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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

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

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

Trenner, J.; Poeschl, Y.; Grau, J.; Gogol-Döring, A.; Quint, M.; Delker, C.; Auxin-induced expression divergence between Arabidopsis species may originate within the TIR1/AFB–AUX/IAA–ARF module J. Exp. Bot. 68, 539-552, (2017) DOI: 10.1093/jxb/erw457

Auxin is an essential regulator of plant growth and development, and auxin signaling components are conserved among land plants. Yet, a remarkable degree of natural variation in physiological and transcriptional auxin responses has been described among Arabidopsis thaliana accessions. As intraspecies comparisons offer only limited genetic variation, we here inspect the variation of auxin responses between A. thaliana and A. lyrata. This approach allowed the identification of conserved auxin response genes including novel genes with potential relevance for auxin biology. Furthermore, promoter divergences were analyzed for putative sources of variation. De novo motif discovery identified novel and variants of known elements with potential relevance for auxin responses, emphasizing the complex, and yet elusive, code of element combinations accounting for the diversity in transcriptional auxin responses. Furthermore, network analysis revealed correlations of interspecies differences in the expression of AUX/IAA gene clusters and classic auxin-related genes. We conclude that variation in general transcriptional and physiological auxin responses may originate substantially from functional or transcriptional variations in the TIR1/AFB, AUX/IAA, and ARF signaling network. In that respect, AUX/IAA gene expression divergence potentially reflects differences in the manner in which different species transduce identical auxin signals into gene expression responses.
Publikation

Ibañez, C.; Poeschl, Y.; Peterson, T.; Bellstädt, J.; Denk, K.; Gogol-Döring, A.; Quint, M.; Delker, C.; Ambient temperature and genotype differentially affect developmental and phenotypic plasticity in Arabidopsis thaliana BMC Plant Biol. 17, 114, (2017) DOI: 10.1186/s12870-017-1068-5

BackgroundGlobal increase in ambient temperatures constitute a significant challenge to wild and cultivated plant species. Forward genetic analyses of individual temperature-responsive traits have resulted in the identification of several signaling and response components. However, a comprehensive knowledge about temperature sensitivity of different developmental stages and the contribution of natural variation is still scarce and fragmented at best.ResultsHere, we systematically analyze thermomorphogenesis throughout a complete life cycle in ten natural Arabidopsis thaliana accessions grown under long day conditions in four different temperatures ranging from 16 to 28 °C. We used Q10, GxE, phenotypic divergence and correlation analyses to assess temperature sensitivity and genotype effects of more than 30 morphometric and developmental traits representing five phenotype classes. We found that genotype and temperature differentially affected plant growth and development with variing strengths. Furthermore, overall correlations among phenotypic temperature responses was relatively low which seems to be caused by differential capacities for temperature adaptations of individual accessions.ConclusionGenotype-specific temperature responses may be attractive targets for future forward genetic approaches and accession-specific thermomorphogenesis maps may aid the assessment of functional relevance of known and novel regulatory components.
Preprints

Trenner, J.; Poeschl, Y.; Grau, J.; Gogol-Döring, A.; Quint, M.; Delker, C.; Auxin-induced expression divergence between Arabidopsis species likely originates within the TIR1/AFB-AUX/IAA-ARF module bioRxiv (2016) DOI: 10.1101/038422

Auxin is an essential regulator of plant growth and development and auxin signaling components are conserved among land plants. Yet, a remarkable degree of natural variation in physiological and transcriptional auxin responses has been described among Arabidopsis thaliana accessions. As intra-species comparisons offer only limited genetic variation, we here inspect the variation of auxin responses between A. thaliana and A. lyrata. This approach allowed the identification of conserved auxin response genes including novel genes with potential relevance for auxin biology. Furthermore, promoter divergences were analyzed for putative sources of variation. De novo motif discovery identified novel and variants of known elements with potential relevance for auxin responses, emphasizing the complex, and yet elusive, code of element combinations accounting for the diversity in transcriptional auxin responses. Furthermore, network analysis revealed correlations of inter-species differences in the expression of AUX/IAA gene clusters and classic auxin-related genes. We conclude that variation in general transcriptional and physiological auxin responses may originate substantially from functional or transcriptional variations in the TIR1/AFB, AUX/IAA, and ARF signaling network. In that respect, AUX/IAA gene expression divergence potentially reflects differences in the manner in which different species transduce identical auxin signals into gene expression responses.
Preprints

Ibañez, C.; Poeschl, Y.; Peterson, T.; Bellstädt, J.; Denk, K.; Gogol-Döring, A.; Quint, M.; Delker, C.; Ambient temperature and genotype differentially affect developmental and phenotypic plasticity in Arabidopsis thaliana bioRxiv (2015) DOI: 10.1101/017285

Background Global increase in ambient temperatures constitute a significant challenge to wild and cultivated plant species. Forward genetic analyses of individual temperature-responsive traits have resulted in the identification of several signaling and response components. However, a comprehensive knowledge about temperature sensitivity of different developmental stages and the contribution of natural variation is still scarce and fragmented at best.Results Here, we systematically analyze thermomorphogenesis throughout a complete life cycle in ten natural Arabidopsis thaliana accessions grown in four different temperatures ranging from 16 to 28 °C. We used Q10, GxE, phenotypic divergence and correlation analyses to assess temperature sensitivity and genotype effects of more than 30 morphometric and developmental traits representing five phenotype classes. We found that genotype and temperature differentially affected plant growth and development with variing strengths. Furthermore, overall correlations among phenotypic temperature responses was relatively low which seems to be caused by differential capacities for temperature adaptations of individual accessions.Conclusion Genotype-specific temperature responses may be attractive targets for future forward genetic approaches and accession-specific thermomorphogenesis maps may aid the assessment of functional relevance of known and novel regulatory components.
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.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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