Characterization of thiol‐based redox modifications of Brassica napusSNF1‐related protein kinase 2.6‐2C

Abstract

Sucrose nonfermenting 1‐related protein kinase 2.6 (SnRK2.6), also known as Open Stomata 1 (OST1) in Arabidopsis thaliana, plays a pivotal role in abscisic acid (ABA)‐mediated stomatal closure. Four SnRK2.6 paralogs were identified in the Brassica napus genome in our previous work. Here we studied one of the paralogs, BnSnRK2.6‐2C, which was transcriptionally induced by ABA in guard cells. Recombinant BnSnRK2.6‐2C exhibited autophosphorylation activity and its phosphorylation sites were mapped. The autophosphorylation activity was inhibited by S‐nitrosoglutathione (GSNO) and by oxidized glutathione (GSSG), and the inhibition was reversed by reductants. Using monobromobimane (mBBr) labeling, we demonstrated a dose‐dependent modification of BnSnRK2.6‐2C by GSNO. Furthermore, mass spectrometry analysis revealed previously uncharacterized thiol‐based modifications including glutathionylation and sulfonic acid formation. Of the six cysteine residues in BnSnRK2.6‐2C, C159 was found to have different types of thiol modifications, suggesting its high redox sensitivity and versatility. In addition, mBBr labeling on tyrosine residues was identified. Collectively, these data provide detailed biochemical characterization of redox‐induced modifications and changes of the BnSnRK2.6‐2C activity.

Plastidial and cytosolic thiol reductases participate in the control of stomatal functioning

“…This system is likely to fulfil several functions through redox interaction with known actors of the signalling network regulating stomatal movements. Thus, cytosolic TRs might control the redox status of OST1/SnRK2.6, BAK1, SnRK2.4 and CPK21, which are likely prone to thiol‐based modifications as they display cysteine thiol redox switches modifying their activity in vitro, or interact in vitro with TRs (Bender et al, 2015; Ma et al, 2018; Wang et al, 2015; Zhu et al, 2017). This hypothesis is further supported by the identification of ~80 potential TRX targets in a fraction enriched in guard cells (Zhang et al, 2016), and of numerous proteins harbouring redox‐sensitive Cys in B. napus guard cells (Zhu et al, 2014).…”

Plasticity in Plastid Redox Networks: Evolution of Glutathione-Dependent Redox Cascades and Glutathionylation Sites

“…Proteins found to be glutathionylated in different proteomic and in vitro studies [14,61,[72][73][74][75][76][77][78][79][80][81][81][82][83][84][85][86][87][88][89][90][91][92][93][94] were retrieved from the literature and used to assemble a list (Additional File 9: Table S2, sheet1). NCBI reference sequences (www.ncbi.nlm.nih.gov) and TAIR (www.arabidopsis.org) accession numbers were used to unambiguously identify proteins.…”

“…biotinylated GSH (biotinylated GSH ethyl ester (BioGEE) [72], biotinylated GSSG (BioGSSG) [73,74], anti-GSH antibodies [75] or radiolabelling of the glutathione pool using 35 S-cysteine [76]. In order to obtain an exhaustive and complete list of S-glutathionylated proteins, we also considered research studies carried out on puri ed proteins in vitro [72,[77][78][79][80][81][81][82][83][84][85][86][87][88][89][90][91][92][93][94] (see Additional File 9: Table S2). Combining all these studies, we compiled a list of 364 proteins known to undergo S-glutathionylation in green eukaryotes (Additional File 9: Table S2, Fig.…”

Proteomics data of SNF1-related protein kinase 2.4 interacting proteins revealed by immunoprecipitation-mass spectrometry

“…Both wild type (WT) Arabidopsis thaliana col-0 (obtained from Arabidopsis Biological Resource Center) and transgenic plants overexpressing a Brassica napus SnRK2.4-1C were used in this study. The transgenic plants were generated by transferring a FLAG-tagged SnRK2.4-1C into the WT A. thaliana as described previously [6 , 7] . Seeds were soaked in 50% bleach for disinfection and were then sown on half-strength Murashige and Skoog medium supplemented with 1 × MS vitamins (Caisson, UT).…”