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.

DNA methylation: an emerging paradigm of gene regulation under drought stress in plants.

Drought is an enormous threat to global crop production. In order to ensure food security for the burgeoning population, we must develop drought tolerant crop varieties. This necessitates the identification of drought-responsive genes and understanding the mechanisms involved in their regulation. DNA methylation is a widely studied mechanism of epigenetic regulation of gene expression, which is known to play vital role in conferring tolerance to various biotic and abiotic stress factors. The recent advances in next-generation sequencing (NGS) technologies, has allowed unprecedented access to genome-wide methylation marks, with single base resolution. The most important roles of DNA methylation have been studied in terms of gene body methylation (gbM), which is associated with regulation of both transcript abundance and its stability. The availability of mutants for the various genes encoding enzymes involved in methylation of DNA has allowed ascertainment of the biological significance of methylation. Even though a vast number of reports have emerged in the recent past, where both genome-wide methylation landscape and locus specific changes in DNA methylation have been studied, a conclusive picture with regards to the biological role of DNA methylation is still lacking. Compounding this, is the lack of sufficient evidence supporting the heritability of these epigenetic changes. Amongst the various epigenetic variations, the DNA methylation changes are observed to be the most stable. This review describes the drought-induced changes in DNA methylation identified across different plant species. We also briefly describe the stress memory contributed by these changes. The identification of heritable, drought-induced methylation marks would broaden the scope of crop improvement in the future.

Unravelling the due importance of pseudogenes and their resurrection in plants.

The complexities of a genome are underpinned to the vast expanses of the intergenic region, which constitutes ∼97-98% of the genome. This region is essentially composed of what is colloquially referred to as the "junk DNA" and is composed of various elements like transposons, repeats, pseudogenes, etc. The latter have long been considered as dead elements merely contributing to transcriptional noise in the genome. Many studies now describe the previously unknown regulatory functions of these genes. Recent advances in the Next-generation sequencing (NGS) technologies have allowed unprecedented access to these regions. With the availability of whole genome sequences of more than 788 different plant species in past 20 years, genome annotation has become feasible like never before. Different bioinformatic pipelines are available for the identification of pseudogenes. However, still little is known about their biological functions. The functional validation of these genes remains challenging and research in this area is still in infancy, particularly in plants. CRISPR/Cas-based genome editing could provide solutions to understand the biological roles of these genes by allowing creation of precise edits within these genes. The possibility of pseudogene reactivation or resurrection as has been demonstrated in a few studies might open new avenues of genetic manipulation to yield a desirable phenotype. This review aims at comprehensively summarizing the progress made with regards to the identification of pseudogenes and understanding their biological functions in plants.

Multivariate analysis and genetic dissection of staygreen and stem reserve mobilisation under combined drought and heat stress in wheat (Triticum aestivum L.).

Introduction: Abiotic stresses significantly reduce crop yield by adversely affecting many physio-biochemical processes. Several physiological traits have been targeted and improved for yield enhancement in limiting environmental conditions. Amongst them, staygreen and stem reserve mobilisation are two important mutually exclusive traits contributing to grain filling under drought and heat stress in wheat. Henceforth, the present study was carried out to identify the QTLs governing these traits and to identify the superiors' lines through multi-trait genotype-ideotype distance index (MGIDI) Methods: A mapping population consisting of 166 recombinant inbred lines (RILs) developed from a cross between HD3086 and HI1500 was utilized in this study. The experiment was laid down in alpha lattice design in four environmental conditions viz. Control, drought, heat and combined stress (heat and drought). Genotyping of parents and RILs was carried out with 35 K Axiom® array (Wheat breeder array). Results and Discussion: Medium to high heritability with a moderate to high correlation between traits was observed. Principal component analysis (PCA) was performed to derive latent variables in the original set of traits and the relationship of these traits with latent variables.From this study, 14 QTLs were identified, out of which 11, 2, and 1 for soil plant analysis development (SPAD) value, leaf senescence rate (LSR), and stem reserve mobilisation efficiency (SRE) respectively. Quantitative trait loci (QTLs) for SPAD value harbored various genes like Dirigent protein 6-like, Protein FATTY ACID EXPORT 3, glucan synthase-3 and Ubiquitin carboxyl-terminal hydrolase, whereas QTLs for LSR were found to contain various genes like aspartyl protease family protein, potassium transporter, inositol-tetrakisphosphate 1-kinase, and DNA polymerase epsilon subunit D-like. Furthermore, the chromosomal region for SRE was found to be associated with serine-threonine protein kinase. Serine-threonine protein kinases are involved in many signaling networks such as ABA mediated ROS signaling and acclimation to environmental stimuli. After the validation of QTLs in multilocation trials, these QTLs can be used for marker-assisted selection (MAS) in breeding programs.

