Ubiquitin, Hormones and Biotic Stress in Plants

INVITED REVIEW

Ubiquitin, Hormones and Biotic Stress in Plants

KATE DREHER and JUDY CALLIS*

Section of Molecular and Cellular Biology, Plant Biology Graduate Group Program, University of California,Davis, One Shields Avenue, Davis, CA 95616, USA

Received: 1 August 2006 Returned for revision: 7 September 2006 Accepted: 3 October 2006 Published electronically: 12 January 2007

† Background The covalent attachment of ubiquitin to a substrate protein changes its fate. Notably, proteinstypically tagged with a lysine48-linked polyubiquitin chain become substrates for degradation by the 26Sproteasome. In recent years many experiments have been performed to characterize the proteins involved in theubiquitylation process and to identify their substrates, in order to understand better the mechanisms that linkspecific protein degradation events to regulation of plant growth and development.† Scope This review focuses on the role that ubiquitin plays in hormone synthesis, hormonal signalling cascadesand plant defence mechanisms. Several examples are given of how targeted degradation of proteins affects down-stream transcriptional regulation of hormone-responsive genes in the auxin, gibberellin, abscisic acid, ethyleneand jasmonate signalling pathways. Additional experiments suggest that ubiquitin-mediated proteolysis may alsoact upstream of the hormonal signalling cascades by regulating hormone biosynthesis, transport and perception.Moreover, several experiments demonstrate that hormonal cross-talk can occur at the level of proteolysis. Themore recently established role of the ubiquitin/proteasome system (UPS) in defence against biotic threats is alsoreviewed.† Conclusions The UPS has been implicated in the regulation of almost every developmental process in plants,from embryogenesis to floral organ production probably through its central role in many hormone pathways. Morerecent evidence provides molecular mechanisms for hormonal cross-talk and links the UPS system to bioticdefence responses.

Key words: Ubiquitin, E3 ligase, RING, U-Box, SCF, CRL, ubiquitylation, regulated proteolysis, plant defence,hormonal signalling, biotic stress, pathogen response.

INTRODUCTION

Plants, as sessile organisms, rely on proteomic plasticity toremodel themselves during periods of developmentalchange, to endure varying environmental conditions, andto respond to biotic and abiotic stresses. Regulatedubiquitin- and proteasome-mediated degradation, therefore,plays a crucial role in enabling plants to alter their pro-teome to maximize their chances of survival under manydifferent circumstances.

In plants, as in all eukaryotes, the ubiquitin/proteasomesystem (UPS) targets proteins for degradation (note thatall abbreviations mentioned in this review are listedand defined in Appendix 1). In this system, the 76-amino-acid protein ubiquitin acts as a covalent molecular tag andits attachment requires three distinct enzymatic activities.Catalysed by E1 [ubiquitin-activating enzyme (UBA)], theubiquitin C-terminal carboxyl group is first activated byadenylation, and then forms a thioester bond with a cystei-nyl sulfhydryl residue on the E1 protein itself. Ubiquitin isthen transferred to a cysteinyl sulfhydryl on another protein,the E2 [ubiquitin-conjugating protein (UBC)], preservingthe thioester bond. Finally, ubiquitin is typically transferredto the substrate with the help of an E3 (ubiquitin ligase,Fig. 1; reviewed in Pickart and Eddins, 2004).

The first ubiquitin normally forms an isopeptide linkagewith a lysyl 1-amino group on the substrate, although asmall subset of substrates are modified at the N-terminal

