Plant Hormone Signaling and the Ubiquitin-proteasome System

Serena J. GregoryMarch 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

Santner, A. & Estelle, M.(2010)Vierstra, R.D. (2009)

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

Vierstra, R.D. (2009)

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 1GA receptor- GA-INSENSITIVE DWARF 1 (GID1)Target protein- Della repressor proteins

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

ethyleneinsensitive

WT

Ethylene Signaling

J. M. Alonso , J. R. Ecker 2001

AIR ETHYLENE

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

AbstractThe 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. catherine.day@otago.ac.nz

AbstractThe 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 ABA-binding properties of these proteins should be carefully re-evaluated 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, 1029-1040.• 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-ABAs-biggest-fans/19135884.html

• Risk J.M. Macknight R.C. (2008). FCA does not bind abscisic acid. Nature 456(7223): E5-6.