Functional Exploration of Chaperonin (HSP60/10) Family Genes and their Abiotic Stress-induced Expression Patterns in Sorghum bicolor.

BACKGROUND: Sorghum, the C4 dry-land cereal, important for food, fodder, feed and fuel, is a model crop for abiotic stress tolerance with smaller genome size, genetic diversity, and bio-energy traits. The heat shock proteins/chaperonin 60s (HSP60/Cpn60s) assist the plastid proteins, and participate in the folding and aggregation of proteins. However, the functions of HSP60s in abiotic stress tolerance in Sorghum remain unclear. METHODS: Genome-wide screening and in silico characterization of SbHSP60s were carried out along with tissue and stress-specific expression analysis. RESULTS: A total of 36 HSP60 genes were identified in Sorghum bicolor. They were subdivided into 2 groups, the HSP60 and HSP10 co-chaperonins encoded by 30 and 6 genes, respectively. The genes are distributed on all the chromosomes, chromosome 1 being the hot spot with 9 genes. All the HSP60s were found hydrophilic and highly unstable. The HSP60 genes showed a large number of introns, the majority of them with more than 10. Among the 12 paralogs, only 1 was tandem and the remaining 11 segmental, indicating their role in the expansion of SbHSP60s. Majority of the SbHSP60 genes expressed uniformly in leaf while a moderate expression was observed in the root tissues, with the highest expression displayed by SbHSP60-1. From expression analysis, SbHSP60-3 for drought, SbHSP60-9 for salt, SbHSP60-9 and 24 for heat and SbHSP60-3, 9 and SbHSP10-2 have been found implicated for cold stress tolerance and appeared as the key regulatory genes. CONCLUSION: This work paves the way for the utilization of chaperonin family genes for achieving abiotic stress tolerance in plants.

Genome-wide Scanning and Characterization of Sorghum bicolor L. Heat Shock Transcription Factors.

A genome-wide scanning of Sorghum bicolor resulted in the identification of 25 SbHsf genes. Phylogenetic analysis shows the ortholog genes that are clustered with only rice, representing a common ancestor. Promoter analysis revealed the identification of different cis-acting elements that are responsible for abiotic as well as biotic stresses. Hsf domains like DBD, NLS, NES, and AHA have been analyzed for their sequence similarity and functional characterization. Tissue specific expression patterns of Hsfs in different tissues like mature embryo, seedling, root, and panicle were studied using real-time PCR. While Hsfs4 and 22 are highly expressed in panicle, 4 and 9 are expressed in seedlings. Sorghum plants were exposed to different abiotic stress treatments but no expression of any Hsf was observed when seedlings were treated with ABA. High level expression of Hsf1 was noticed during high temperature as well as cold stresses, 4 and 6 during salt and 5, 6, 10, 13, 19, 23 and 25 during drought stress. This comprehensive analysis of SbHsf genes will provide an insight on how these genes are regulated in different tissues and also under different abiotic stresses and help to determine the functions of Hsfs during drought and temperature stress tolerance.

Genome-Wide Association Study for Major Biofuel Traits in Sorghum Using Minicore Collection.

BACKGROUND: Production of biofuels from lignocellulosic crop biomass is an alternative to reduce greenhouse gas emissions. The biofuel production involves collecting biomass, breaking down cell wall components followed by the conversion of sugars to ethanol. The lingo-cellulosic biomass comprises 40-50% cellulose, 20-30% hemicellulose, and 10-25% lignin. Sorghum is a widely adapted energy crop for biofuel production. Biomass with low lignin, high cellulose, and high hemicellulose contents are exploited to attain maximum biofuel production efficiency. Resistance to lodging, pest, disease, and abiotic stresses related to cell wall components is well documented, and quantitative trait loci were identified to understand these traits' genetic correlation. Selection for reduced lignin and increased cellulose content in stover can increase the ethanol yield. The Genome-Wide Association Studies (GWAS) is a complementary approach to evaluating the marker and phenotype associations among large diversity panels. Single nucleotide polymorphisms were scanned to identify loci associated with the traits of interest. In this study, the GWAS was performed on 245 sorghum minicore genotypes to analyze agronomic traits (days to 50%flowering, fresh biomass yield, dry biomass yield) and cell wall components (cellulose, hemicellulose, and lignin). Further, in-silico validation of the candidate genes was performed in a global gene expression data from large-scale RNA sequencing studies in sorghum available in the NCBI GEO database was used. OBJECTIVE: The objectives of this study are to evaluate native variations in biofuel related agronomic traits and stalk cell wall components and to identify significant SNPs or loci related to the cell wall components. METHODS: In this article, an association mapping panel, comprising of 245 sorghum minicore germplasm accessions, was evaluated during two post rainy seasons of 2013 and 2014, and observations were recorded on the whole plot- for days to 50% flowering, fresh biomass yield (tha-1, and dry biomass yield (tha-1). The biomass of sun-dried plants from both seasons was collected separately, chopped, dried, and ground to powder. The cellulose, hemicellulose, and lignin contents were determined in the powdered. The content of each of these three components in sorghum was expressed in percent of dry matter. The data on agronomic traits and composition analysis was subjected to Analysis of Variance. For the current study, we remapped the raw GBS data with the sorghum assembly version v3.1. A total of 27,589 SNPs were obtained with a minor allele frequency (MAF) >1% and missing data <50%. The GWAS was performed in a single minicore population using FarmCPU, in R software. The synteny positions of the identified significant SNPs between sorghum and other model crop species viz., maize, switchgrass, and Arabidopsis were represented using CIRCOS software for traits viz., dry biomass yield, cellulose, hemicellulose, and lignin. The transcriptome dataset from where sorghum gene atlas studies of grain, sweet, and bioenergy sorghums are available through NCBI's Gene Expression Omnibus (GEO) under accession number GSE49879, was used to cross-validate the identified SNPs for cellulose, hemicellulose, and lignin through GWAS. RESULTS: High broad-sense heritability was exhibited for all the traits in individual seasons along with significant genotype × environment interaction across seasons except lignin. Association mapping with a P < 1×10-4 revealed genomic regions associated with the- (i) agronomic traits (days to 50% flowering, fresh and dry biomass), and (ii) biochemical traits (cellulose, hemicellulose, and lignin) associated with biofuels production, in individual seasons. Twelve significant SNPs for flowering time, 30 fresh biomass yields, and 24 for dry biomass yield, 25 for cellulose, 7 for hemicellulose, and 21 for lignin were identified. CIRCOS plot was constructed to identify and analyze similarities and differences while comparing the sorghum genome with different crops. For cellulose high similarity of >80% was observed for all sorghum gene sequences with the maize homologs. The overall similarity of sorghum homologs with foxtail millet was >65%, for Arabidopsis from 30.6% to 48.6%, and rice from 28.2% to 92.8%. SNPs for hemicellulose displayed maximum similarity to foxtail millet followed by maize. The sequence similarity of lignin SNPs in sorghum was highest with the maize genome followed by Arabidopsis. Both rice and foxtail millet showed >55% similarity to the sorghum genome. CONCLUSION: This study reports large variability for agronomic and biofuel traits in the sorghum minicore collection with high heritability. The genetic architecture of cell wall components using the GWAS approach was studied and candidate genes for each component were annotated. These results give a better understanding of the genetic basis of the sorghum cell wall composition. The association analysis identified regions of the genome that could be targeted to enhance the quality of biomass and yield along with the desired composition promoting breeding efficiency for enhanced biofuel yield.

