Functional characterization and reconstitution of ABA signaling components using transient gene expression in rice protoplasts

Abstract

The core components of ABA-dependent gene expression signaling have been identified in Arabidopsis and rice. This signaling pathway consists of four major components; group A OsbZIPs, SAPKs, subclass A OsPP2Cs and OsPYL/RCARs in rice. These might be able to make thousands of combinations through interaction networks resulting in diverse signaling responses. We tried to characterize those gene functions using transient gene expression for rice protoplasts (TGERP) because it is instantaneous and convenient system. Firstly, in order to monitor the ABA signaling output, we developed reporter system named pRab16A-fLUC which consists of Rab16A promoter of rice and luciferase gene. It responses more rapidly and sensitively to ABA than pABRC3-fLUC that consists of ABRC3 of HVA1 promoter in TGERP. We screened the reporter responses for over-expression of each signaling components from group A OsbZIPs to OsPYL/RCARs with or without ABA in TGERP. OsbZIP46 induced reporter most strongly among OsbZIPs tested in the presence of ABA. SAPKs could activate the OsbZIP46 even in the ABA independence. Subclass A OsPP2C6 and -8 almost completely inhibited the OsbZIP46 activity in the different degree through the SAPK9. Lastly, OsPYL/RCAR2 and -5 rescued the OsbZIP46 activity in the presence of SAPK9 and OsPP2C6 dependent on ABA concentration and expression level. By using TGERP, we could characterize successfully the effects of ABA dependent gene expression signaling components in rice. In conclusion, TGERP represents very useful technology to study systemic functional genomics in rice or other monocots.

Feedback Regulation of ABA Signaling and Biosynthesis by a bZIP Transcription Factor Targets Drought-Resistance-Related Genes

“…We found the transcriptional activity of OsbZIP23 was significantly enhanced even without ABA (Fig. 1B), suggesting that SAPK2 can activate OsbZIP23, which is consistent with a previous study (Kim et al, 2015).…”

“…Recently, OsbZIP46 was found to be phosphorylated by SAPK2 and SAPK6 in vitro, and cotransformation of OsbZIP46 with SAPK2, SAPK6, or SAPK9 into rice protoplasts can significantly induce the transcriptional activation activity of OsbZIP46 (Kim et al, 2015). OsbZIP23 shows high similarity with OsbZIP46 (Nijhawan et al, 2008).…”

“…The subgroup A bZIP transcription factors such as TRAB1, OsbZIP46, and OsbZIP12 in rice and ABI5, ABRE1, ABRE2, and ABF1 in Arabidopsis generally require ABA for transcriptional activation (Gampala et al, 2002;Kobayashi et al, 2005;Yoshida et al, 2010;Kim et al, 2015). Recently, OsbZIP46 was found to be phosphorylated by SAPK2 and SAPK6 in vitro, and cotransformation of OsbZIP46 with SAPK2, SAPK6, or SAPK9 into rice protoplasts can significantly induce the transcriptional activation activity of OsbZIP46 (Kim et al, 2015).…”

“…Generally, subgroup A bZIP transcription factors need SnRKs to fully activate their transcriptional activation activity (Kagaya et al, 2002;Kobayashi et al, 2005;Chae et al, 2007;Fujii et al, 2007;Yoshida et al, 2010;Kim et al, 2015). In rice, 11 subgroup A bZIP transcription factors have been identified, and 6 of them have been associated with diverse biological functions (Hobo et al, 1999;Zou et al, 2007;Xiang et al, 2008;Zou et al, 2008;Lu et al, 2009;Amir Hossain et al, 2010;Hossain et al, 2010;Yang et al, 2011;Tang et al, 2012).…”

“…The OSRK1 (SAPK6) can phosphorylate and activate the bZIP transcription factor OREB1, and ectopic expression of OSRK1 in tobacco (Nicotiana tabacum) reduced the sensitivity to ABA in germinating seeds as well as root elongation (Chae et al, 2007). A more recent work using an in vitro phosphorylation assay and in vivo dual-luciferase assay indicated that SAPK2, SAPK6, and SAPK9 can phosphorylate and activate OsbZIP46, a subgroup A bZIP transcription factor (Tang et al, 2012;Kim et al, 2015).…”