amino group (reviewed in Ciechanover and Ben-Saadon,2004), and at least one substrate has been reported to bindubiquitin on a cysteinyl sulfhydryl group (Cadwell andCoscoy, 2005). If additional ubiquitins are added, theymay be attached to different substrate lysyl 1-aminogroups or to one of seven lysyl 1-amino groups on thebound ubiquityl moiety. All seven lysyl residues can beused to form ubiquitin–ubiquitin linkages in buddingyeast (Saccharomyces cerevisiae; Peng et al., 2003).Previous work on model substrates established that for asubstrate to be recognized and then degraded by theproteasome, a chain of at least four ubiquitins linked viatheir lysyl residue 48 must be generated on a single sub-strate lysyl 1-amino group (Chau et al., 1989; Finley et al.,1994; Thrower et al., 2000). But experiments in mamma-lian cell extracts using an E2 that forms polyubiquitinchains connected through ubiquitin lysyl residue 11(Baboshina and Haas, 1996) or assays performed withpurified proteasomes using an endogenous substrate, yeastcyclin B, ubiquitylated in vitro with lysine 11 and 63linkages (Kirkpatrick et al., 2006) suggest that other ubi-quitin–ubiquitin linkages can serve as proteolytic signals,even in the complete absence of lysine 48 linkages. Inaddition, the attachment of a lysine-63-linked tetraubi-quitin chain to lysine 48 of the ubiquitin portion of theUbDHFR fusion protein permits degradation of this modelsubstrate by purified proteasomes (Hofmann and Pickart,2001). Furthermore, multiple short chains of mixed lysyllinkages are formed on cyclin B in vitro by the APC

* For correspondence. E-mail [email protected]

# The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.

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Annals of Botany 99: 787–822, 2007

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(anaphase-promoting complex) E3 ubiquitin ligase. Theseubiquitylated forms of cyclin B interact with substrate rec-ognition proteins and are substrates for in vitro degradationby purified proteasomes (Kirkpatrick et al., 2006),suggesting that ubiquitin chain formation may be morecomplex, and that the requirements for recognition by pro-teasomes and associated proteins may be more relaxedthan previously appreciated.

After delivery to the proteasome mediated in part byubiquitin binding proteins, the polyubiquitylated substratecan then be deubiquitylated by the proteasome’s regulatorycap or associated proteases. The free ubiquitin becomesavailable again for attachment, i.e. it is not degraded con-comitant with the substrate. The deubiquitylated substrateis fed into the proteolytic core of the proteasome where itis cleaved into small peptides. After release, these peptidefragments are further hydrolysed to free amino acids bymultiple separate proteases and/or proteolytic complexes.One of the few characterized examples is tripeptidyl pepti-dase II, a homooligomeric complex that hydrolyses largerpeptides to tripeptides and could function to process pro-teasome products (Book et al., 2005).

Although the best-characterized function of ubiquitylationis to target substrates for proteolysis by the proteasome,ubiquitylation has also been linked to non-proteasomalfunctions, currently mostly in fungi and vertebrates.Ubiquitin chains linked via ubiquitin lysine 63 (UbK63) areinvolved in several processes including DNA repair,protein activation (reviewed in Schnell and Hicke, 2003),

endolysosomal degradation (Duncan et al., 2006) and ribo-somal regulation (Spence et al., 2000), and there is someevidence that UbK6-linked chains synthesized by the het-erodimeric E3 ligase BRCA1/BARD1 mediate regulationof DNA replication and repair (Morris and Solomon, 2004;Nishikawa et al., 2004). Although there is also evidence forthe formation of chains using lysines 11, 27, 29 and 33 invivo (Peng et al., 2003), knowledge of the functionalimportance of these alternative polyubiquitin chains islacking. Meanwhile, monoubiquitylation is required forreceptor endocytosis, transcriptional regulation and intra-cellular sorting (reviewed in Schnell and Hicke, 2003). Thenon-degradative functions of ubiquitylation are ripe areasfor future research in plants.

Components of the UPS have been identified in manyspecies of plants through mutant screens and bioinformaticsearches (reviewed in Moon et al., 2004). Strikingly, over6 % of the predicted arabidopsis (Arabidopsis thalianaecotype ‘Columbia’) genome encodes proteins involved inthe UPS (Downes and Vierstra, 2005; with selectedexamples in Table 1), and analyses of other plant genomesdemonstrate a similar abundance of UPS-related genes.Not surprisingly, components of the UPS have been impli-cated in countless processes including organ initiation andpatterning (Samach et al., 1999; Shen et al., 2002;Imaizumi et al., 2005), light signalling (reviewed inHoecker, 2005) and circadian clock regulation (Han et al.,2004), but this review will focus on the role of UPS inhormone production, perception and signal transduction,and in plant defence. Although the relationship betweenthe UPS and plant signalling became evident about 20years ago, when the light-signalling protein phytochromeA was shown to be ubiquitylated (Shanklin et al., 1987),the broad scope of ubiquitin’s involvement in plantbiology was not predicted. On-going discovery, annotationand functional characterization of UPS genes continues toprovide insight into the mechanisms that plants use toregulate their growth and development through targetedprotein degradation.

