Redox Modulation of Plant Developmental Regulators from the Class I TCP Transcription Factor Family 1[W][OA] Ivana L. Viola, Leandro N. Güttlein, and Daniel H. Gonzalez* Instituto de Agrobiotecnología del Litoral, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina 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 DNA- binding 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 do- main 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 (Luo et al., 1996; Doebley et al., 1997; Nath et al., 2003; Palatnik et al., 2003; Aguilar-Martínez et al., 2007; Koyama et al., 2007; Nag et al., 2009). The role of class I proteins is less clear, but several studies indicated that they participate in developmental processes probably asso- ciated with the regulation of cell growth and prolifera- tion. TCP20 has been linked to the regulation of the expression of the cyclin CYCB1;1 and ribosomal protein genes, thus establishing a link between cell growth and cell cycle control (Li et al., 2005). Accordingly, alteration of the function of TCP20 causes dramatic changes in plant development (Hervé et al., 2009). TCP15 also modulates the expression of cell cycle genes (Li et al., 2012) and is involved in leaf and inflorescence devel- opment and the regulation of auxin and cytokinin ho- meostasis (Kieffer et al., 2011; Steiner et al., 2012; Uberti Manassero et al., 2012). TCP16 and TCP11 participate in pollen development (Takeda et al., 2006; Viola et al., 2011). Other roles proposed for class I proteins include the coordination of mitochondrial biogenesis (Welchen and Gonzalez, 2006; Gonzalez et al., 2007; Giraud et al., 2010), the regulation of embryonic growth potential during germination (Tatematsu et al., 2008), and the regulation of the circadian clock. Particularly, TCP21 is known to interact with TOC1 and to bind to the CCA1 promoter, thus establishing a link between these core components of the clock (Pruneda-Paz et al., 2009). 1 This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica, Argentina, and the Universidad Nacional del Litoral. * Corresponding author; e-mail dhgonza@fbcb.unl.edu.ar. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Daniel H. Gonzalez (dhgonza@fbcb.unl.edu.ar). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscrip- tion. www.plantphysiol.org/cgi/doi/10.1104/pp.113.216416 1434 Plant Physiology Ò , July 2013, Vol. 162, pp. 1434–1447, www.plantphysiol.org Ó 2013 American Society of Plant Biologists. All Rights Reserved. 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