Title: E3 Ubiquitin Ligases: Key Regulators of Hormone Signaling in Plants
Abstract: Ubiquitin-mediated control of protein stability is central to most aspects of plant hormone signaling. Attachment of ubiquitin to target proteins occurs via an enzymatic cascade with the final step being catalyzed by a family of enzymes known as E3 ubiquitin ligases, which have been classified based on their protein domains and structures. Although E3 ubiquitin ligases are conserved among eukaryotes, in plants they are well-known to fulfill unique roles as central regulators of phytohormone signaling, including hormone perception and regulation of hormone biosynthesis. This review will highlight up-to-date findings that have refined well-known E3 ligase-substrate interactions and defined novel E3 ligase substrates that mediate numerous hormone signaling pathways. Additionally, examples of how particular E3 ligases may mediate hormone crosstalk will be discussed as an emerging theme. Looking forward, promising experimental approaches and methods that will provide deeper mechanistic insight into the roles of E3 ubiquitin ligases in plants will be considered. Ubiquitin-mediated control of protein stability is central to most aspects of plant hormone signaling. Attachment of ubiquitin to target proteins occurs via an enzymatic cascade with the final step being catalyzed by a family of enzymes known as E3 ubiquitin ligases, which have been classified based on their protein domains and structures. Although E3 ubiquitin ligases are conserved among eukaryotes, in plants they are well-known to fulfill unique roles as central regulators of phytohormone signaling, including hormone perception and regulation of hormone biosynthesis. This review will highlight up-to-date findings that have refined well-known E3 ligase-substrate interactions and defined novel E3 ligase substrates that mediate numerous hormone signaling pathways. Additionally, examples of how particular E3 ligases may mediate hormone crosstalk will be discussed as an emerging theme. Looking forward, promising experimental approaches and methods that will provide deeper mechanistic insight into the roles of E3 ubiquitin ligases in plants will be considered. Ubiquitin E3 ligases are conserved among eukaryotes and fulfill a myriad of regulatory functions by facilitating the covalent attachment of ubiquitin to target proteins. Attachment typically occurs on lysine residues and can occur singly (monoubiquitination) or in chains (polyubiquitination). The types of attachments and degree of ubiquitination varies considerably among substrates and may be context dependent. This enzymatic reaction is carried out by a family of proteins called E3 ubiquitin ligases, which act at the end of a three-enzyme cascade to transfer ubiquitin from an E2 ubiquitin conjugating enzyme to a specific substrate(s). Ubiquitination of substrates is a tightly regulated process and can result in several functional outcomes, including protein degradation, changes in subcellular localization, and protein activation. In plants, numerous ubiquitin E3 ligases act as central regulators in phytohormone signaling pathways including auxin, brassinosteroid (BR), cytokinin (CK), ethylene, gibberellic acid (GA), jasmonate (JA), salicylic acid (SA), and strigolactone (SL) (for recent detailed reviews see (1.Kelley D.R. Estelle M. Ubiquitin-mediated control of plant hormone signaling.Plant Physiol. 2012; 160: 47-55Crossref PubMed Scopus (116) Google Scholar, 2.Guerra D.D. Callis J. Ubiquitin on the Move: The Ubiquitin Modification System Plays Diverse Roles in the Regulation of Endoplasmic Reticulum- and Plasma Membrane-Localized Proteins.Plant Physiol. 2012; 160: 56-64Crossref PubMed Scopus (49) Google Scholar, 3.Shabek N. Zheng N. Plant ubiquitin ligases as signaling hubs.Nat. Struct. Mol. Biol. 2014; 21: 293-296Crossref PubMed Scopus (43) Google Scholar, 4.Vierstra R.D. The Expanding Universe of Ubiquitin and Ubiquitin-Like Modifiers.PLANT Physiol. 