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Publikation

Terrile, M. C.; Tebez, N. M.; Colman, S. L.; Mateos, J. L.; Morato-López, E.; Sánchez-López, N.; Izquierdo-Álvarez, A.; Marina, A.; Calderón Villalobos, L. I. A.; Estelle, M.; Martínez-Ruiz, A.; Fiol, D. F.; Casalongué, C. A.; Iglesias, M. J.; S-Nitrosation of E3 ubiquitin ligase complex components regulates hormonal signalings in Arabidopsis Front. Plant Sci. 12, 794582, (2022) DOI: 10.3389/fpls.2021.794582

E3 ubiquitin ligases mediate the last step of the ubiquitination pathway in the ubiquitin-proteasome system (UPS). By targeting transcriptional regulators for their turnover, E3s play a crucial role in every aspect of plant biology. In plants, SKP1/CULLIN1/F-BOX PROTEIN (SCF)-type E3 ubiquitin ligases are essential for the perception and signaling of several key hormones including auxins and jasmonates (JAs). F-box proteins, TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and CORONATINE INSENSITIVE 1 (COI1), bind directly transcriptional repressors AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) and JASMONATE ZIM-DOMAIN (JAZ) in auxin- and JAs-depending manner, respectively, which permits the perception of the hormones and transcriptional activation of signaling pathways. Redox modification of proteins mainly by S-nitrosation of cysteines (Cys) residues via nitric oxide (NO) has emerged as a valued regulatory mechanism in physiological processes requiring its rapid and versatile integration. Previously, we demonstrated that TIR1 and Arabidopsis thaliana SKP1 (ASK1) are targets of S-nitrosation, and these NO-dependent posttranslational modifications enhance protein-protein interactions and positively regulate SCFTIR1 complex assembly and expression of auxin response genes. In this work, we confirmed S-nitrosation of Cys140 in TIR1, which was associated in planta to auxin-dependent developmental and stress-associated responses. In addition, we provide evidence on the modulation of the SCFCOI1 complex by different S-nitrosation events. We demonstrated that S-nitrosation of ASK1 Cys118 enhanced ASK1-COI1 protein-protein interaction. Overexpression of non-nitrosable ask1 mutant protein impaired the activation of JA-responsive genes mediated by SCFCOI1 illustrating the functional relevance of this redox-mediated regulation in planta. In silico analysis positions COI1 as a promising S-nitrosation target, and demonstrated that plants treated with methyl JA (MeJA) or S-nitrosocysteine (NO-Cys, S-nitrosation agent) develop shared responses at a genome-wide level. The regulation of SCF components involved in hormonal perception by S-nitrosation may represent a key strategy to determine the precise time and site-dependent activation of each hormonal signaling pathway and highlights NO as a pivotal molecular player in these scenarios.
Publikation

Bagchi, R.; Melnyk, C. W.; Christ, G.; Winkler, M.; Kirchsteiner, K.; Salehin, M.; Mergner, J.; Niemeyer, M.; Schwechheimer, C.; Calderón Villalobos, L. I. A.; Estelle, M.; The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases EMBO J. 37, 255-268, (2018) DOI: 10.15252/embj.201797159

The cullin‐RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin‐conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.
Publikation

Iglesias, M. J.; Terrile, M. C.; Correa-Aragunde, N.; Colman, S. L.; Izquierdo-Álvarez, A.; Fiol, D. F.; París, R.; Sánchez-López, N.; Marina, A.; Calderón Villalobos, L. I. A.; Estelle, M.; Lamattina, L.; Martínez-Ruiz, A.; Casalongué, C. A.; Regulation of SCFTIR1/AFBs E3 ligase assembly by S-nitrosylation of Arabidopsis SKP1-like1 impacts on auxin signaling Redox Biol. 18, 200-210, (2018) DOI: 10.1016/j.redox.2018.07.003

