To that showed presence of an appropriate

Togain an insight into the role of OsbZIP16transcription factor in stress responses, Arabidopsisplants with over-expression of OsbZIP16were raised up to the homozygous T4 generation before being assayed (Figure 3).Confirmation of the transgene in Arabidopsiswas done by PCR amplification that showed presence of an appropriate size band inthe over-expressor lines but not in the WT plants (Figure 3A). The transcriptlevel of OsbZIP16 was also checked byRT-qPCR analysis which showed much higher expression of OsbZIP16 in the OsbZIP16ox plants (Figure 3B). Thephenotypic analysis of OsbZIP16ox plants showed similar growthpattern and morphology under normal conditions (Supplementary Figure 3).  As the expression of OsbZIP16 was induced by dehydration, salt and ABA, thus the germinationassay was performed on OsbZIP16ox seeds under abiotic stressconditions (on MS Agar + Mannitol/NaCl/ABA) and checked for their stresstolerance.

The results showed that the OsbZIP16ox seeds have highergermination percentage than WT under all three stress conditions – mannitol,NaCl and ABA in MS Agar media (Figure 4). The response of OsbZIP16oxplants towards drought stress conditions at vegetative stage was also assayedby water withdrawal for 14 days. .

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The assay revealed that OsbZIP16oxplants were tolerant to drought as the transgenic plants were able to survivethe stress and able to complete the normal course of their life-cycle bybolting as well as setting seeds after revival. This assay coupled withprevious stress assays performed using seeds and seedlings gives a clear roleof OsbZIP16 in alleviating dehydration and salt induced stress in plants. Sincethe OsbZIP16ox seedlings showed reduced sensitivity towards ABA, it couldbe speculated that the tolerance mechanism by OsbZIP16 also involves ABA-independent pathways. Many members ofthe 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 etal. 2012; Liu et al.

2014)

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