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.

Seasonal prevalence of dengue vector mosquito Aedes aegypti Linn in Jaipur city, Rajasthan, India.

Background & objectives: Vector-borne diseases are a significant issue for public health worldwide, especially in India. In recent years, high number of dengue and chikungunya cases have been reported from Rajasthan state of India, those are principally transmitted by Aedes aegypti. These vectors are extremely intrusive and can thrive in practically any climate. However, vector mosquitos' prevalence in Jaipur district is not properly documented. Therefore, current research was carried out to ascertain the seasonal fluctuations of Aedes aegypti in Jaipur city, Rajasthan, India. Methods: In order to ascertain the seasonal variation, monitoring of Aedes mosquitoes was conducted from August 2021 to July 2022 at nine selected regions in the Jaipur city. The breeding capacity of vectors was evaluated using three vector indices: the House Index, Breteau Index, and Container Index. Results: A total of 2172 out of 6336 breeding sites and 3735 out of 7477 containers were found positive for Aedes species. Three important species of Aedes vectors were collected in which Aedes aegypti was reported as the most prevalent. The highest values for House Index (57.60%) and Container Index (54.95%) were observed in October and the least rate was observed in March. Interpretation & conclusion: This survey was carried out to investigate the seasonal prevalence of dengue vectors and the findings revealed seasonal fluctuations in the indices of Aedes reproducing potential. This calls for precautionary actions to avoid infection rates and epidemic emergence. Therefore, to stop epidemics and eradicate vector-borne infections, the current study recommends close monitoring and further vector management efforts.

Genome-wide identification, in silico characterization and expression analysis of the RNA helicase gene family in chickpea (C. arietinum L.).

The RNA helicases are an important class of enzymes which are known to influence almost every aspect of RNA metabolism. The majority of RNA helicases belong to the SF2 (superfamily 2) superfamily, members of which are further categorized into three separate subfamilies i.e., the DEAD, DEAH and DExD/H-box subfamilies. In chickpea, these RNA helicases have not been characterized until now. A genome-wide analysis across the chickpea genome led to the identification of a total of 150 RNA helicase genes which included 50 DEAD, 33 DEAH and 67 DExD/H-box genes. These were distributed across all the eight chromosomes, with highest number on chromosome 4 (26) and least on chromosome 8 (8). Gene duplication analysis resulted in identification of 15 paralogous gene pairs with Ka/Ks values < 1, indicating towards the genes being under purifying selection during the course of evolution. The promoter regions of the RNA helicase genes were enriched in cis-acting elements like the light and ABA-responsive elements. The drought responsiveness of the genes was analysed by studying the expression profiles of few of these genes, in two different genotypes, the cultivated variety ICC 8261 (kabuli, C. arietinum) and the wild accession ILWC 292 (C. reticulatum), through qRT-PCR. These genotypes were selected based on their drought responsiveness in a field experiment, where it was observed that the percentage (%) reduction in relative water content (RWC) and membrane stability index (MSI) for the drought stressed plants after withholding water for 24 days, over the control or well-watered plants, was least for both the genotypes. The genes CaDEAD50 and CaDExD/H66 were identified as drought-responsive RNA helicase genes in chickpea. The protein encoded by the CaDExD/H66 gene shares a high degree of homology with one of the CLSY (CLASSY) proteins of A. thaliana. We hypothesize that this gene could possibly be involved in regulation of DNA methylation levels in chickpea by regulating siRNA production, in conjunction with other proteins like the Argonaute, RNA dependent RNA polymerases and Dicer-like proteins.