There are other excellent reviews covering the UPS inplants (Moon et al., 2004; Smalle and Vierstra, 2004),hormone signalling (auxin: Schwechheimer and Schwager,2004; Woodward and Bartel, 2005; GA: Sun andGubler, 2004; Fleet and Sun, 2005; ABA: Himmelbachet al., 2003; BR: Vert and Chory, 2006; Z-Y Wang et al.,2006; ethylene: Chen et al., 2005; Etheridge et al., 2006;JA: Turner et al., 2002; Devoto and Turner, 2005;Lorenzo and Solano, 2005) and plant defence mechanisms(Dangl and Jones, 2001; Thordal-Christensen, 2003;Nurnberger et al., 2004; Schulze-Lefert and Bieri, 2005;Zeng et al., 2006), which cogently describe the exper-imental underpinnings of our current understanding ofthese processes. But, with the rapid pace of scientificprogress in these exciting areas, new information continuesto emerge. This review seeks to build upon the foundationof the previous reviews by drawing together informationconcerning both internally and externally regulated plantdevelopmental processes that depend upon the UPS bothto highlight some common themes and to present the mostrecent published findings.

E1

E1

E2

E2

E2E2

E2

E3E3

Substrate

Substrate

Substrate

Substrate

CULLIN

RBXRecognition/

adaptorproteins

OR

UbUb UbUb CRL

Ubiquitin

RING orU-box

ATP

AMP + PPi

HECT

s

s

s

s

s

FI G. 1. An overview of the ubiquitylation process. RING- andU-box-domain-containing E3 ligases facilitate direct transfer of ubiquitinfrom the E2 to the substrate, as shown on the right branch of thepathway. In the case of CRLs, the E2 binds to the RBX RING domain-containing subunit that is linked to the substrate through a cullin andvarious adaptor proteins. HECT domain-containing E3 ligases form athioester bond with the ubiquitin delivered by the E2 before passing the

ubiquitin to the substrate, as shown on the left branch of the pathway.

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TABLE 1. Putative E3 ubiquitin ligase components that recognize substrates involved in hormonal signalling pathways and plant–pathogen interactions

E3 ligase substrate specificity factor Organism Pathway Predicted substrate(s) References

SCF F-boxAFB1, AFB2, AFB3, AFB5 A. thaliana Auxin signalling Aux/IAA proteins with Domain II (Dharmasiri et al., 2005; Walsh

et al., 2006)ACRE189 N. tabacum Plant defence (Rowland et al., 2005)CEGENDUO A. thaliana Lateral root formation (Dong et al., 2006)CLINK Faba bean necrotic yellow virus Viral pathogenesis (Aronson et al., 2000)COI1 A. thaliana/S. lycopersicum JA signalling HDAC6?, RUBISCO small

subunit 1b?(Devoto et al., 2002; Xu et al.,

2002; Li et al., 2004)EBF1, EBF2 A. thaliana Ethylene signalling EIN3, EIL1 and homologues (Guo and Ecker, 2003; Potuschak

et al., 2003; Gagne et al., 2004)SLY1/GID2 A. thaliana/O. sativa GA signalling DELLA proteins RGA, GAI, RGL1,

RGL2, RGL3 (At); SLR1 (Os);SLN1 (Hv)

(reviewed in Fleet and Sun, 2005)

SNE A. thaliana GA signalling DELLA proteins RGA, GAI, RGL1,RGL2, RGL3

(Strader et al., 2004)

