StCDPK3 Phosphorylates In Vitro Two Transcription Factors Involved in GA and ABA Signaling in Potato: StRSG1 and StABF1.

Carolina Grandellis,Magalí Graciela Bialer,Elisa Fantino,Daniela Andrea Capiati,Rita María Ulloa,María Noelia Muñiz García,Franco Santin,Ing-Feng Chang

doi:10.1371/journal.pone.0167389

Abstract

Calcium-dependent protein kinases, CDPKs, decode calcium (Ca2+) transients and initiate downstream responses in plants. In order to understand how CDPKs affect plant physiology, their specific target proteins must be identified. In tobacco, the bZIP transcription factor Repression of Shoot Growth (NtRSG) that modulates gibberellin (GA) content is a specific target of NtCDPK1. StCDPK3 from potato is homologous (88% identical) to NtCDPK1 even in its N-terminal variable domain. In this work, we observe that NtRSG is also phosphorylated by StCDPK3. The potato RSG family of transcription factors is composed of three members that share similar features. The closest homologue to NtRSG, which was named StRSG1, was amplified and sequenced. qRT-PCR data indicate that StRSG1 is mainly expressed in petioles, stems, lateral buds, and roots. In addition, GA treatment affected StRSG1 expression. StCDPK3 transcripts were detected in leaves, petioles, stolons, roots, and dormant tubers, and transcript levels were modified in response to GA. The recombinant StRSG1-GST protein was produced and tested as a substrate for StCDPK3 and StCDPK1. 6xHisStCDPK3 was able to phosphorylate the potato StRSG1 in a Ca2+-dependent way, while 6xHisStCDPK1 could not. StCDPK3 also interacts and phosphorylates the transcription factor StABF1 (ABRE binding factor 1) involved in ABA signaling, as shown by EMSA and phosphorylation assays. StABF1 transcripts were mainly detected in roots, stems, and stolons. Our data suggest that StCDPK3 could be involved in the cross-talk between ABA and GA signaling at the onset of tuber development.

Highlights

Plant signaling involves the interaction of several components and second messengers such as calcium (Ca2+)
We show that 6xHisStCDPK3 phosphorylates the potato basic region/leucine zipper (bZIP) transcription factors (TFs) StRSG1 and StABF1 in a Ca2+-dependent manner suggesting that StCDPK3 could be an important point of cross-talk between the abscisic acid (ABA) and GA signaling pathways mediated by both TFs
In order to identify the RSG TF family in potato, the tobacco Nicotiana tabacum Repression of Shoot Growth (NtRSG) coding sequence was blasted against the Solanum phureja database

Summary

Introduction

Plant signaling involves the interaction of several components and second messengers such as calcium (Ca2+). CDPKs exhibit overlapping and distinct expression patterns, subcellular localizations, substrate specificities, and Ca2+ sensitivities These kinases regulate the activity, localization, and lifetime of enzymes, channels, and transcription factors (TFs) by phosphorylating specific serine and threonine residues on their target sequences [2,3]. The 14-3-3 proteins emerged as phosphorylation-dependent regulators of hormone and light signaling in plants [22,23,24]. CDPKs have been implicated in abscisic acid (ABA) signaling, and overexpression of CDPKs has been shown to activate ABA-regulated promoters [26]. We show that 6xHisStCDPK3 phosphorylates the potato bZIP TFs StRSG1 and StABF1 in a Ca2+-dependent manner suggesting that StCDPK3 could be an important point of cross-talk between the ABA and GA signaling pathways mediated by both TFs

Results

Discussion

Materials and Methods