Plant Science 254 (2017) 48–59 Contents lists available at ScienceDirect Plant Science j ourna l ho me pa g e: www.elsevier.com/locate/plantsci Arabidopsis CBL interacting protein kinase 3 interacts with ABR1, an APETALA2 domain transcription factor, to regulate ABA responses Sibaji K. Sanyal, Poonam Kanwar, Akhilesh K. Yadav, Cheshta Sharma, Ashish Kumar, Girdhar K. Pandey ∗ Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi, 110021, India a r t i c l e i n f o Article history: Received 22 June 2016 Received in revised form 4 November 2016 Accepted 7 November 2016 Available online 9 November 2016 Keywords: ABA Abiotic stress CBL CIPK Drought Signaling a b s t r a c t Calcium (Ca 2+ ) plays a vital role as a second messenger in several signaling pathways in plants. The calcineurin B-like proteins (CBLs) represent a family of plant calcium-binding proteins that function in propagating Ca 2+ signals by interacting with CBL interacting protein kinases (CIPKs). Phosphorylation of CBL by CIPK is essential for the module to display full activity towards its target protein. Previous genetic analysis showed that the function of CBL9-CIPK3 module was implicated in negatively regulating seed germination and early development. In the present study, we have biochemically investigated the interaction of CBL9-CIPK3 module and our findings show that CBL9 is phosphorylated by CIPK3. Moreover, Abscisic acid repressor 1 (ABR1) is identified as the downstream target of CIPK3 and CIPK3-ABR1 function to regulate ABA responses during seed germination. Our study also indicates that the role of ABR1 is not limited to seed germination but it also regulates the ABA dependent processes in the adult stage of plant development. Combining our results, we conclude that the CBL9-CIPK3-ABR1 pathway functions to regulate seed germination and ABA dependent physiological processes in Arabidopsis. © 2016 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Calcium (Ca 2+ ) is one of the major signaling molecules in plants [1–7] and is involved in a number of signaling pathways that trans- duce signals generated by biotic, abiotic as well as developmental stimuli [8,9]. In response to various stimuli, there is a fluctuation in the level of cytosolic Ca 2+ and hence a typical Ca 2+ signature is generated in response to a particular signal [10]. The Ca 2+ signa- ture can originate from either by influx of Ca 2+ from extracellular space or by release from endomembrane compartments such as vacuole [11]. The plant cell has several toolkits to decode these diverse Ca 2+ signatures to elicit spatio-temporal responses [5,12]. CBL-CIPK network is one of the member of this toolkit group [4]. The CBL-CIPK module is precisely targeted to the plasma membrane (PM) and vacuolar membrane (tonoplast) and modulates several downstream targets [13–17]. Till date studies have identified var- ious downstream targets such as protein phosphatase 2C (PP2C), channels/transporters and transcription factors [18], and a recently identified target is Snrk2 [19]. Several Ca 2+ related signaling path- ∗ Corresponding author. E-mail addresses: gkpandey@south.du.ac.in, giridhar98@gmail.com (G.K. Pandey). ways like nutrient uptake, abiotic stress response, modulation of ion flux and Abscisic acid (ABA) signaling are regulated by this module [8,12,20–25]. Remarkably for ABA signaling, till date, many transcription factors have been identified as potential downstream signaling component of the CBL-CIPK module. Notable in this regard are the regulation of Ethylene Responsive Factor 7 (ERF7) by CIPK15 [26], ABI5 by CIPK26 [27] and CIPK11/PKS5 [28]. As an exception, CBL1/SCaBP5 and CIPK15/PKS3 interact to regulate ABA signaling in Arabidopsis but the downstream targets of this pair are ABA insen- sitive 1 and 2 (ABI1 and ABI2), which are protein phosphatases [29]. The Ca 2+ sensor, CBL9 was identified as a negative regulator of ABA signaling and biosynthesis in Arabidopsis [24]. Kim and co- workers reported that CIPK3 also regulate ABA responses during seed germination and ABA induced gene expression in Arabidopsis [23]. Later by genetic analysis, Pandey et al. [41] proved that CBL9 and CIPK3 form a module and regulate ABA responses during seed germination. The biochemical characterization of a kinase yields valuable clues regarding its function, and this facet was not probed for CBL9-CIPK3 module. Several other CIPKs have been extensively characterized and many recent reports have proved that CBLs are phosphorylated by interacting CIPKs [30–35]. It is speculated to be an important mechanism that fully activates the CBL-CIPK module http://dx.doi.org/10.1016/j.plantsci.2016.11.004 0168-9452/© 2016 Elsevier Ireland Ltd. All rights reserved.