2012; 160: 2-14Crossref PubMed Scopus (141) Google Scholar, 5.Yan S. Dong X. Perception of the plant immune signal salicylic acid.Curr. Opin. Plant Biol. 2014; 20: 64-68Crossref PubMed Scopus (157) Google Scholar)). This review will highlight up-to-date findings that have defined new E3 ligase-substrate interactions that mediate phytohormone signaling pathways, discuss examples of how some E3 ligases mediate hormone crosstalk, and touch on emerging approaches that will help us gain deeper mechanistic insight into these proteins. There are over 1500 E3 ubiquitin ligase proteins encoded by the Arabidopsis genome which can be subdivided into different families (6.Hua Z. Vierstra R.D. The Cullin-RING Ubiquitin-Protein Ligases.Annu. Rev. Plant Biol. 2011; 62: 299-334Crossref PubMed Scopus (350) Google Scholar). This includes the HECT (homologous to the E6AP carboxyl terminus) type, RING (really interesting new gene) 1The abbreviations used are: RING, really interesting new gene; CRL, Cullin-RING ligase; AFB, auxin F-box; AID, auxin-inducible degradation; NPR, nonexpression of pathogenesis-related genes. family, Kelch-type and U-box containing ubiquitin protein ligases. The Cullin-RING ligase (CRL) family can be further subdivided into five subfamilies based on subunit organization and conserved domains: (1) SKP1-Cullin-F-box (SCF) type, (2) broad complex/tramtrack/bric-a-brac (BTB) type, (3) DDB1-binding/WD-40 domain containing proteins (DWD) type, (4) VON-HIPPEL LINDAU (VHL) type, and (5) SUPRESSOR OF CYTOKINE SIGNALING (SOCS) type (6.Hua Z. Vierstra R.D. The Cullin-RING Ubiquitin-Protein Ligases.Annu. Rev. Plant Biol. 2011; 62: 299-334Crossref PubMed Scopus (350) Google Scholar). While CRLs have been called “molecular hubs” in plant hormone signaling pathways because of their central roles in hormone perception mechanisms and far-reaching cellular signaling effects, there are several examples of other types of E3 ubiquitin ligases playing roles in phytohormone signaling. Regardless of E3 ligase type, one common theme among these plant E3 proteins is that they interact with their substrates in a hormone-dependent manner (3.Shabek N. Zheng N. Plant ubiquitin ligases as signaling hubs.Nat. Struct. Mol. Biol. 2014; 21: 293-296Crossref PubMed Scopus (43) Google Scholar). This is especially interesting given that both E3 ubiquitin ligases and plant hormones are diverse chemical structures with complex evolutionary histories. The exact roles of E3 ligases in hormone perception and/or signaling have been well established for most known cases (Fig. 1). The first hormone discovered to utilize ubiquitin E3 ligases as receptor molecules was auxin, which is perceived by SCFTIR1 and related Auxin F-box (AFB) family members (7.Dharmasiri N. Dharmasiri S. Estelle M. The F-box protein TIR1 is an auxin receptor.Nature. 2005; 435: 441-445Crossref PubMed Scopus (1545) Google Scholar, 8.Kepinski S. Leyser O. The Arabidopsis F-box protein TIR1 is an auxin receptor.Nature. 2005; 435: 446-451Crossref PubMed Scopus (1288) Google Scholar). Perception of auxin by SCFTIR1/AFB triggers rapid degradation of the Aux/IAA family of transcriptional repressors (9.Santner A. Calderon-Villalobos L.I.A. Estelle M. Plant hormones are versatile chemical regulators of plant growth.Nat. Chem. Biol. 2009; 5: 301-307Crossref PubMed Scopus (521) Google Scholar, 10.Dharmasiri N. Dharmasiri S. Weijers D. Lechner E. Yamada M. Hobbie L. Ehrismann J.S. Jürgens G. Estelle M. Plant development is regulated by a family of auxin receptor F box proteins.Dev. 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Other types of E3 ligases have also been linked to hormone signaling, including BTB type regulating ethylene biosynthesis (26.Wang K.L.C. Yoshida H. Lurin C. Ecker J.R. Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein.Nature. 2004; 428: 945-950Crossref PubMed Scopus (312) Google Scholar, 27.Yoshida H. Nagata M. Saito K. Kevin W.L.C. Ecker J.R. Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases.BMC Plant Biol. 2005; 5Crossref PubMed Scopus (124) Google Scholar, 28.Christians M.J. Gingerich D.J. Hansen M. Binder B.M. Kieber J.J. Vierstra R.D. The BTB ubiquitin ligases ETO1, EOL1 and EOL2 act collectively to regulate ethylene biosynthesis in Arabidopsis by controlling type-2 ACC synthase levels.Plant J. 2009; 57: 332-345Crossref PubMed Scopus (131) Google Scholar) and CRL3-based E3 ligases modulating ABA signaling (Fig. 1B). 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Identification of a novel E3 ubiquitin ligase that is required for suppression of premature senescence in Arabidopsis.Plant J. 2009; 59: 39-51Crossref PubMed Scopus (107) Google Scholar, 36.Samuel M.A. Mudgil Y. Salt J.N. Delmas F. Ramachandran S. Chilelli A. Goring D.R. Interactions between the S-Domain Receptor Kinases and AtPUB-ARM E3 Ubiquitin Ligases Suggest a Conserved Signaling Pathway in Arabidopsis.Plant Physiol. 2008; 147: 2084-2095Crossref PubMed Scopus (131) Google Scholar, 37.Luo J. Shen G. Yan J. He C. Zhang H. AtCHIP functions as an E3 ubiquitin ligase of protein phosphatase 2A subunits and alters plant response to abscisic acid treatment.Plant J. 2006; 46: 649-657Crossref PubMed Scopus (107) Google Scholar) (Fig. 1D). Finally, an E3 ligase and autophagy receptor protein have recently described to regulate BR signaling during stress (38.Yang M. Li C. Cai Z. Hu Y. Nolan T. Yu F. Yin Y. Xie Q. Tang G. Wang X. SINAT E3 Ligases Control the Light-Mediated Stability of the Brassinosteroid-Activated Transcription Factor BES1 in Arabidopsis.Dev. Cell. 2017; 41: 47-58.e4Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 39.Nolan T.M. Brennan B. Yang M. Chen J. Zhang M. Li Z. Wang X. Bassham D.C. Walley J. Yin Y. Selective Autophagy of BES1 Mediated by DSK2 Balances Plant Growth and Survival.Dev. Cell. 2017; 41: 33-46.e7Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) (Fig. 1E). Overall, the number of E3 ligases linked to ABA signaling thus far is greater than for any other phytohormone, demonstrating the diversity and extent to which substrate ubiquitination can regulate ABA biosynthesis, signaling and downstream responses (1.Kelley D.R. Estelle M. Ubiquitin-mediated control of plant hormone signaling.Plant Physiol. 2012; 160: 47-55Crossref PubMed Scopus (116) Google Scholar). Detailed biochemical studies have provided new insights into E3-substrate complex assembly and composition. For instance, protein structure studies on TIR1-auxin-AUX/IAA and JA-COI1-JAZ (JASMONATE ZIM-DOMAIN) complexes have revealed that small molecule co-factors are directly involved in SCFTIR1 and SCFCOI1 complexes (inositol hexakisphosphate (InsP6) and inositol pentakisphosphate (InsP5) respectively) (14.Sheard L.B. Tan X. Mao H. Withers J. Ben-Nissan G. Hinds T.R. Kobayashi Y. Hsu F.-F. Sharon M. Browse J. He S.Y. Rizo J. Howe G.A. Zheng N. Jasmonate perception by inositol-phosphate-potentiated COI1–JAZ co-receptor.Nature. 2010; 468: 400-405Crossref PubMed Scopus (946) Google Scholar, 40.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. 2007; 446: 640-645Crossref PubMed Scopus (1112) Google Scholar). Additionally, several of the key E3 ligases involved in auxin, JA and SL perception have been proposed to function as “co-receptor” complexes, whereby high-affinity hormone binding is facilitated by both the E3 ubiquitin ligase and substrate. This has been observed for the TIR1-auxin-AUX/IAA complex, the COI1-JA-Ile-JAZ complex, the ABI1 PUB12/13/U-box E3 ligase complex and the MAX2/D3-SL-D14 complex (11.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. 2012; 8: 477-485Crossref PubMed Scopus (383) Google Scholar, 14.Sheard L.B. Tan X. Mao H. Withers J. Ben-Nissan G. Hinds T.R. Kobayashi Y. Hsu F.-F. Sharon M. Browse J. He S.Y. Rizo J. Howe G.A. Zheng N. 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Substrate recognition has been mapped to minimal amino acid sequence motifs for several E3 targets related to auxin and JA signaling through careful biochemical studies; such sequences are not identifiable based solely on primary amino acid sequence and thus are not amenable to bioinformatics-based approaches. For instance, all Aux/IAAs contain a short consensus recognition motif, or “degron,” which directly engages with auxin-loaded TIR1 (13.Dreher K.A. The Arabidopsis Aux/IAA protein family has diversified in degradation and auxin responsiveness.Plant Cell Online. 