI active against both trypsin and chymotrypsin was purified from Dolichos biflorus to -14-fold with -84% recovery using an immobilized metal affinity chromatography (IMAC) medium consisting of Zn-alginate beads. The method was single-step, fast, simple, reliable and economical. The purified inhibitor showed a single band on SDS-PAGE corresponding to molecular mass of 16 kDa and was stable over a pH range of 2.0-12.0 and up to a temperature of 100 degrees C for 20 min. The optimum temperature for trypsin and chymotrypsin inhibitor was observed to be 50 degrees C and 37 degrees C, respectively and pH optimum was pH 7.0 and 8.0, respectively. Thus, IMAC using Zn-alginate beads was useful in simultaneous purification and removal of an anti-nutritional factor from horse gram flour in single step. This procedure may also be employed for purification of other plant PIs in one step.

The first draft of the pigeonpea genome sequence.

Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety 'Asha'. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550 bp and >10-fold genome coverage, resulting in 510,809,477 bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable 'Arhar' simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement.

Improved and convenient method of RNA isolation from polyphenols and polysaccharide rich plant tissues.

It has been difficult to extract a good quality total RNA from the plant parts (such as seeds) which contain high levels of phenolic compounds, carbohydrates and other compounds that bind and/or co-precipitate with RNA. A simple, rapid and efficient method for isolating total RNA from polyphenols and polysaccharide rich plant tissues has been developed. Seeds of leguminosae family were chosen for the study. The good quality and high yield of total RNA was achieved with A260/A280 ratio of 1.9. Seeds of three different crops (Cajanus cajan, Dolichos biflorus and Vigna mungo) at different developmental stages were evaluated for total RNA extraction using standardized protocol. Seeds at 21 days after flowering (DAF) gave the best results among others (7 DAF and dry seeds). Quality of isolated RNA from all the three crops was further checked by cDNA synthesis. The extracted RNA was found suitable for further molecular applications such as reverse transcription and cDNA library construction.