SON1 A. thaliana Plant defence (Kim and Delaney, 2002)TIR1 A. thaliana Auxin signalling Aux/IAA proteins with Domain II (Gray et al., 1999)TLP9 A. thaliana ABA signalling (Lai et al., 2004)VirF A. tumefaciens Bacterial infection VirE2, VIP1 (Tzfira et al., 2004)

BTBARIA A. thaliana ABA signalling ABF2 (Kim et al., 2004)ETO1, EOL1, EOL2 A. thaliana Ethylene synthesis ACC synthase 5 (ACS5) (Wang et al., 2004)GMPOZ H. vulgare GA and ABA signalling (Woodger et al., 2004)NPR1/NIM1 A. thaliana Plant defence (reviewed in Dong, 2004)

U-boxACRE276/PUB17 N. tobacum,

S. lycopersicum/A. thalianaPlant defence (Yang et al., 2006)

AvrPtoB P. syringae Plant defence (Abramovitch et al., 2006;Janjusevic et al., 2006)

CHIP A. thaliana ABA signalling protein phosphatase 2A subunit (Luo et al., 2006)CMPG1/ELI17 CMPG1/ACRE74

Cmpg1 PUB20/PUB21P. crispum, N. tabacum

S. lycopersicum, A. thalianaPlant defence (Kirsch et al., 2001;

Gonzalez-Lamothe et al., 2006)PHOR1 S. tuberosum GA signalling (Monte et al., 2003)PUB5, PUB12 A. thaliana Plant defence (Navarro et al., 2004)PUB27, PUB28, PUB29 A. thaliana GA signalling? (Monte et al., 2003)SPL11 O. sativa Plant defence (Zeng et al., 2004)

HECTUPL3 A. thaliana GA signalling (Downes et al., 2003)

APCHOBBIT A. thaliana Auxin signalling Aux/IAA protein IAA17/AXR3? (Blilou et al., 2002)

Continued

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TABLE 1. Continued

E3 ligase substrate specificity factor Organism Pathway Predicted substrate(s) References

RINGACRE132 N. tabacum Plant defence (Durrant et al., 2000)AIP2 A. thaliana ABA signalling ABI3 (Zhang et al., 2005)ATL2 A. thaliana Plant defence (Salinas-Mondragon et al., 1999)ATL6 A. thaliana Plant defence (Salinas-Mondragon et al., 1999)ATL43 A. thaliana ABA signalling (Serrano et al., 2006)BRH1 A. thaliana BR signalling/plant defence (Molnar et al., 2002)EL5 O. sativa Plant defence (Takai et al., 2002)RHA1b, RHA3b, RMA1,

At2g44410, At2g42360(ATL41), At4g26400,At2g35000 (ATL9), At2g42350(ATL40),

A. thaliana Plant defence (Navarro et al., 2004)

RIN2/RIN3 A. thaliana Plant defence RPM1? (Kawasaki et al., 2005)SINAT5 A. thaliana Auxin signalling NAC1 (Xie et al., 2002)XBAT32 A. thaliana Auxin signalling (Nodzon et al., 2004)XERICO A. thaliana ABA synthesis (Ko et al., 2006)

Interacting proteinsSIP A. thaliana Plant defence (Kim et al., 2006)