2006; 18: 699-714Crossref PubMed Scopus (229) Google Scholar, 40.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. 2007; 446: 640-645Crossref PubMed Scopus (1112) Google Scholar, 46.Moss B.L. Mao H. Guseman J.M. Hinds T.R. Hellmuth A. Kovenock M. Noorassa A. Lanctot A. Villalobos L.I. A.C. Zheng N. Nemhauser J.L. Rate motifs tune auxin/indole-3-acetic acid degradation dynamics.Plant Physiol. 2015; 169: 803-813Crossref PubMed Scopus (47) Google Scholar). However, regions outside the degron appear to contribute to differential hormone binding affinity among Aux/IAAs and F-box proteins (11.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. 2012; 8: 477-485Crossref PubMed Scopus (383) Google Scholar, 46.Moss B.L. Mao H. Guseman J.M. Hinds T.R. Hellmuth A. Kovenock M. Noorassa A. Lanctot A. Villalobos L.I. A.C. Zheng N. Nemhauser J.L. Rate motifs tune auxin/indole-3-acetic acid degradation dynamics.Plant Physiol. 2015; 169: 803-813Crossref PubMed Scopus (47) Google Scholar). JAZ proteins also contain a condensed degron sequence, which is a variable region in direct contact with the COI1-anchored JA-Ile molecule (14.Sheard L.B. Tan X. Mao H. Withers J. Ben-Nissan G. Hinds T.R. Kobayashi Y. Hsu F.-F. Sharon M. Browse J. He S.Y. Rizo J. Howe G.A. Zheng N. Jasmonate perception by inositol-phosphate-potentiated COI1–JAZ co-receptor.Nature. 2010; 468: 400-405Crossref PubMed Scopus (946) Google Scholar). Notably, this mechanism of interaction has been co-opted into a yeast assay for monitoring ubiquitin-mediated protein degradation in yeast called the auxin-inducible degradation (AID) system (46.Moss B.L. Mao H. Guseman J.M. Hinds T.R. Hellmuth A. Kovenock M. Noorassa A. Lanctot A. Villalobos L.I. A.C. Zheng N. Nemhauser J.L. Rate motifs tune auxin/indole-3-acetic acid degradation dynamics.Plant Physiol. 2015; 169: 803-813Crossref PubMed Scopus (47) Google Scholar, 47.Havens K.A. Guseman J.M. Jang S.S. Pierre-Jerome E. Bolten N. Klavins E. Nemhauser J.L. 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One of the most surprising recent findings describes how BES1 (BRI1 EMS SUPPRESSOR 1) can be ubiquitinated by SINAT (SINA of Arabidopsis thaliana) E3 ligases, which promotes BES1-DSK2 (DOMINANT SUPPRESSOR OF KAR 2) interactions and subsequent degradation via selective autophagy (38.Yang M. Li C. Cai Z. Hu Y. Nolan T. Yu F. Yin Y. Xie Q. Tang G. Wang X. SINAT E3 Ligases Control the Light-Mediated Stability of the Brassinosteroid-Activated Transcription Factor BES1 in Arabidopsis.Dev. Cell. 2017; 41: 47-58.e4Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 39.Nolan T.M. Brennan B. Yang M. Chen J. Zhang M. Li Z. Wang X. Bassham D.C. Walley J. Yin Y. Selective Autophagy of BES1 Mediated by DSK2 Balances Plant Growth and Survival.Dev. Cell. 2017; 41: 33-46.e7Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Thus, both the proteasome and selective autophagy are involved in degrading BES1 while SINAT E3s ubiquitinate BES1 during starvation and light response. Although it is not clear why a single transcription factor would need to be degraded through several independent pathways, although it has been proposed that such multiple regulatory checkpoints could regulate BES1 levels to allow for integration of morphogenesis with distinct environmental cues such as light or stress. This work also points to an interesting possibility that other E3 ubiquitin ligase substrates could be degraded via selective autophagy mechanisms, but the signals that direct ubiquitinated proteins to autophagy versus proteasome mediated degradation need to be studied further. A novel F-box protein, KINK SUPPRESSED IN BZR1–1D (KIB1) was recently shown to mediate BR-induced ubiquitination and degradation of