To gain an insight into the role OsbZIP16 transcription factor in stress responses, Arabidopsis plants over-expressing OsbZIP16 were raised up to the homozygous T4 generation before being assayed (Figure 3). Confirmation of the transgene in Arabidopsis was done by PCR amplification that showed presence of an appropriate size band in the over-expressor lines but not in the WT plants (Figure 3A).
The transcript level of OsbZIP16 was also checked by RT-qPCR analysis which showed much higher expression of OsbZIP16 in the OsbZIP16ox plants (Figure 3B). The phenotypic analysis of OsbZIP16ox plants showed similar growth pattern and morphology under normal conditions (Supplementary Figure 3). As the expression of OsbZIP16 was induced by dehydration, salt and ABA, thus the OsbZIP16ox seeds were analysed for their germination ability under abiotic stress conditions (on MS Agar + Mannitol/NaCl/ABA) and checked for their stress tolerance. The results showed that the OsbZIP16ox seeds have higher germination percentage than WT under all three stress conditions – mannitol, NaCl and ABA in MS Agar media (Figure 4). The response of OsbZIP16ox plants towards drought stress conditions at vegetative stage was also assayed by water withdrawal for 14 days. .
The assay revealed that OsbZIP16ox plants were tolerant to drought as the transgenic plants were able to survive the stress and able to complete the normal course of their life-cycle by bolting as well as setting seeds after revival. This assay coupled with previous stress assays performed using seeds and seedlings gives a clear role of OsbZIP16 in alleviating dehydration and salt induced stress in plants. Since the OsbZIP16ox seedlings showed reduced sensitivity towards ABA, it could be speculated that the tolerance mechanism by OsbZIP16 also involves ABA-independent pathways. Many members of the bZIP TFs have been well characterized in both ABA dependent (OsABF1/OsbZIP12, OsbZIP23, OsbZIP46, OsbZIP71) as well as ABA independent (RISBZ5/OsbZIP52) pathways involved in stress signalling in plants (Amir Hossain et al. 2010; Liu et al.
2012; Tang et al. 2012; Liu et al. 2014)