Analysis of thermotolerance
To check if the TaHSFA6b overexpression plants are showing increased tolerance towards high temperature, we grow plants of 3 transgenic lines and WT in temperature controlled environment of 35 ºC right from germination. We found that initially all seeds germinated and grow normally during first 5-6 days (1 leaf stage), however, afterwards WT plants started showing reduced growth and with flaccid leaves, while plants of transgenic lines keep growing and increasing in height and making new leaves. After 3 weeks of providing continuous heat stress environment, all the plants of WT either showed senescence or died, while those of 3 transgenic lines were healthier and greener, and making new leaves (Figure 2).
Since elevated temperature results in production of reactive oxygen species we checked the status of free oxygen radicals in plant grown under constant temperature by NBT staining method (Wohlgemuth et al. 2002). We found marked difference in staining in leaf of WT and transgenic lines. While there is not much difference in the leaves of WT plants and transgenic lines under control conditions, however, marked difference is observed in NBT staining under heat stress condition. It was much less in the transgenic lines AP2-2 and AP6-2 (Figure 3A) compared to wild type. Further, to quantify the activity of Reactive Oxygen Species (ROS), we measured the activity of 3 enzymes: Ascorbate peroxidase, Catalase and Superoxide dismutase. We found the all these enzymes showed higher activity in transgenic plants as compared to WT plants. Ascorbate peroxidase and catalase showing significantly higher activity under heat stress conditions (Figure 3B).
It has been shown that HSFs and specially A type have profound effect on global transcriptome of plant. We proceeded for transcriptomics through RNA-sequencing of WT and plants from two transgenic lines (AP 2-1 and AP 2-2). A total of 103 million reads each were obtained from WT and trangenic plants. Principal component analysis (PCA) revealed a clear separation between replicates of WT and transgenic plants (Figure 4A). Analysis of differential gene expression revealed that a total of 1565 transcripts differentially expressed with a log2 fold change of> 2 and p-value of 0.05, these represent 663 upregulated and 902 downregulated genes (Supplementary Table S2).
To get an idea what the top upregulated genes are involved in, we used signature search option in Genvestigator omics analysis tool (https://genevestigator.com/gv/doc/intro_plant.jsp) by choosing available RNA sequencing experiments for barley. As can be seen from supplementary figure S4, our upregulated genes are mostly involved in plants responses to abiotic stresses such as heat, drought and salinity. A careful analysis of upregulated genes revealed that these genes belong to heat shock proteins and other molecular chaperones, genes related to water and oxidative stress and biosynthesis of stress related metabolites (Supplementary Table S2). To confirm RNA sequencing data, we performed qRT-PCR expression analysis on some selected genes representing HSPs, ROS scavenging enzymes, drought response and stress signaling components. As can be seen in figure 5, all these genes showed significantly higher expression in terms of relative fold change in different transgenic lines as compared to wild type plants. Further, we checked the performance of wild type and transgenic plants from line AP2-2 under simulated water stress. For this, seeds were sown in nursery pots and water was given till filed capacity and the pots were kept in a greenhouse kept at 26°C temperature and 16:8 hour photoperiod. No water was given after first instance and the plants were observed for four weeks from date of sowing. All the seeds germinated well and start growing normally, however after three weeks, signs of water stress were visible as the leaves start rolling and limping. At four week stage, all the wild type plants showed severe limping of leaves, on the other hand plants from transgenic line AP2-2 were still showing erect and longer leaves (Figure 6A). We measured relative water content in last fully opened leaves of both wild type and transgenic plants and found that transgenic plants had around 50% more relative water content than wild type plants (Figure 6B).