Genome-wide identification and expression analysis of the GRAS gene family in response to drought stress in chickpea (Cicer arietinum L.).

The GRAS (gibberellic acid insensitive, repressor of GAI and scarecrow) transcription factors (TFs) regulate diverse biological processes involved in plant growth and development. These TFs are also known to regulate gene expression in response to various abiotic stress factors like cold, drought, etc. In chickpea one of the most devastating abiotic stress factors is terminal drought. The GRAS TF family has not been characterized in chickpea (Cicer arietinum L.) until now. In this study, we report 46 GRAS TF genes (CaGRAS genes) in the chickpea genome. The CaGRAS proteins were categorized into nine subfamilies based on their phylogenetic relationship with known GRAS members of Arabidopsis and soybean. The PAT subfamily was the largest consisting of ten CaGRAS members whereas the LAS subfamily was the smallest with only one member. Gene duplication analysis revealed that segmental duplication was the primary reason for the expansion of this gene family within the chickpea genome. The gene expression levels of CaGRAS genes were analysed using two different chickpea varieties contrasting for drought tolerance trait, i.e., ICC 4958 (drought tolerant) and ICC 1882 (drought sensitive). On exposure to drought stress, the two chickpea genotypes, exhibited differential drought response, which was quantified and estimated in terms of differences in leaf relative water content (RWC). The well-watered or control plants of the drought tolerant variety were able to maintain a higher leaf RWC by the end of the drought stress period, whereas the control plants of the drought sensitive variety continued to show a decline in leaf RWC. The two genotypes also differed in their root morphologies, under well-watered and drought stress conditions. The gene expression analysis revealed a potential role of PAT, SCR, SCL3 and SHR GRAS members in the regulation of differential response to drought, in the root tissues, for both the genotypes. CaGRAS 12 (SCR) was identified as a drought-responsive GRAS TF gene, which could serve as a potential candidate gene for utilization in developing chickpea varieties with improved drought tolerance. This study demonstrates the drought-responsive expression of CaGRAS genes in chickpea and also describes the morpho-physiological response of chickpea plants to drought stress conditions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03104-z.

Understanding the Role of Gibberellic Acid and Paclobutrazol in Terminal Heat Stress Tolerance in Wheat.

Understanding the physiological mechanism of tolerance under stress conditions is an imperative aspect of the crop improvement programme. The role of plant hormones is well-established in abiotic stress tolerance. However, the information on the role of gibberellic acid (GA) in abiotic stress tolerance in late sown wheat is still not thoroughly explored. Thus, we aimed to investigate the role of endogenous GA3 level in stress tolerance in contrasting wheat cultivars, viz., temperature-tolerant (HD 2643 and DBW 14) and susceptible (HD 2189 and HD 2833) cultivars under timely and late sown conditions. We created the variation in endogenous GA3 level by exogenous spray of GA3 and its biosynthesis inhibitor paclobutrazol (PBZ). Tolerant genotypes had higher antioxidant enzyme activity, membrane stability, and photosynthesis rate, lower lipid peroxidase activity, and better growth and yield traits under late sown conditions attributed to H2O2 content. Application of PBZ escalated antioxidant enzymes activity and photosynthesis rate, and reduced the lipid peroxidation and ion leakage in stress, leading to improved thermotolerance. GA3 had a non-significant effect on antioxidant enzyme activity, lipid peroxidation, and membrane stability. However, GA3 application increased the test weight in HD 2643 and HD 2833 under timely and late sown conditions. GA3 upregulated GA biosynthesis and degradation pathway genes, and PBZ downregulated kaurene oxidase and GA2ox gene expression. GA3 also upregulated the expression of the cell expansins gene under both timely and late sown conditions. Exogenous GA3 did not increase thermotolerance but positively affected test weight and cell expansins gene expression. No direct relationship existed between endogenous GA3 content and stress tolerance traits, indicating that PBZ could have conferred thermotolerance through an alternative mechanism instead of inhibiting GA3biosynthesis.