To by OsbZIP16 also involves ABA-independent pathways. Many

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)


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