Redox Modulation of Plant Developmental Regulators from the Class I TCP Transcription Factor Family    

Abstract

TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR1 (TCP) transcription factors participate in plant developmental processes associated with cell proliferation and growth. Most members of class I, one of the two classes that compose the family, have a conserved cysteine at position 20 (Cys-20) of the TCP DNA-binding and dimerization domain. We show that Arabidopsis (Arabidopsis thaliana) class I proteins with Cys-20 are sensitive to redox conditions, since their DNAbinding activity is inhibited after incubation with the oxidants diamide, oxidized glutathione, or hydrogen peroxide or with nitric oxide-producing agents. Inhibition can be reversed by treatment with the reductants dithiothreitol or reduced glutathione or by incubation with the thioredoxin/thioredoxin reductase system. Mutation of Cys-20 in the class I protein TCP15 abolished its redox sensitivity. Under oxidizing conditions, covalently linked dimers were formed, suggesting that inactivation is associated with the formation of intermolecular disulfide bonds. Inhibition of class I TCP protein activity was also observed in vivo, in yeast (Saccharomyces cerevisiae) cells expressing TCP proteins and in plants after treatment with redox agents. This inhibition was correlated with modifications in the expression of the downstream CUC1 gene in plants. Modeling studies indicated that Cys-20 is located at the dimer interface near the DNA-binding surface. This places this residue in the correct orientation for intermolecular disulfide bond formation and explains the sensitivity of DNA binding to the oxidation of Cys-20. The redox properties of Cys-20 and the observed effects of cellular redox agents both in vitro and in vivo suggest that class I TCP protein action is under redox control in plants.TCP transcription factors constitute a family of plant developmental regulators (Martín-Trillo and Cubas, 2010;Uberti Manassero et al., 2013). The name of the family stands for the three first characterized members: TEOSINTE BRANCHED1, CYCLOIDEA, and PRO-LIFERATING CELL FACTOR1 (Cubas et al., 1999). These factors bind DNA through the TCP domain, a conserved region of approximately 60 amino acids that also participates in protein dimerization. The TCP domain contains a basic N-terminal region involved in DNA recognition, followed by a helix-loop-helix (HLH) motif similar to the one present in basic helix-loop-helix (bHLH) transcription factors (Aggarwal et al., 2010;Martín-Trillo and Cubas, 2010). Target sites recognized by TCP proteins are different from those bound by bHLH proteins (Kosugi and Ohashi, 2002;Viola et al., 2012). This is probably due to the fact that the respective basic regions differ markedly in composition and structure. Based on sequence homology, two main classes of TCP domains can be described. Class II proteins affect leaf and petal development and also influence shoot branching, among other processes

TheArabidopsis thalianaTCP transcription factors: A broadening horizon beyond development

“…Single residues in the TCP domain not only operate as determinants of DNA-binding specificities for both classes of TCP factors but also confer redox modulation of DNA-binding activity for class I TCP proteins. 18,112,119 The conserved Cys-20 localized in the TCP domain of class I TCP proteins functions as a redox switch to trigger transcriptional reprogramming in response to cellular redox changes, thus adding another layer of complexity to the signaling pathways related to class I TCP factors. Further identification of TCP target genes, which are involved in redox metabolism or whose expression is affected by redox conditions, will favor the dissection of the molecular link between redox regulation, developmental events and defense signaling.…”

“…109 EMSA experiments show that Arabidopsis class I TCP factors with Cys-20 are sensitive to redox conditions, as their DNA-binding activity is inhibited by different types of oxidants examined. 119 Nevertheless, this inhibition can be reversed by treatment with reductants or by incubation with a TRX/TRX reductase system, 119 suggesting that a reduced state is required for class I TCP proteins to interact with their cognate DNA sequences. Mutagenesis of Cys-20 in class I TCP proteins renders their DNA-binding activity insensitive to changes in redox status.…”

“…Mutagenesis of Cys-20 in class I TCP proteins renders their DNA-binding activity insensitive to changes in redox status. 119 Under oxidative conditions, class I TCP proteins harboring Cys-20 are found to exist as covalently linked homodimers, 119 hinting at the formation of intermolecular disulfide bridges in vitro. Furthermore, the DNA-binding activity of class I TCP proteins with Cys-20 is also observed to be inhibited in vivo after pretreatment with redox agents.…”

“…Furthermore, the DNA-binding activity of class I TCP proteins with Cys-20 is also observed to be inhibited in vivo after pretreatment with redox agents. 119 Homology modeling using eukaryotic bHLH proteins as a template reveals that Cys-20 resides at the dimer interface near the DNA-binding surface. 119 This spatial orientation allows for the formation of intermolecular disulfide bonds and accounts for the sensitivity of DNA binding to the oxidation of Cys-20.…”

“…119 Homology modeling using eukaryotic bHLH proteins as a template reveals that Cys-20 resides at the dimer interface near the DNA-binding surface. 119 This spatial orientation allows for the formation of intermolecular disulfide bonds and accounts for the sensitivity of DNA binding to the oxidation of Cys-20. Taken together, these observations suggest that the conserved Cys-20 acts as a redox switch, enabling a subset of TCP proteins with such a residue to participate in redox-dependent developmental processes.…”