Genome-wide identification and transcriptional profiling of small heat shock protein gene family under diverse abiotic stress conditions in Sorghum bicolor (L.).

The small heat shock proteins (sHsps/Hsp20s) are the molecular chaperones that maintain proper folding, trafficking and disaggregation of proteins under diverse abiotic stress conditions. In the present investigation, a genome-wide scan revealed the presence of a total of 47 sHsps in Sorghum bicolor (SbsHsps), distributed across 10 subfamilies, the major subfamily being P (plastid) group with 17 genes. Chromosomes 1 and 3 appear as the hot spot regions for SbsHsps, and majority of them were found acidic, hydrophilic, unstable and intron less. Interestingly, promoter analysis indicated that they are associated with both biotic and abiotic stresses, as well as plant development. Sorghum sHsps exhibited 15 paralogous and 20 orthologous duplications. Expression analysis of 15 genes selected from different subfamilies showed high transcript levels in roots and leaves implying that they are likely to participate in the developmental processes. SbsHsp genes were highly induced by diverse abiotic stresses inferring their critical role in mediating the environmental stress responses. Gene expression data revealed that SbsHsp-02 is a candidate gene expressed in all the tissues under varied stress conditions tested. Our results contribute to the understanding of the complexity of SbsHsp genes and help to analyse them further for functional validation.

Genome-scale identification, classification, and tissue specific expression analysis of late embryogenesis abundant (LEA) genes under abiotic stress conditions in Sorghum bicolor L.

Late embryogenesis abundant (LEA) proteins, the space fillers or molecular shields, are the hydrophilic protective proteins which play an important role during plant development and abiotic stress. The systematic survey and characterization revealed a total of 68 LEA genes, belonging to 8 families in Sorghum bicolor. The LEA-2, a typical hydrophobic family is the most abundant family. All of them are evenly distributed on all 10 chromosomes and chromosomes 1, 2, and 3 appear to be the hot spots. Majority of the S. bicolor LEA (SbLEA) genes are intron less or have fewer introns. A total of 22 paralogous events were observed and majority of them appear to be segmental duplications. Segmental duplication played an important role in SbLEA-2 family expansion. A total of 12 orthologs were observed with Arabidopsis and 13 with Oryza sativa. Majority of them are basic in nature, and targeted by chloroplast subcellular localization. Fifteen miRNAs targeted to 25 SbLEAs appear to participate in development, as well as in abiotic stress tolerance. Promoter analysis revealed the presence of abiotic stress-responsive DRE, MYB, MYC, and GT1, biotic stress-responsive W-Box, hormone-responsive ABA, ERE, and TGA, and development-responsive SKn cis-elements. This reveals that LEA proteins play a vital role during stress tolerance and developmental processes. Using microarray data, 65 SbLEA genes were analyzed in different tissues (roots, pith, rind, internode, shoot, and leaf) which show clear tissue specific expression. qRT-PCR analysis of 23 SbLEA genes revealed their abundant expression in various tissues like roots, stems and leaves. Higher expression was noticed in stems compared to roots and leaves. Majority of the SbLEA family members were up-regulated at least in one tissue under different stress conditions. The SbLEA3-2 is the regulator, which showed abundant expression under diverse stress conditions. Present study provides new insights into the formation of LEAs in S. bicolor and to understand their role in developmental processes under stress conditions, which may be a valuable source for future research.