Genome-wide characterization and functional analysis of class III peroxidase gene family in soybean reveal regulatory roles of GsPOD40 in drought tolerance.

Class III peroxidases (PODs) are plant-specific glycoproteins, that play essential roles in various plant physiological processes and defence responses. To date, scarce information is available about the POD gene family in soybean. Hence, the present study is the first comprehensive report about the genome-wide characterization of GmPOD gene family in soybean (Glycine max L.). Here, we identified a total of 124 GmPOD genes in soybean, that are unevenly distributed across the genome. Phylogenetic analysis classified them into six distinct sub-groups (A-F), with one soybean specific subgroup. Exon-intron and motif analysis suggested the existence of structural and functional diversity among the sub-groups. Duplication analysis identified 58 paralogous gene pairs; segmental duplication and positive/Darwinian selection were observed as the major factors involved in the evolution of GmPODs. Furthermore, RNA-seq analysis revealed that 23 out of a total 124 GmPODs showed differential expression between drought-tolerant and drought-sensitive genotypes under stress conditions; however, two of them (GmPOD40 and GmPOD42) revealed the maximum deregulation in all contrasting genotypes. Overexpression (OE) lines of GsPOD40 showed considerably higher drought tolerance compared to wild type (WT) plants under stress treatment. Moreover, the OE lines showed enhanced photosynthesis and enzymatic antioxidant activities under drought stress, resulting in alleviation of ROS induced oxidative damage. Hence, the GsPOD40 enhanced drought tolerance in soybean by regulating the key physiological and biochemical pathways involved in the defence response. Lastly, the results of our study will greatly assist in further functional characterization of GsPODs in plant growth and stress tolerance in soybean.

Comprehensive RNA-seq analysis revealed molecular pathways and genes associated with drought tolerance in wild soybean (Glycine soja Sieb. and Zucc.).

Drought stress at the germination stage is an important environmental stress limiting crop yield. Hence, our study investigated comparative root transcriptome profiles of four contrasting soybean genotypes viz., drought-tolerant (PI342618B/DTP and A214/DTL) and drought-sensitive (NN86-4/DSP and A195/DSL) under drought stress using RNA-Seq approach. A total of 4850 and 6272 differentially expressed genes (DEGs) were identified in tolerant (DTP and DTL) and sensitive (DSP and DSL) genotypes, respectively. Principle component analysis (PCA) and correlation analysis revealed higher correlation between DTP and DTL. Both gene ontology (GO) and MapMan analyses showed that the drought response was enriched in DEGs associated with water and auxin transport, cell wall/membrane, antioxidant activity, catalytic activity, secondary metabolism, signaling and transcription factor (TF) activities. Out of 981 DEGs screened from above terms, only 547 showed consistent opposite expression between contrasting genotypes. Twenty-eight DEGs of 547 were located on Chr.08 rich in QTLs and "Hotspot regions" associated with drought stress, and eight of them showed non-synonymous single nucleotide polymorphism. Hence, 10 genes (including above eight genes plus two hub genes) were predicated as possible candidates regulating drought tolerance, which needs further functional validation. Overall, the transcriptome profiling provided in-depth understanding about the genetic mechanism and candidate genes underlying drought tolerance in soybean.

Omics and CRISPR-Cas9 Approaches for Molecular Insight, Functional Gene Analysis, and Stress Tolerance Development in Crops.

Plants are regularly exposed to biotic and abiotic stresses that adversely affect agricultural production. Omics has gained momentum in the last two decades, fueled by statistical methodologies, computational capabilities, mass spectrometry, nucleic-acid sequencing, and peptide-sequencing platforms. Functional genomics-especially metabolomics, transcriptomics, and proteomics-have contributed substantially to plant molecular responses to stress. Recent progress in reverse and forward genetics approaches have mediated high-throughput techniques for identifying stress-related genes. Furthermore, web-based genetic databases have mediated bioinformatics techniques for detecting families of stress-tolerant genes. Gene ontology (GO) databases provide information on the gene product's functional features and help with the computational estimation of gene function. Functional omics data from multiple platforms are useful for positional cloning. Stress-tolerant plants have been engineered using stress response genes, regulatory networks, and pathways. The genome-editing tool, CRISPR-Cas9, reveals the functional features of several parts of the plant genome. Current developments in CRISPR, such as de novo meristem induction genome-engineering in dicots and temperature-tolerant LbCas12a/CRISPR, enable greater DNA insertion precision. This review discusses functional omics for molecular insight and CRISPR-Cas9-based validation of gene function in crop plants. Omics and CRISPR-Cas9 are expected to garner knowledge on molecular systems and gene function and stress-tolerant crop production.

Genome-wide association study provides new insight into the underlying mechanism of drought tolerance during seed germination stage in soybean.