The F-box proteins (FBPs) TIR1/AFBs are the substrate recognition subunits of SKP1–cullin–F-box (SCF) ubiquitin ligase complexes and together with Aux/IAAs form the auxin co-receptor. Although tremendous knowledge on auxin perception and signaling has been gained in the last years, SCFTIR1/AFBs complex assembly and stabilization are emerging as new layers of regulation. Here, we investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is involved in SCFTIR1/AFBs assembly. We demonstrate that ASK1 is S-nitrosylated and S-glutathionylated in cysteine (Cys) 37 and Cys118 residues in vitro. Both, in vitro and in vivo protein-protein interaction assays show that NO enhances ASK1 binding to CUL1 and TIR1/AFB2, required for SCFTIR1/AFB2 assembly. In addition, we demonstrate that Cys37 and Cys118 are essential residues for proper activation of auxin signaling pathway in planta. Phylogenetic analysis revealed that Cys37 residue is only conserved in SKP proteins in Angiosperms, suggesting that S-nitrosylation on Cys37 could represent an evolutionary adaption for SKP1 function in flowering plants. Collectively, these findings indicate that multiple events of redox modifications might be part of a fine-tuning regulation of SCFTIR1/AFBs for proper auxin signal transduction.
Publikation

Calderón Villalobos, L. I. A.; Lee, S.; De Oliveira, C.; Ivetac, A.; Brandt, W.; Armitage, L.; Sheard, L. B.; Tan, X.; Parry, G.; Mao, H.; Zheng, N.; Napier, R.; Kepinski, S.; Estelle, M.; A combinatorial TIR1/AFB–Aux/IAA co-receptor system for differential sensing of auxin Nat. Chem. Biol. 8, 477-485, (2012) DOI: 10.1038/nchembio.926

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding the F-box protein transport inhibitor response 1 (TIR1) and promotes the degradation of the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressors. Here we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein. Heterologous experiments in yeast and quantitative IAA binding assays using purified proteins showed that different combinations of TIR1 and Aux/IAA proteins form co-receptor complexes with a wide range of auxin-binding affinities. Auxin affinity seems to be largely determined by the Aux/IAA. As there are 6 TIR1/AUXIN SIGNALING F-BOX proteins (AFBs) and 29 Aux/IAA proteins in Arabidopsis thaliana, combinatorial interactions may result in many co-receptors with distinct auxin-sensing properties. We also demonstrate that the AFB5–Aux/IAA co-receptor selectively binds the auxinic herbicide picloram. This co-receptor system broadens the effective concentration range of the hormone and may contribute to the complexity of auxin response.
Publikation

Terrile, M. C.; París, R.; Calderón-Villalobos, L. I. A.; Iglesias, M. J.; Lamattina, L.; Estelle, M.; Casalongué, C. A.; Nitric oxide influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor Plant J. 70, 492-500, (2011) DOI: 10.1111/j.1365-313X.2011.04885.x

Previous studies have demonstrated that auxin (indole‐3‐acetic acid) and nitric oxide (NO) are plant growth regulators that coordinate several plant physiological responses determining root architecture. Nonetheless, the way in which these factors interact to affect these growth and developmental processes is not well understood. The Arabidopsis thaliana F‐box proteins TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F‐BOX (TIR1/AFB) are auxin receptors that mediate degradation of AUXIN/INDOLE‐3‐ACETIC ACID (Aux/IAA) repressors to induce auxin‐regulated responses. A broad spectrum of NO‐mediated protein modifications are known in eukaryotic cells. Here, we provide evidence that NO donors increase auxin‐dependent gene expression while NO depletion blocks Aux/IAA protein degradation. NO also enhances TIR1‐Aux/IAA interaction as evidenced by pull‐down and two‐hybrid assays. In addition, we provide evidence for NO‐mediated modulation of auxin signaling through S‐nitrosylation of the TIR1 auxin receptor. S‐nitrosylation of cysteine is a redox‐based post‐translational modification that contributes to the complexity of the cellular proteome. We show that TIR1 C140 is a critical residue for TIR1–Aux/IAA interaction and TIR1 function. These results suggest that TIR1 S‐nitrosylation enhances TIR1–Aux/IAA interaction, facilitating Aux/IAA degradation and subsequently promoting activation of gene expression. Our findings underline the importance of NO in phytohormone signaling pathways.
Publikation

Calderon-Villalobos, L. I.; Tan, X.; Zheng, N.; Estelle, M.; Auxin Perception—Structural Insights Cold Spring Harb. Perspect. Biol. 2, a005546, (2010) DOI: 10.1101/cshperspect.a005546

The identity of the auxin receptor(s) and the mechanism of auxin perception has been a subject of intense interest since the discovery of auxin almost a century ago. The development of genetic approaches to the study of plant hormone signaling led to the discovery that auxin acts by promoting degradation of transcriptional repressors called Aux/IAA proteins. This process requires a ubiquitin protein ligase (E3) called SCFTIR1 and related SCF complexes. Surprisingly, auxin works by directly binding to TIR1, the F-box protein subunit of this SCF. Structural studies demonstrate that auxin acts like a “molecular glue,” to stabilize the interaction between TIR1 and the Aux/IAA substrate. These exciting results solve an old problem in plant biology and reveal new mechanisms for E3 regulation and hormone perception.
Publikation

