Raj D. Rai

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

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. Sangers 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.

miR430: the novel heat-responsive microRNA identified from miRNome analysis in wheat (Triticum aestivum L.)

AbstractmiRNAs are 21–24 nt non-coding RNAs involved in negative regulation of the target gene expression in response to stress and developmental cues. Wheat, the major staple food crop, is highly sensitive to heat stress (HS). Even moderate high temperatures during critical stages causes reduction in yield and quality of the grains. Here, we identified a novel miRNA (candidate miR430) from wheat using de novo assembly, and cloned it from wheat cv. HD2985 using universal adaptor. The identified miRNA was mapped on to the Chromosome 3B and predicted to have more than 30 target genes. Most of the targets identified were associated with heat shock proteins. Expression analysis of miR430 in contrasting wheat cultivars by quantitative real-time PCR revealed significant genotypic variations under HS. Tissue specific expression analysis showed relatively low expression in leaves, as compared with stem and root under HS. A negative correlation was found between the expression of miR430 and their respective target genes under HS. MiR430 can be used to manipulate the expression of target genes under HS towards enhancing HS tolerance for the development of ‘climate-smart’ wheat crop.