Heat stress transcripts, differential expression, and profiling of heat stress tolerant gene <i>TaHsp90</i> in Indian wheat (<i>Triticum aestivum</i> L.) cv C306

<div><p>To generate a genetic resource of heat stress responsive genes/ESTs, suppression subtractive hybridization (SSH) library was constructed in a heat and drought stress tolerant Indian bread wheat cultivar C306. Ninety three days old plants during grain filling stage were subjected to heat stress at an elevated temperature of 37°C and 42°C for different time intervals (30 min, 1h, 2h, 4h, and 6h). Two subtractive cDNA libraries were prepared with RNA isolated from leaf samples at 37°C and 42°C heat stress. The ESTs obtained were reconfirmed by reverse northern dot blot hybridization. A total of 175 contigs and 403 singlets were obtained from 1728 ESTs by gene ontology analysis. Differential expression under heat stress was validated for a few selected genes (10) by qRT-PCR. A transcript showing homology to <i>Hsp90</i> was observed to be upregulated (7.6 fold) under heat stress in cv. C306. CDS of <i>TaHsp90</i> (Accession no. MF383197) was isolated from cv. C306 and characterized. Heterologous expression of <i>TaHsp90</i> was validated in <i>E</i>. <i>coli</i> BL21 and confirmed by protein gel blot and MALDI-TOF analysis. Computational based analysis was carried out to understand the molecular functioning of TaHsp90. The heat stress responsive SSH library developed led to identification of a number of heat responsive genes/ESTs, which can be utilized for unravelling the heat tolerance mechanism in wheat. Gene <i>TaHsp90</i> isolated and characterized in the present study can be utilized for developing heat tolerant transgenic crops.</p></div

Subcellular location of TaHsp90 as predicted by CELLO v.2.5 server.

<p>Subcellular location of TaHsp90 as predicted by CELLO v.2.5 server.</p

List of TaHsp90 genes retrieved from NCBI database for analyzing nucleotides and amino acid sequences.

<p>List of TaHsp90 genes retrieved from NCBI database for analyzing nucleotides and amino acid sequences.</p

3D structure prediction of TaHsp90.

<p>(A) Modelling of TaHsp90 based on template-c5fwkA. (B) TaHsp90 docking to a predicted ligand (colored portion shows active site interaction with ligand). (C) Ramachandran plot showing the favourable region of TaHsp90 protein.</p

TaHsp90 phosphorylated sites and secondary structure prediction.

<p>(A) Phosphorylated sites prediction in TaHsp90 using Netphos 3.1a server. (B) Secondary structure map of TaHsp90 shows helices, sheets and disorderness. (C) Amino acid composition percentage along with the predicted significance p value. Feature predictions are color coded according to the sequence feature key shown below.</p

Gene expression analysis by qRT-PCR in contrasting wheat cultivars C306 (heat tolerant) and HD2967 (heat susceptible).

<p>(A) Fold change expression of Hsp90, <i>Hsp-Sti</i>, hypothetical <i>Dnaj</i>, ClpB1/Hsp100, PPIase, <i>GAPDH</i>, AAA ATPase, FKBP, PSBR, hypothetical <i>Hsp</i> at 37°C. (B) Fold change expression at 42°C. (C) Fold change expression of <i>Hsp90</i> in cv. C306. (D) Fold change expression of <i>Hsp-Sti</i> in wheat cv. C306. (E) Fold change expression of hyp <i>Dnaj</i> in wheat cv. C306. Statistical significance has been shown by asterisk (*) at p≤0.05 (n = 3).</p

Additional file 8: Figure S7. of Transcriptome analysis of lentil (Lens culinaris Medikus) in response to seedling drought stress

HeatMap of Top Down regulated between samples with p value < 0.05 in 1C_Control_1T_Treated. (TIF 974 kb

Additional file 12: Figure S11. of Transcriptome analysis of lentil (Lens culinaris Medikus) in response to seedling drought stress

Top 25 GO terms for down-regulated and up-regulated transcripts in ‘1 T’ as compared to ‘1C. (TIF 2218 kb

Evolutionay relationship of <i>T</i>. <i>aestivum</i> cv. C306 <i>TaHsp90</i> with other plant species.

<p>(A) Based on the nucleotide sequence. (B) Based on the amino acid sequence. Evolutionary analyses were conducted in MEGA7 using Neighbour-joining method with boot strap value of 1500.</p

Potential N-linked glycosylated sites found in TaHsp90.

<p>The result was generated using NetNGlyc 1.2 server.</p