Genetic Conservation of CBS Domain Containing Protein Family in Oryza Species and Their Association with Abiotic Stress Responses.

Crop Wild Relatives (CWRs) form a comprehensive gene pool that can answer the queries related to plant domestication, speciation, and ecological adaptation. The genus 'Oryza' comprises about 27 species, of which two are cultivated, while the remaining are wild. Here, we have attempted to understand the conservation and diversification of the genes encoding Cystathionine ß-synthase (CBS) domain-containing proteins (CDCPs) in domesticated and CWRs of rice. Few members of CDCPs were previously identified to be stress-responsive and associated with multiple stress tolerance in rice. Through genome-wide analysis of eleven rice genomes, we identified a total of 36 genes encoding CDCPs in O. longistaminata, 38 in O. glaberrima, 39 each in O. rufipogon, O. glumaepatula, O. brachyantha, O. punctata, and O. sativa subsp. japonica, 40 each in O. barthii and O. meridionalis, 41 in O. nivara, and 42 in O. sativa subsp. indica. Gene duplication analysis as well as non-synonymous and synonymous substitutions in the duplicated gene pairs indicated that this family is shaped majorly by the negative or purifying selection pressure through the long-term evolution process. We identified the presence of two additional hetero-domains, namely TerCH and CoatomerE (specifically in O. sativa subsp. indica), which were not reported previously in plant CDCPs. The in silico expression analysis revealed some of the members to be responsive to various abiotic stresses. Furthermore, the qRT-PCR based analysis identified some members to be highly inducive specifically in salt-tolerant genotype in response to salinity. The cis-regulatory element analysis predicted the presence of numerous stress as well as a few phytohormone-responsive elements in their promoter region. The data presented in this study would be helpful in the characterization of these CDCPs from rice, particularly in relation to abiotic stress tolerance.

Identification and validation of reference genes for qRT-PCR based studies in horse gram (Macrotyloma uniflorum).

The quantitative real-time polymerase chain reaction (qRT-PCR) is the most sensitive and commonly used technique for gene expression studies in biological systems. However, the reliability of qRT-PCR results depends on the selection of reference gene(s) for data normalization. Horse gram (Macrotyloma uniflorum) is an important legume crop on which several molecular studies have been reported. However, the stability of reference genes has not been evaluated. In the present study, nine candidate reference genes were identified from horse gram RNA-seq data and evaluated in two horse gram genotypes, HPK4 and HPKM317 under six abiotic stresses viz. cold, drought, salinity, heat, abscisic acid and methyl viologen-induced oxidative stress. The results were evaluated using geNorm, Bestkeeper, Normfinder and delta-delta Ct methods and comprehensive ranking was assigned using RefFinder and RankAggreg software. The overall result showed that TCTP was one of the most stable genes in all samples and in genotype HPK4, while in HPKM317 profilin was most stably expressed. However, PSMA5 was identified as least stable in all the experimental conditions. Expression of target genes dehydrin and early response to dehydration 6 under drought stress was also validated using TCTP and profilin for data normalization, either alone or in combination, which confirmed their suitability for qRT-PCR data normalization. Thus, TCTP and profilin genes may be used for qRT-PCR data normalization for molecular and genomic studies in horse gram. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01104-0.

Silicon-mediated abiotic and biotic stress mitigation in plants: Underlying mechanisms and potential for stress resilient agriculture.

Silicon (Si) is a beneficial macronutrient for plants. The Si supplementation to growth media mitigates abiotic and biotic stresses by regulating several physiological, biochemical and molecular mechanisms. The uptake of Si from the soil by root cells and subsequent transport are facilitated by Lsi1 (Low silicon1) belonging to nodulin 26-like major intrinsic protein (NIP) subfamily of aquaporin protein family, and Lsi2 (Low silicon 2) belonging to putative anion transporters, respectively. The soluble Si in the cytosol enhances the production of jasmonic acid, enzymatic and non-enzymatic antioxidants, secondary metabolites and induces expression of genes in plants under stress conditions. Silicon has been found beneficial in conferring tolerance against biotic and abiotic stresses by scavenging the reactive oxygen species (ROS) and regulation of different metabolic pathways. In the present review, Si transporters identified in various plant species and mechanisms of Si-mediated abiotic and biotic stress tolerance have been presented. In addition, role of Si in regulating gene expression under various abiotic and biotic stresses as revealed by transcriptome level studies has been discussed. This provides a deeper understanding of various mechanisms of Si-mediated stress tolerance in plants and may help in devising strategies for stress resilient agriculture.

Methods of Gene Expression Profiling to Understand Abiotic Stress Perception and Response in Legume Crops.

Legume crops offer a wide genetic diversity that can be exploited to raise improved crop varieties with higher tolerance against adverse climatic conditions. In order to achieve food and nutritional security, legume breeding programs should also incorporate advanced genomics tools. Genomes of many model and nonmodel legume crops have been sequenced, which provide opportunities to identify and characterize candidate genes to develop abiotic stress tolerant crops. Gene expression profiling is a powerful tool to identify candidate genes and understand their function. The present chapter describes two such strategies, that is, candidate gene expression profiling approach and global transcriptome profiling approach. The methods like RT-PCR and qRT-PCR that are being traditionally used to study expression of target genes under defined experimental conditions are discussed. In addition, global transcriptome analysis approach and its advancements are discussed. Details of next-generation sequencing (NGS) based RNA-sequencing (RNA-seq) and associated advanced bioinformatics tools to identify differentially expressing genes at a global level are also described.

Methods for Screening Legume Crops for Abiotic Stress Tolerance through Physiological and Biochemical Approaches.

Legume crops are subjected to a wide range of abiotic stresses, which stimulate an array of physiological, biochemical, and molecular responses. However, different genotypes may exhibit significant variations between individual responses, which can determine their tolerance or susceptibility to these stresses. The present chapter suggests a broad range of assays that can help in understanding stress perception by plants at cellular and molecular levels. The genotypes may be sorted depending on their tolerance potential, by broadly analysing morphological, physiological, biochemical, and enzyme kinetics parameters. These assays are very beneficial in revealing the mechanism of stress perception and response in varied plant types, and have helped in discriminating contrasting genotypes. Here, we have described detailed protocols of assays which may be carried out to assess tolerance or susceptibility to abiotic stresses. The analysis, as a whole, can help researchers understand the effect of abiotic stresses on plant biochemical pathways, be it photosynthesis, redox homeostasis, metabolite perturbation, signaling, transcription, and translation. These protocols may be beneficial in identification of suitable donors for breeding programs, as well as for identifying promising candidate genes or pathways for developing stress tolerant legume crops through genetic engineering.