Drought is one of the major environmental issues that reduce crop yield. Seed germination is a crucial stage of plant development in all crop plants, including soybean. In soybean breeding, information about genetic mechanism of drought tolerance has great importance. However, at germination stage, there is relatively little knowledge on the genetic basis of soybean drought resistance. The objective of this work was to find the quantitative trait nucleotides (QTNs) linked to drought tolerance related three traits using a genome-wide association study (GWAS), viz., germination rate (GR), root length (RL), and whole seedling length (WSL), using germplasm population of 240 soybean PIs with 34,817 SNPs genotype data having MAF > 0.05. It was observed that heritability (H2) for GR, WSL, and RL across both environments (2020, and 2019) were high in the range of 0.76-0.99, showing that genetic factors play a vital role in drought tolerance as compared to environmental factors. A number of 23 and 27 QTNs were found to be linked to three traits using MLM and mrMLM, respectively. Three significant QTNs, qGR8-1, qWSL13-1, and qRL-8, were identified using both MLM and mrMLM methods among these QTNs. QTN8, located on chromosome 8 was consistently linked to two traits (GR and RL). The area (± 100 Kb) associated with this QTN was screened for drought tolerance based on gene annotation. Fifteen candidate genes were found by this screening. Based on the expression data, four candidate genes i.e. Glyma08g156800, Glyma08g160000, Glyma08g162700, and Glyma13g249600 were found to be linked to drought tolerance regulation in soybean. Hence, the current study provides evidence to understand the genetic constitution of drought tolerance during the germination stage and identified QTNs or genes could be utilized in molecular breeding to enhance the yield under drought stress.

Genome-Wide Identification and Analysis of the Hsp40/J-Protein Family Reveals Its Role in Soybean (Glycine max) Growth and Development.

The J-protein family comprises molecular chaperones involved in plant growth, development, and stress responses. Little is known about this gene family in soybean. Hence, we characterized J-protein genes in soybean, with the most highly expressed and responsive during flower and seed development. We also revealed their phylogeny, structure, motif analysis, chromosome location, and expression. Based on their evolutionary links, we divided the 111 potential soybean J-proteins into 12 main clades (I-XII). Gene-structure estimation revealed that each clade had an exon-intron structure resembling or comparable to others. Most soybean J-protein genes lacked introns in Clades I, III, and XII. Moreover, transcriptome data obtained from a publicly accessible soybean database and RT-qPCR were used to examine the differential expression of DnaJ genes in various soybean tissues and organs. The expression level of DnaJ genes indicated that, among 14 tissues, at least one tissue expressed the 91 soybean genes. The findings suggest that J-protein genes could be involved in the soybean growth period and offer a baseline for further functional research into J-proteins' role in soybean. One important application is the identification of J-proteins that are highly expressed and responsive during flower and seed development in soybean. These genes likely play crucial roles in these processes, and their identification can contribute to breeding programs to improve soybean yield and quality.

Whole-Genome Identification of APX and CAT Gene Families in Cultivated and Wild Soybeans and Their Regulatory Function in Plant Development and Stress Response.

Plants coevolved with their antioxidant defense systems, which detoxify and adjust levels of reactive oxygen species (ROS) under multiple plant stresses. We performed whole-genome identification of ascorbate peroxidase (APX) and catalase (CAT) families in cultivated and wild soybeans. In cultivated and wild soybean genomes, we identified 11 and 10 APX genes, respectively, whereas the numbers of identified CAT genes were four in each species. Comparative phylogenetic analysis revealed more homology among cultivated and wild soybeans relative to other legumes. Exon/intron structure, motif and synteny blocks are conserved in cultivated and wild species. According to the Ka/Ks value, purifying selection is a major force for evolution of these gene families in wild soybean; however, the APX gene family was evolved by both positive and purifying selection in cultivated soybean. Segmental duplication was a major factor involved in the expansion of APX and CAT genes. Expression patterns revealed that APX and CAT genes are differentially expressed across fourteen different soybean tissues under water deficit (WD), heat stress (HS) and combined drought plus heat stress (WD + HS). Altogether, the current study provides broad insights into these gene families in soybeans. Our results indicate that APX and CAT gene families modulate multiple stress response in soybeans.

Characterization of SOD and GPX Gene Families in the Soybeans in Response to Drought and Salinity Stresses.

Plant stresses causing accumulation of reactive oxidative species (ROS) are scavenged by effective antioxidant defense systems. Therefore, the present study performed genome-wide identification of superoxide dismutase (SOD) and glutathione peroxidase (GPX) gene families in cultivated and wild soybeans, and 11 other legume species. We identified a total of 101 and 95 genes of SOD and GPX, respectively, across thirteen legume species. The highest numbers of SODs and GPXs were identified in cultivated (Glycine max) and wild (Glycine soja). A comparative phylogenetic study revealed highest homology among the SODs and GPXs of cultivated and wild soybeans relative to other legumes. The exon/intron structure, motif and synteny blocks were conserved in both soybean species. According to Ka/Ks, purifying the selection played the major evolutionary role in these gene families, and segmental duplication are major driving force for SODs and GPXs expansion. In addition, the qRT-PCR analysis of the G. max and G. soja SOD and GPX genes revealed significant differential expression of these genes in response to oxidative, drought and salinity stresses in root tissue. In conclusion, our study provides new insights for the evolution of SOD and GPX gene families in legumes, and provides resources for further functional characterization of these genes for multiple stresses.