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OVERVIEW OF UPS COMPONENTS

Before discussing the specific function of the UPS invarious hormone signal transduction and plant defencepathways, it is useful to highlight some of the many com-ponents that are required to regulate properly the attach-ment and removal of ubiquitin from potential substrates. Awebsite dedicated to the ubiquitin pathway in arabidopsislists the identified components (http://plantsubq.genomics.purdue.edu). In the genome of arabidopsis only two genesencode an E1, the first enzyme in the ubiquitylationpathway (Hatfield et al., 1997), whereas the E2 genefamily is predicted to contain 37 members (Kraft et al.,2005). The precise function of individual members of thisexpanded family of ubiquitin-conjugating enzymesremains to be determined. Eight additional genes code forputative UEV (ubiquitin-conjugating E2 enzyme variant)proteins in arabidopsis (Kraft et al., 2005). UEVs resembleubiquitin-conjugating enzymes, but lack the catalyticcysteine (Broomfield et al., 1998; Sancho et al., 1998).Not surprisingly, human UEV1 fails to promote polyubi-quitylation in the presence of an E1 and cellular fractionscontaining E3s (Sancho et al., 1998), but UEVs do appearto function in UPS-related processes. For instance,Mms2p, a UEV from budding yeast, interacts with a cano-nical E2, Ubc13p, to direct the formation of UbK63-linkedpolyubiquitin chains (Hofmann and Pickart, 1999),whereas Tsg101 in humans binds ubiquitin and partici-pates in endosomal sorting and HIV budding (Garruset al., 2001; Katzmann et al., 2001; Sundquist et al.,2004). By contrast, the COP10 UEV, first identified in ara-bidopsis, appears to interact with a number of E2s andenhances the formation of UbK48- and UbK63-linkedpolyubiquitin chains in vitro, perhaps through its ability toincrease E2/ubiquitin thioester formation (Yanagawaet al., 2004). Additionally, COP10 forms complexes withcomponents of Cullin4-based E3 ligases described below(Chen et al., 2005).

The vast bulk of the arabidopsis UPS-related genesencode components of E3 ubiquitin ligases (Smalle andVierstra, 2004). Two mechanistic classes of E3 ligasesfacilitate the transfer of ubiquitin from the E2 to the sub-strate. In the case of the HECT domain E3 ligases, ubiqui-tin forms a covalent thioester linkage with a cysteinylsulfhydryl group on the HECT protein before being trans-ferred to a lysine on the substrate. Using the conserveddomain containing this cysteinyl residue, seven HECT pro-teins have been identified in arabidopsis (Downes et al.,2003).

The other E3 ligase class does not covalently link withubiquitin, but rather non-covalently interacts with an E2protein carrying ubiquitin. Currently, there are two groupswithin this class, the U-box domain- and RING domain-containing proteins and both domains are thought to bestructurally and functionally similar. The U-box and RINGdomains are required for E2 interaction in their respectiveproteins, utilizing H-bonds or zinc chelation, respectively,to build a platform and expose the appropriate interface(Zheng et al., 2000; Ohi et al., 2003; Andersen et al.,2004). There are approximately 61 predicted U-box

proteins in arabidopsis (http://www.arabidopsis.org/info/genefamily/pub.html). By contrast, over 450 proteins inarabidopsis contain one or more RING domains (Stoneet al., 2005).

Cullin RING ligases in plants

RING proteins may act alone, as homo- or heterodi-mers, or in complex assemblies. Two RING familymembers, RBX1a and RBX1b (in arabidopsis; Gray et al.,2002), function redundantly to play an essential role inpromoting ubiquitylation as part of the multi-subunit E3ligases called cullin RING ligases (CRLs) (reviewed inPetroski and Deshaies, 2005) – a type universally presentin eukaryotes (Willems et al., 2004; Petroski andDeshaies, 2005). Each CRL includes a cullin (CUL in ara-bidopsis) protein that contains a C-terminal RBX1 bindingsite. Distinct cullin proteins that define the different typesof CRLs bind different adaptors and substrate-recruitingsubunits at the cullin N-terminus (see figure inAppendix 2). Eleven CUL-like proteins have been ident-ified in arabidopsis based on identity to vertebrate cullins(Shen et al., 2002) and six have been implicated in CRLs.

