Plant Hormone Signaling and the Ubiquitin-proteasome System Serena J. Gregory March 30th, 2011 Objectives • Introduction • Overview of ubiquitin and the Ub/proteasome pathway • Hormone signaling – Auxin signaling – Ethylene signaling – Abscisic Acid signaling • Summary Growth and development in plants Hormone signaling through protein destruction The ubiquitin molecule • 76 amino acid protein • Contains a b-grasp fold • Di-glycine tail on the C-terminus • Covalently attached to target proteins Vierstra, R.D. (2009) Schematic overview of the ubiquitin pathway Vierstra, R.D. (2009) The Ubiquitin Code Ye Y and Rape M. (2009). Predicted number of genes that encode each UPS component in plants • E1- 2 genes • E2- 37 genes • E3- >1400 Vierstra, R.D. (2009) Target protein specificity is achieved through E3s • Three types of E3s – RING – U-box – HECT • Structure can be either single subunit or multi subunit Santner, A. & Estelle, M.(2010) RING E3s • RING (Really Interesting New Gene) • 465 RING proteins • 70 aa motif characterized as the ring finger • Zinc-binding motif that binds to E2s Vierstra, R.D. (2009) Santner, A. & Estelle, M.(2010) Multi subunit RING E3s • Components – – – – RING protein RBX1 (RING Box 1) Cullin scaffold-like protein Adaptor subunits Additional substrate-recognition protein • Types – SCF (Skp-Cullin-F-box) – CUL3-BTB (Broad-complex, Tramtrack, Bric-a-Brac) – CUL4-DDB1 (DNA-DAMAGE BINDING 1) Santner, A. & Estelle, M.(2010) U-box E3s • Modified RING-finger domain • 64 U-Box proteins • 70 aa U-box domain • Does not use zinc ions to stabilize structure Santner, A. & Estelle, M.(2010) HECT E3s • Homology to E6-AP C Terminus (HECT) • Smallest E3 subfamily • 20 HECT proteins • 350 aa motif • Contains an Ub-binding and E2 binding site Santner, A. & Estelle, M.(2010) SCF complex assembly and hormone signaling • SCF-most common E3 • Discovered through mutant screens • Exposed to auxin or auxin transport inhibitors • Disruptions of components of the SCF E3s • CUL1 mutants were discovered that are auxin resistant Mark Estelle Objectives • Introduction • Overview of ubiquitin and the Ub/proteasome pathway • Hormone signaling – Auxin signaling – Ethylene signaling – Absicic Acid signaling • Summary Hormone signaling in plants • Controls communication – Internally – Neighboring cells – Long distance between different organ systems • Three step process – Signal perception, signal transduction and response • Phytohormones – Produced by individual cells instead of gland secretion Structures of Phytohormones Functional Interactions of Phytohormones Jailais, Y. & Chory, J. (2010). Plant hormone receptors • • • • • • • Ethylene: ETR1 (histidine kinases) Cytokinins: CRE1 (histidine kinases) Auxin: TIR1 and IAA proteins GA: GID1 (regulates GID2 and DELLA) Jasmonic acid: COI1 and JAZs ABA: GTG1 and GTG2 BR: BRI1 (LRR-RLK) Sites of plant hormone perception Santner, A. & Estelle, M. (2009). Objectives • Introduction • Overview of the ubiquitin and the Ub/proteasome pathway • Hormone signaling – Auxin signaling – Ethylene signaling – Absicic Acid signaling • Summary Auxin Signaling • From the Greek auxein, meaning to grow. • Described by Charles Darwin in 1880 as a growth promoting substance that moves in plants • Responsible for – Cell division/elongation – Organogenesis – Prevents senescence • Transported via influx transporter proteins and efflux transporter proteins • Act primarily through Auxin Response Transcription Factors (ARFs) Auxin signaling with ARFs F-box protein TIR1 is the auxin receptor Vierstra, R.D. (2009) Crystal structure of TIR1 Vierstra, R.D. (2009) Jasmonic Acid and Gibberellic Acid F-box protein- CORONATE INSENSITIVE 1 (COI1) Target protein- JA-ZIM domain repressor proteins (JAZ) Transcription factor- MYB DOMAIN PROTEIN 2 (MYB2) F-box proteins- SLEEPY 1 AND SNEEZY 1 GA receptor- GA-INSENSITIVE DWARF 1 (GID1) Target protein- Della repressor proteins Vierstra, R.D. (2009) Ethylene Signaling • Gaseous hormone – Promote fruit ripening and leaf abscission and plant senescence – Resistance to pathogens and stress response • Triple Response Phenotype – Dark grown seedlings exposed to ethylene – Very distinctive phenotype The Ethylene Triple-Response Phenotype The Ethylene Triple-Response Phenotype Mutant testing ethylene insensitive WT Ethylene Signaling AIR ETHYLENE J. M. Alonso , J. R. Ecker 2001 Abscisic Acid Signaling • Isoprenoid compound • Responsible for – Control of seed dormancy – Drought response • Controversial receptors – CHLH (GUN5 in Arabidopsis) – GCR2 • Established receptors – GTG1 and GTG2 The RNA-binding protein FCA is an abscisic acid receptor Razem FA, El-Kereamy A, Abrams SR, Hill RD. Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada Abstract The phytohormone abscisic acid (ABA) regulates various physiological processes in plants. The molecular mechanisms by which this is achieved are not fully understood. Genetic approaches have characterized several downstream components of ABA signaling, but a receptor for ABA has remained elusive. Although studies indicate that several ABA response genes encode RNA-binding or RNA-processing proteins, none has been found to be functional in binding ABA. Here we show that FCA, an RNA-binding protein involved in flowering, binds ABA with high affinity in an interaction that is stereospecific and follows receptor kinetics. The interaction between FCA and ABA has molecular effects on downstream events in the autonomous floral pathway and, consequently, on the ability of the plant to undergo transition to flowering. We further show that ABA binding exerts a direct control on the FCA-mediated processing of precursor messenger RNA. Our results indicate that FCA is an ABA receptor involved in RNA metabolism and in controlling flowering time. FCA does not bind abscisic acid Risk JM, Macknight RC, Day CL. Biochemistry Department, University of Otago, Dunedin 9054, New Zealand. email@example.com Abstract The RNA-binding protein FCA promotes flowering in Arabidopsis. Razem et al. reported that FCA is also a receptor for the phytohormone abscisic acid (ABA). However, we find that FCA does not bind ABA, suggesting that the quality of the proteins assayed and the sensitivity of the ABA-binding assay have led Razem et al. to erroneous conclusions. Because similar assays have been used to characterize other ABA receptors, our results indicate that the ABAbinding properties of these proteins should be carefully reevaluated and that alternative ABA receptors are likely to be discovered. Sites of plant hormone perception Santner, A. & Estelle, M. (2009) Abscisic Acid Signaling • Two-fold process • Blocks degradation of ABI5 preventing ubiquitination by KEG • Promotes degradation of ABI3 by increasing AIP2 expression Vierstra, R.D. (2009) Effects of abscisic acid signaling on stomata Summary • The ubiquitin-26S proteasome system plays a prominent regulatory role in plant hormone signaling • E3 ubiquitin ligases represent a new class of hormone signaling receptors • E3 activities can be manipulated by small molecules could lead to new pharmacological treatments • Elucidation of all E3 ligases will potentially uncover other interesting signaling pathways and mechanisms References • Vierstra, R.D. (2009). The ubiquitin-26S proteasome system at the nexus of plant biology. Nat. Rev. Mol. Cell Biol. 10, 385–397. • Ye Y and Rape M. (2009). Building ubiquitin chains: E2 enzymes at work. Nat. Rev. Mol. Cell Biol. 10(11):755-64. • Santner, A. & Estelle, M. (2009). Recent advances and emerging trends in plant hormone signaling. Nature 459, 1071–1078. • Santner, A. & Estelle, M.(2010). The ubiquitin-proteasome system regulates plant hormone signaling. The Plant Journal. 61, 10291040. • Jailais, Y. & Chory, J. (2010). Unraveling the paradoxes of plant hormone signaling integration. Nat. Structural & Mol. Biol. 17(6): 642-645. • Benavente, L. M. & Alonso, J. M. (2006). Molecular mechanisms of ethylene signaling in Arabidopsis. Mol. BioSyst. 2, 165-173. References • J. M. Alonso , J. R. Ecker , The Ethylene Pathway: A Paradigm for Plant Hormone Signaling and Interaction. Science's STKE (2001), http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/70/re1. • Tan, X. et al. (2007). Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446, 640–645. • Pandey, S., Nelson, D. C. & Assmann, S. M. (2009). Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis. Cell 136, 136–148. • Christmann, Alexander & Grill, Erwin. (2009). Are GTGs ABA's biggest fans?. Cell, 136. Retrieved from http://www.biomedsearch.com/nih/Are-GTGs-ABAsbiggest-fans/19135884.html • Risk J.M. Macknight R.C. (2008). FCA does not bind abscisic acid. Nature 456(7223): E5-6.