DECIPHERING THE DIFFERENTIALLY EXPRESSED GENES IN FOXTAIL MILLET (Setaria italica L.) IN RESPONSE TO WATER STRESS.
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Date
2017-06-15
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Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola, Maharashtra.
Abstract
The present investigation entitled “Deciphering the differentially expressed genes in Foxtail millet (Setaria italica L.) in response to water stress” was carried out at the Biotechnology Centre, Department of Agricultural Botany, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola during 2013-2016.
In the present study, sixty two accessions of foxtail millet (Setaria italica L.) were investigated for physiological, biochemical and molecular responses for water stress. Water stress was imposed under laboratory conditions by using PEG-6000 and by withholding water and performances of various genotypes were monitored against a control. Seedling traits such as germination percentage, root length, shoot length and seedling dry weight, various physiological parameters like relative water content (RWC), leaf water potential (LWP) and chlorophyll content (CC) were studied in unstressed and stressed experiments. The results showed that water deficit stress has exerted negative effect on all the physiological parameters considered. Based on these observations, most drought tolerant and susceptible accessions were selected. IC97087, IC97189, IC120159 and IC120239 were recorded as the most drought tolerant accessions, whereas, IC97109, IC120234, IC120346 and Lepakshi were recorded as susceptible accessions.
The selected core set of accessions were then screened for their water stress tolerance on the basis of some primary metabolites like proline, total carbohydrates, starch and antioxidant enzymes like superoxide dismutase, peroxidase, catalase and glutathione reductase. Osmolytes viz. proline and carbohydrates increased with increasing drought stress. The proline content in unstressed tolerant plant is found to be much higher as compared to that in unstressed susceptible plants suggesting its important role in drought tolerance of plants. Photosynthetic pigment decreased with increasing drought stress. Activities of antioxidant enzymes increased with drought stress in most of the accessions. The activity of SOD was found to be highest in IC120239 which was found to be a tolerant accession in prior physiological screening. The activity of GR was high in all tolerant accession than in susceptible accessions. Catalase and POD activity was found to be highest in Prasad. Drought stress preferentially enhanced the activities of enzymatic antioxidants and accumulation of osmolytes.
Further, gene expression studies were carried out using the two contrasting accessions IC97189 (tolerant) and IC97109 (susceptible), for deciphering the transcriptional regulation of abiotic stress-related genes. cDNA SRAP analysis showed a relatively higher number of genes expressed in IC97189. The differential gene expression of drought responsible transcriptional factors like DREB1, DREB2, Aquaporins and C2H2 was studied in selected drought tolerant and susceptible plants each one showing considerable difference gene expression. Genome–wide investigation of plant aquaporin protein from foxtail millet was carried out to show that aquaporins comprise of MIP superfamily with conserved motif. Expression profiling of PIPs showed differences in gene expression pattern. Further an attempt was made to characterise full length sequence of PIP 2;7 to isolate a 770bp long DNA stretch out of the 857 base pair CDS having high homology to a UNPRIDICTED PIP2;7 gene in Setaria italica L. with an identity of 99%. Theoretical model of the tertiary structure shows that it has a highly conserved containing the NPA motif which was further validated by Ramachandran plot.
Analysis for cis regulatory elements identified more than 250 cis-regulatory elements present in the promoter region of PIP2;7 aquaporin gene in foxtail millet which revealed the presence of a number of abiotic stress responsive acting elements such as, ABRE binding site, MYB binding site, AP2/ERF binding site, LEA-5 binding site, NF-YB/A/C binding site, Trihelix binding site, EIN3:EIL binding site etc. Promoter analysis also revealed the presence of binding site for a sequence-specific DNA-binding domain (designated CG-1) present in calmodulin-binding transcription activators (CAMTAs) in the upstream region of PIP2;7 aquaporin. This domain could bind DNA directly and activate transcription, or interact with other transcription factors, not through DNA binding, thus acting as a co-activator of transcription. This might be the reason for high expression of PIP2;7 aquaporin.
Applying different physiological and biochemical tests to appreciate drought tolerance in plant leads to faster selection methods. Therefore, these characters can be used as an indirect selection criterion for screening drought tolerance plant materials which will lead to new cultivars with high yield potential and high yield stability that in turn will result in superior performance in dry environments. Hence, this research can provide documentation for breeding/selection of higher drought resistant foxtail millet in arid regions and acquisition of good information for future molecular research. The physio-biochemically screened accessions can further prove to be useful in studies involving transcriptome changes to fetch out the molecular mechanism underneath of its drought adaptation.
Accomplishments of these goals permit better understanding of molecular and physiological mechanisms utilized by plants during adverse growth conditions and define the biomarkers of tolerance against stresses. The research provides a possible point of integrating various molecular and biological pathways with water stress regulated gene expression.
The study can prove helpful to the farmers in selecting foxtail millet cultivars for unaffected yields in diverse agronomic conditions. These findings provide insight for further investigation of CG-1 domain in plant aquaporins in opening new perspectives for improving drought tolerance which could eventually lead to better crop production. The identified aquaporin could be explored in development of tolerant lines by MAS using PIP 2;7 as a functional marker and also in transgenic development in other related crops.
Description
Understanding drought tolerance at molecular level particularly identification of relevant genes in foxtail millet is likely to pave the ways for mitigating the drought stress losses in major crops. The adaptation of foxtail millet to low water conditions has been ascribed to its relatively small leaf area, the cell arrangement in its epidermis, its thick cell walls, and its ability to form a dense root system. Also, the water use efficiency of foxtail millet has been shown to be higher than that of maize, wheat, and sorghum. However, the molecular mechanism underneath of its drought adaptation is still not clear. The genome size of foxtail millet is very much similar to that of rice and is also one of the smallest among the Panicoid grasses. An extensive germplasm collection of the crop is available, providing opportunities to study the various biological processes and to fetch out the molecular mechanism underneath its tolerance.
All these characteristics make the plant an ideal choice to explore candidate genes involved in drought tolerance. Unfortunately, the crop has remained neglected with little or no importance in today’s world and has hence lagged behind in genetic and molecular studies.
Keywords
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Citation
GAWAI, DIPTI CHANDRABHA. (2017). Deciphering the differentially expressed genes in foxtail millet (Setaria italica L.) in response to water stress. Biotechnology centre. Department of Agricultural Botany. Dr. Panjabrao Deshmukh Krishi Vidhyapeeth, Akola. Ph. D. 2017. Print. xxviii, 197p. (Unpublished).