Santner, A.; Calderon-Villalobos, L. I. A.; Estelle, M.; Plant hormones are versatile chemical regulators of plant growth Nat. Chem. Biol. 5, 301-307, (2009) DOI: 10.1038/nchembio.165

The plant hormones are a structurally unrelated collection of small molecules derived from various essential metabolic pathways. These compounds are important regulators of plant growth and mediate responses to both biotic and abiotic stresses. During the last ten years there have been many exciting advances in our understanding of plant hormone biology, including new discoveries in the areas of hormone biosynthesis, transport, perception and response. Receptors for many of the major hormones have now been identified, providing new opportunities to study the chemical specificity of hormone signaling. These studies also reveal a surprisingly important role for the ubiquitin-proteasome pathway in hormone signaling. In addition, recent work confirms that hormone signaling interacts at multiple levels during plant growth and development. In the future, a major challenge will be to understand how the information conveyed by these simple compounds is integrated during plant growth.
Publikation

Parry, G.; Calderon-Villalobos, L. I.; Prigge, M.; Peret, B.; Dharmasiri, S.; Itoh, H.; Lechner, E.; Gray, W. M.; Bennett, M.; Estelle, M.; Complex regulation of the TIR1/AFB family of auxin receptors Proc. Natl. Acad. Sci. U.S.A. 106, 22540-22545, (2009) DOI: 10.1073/pnas.0911967106

Auxin regulates most aspects of plant growth and development. The hormone is perceived by the TIR1/AFB family of F-box proteins acting in concert with the Aux/IAA transcriptional repressors. Arabidopsis plants that lack members of the TIR1/AFB family are auxin resistant and display a variety of growth defects. However, little is known about the functional differences between individual members of the family. Phylogenetic studies reveal that the TIR1/AFB proteins are conserved across land plant lineages and fall into four clades. Three of these subgroups emerged before separation of angiosperms and gymnosperms whereas the last emerged before the monocot-eudicot split. This evolutionary history suggests that the members of each clade have distinct functions. To explore this possibility in Arabidopsis, we have analyzed a range of mutant genotypes, generated promoter swap transgenic lines, and performed in vitro binding assays between individual TIR1/AFB and Aux/IAA proteins. Our results indicate that the TIR1/AFB proteins have distinct biochemical activities and that TIR1 and AFB2 are the dominant auxin receptors in the seedling root. Further, we demonstrate that TIR1, AFB2, and AFB3, but not AFB1 exhibit significant posttranscriptional regulation. The microRNA miR393 is expressed in a pattern complementary to that of the auxin receptors and appears to regulate TIR1/AFB expression. However our data suggest that this regulation is complex. Our results suggest that differences between members of the auxin receptor family may contribute to the complexity of auxin response.
Publikation

Tan, X.; Calderon-Villalobos, L. I. A.; Sharon, M.; Zheng, C.; Robinson, C. V.; Estelle, M.; Zheng, N.; Mechanism of auxin perception by the TIR1 ubiquitin ligase Nature 446, 640-645, (2007) DOI: 10.1038/nature05731

Auxin is a pivotal plant hormone that controls many aspects of plant growth and development. Perceived by a small family of F-box proteins including transport inhibitor response 1 (TIR1), auxin regulates gene expression by promoting SCF ubiquitin-ligase-catalysed degradation of the Aux/IAA transcription repressors, but how the TIR1 F-box protein senses and becomes activated by auxin remains unclear. Here we present the crystal structures of the Arabidopsis TIR1–ASK1 complex, free and in complexes with three different auxin compounds and an Aux/IAA substrate peptide. These structures show that the leucine-rich repeat domain of TIR1 contains an unexpected inositol hexakisphosphate co-factor and recognizes auxin and the Aux/IAA polypeptide substrate through a single surface pocket. Anchored to the base of the TIR1 pocket, auxin binds to a partially promiscuous site, which can also accommodate various auxin analogues. Docked on top of auxin, the Aux/IAA substrate peptide occupies the rest of the TIR1 pocket and completely encloses the hormone-binding site. By filling in a hydrophobic cavity at the protein interface, auxin enhances the TIR1–substrate interactions by acting as a ‘molecular glue’. Our results establish the first structural model of a plant hormone receptor.
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