SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat

Abstract

Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat—a novel step toward the development of “climate-smart” wheat.

Functional Annotation of Hypothetical Proteins Derived from Suppressive Subtraction Hybridization (SSH) Analysis Shows NPR1 (Non-Pathogenesis Related)-Like Activity

“…Transcriptome refers to the comprehensive listing of transcripts along with their splice variants. SSH analysis has been adopted as an economical mode for the identification of genes with ease and looks out for novel genes in a genome [38]. Bioinformatic analysis, coupled with SSH, has been utilized for the identification of differentially expressed immune response in insects [39].…”

Rapid induction of small heat shock proteins improves physiological adaptation to high temperature stress in peanut

“…This ability of the tolerant genotypes for prompt induction of HSPs probably helped them to counteract HT stress better by binding to the thermolabile proteins and macromolecules and preventing them from misfolding or other heat injuries. Definite role of small HSP for preventing wrong aggregation or irreversible unfolding of partially denatured proteins was established in many crops under heat stress (Goswami et al., ; Lee et al., ; Xu, Zhan, & Huang, ). Besides, both HSP 70 and 90 play key role as molecular chaperon and known to modulate downstream gene expression under heat stress (Duan et al., ; Zhang et al., ).…”

“…Thermo-stability of existing proteins and other macromolecular structures and prevention of newly synthesized proteins from denaturation were thought to be essential role of both small and large HSPs (Usman et al, 2014). In addition, larger HSPs, especially HSP 70 and HSP 90, were reported to act as molecular chaperone as they involved in upregulation of several downstream genes associated with heat tolerance in plants (Kumar et al 2016;Zhang et al, 2013).…”

Overexpression of V-type H+ pyrophosphatase gene EdVP1 from Elymus dahuricus increases yield and potassium uptake of transgenic wheat under low potassium conditions

“…A lane of driver (control) and one of tester (heat stress) were used for electrophoresis on a 1.0% formaldehyde agarose gel; the samples were then transferred onto a nylon membrane using iBlotter (Invitrogen, UK). Experimental procedures were performed as previously described 25 .…”