Arabidopsis CUL1 (Gray et al., 1999) forms a CRLcalled an SCF complex (named for the subunits Skp1,cullin1 and F-box). In arabidopsis cells, CUL1co-immunoprecipitates with HA-tagged ASK1, one of 21predicted arabidopsis SKP family members (called ASKsfor Arabidopsis SKP1-like) (Farras et al., 2001). BothASK1 and ASK2 interact with several F-box proteins inyeast-two-hybrid assays, including COI1, EID1, TIR1 andUFO (Gray et al., 1999; Samach et al., 1999; Dieterleet al., 2001; Xu et al., 2002) and many of theseinteractions have been confirmed in vitro or byco-immunoprecipitation assays using arabidopsis extracts.The phenotype of plants with single mutations in thesefamily members suggests that ASK1 and ASK2 performoverlapping but not totally redundant functions. ask2-1null mutants grow quite normally (Liu et al., 2004).ask1-1 mutants display some defects in vegetative andfloral development, such as smaller rosette size, reducedfilament length and male sterility (Zhao et al., 1999)owing to a defect in homologous chromosome separationin meiosis (Yang et al., 1999). Meanwhile, ask1 ask2double mutants exhibit severe defects in embryogenesisand die as seedlings, plus they have higher levels of cyclinD3 (Liu et al., 2004). D-type cyclins are degraded via anSCF in humans (Yu et al., 1998) and cyclin D3 levels risein arabidopsis seedlings with lower level of RBX1(Lechner et al., 2002), further implicating ASK1 andASK2 in SCF-mediated degradation. The biochemicalfunction of the other ASKs has not been elucidated, butthe varying RNA expression patterns (Zhao et al., 2003;Takahashi et al., 2004) and RNAi-induced phenotypesobserved for different ASK family members suggests thatthey could regulate particular processes in different organsor tissues (Zhao et al., 2003), although there is also evi-dence that single knock-outs are often insufficient to causephenotypic abnormalities (Takahashi et al., 2004).

Dreher and Callis — Ubiquitin, Hormones and Biotic Stress in Plants 791


The SKP/ASK proteins in turn serve as adaptor proteinsby interacting with one or more of the 700-plus F-box(substrate-recruiting) proteins encoded by the arabidopsisgenome (Gray et al., 1999; Gagne et al., 2002; Kurodaet al., 2002; Risseeuw et al., 2003; Takahashi et al.,2004). The F-box domain derives its name from the SKP/ASK interaction motif found in mammalian cyclin F (Baiet al., 1996). F-box proteins bind an SKP/ASK proteinand a UPS substrate simultaneously, thus bringing the sub-strate in proximity to the CUL1-RBX1-tethered E2enzyme with an attached activated ubiquityl group. Theclose proximity presumably facilitates ubiquitin transferto the substrate, although E2 binding to the RINGdomain may additionally allosterically activate the E2 forubiquitin transfer (Ozkan et al., 2005). Another protein,SGT1b, can also associate with this complex (Azevedoet al., 2002; Y. Liu et al., 2002; Gray et al., 2003), andhas been shown to reduce the accumulation of at least oneSCF substrate in arabidopsis (Gray et al., 2003), but itsfull role in plant SCFs and proteolysis in general(Holt et al., 2005; Azevedo et al., 2006) is not completelyclear.

The other arabidopsis cullin most closely related toCUL1, CUL2, also shows enhanced interaction with oneF-box protein when ASK1 is over-expressed in ayeast-two-hybrid assay (Risseeuw et al., 2003), and soprobably forms a CRL similar to that of CUL1, but thereare as yet no in planta data.

Another class of CRL utilizes the CUL3a or CUL3bprotein as the primary scaffold component. Like CUL1,CUL3a and CUL3b associate with the RBX1 E2-recruitingprotein, as demonstrated through yeast-two-hybrid(Dieterle et al., 2005; Gingerich et al., 2005; Weber et al.,2005), in vitro pull-down (Weber et al., 2005) andplant extract-based co-immunoprecipitation experiments(Figueroa et al., 2005). But, unlike CUL1, CUL3a andCUL3b do not interact with ASK1; instead they interact inyeast-two-hybrid and pull-down assays with BTB domain-containing proteins (Dieterle et al., 2005; Figueroa et al.,2005; Gingerich et al., 2005; Weber et al., 2005). Theseinteractions depend on residues within the N-terminus ofCUL3 and for BTB proteins, residues within the BTBdomain of several family members that have been studied(Figueroa et al., 2005; Weber et al., 2005). BTB proteinsassociate directly with targets of the CUL3-based CRLs,combining adaptor and substrate-recruiting functions inone protein (Pintard et al., 2003b). Thus, it is not surpris-ing that the 80 putative BTB proteins found in arabidopsiscontain a number of additional domains such as ankyrinrepeats (reviewed in Michaely and Bennett, 1992) and theMATH domain (Xu et al., 2003) implicated in protein–protein interactions (Dieterle et al., 2005; Figueroa et al.,2005; Gingerich et al., 2005; Weber et al., 2005) thatare predicted to tether substrates to the CRL forubiquitylation.

