Next Generation Sequencing (Ngs) Assisted Identification and Functional Characterization of Multi-Stress Tolerant Genes in Arabidopsis Thaliana

Abstract

The demand for global food productivity has been greatly increased with continuously rising population and varying climatic conditions; as a result there is dire need for the development of stress-tolerant crop varieties. To cope with this condition, it is essential to understand the mechanism of plant response to a combination of stresses, as revealed by stress induced transcriptomics. Therefore, this study was aimed at utilizing the publicly available specific transcriptomic data sets of plants exposed to different abiotic stresses in order to identify potential stress tolerant genes. Genes were identified that were differentially expressed between control plants and stressed. Based on the RNAseq analysis of Filichkin et al. (2010), a total of 12,606 12,594 and 12,530 differentially expressed genes (DEGs) were detected under heat, drought and salt stress conditions. This included 7145, 6336 and 6551 genes up-regulated and 5461, 6258 and 5979 genes down regulated under heat, drought and salt stress, respectively. A total of 2058 and 7543 and 8056 DEGs were detected under salt, heat and combined salt and heat stress conditions in the data set of Suzuki et al. (2016). This included 931, 4092 and 4492 genes up-regulated while 1127, 3451 and 3564 genes down regulated under salt, heat and combined salt and heat stress conditions, respectively. Analysis of RNA-seq data of Ding et al. (2014) revealed a total 30,941 DEGs (17318 up-regulated and 13623 down regulated) under salt stress. A total number of 14, 12 and 7 genes were short-listed for drought, heat and salt stresses, respectively on the basis of fold change and lowest p value (p<0.01). Subsequently, on the basis of the expression pattern of the shortlisted genes, a total of six multi-stress tolerant genes were selected, two from each stress condition. The RNA-seq predicted expression of the selected genes was confirmed in lab by qRT-PCR which showed significantly comparable results. Arabidopsis (Col-0) transgenic plants expressing the selected multi-stress tolerant genes were confirmed at transcript and protein level, the functional assessment of these transgenic plants was done compared to WT (Col-0) plants. The transgenic plants over expressed with AT4G18280 gene showed tolerance to heat, drought and salinity. At 100 mM NaCl, the germination rate of the transgenic lines varied from 70 % to 90 % as compared to WT (Col-0) plants which was recorded only 55 %. Similarly, at 200 mM NaCl the range varied from 65 % to 52 % for the transgenic lines as compared to the WT plant (40 %). The relative root length of the transgenic lines varied in the range of 70 % (OX-L#1 and WT) to 100 % (OX-L#3) at 100 mM NaCl. However, at 200 mM NaCl the range varied from 40 % (OX-L#1 and WT) to 75 % (OX-L#3). Chlorophyll content of the transgenic lines varied in the range of 0.1 mg/g fresh weight (FW) (OX-L#1 and WT) to 0.4 mg/g FW (OX-L#2 and #3) at 100 mM NaCl. While at 200 mM NaCl, almost negligible chlorophyll content was recorded (OX-L#1 and WT), however, significantly higher chlorophyll content of 0.1 mg/g FW was observed (OX-L#2 and #3). Under salt x stress, the melondialdehyde (MDA) content recorded for the over-expressed transgenic lines varied in the range of 6 to 8 nmol/g FW as compared to WT (17 nmol/g FW). Under salt stress the wild type plant showed more electrolyte leakage (45%), while significantly reduced relative electrolyte leakage in the range of 16 % to 24 % was recorded for all of the over-expressed transgenic lines. Under salt stress, the proline contents recorded for the transgenic lines varied in the range of 380 to 410 µg/g FW compared to the WT plant which was only 120 µg/g FW. The fresh weights determined for the transgenic plants varied in the range of 15 to 18 mg/plant as compared to the wild type plant which was 8 mg/plant only. The survival rate was only 20 % in WT plants, while the transgenic lines showed more survival rate in the range of 50 % to 65 % at 200 mM NaCl. Similarly, the transgenic lines with AT4G18280 gene showed tolerance to drought stress. The survival rate of the transgenic lines varied in the range of 31 % to 39 % under drought stress as compared to WT plants which was recorded only 24 %. The transgenic lines showed significantly less water loss at different hours of dehydration compared to WT plants. The survival percentage recorded for the transgenic lines varied from 35 % to 45 % under heat stress condition compared to the WT plants which showed only 20 % survival rate. HSP17.6B (AT2G29500) overexpression increased tolerance to heat stress only. The survival rate of the transgenic lines varied in the range of 40 % to 45 % than the WT plants which showed only 20% survival rate under heat stress (45 °C for 1hr). The transgenic lines showed lower electrolyte leakage (25 % to 35 %) compared to the WT plants which showed 50 % relative electrolyte leakage. Chlorophyll content recorded for the transgenic lines varied in the range of ~ 0.38 to ~ 0.4 mg/g FW than WT plants which showed only ~ 0.2 mg/g FW. Higher root elongation recorded for the transgenic lines in the range of 23 % to 30 % as compared to the WT plants which was only 0.9 % under heat stress. Overexpression of the remaining genes (HOP3, LSU1, GRP9 and AT2G16586) had no significant effect on transgenic plants response to different abiotic stresses. AT4G18280 and HSP 17.6B may be used to identify orthologues in other non-model crops for the development of multi- stress and heat stress tolerant crop varieties, respectively.