Differential modulation of heat inducible genes across diverse genotypes and molecular cloning of a sHSP from Pearl millet [Pennisetum glaucum (L.) R. Br.]

Environmental stresses negatively influence survival, biomass and grain yield of most crops. Towards functionally clarifying the role of heat responsive genes in Pearl millet under high temperature stress, the present study were carried out using semi quantitative RT- PCR for transcript expression profiling of hsf and hsps in 8 different inbred lines at seedling stage, which was earlier identified as thermo tolerant/susceptible lines through initial screening for thermo tolerance using membrane stability index among 38 elite genotypes. Transcript expression pattern suggested existence of differential response among different genotypes in response to heat stress in the form of accumulation of heat shock responsive gene transcripts. Genotypes WGI 126, TT-1 and MS 841B responded positively towards high temperature stress for transcript accumulation for both Pgcp 70 and Pghsf and also had better growth under heat stress, whereas PPMI 69 showed the least responsiveness to transcript induction supporting the membrane stability index data for scoring thermotolerance, suggesting the efficacy of transcript expression profiling as a molecular based screening technique for identification of thermotolerant genes and genotypes at particular crop growth stages. As to demonstrate this, a full length cDNA of Pghsp 16.97 was cloned from the thermotolerant cultivar, WGI 126 and characterized for thermotolerance. The results of demonstration set forth the transcript profiling for heat tolerant genes can be a very useful technique for high throughput screening of tolerant genotypes at molecular level from large cultivar collections at seedling stage.


96
Howeverthe picture of molecular mechanism of thermo-tolerance is highly complex consists of poorly 97 understood, various interlinking gene networks [22]. Henceit became a felt need to draw the detailed aspects of 98 the correlated gene expression activities and identifying those genes or gene product which have maximum 99 potency in imparting thermo-tolerance among genotypes at various crop growth stages, for which gene based 100 genotype screening come to be inevitable. The above said facts more significant as the threat of climate change 101 and global warming become a matter of reality as according to the report of the United States Environment 102 Protection Agency, the global average temperature has risen by about 1.4°F in the past century and is expected 103 to increase by 2°F to 11.5°F by 2100 [23]. Rising temperatures may lead to altered geographical distribution 104 and growing season of agricultural crops by allowing the threshold temperature for the start of the season and 105 crop maturity to reach earlier [24]. There is a constant need for the identification, isolation and characterisation 106 of increasing number of stress induced genes unravelling their functions for enhancing agricultural productivity.

107
A major challenge in conventional breeding for heat tolerance is the identification of reliable and 108 effective screening methods to facilitate detection of heat-tolerant plants and the genes responsible for thermo-109 tolerance. In present study, we performed semi quantitative RT-PCR (Reverse Transcription Polymerase Chain 110 Reaction) for expression profiling of Pennisetum glaucum heat shock factor (Pghsf) andPennisetum glaucum 111 chloroplast localized HSP 70 (Pgcp70) transcripts in different cultivar at seedling stage, screened for 112 thermotolerance. The correlated expression pattern of Pghsf and Pgcp70 were also studied which revealed the 113 initiator role of Pghsf and early response role of Pgcp70 towards high temperature stress. We also carried out 114 isolation and characterization of full length transcript of PgHSP16.97, a gene encoding alpha-crystalline sHSP 115 shows specific expression patterns during water and high temperature stress. This paper give validation on the 116 effectiveness of RT-PCR based screening methods for the identification and utilisation of thermotolerance 117 genes from superior heat tolerant genotypes for bridging supra-optimal temperature tolerance with high 118 productivity in Pearl millet.

Isolation and cloning of a full length Pg HSP
Based on transcript expression profiling studies, the best thermotolerant genotype was used to isolate a 174 full length cDNA of one small heat shock protein Pghsp17.0 (Acc. No. X94191.1). Primers were designed 175 (Table.2) to carried out RT-PCR as described above and the fragment obtained was purified and sequenced.    243 Comparative expression studies between two genes Pghsf and Pgcp70 among genotypes (Fig 4) suggested, even 244 though Pghsf showed lower expression under induction of heat stress during initial phase (2hrs), its expression 245 got a steady increase (4.6% to 12.3% and 11.0% to 13.3% at 7 & 10D old seedling respectively) upon 246 prolonging the heat stress upto 6hrs. In contrast to Pghsf, Pgcp70 had relatively faster response kinetics and 247 reached its peak expression at early stages of heat stress (2hrs) and its level diminished (21.5% to 14.2% and 248 28.2% to 15.4% at 7 and 10D old seedling respectively) after a prolonged treatment of 6hrs. The transcript 249 expression upon heat stress increased for corresponding differential heat treatment as the age of seedling 250 progressed, as the 10D old seedling shown to have more accumulation of transcript than that in 7D old seedling.  322 Client program was used to depict the PgHsp16.97 molecular model (Fig 10a). Superimposition of the model 323 with the template and root mean square deviation (RMSD) calculation was done using the program STRAP 324 (http://www.bioinformatics.org/strap/) The RMSD value of the selected Pghsp 16.97 model structure is 1.60A° 325 with respect to the template 1GME. The structural superimposition was done using STRAP interface observed 326 to have better level of model superimposition onto template which is shown in (Fig 10b).

360
There was differential expression pattern for these two heat responsive genes among different 361 genotypes, in which WGI 126 have shown the positive response to heat stress by accumulating more transcript, 362 while PPMI 69 with low accumulation of transcript as expected from the MSI data [26] which suggest the 363 transcript expression profiling can be used for screening thermotolerant and susceptible lines in a large pool of 364 genotypes along with identifying the potential genes responsible for thermotolerance at various stages of crop 365 growth.

366
Isolation and Cloning of full length cDNA of Pg HSP from Pearl millet

367
Of the molecular chaperones, the sHSPs were diverse and found in both prokaryotes and eukaryotes, shock proteins in young grape leaves during cross-adaptation to temperature stresses. SciHortic. 2008; 117: 231-519 240.