Reactive oxygen species‐mediatedBIN2 activity revealed by single‐molecule analysis

Abstract

Summary Much evidence has shown that reactive oxygen species (ROS) regulate several plant hormone signaling cascades, but little is known about the real‐time kinetics and the underlying molecular mechanisms of the target proteins in the brassinosteroid (BR) signaling pathway. In this study, we used single‐molecule techniques to investigate the true signaling timescales of the major BR signaling components BRI1‐EMS‐SUPPRESSOR 1 (BES1) and BRASSINOSTEROID INSENSITIVE 2 (BIN2) of Arabidopsis thaliana. The rate constants of BIN2 associating with ATP and phosphorylating BES1 were determined to be 0.7 ± 0.4 mM−1 s−1 and 2.3 ± 1.4 s−1, respectively. Interestingly, we found that the interaction of BIN2 and BES1 was oxygen‐dependent, and oxygen can directly modify BIN2. The activity of BIN2 was switched on via modification of specific cysteine (Cys) residues, including C59, C95, C99 and C162. The mutation of these Cys residues inhibited the BR signaling outputs. These findings demonstrate the power of using single‐molecule techniques to study the dynamic interactions of signaling components, which is difficult to be discovered by conventional physiological and biochemical methods.

Brassinosteroids: Multidimensional Regulators of Plant Growth, Development, and Stress Responses

“…Several posttranslational modifications also affect the activity of BIN2. HISTONE DEACETYLASE6 (HDA6) deacetylates BIN2, thereby repressing its kinase activity (Hao et al, 2016), whereas BIN2 is activated via oxidation at specific Cys residues (Song et al, 2018b). Dephosphorylation by RELATED TO ABSCISIC ACID INSENSITIVE1 (ABI1) and ABI2 (which negatively regulate abscisic acid [ABA] signaling) activates BIN2 in the presence of ABA (Wang et al, 2018a;Jiang et al, 2019).…”

Identification of Sulfenylated Cysteines in Arabidopsis thaliana Proteins Using a Disulfide-Linked Peptide Reporter

“…Protein S-sulfenylation can directly regulate protein functions. For instance, in Arabidopsis, H 2 O 2 -dependent S-sulfenylation of BRASSINAZOLE-RESISTANT 1 (BZR1) promotes its interaction with transcriptional factors (TFs) and drives gene expression (Tian et al, 2018), whereas, S-sulfenylation of BRASSINOSTEROID INSENSITIVE 2 (BIN2) enhances the formation of phosphorylated BRI1-EMS-SUPPRESSOR 1 (BES1), which cannot transport and bind TFs in nuclei, thereby decreasing brassinosteroid (BR) signaling outputs (Song et al, 2019). Another example is the impact on the levels of the auxin biosynthetic precursor tryptophan by S-sulfenylation of a tryptophan synthetase β subunit 1 (Yuan et al, 2017).…”

Molecular Mechanisms of Brassinosteroid-Mediated Responses to Changing Environments in Arabidopsis

“…At the receptor level, the function of BRI1 can be post-translationally regulated by PUB12/13-directed ubiquitination, PP2A-mediated dephosphorylation, BKI1-mediated kinase inhibition, and BIK1-and BIR3-mediated competition for the coreceptor BAK1 [69][70][71][72][73]. At the BIN2 level, it has been reported that BIN2 is regulated by OCTOPUSor POLAR-mediated membrane sequestration, HDA6-mediated deacetylation, KIB1-mediated ubiquitination, TTL-enhanced interaction with BSU1, ABI1/2-mediated dephosphorylation, and ROS (reactive oxygen species)-mediated oxidation [74][75][76][77][78][79][80]. At the transcription level, it has been reported that PP2A phosphatases can promote BR signaling by dephosphorylating BES1 and BZR1, whereas 14-3-3 and BRZ-SENSITIVE-SHORT HYPOCOTYL1 (BSS1) negatively regulate BR signaling by inhibiting the translocation of BES1 and BZR1 from the cytosol to the nucleus [81][82][83].…”