the glycogen synthase kinase-3 (GSK3)-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) (50.Zhu J.Y. Li Y. Cao D.M. Yang H. Oh E. Bi Y. Zhu S. Wang Z.Y. The F-box protein KIB1 mediates brassinosteroid-induced inactivation and degradation of GSK3-like kinases in Arabidopsis.Mol. Cell. 2017; 66: 648-657.e4Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Also, plasma membrane localization of the BRI1 receptor is regulated by ubiquitination but the E3 ligase(s) responsible are still unknown (51.Martins S. Dohmann E.M.N. Cayrel A. Johnson A. Fischer W. Pojer F. Satiat-Jeunemaître B. Jaillais Y. Chory J. Geldner N. Vert G. Internalization and vacuolar targeting of the brassinosteroid hormone receptor BRI1 are regulated by ubiquitination.Nat. Commun. 2015; 6Crossref PubMed Scopus (111) Google Scholar). Additionally, two different U-box type E3 ubiquitin ligases have been implicated as positive regulators of BR signaling in rice, ERECT LEAF 1 (ELF1) (52.Sakamoto T. Kitano H. Fujioka S. An E3 ubiquitin ligase, ERECT LEAF1, functions in brassinosteroid signaling of rice.Plant Signal. Behav. 2013; 8: e27117Crossref PubMed Scopus (19) Google Scholar) and Taihu Dwarf (TUD1) (53.Hu X. Qian Q. Xu T. Zhang Y. Dong G. Gao T. Xie Q. Xue Y. The U-Box E3 ubiquitin ligase TUD1 functions with a heterotrimeric G α subunit to regulate brassinosteroid-mediated growth in rice.PLoS Genet. 2013; 9Crossref Scopus (107) Google Scholar) but further studies are required to identify the substrates of these ligases and their role(s) in BR-mediated plant growth. Recent studies have continued to build on our understanding of how ABA pathways are regulated post-translationally. For example, the RING E3 SDIR targets SDIR1-INTERACTING PROTEIN1 for degradation to modulate ABA signaling (54.Zhang H. Cui F. Wu Y. Lou L. Liu L. Tian M. Ning Y. Shu K. Tang S. Xie Q. The RING finger ubiquitin E3 ligase SDIR1 targets SDIR1-INTERACTING PROTEIN1 for degradation to modulate the salt stress response and ABA signaling in Arabidopsis.Plant Cell Online. 2015; 27: 214-227Crossref PubMed Scopus (119) Google Scholar). Additionally, degradation of the ABA receptor ABI1 occurs by the PUB12/13 U-box E3 ligases (41.Kong L. Cheng J. Zhu Y. Ding Y. Meng J. Chen Z. Xie Q. Guo Y. Li J. Yang S. Gong Z. Degradation of the ABA co-receptor ABI1 by PUB12/13 U-box E3 ligases.Nat. Commun. 2015; 6: 8630Crossref PubMed Scopus (180) Google Scholar). Perception of ABA by the pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) proteins with the co-receptor protein phosphatase type 2Cs facilitates activation of Snf1-related protein kinase 2 (SnRK2) kinases, which are in turn ubiquitinated and degraded by SNFAtPP2-B11 (55.Cheng C. Wang Z. Ren Z. Zhi L. Yao B. Su C. Liu L. Li X. SCFAtPP2-B11modulates ABA signaling by facilitating SnRK2.3 degradation in Arabidopsis thaliana.PLoS Genet. 2017; 13Crossref Scopus (64) Google Scholar). AtPP2-B11 is an newly described F-box protein that functions as part of a canonical SCF E3 ligase complex to negatively regulate plant responses to ABA (55.Cheng C. Wang Z. Ren Z. Zhi L. Yao B. Su C. Liu L. Li X. SCFAtPP2-B11modulates ABA signaling by facilitating SnRK2.3 degradation in Arabidopsis thaliana.PLoS Genet. 2017; 13Crossref Scopus (64) Google Scholar). Adding to the complexity of this pathway is ECERIFERUM9 (CER9), which encodes a putative RING domain-containing E3 ubiquitin ligase that is a novel negative regulator of ABA biosynthesis and ABA signaling during seed germination. CER9 is like Doa10 in S. cerevisiae, which targets substrates for degradation via the UPS. Further work on CER9 will be required to identify target protein(s) and their role in ABA signaling (56.Zhao H. Zhang H. Cui P. Ding F. Wang G. Li R. Jenks M.A. Lu S. Xiong L. The putative E3 ubiquitin ligase ECERIFERUM9 regulates abscisic acid biosynthesis and response during seed germination and postgermination growth in Arabidopsis.Plant Physiol. 2014; 165: 1255-1268Crossref PubMed Scopus (37) Google Scholar). Other examples of recent ligase-s