Forward genetic identification of CUL1 (Hellmannet al., 2003; Quint et al., 2005) and reverse genetic studieson CUL1 (Shen et al., 2002; Hellmann et al., 2003) andCUL3a and b (Dieterle et al., 2005; Figueroa et al., 2005;Gingerich et al., 2005; Thomann et al., 2005) have

established their essential nature. cul1 loss-of-functionmutants are early embryo lethals (Shen et al., 2002;Hellmann et al., 2003), suggesting that CUL2 cannot func-tionally replace CUL1. By contrast, CUL3a and CUL3bappear to be redundant as mutation of either CUL3 genedoes not obviously compromise plant growth (Figueroaet al., 2005) and only mild phenotypic changes such asslightly delayed flowering and reduced hypocotyl responseto far red light are reported for the cul3a-1 mutant(Dieterle et al., 2005). However, CUL3 activity is requiredfor viability given that cul3a/cul3b double mutants areearly embryonic lethals (Dieterle et al., 2005; Figueroaet al., 2005; Gingerich et al., 2005; Thomann et al.,2005). These findings suggest that both CUL1 andCUL3a/b broadly influence a wide range of essential path-ways. By contrast, based on phenotypes of mutants, theindividual F-box and BTB family members that potentiallybring substrates to CUL1 and CUL3, respectively, appearto regulate more specific aspects of plant development andgrowth responses during the plant life cycle, ranging fromseedling blue-light-mediated phototropism (Motchoulskiand Liscum, 1999; Inada et al., 2004) to leaf senescence(Woo et al., 2001; also see below and reviewed in Moonet al., 2004; Gingerich et al., 2005), and these phenotypesare consistent with their functioning in a specific CRL. Itis worth mentioning that non-CRL functions, although notdocumented in plants, are possible for F-box and BTBproteins. Two F-box proteins in budding yeast, Rcy1p andCtf13p (Kaplan et al., 1997; Russell et al., 1999; Galanet al., 2001), and one BTB protein in Caenorhabditiselegans, MEL-26 (Luke-Glaser et al., 2005), appear toperform CRL-independent functions. However, the stab-ility of the Ctf13p (Kaplan et al., 1997) and MEL-26(Pintard et al., 2003b) proteins still depends on CRLs,suggesting that the biological function of these proteinsremains influenced by the UPS.

The most recently characterized plant CRL uses CUL4and is similar to animal CUL4 CRLs (McCall et al., 2005;Bernhardt et al., 2006; Chen et al., 2006). Two proteins,AtCUL4-L (long) and AtCUL4-S (short), encoded by thearabidopsis CUL4 gene differ in length by 50 amino acids,but both contain the N- and C-terminal cullin functionaldomains (Chen et al., 2006). To date, studies have focusedon the long version of CUL4. AtCUL4-L interacts withRBX1 at its C-terminus, binds directly to DDB1a (fromhuman Damaged DNA Binding-1) at its N-terminus(Bernhardt et al., 2006; Chen et al., 2006), and connectsindirectly with DDB2 or DET1 via DDB1a (Bernhardtet al., 2006). One reasonable interpretation of these obser-vations is that DDB1 serves as the adaptor subunit (analo-gous to ASK/SKP in CUL1 CRLs) and DDB2 and DET1are alternative substrate-recruiting subunits (analogous toF-box proteins). However, there is also evidence for theformation of larger CUL4-based complexes. In humans,degradation of the c-Jun transcription factor requires acomplex containing CUL4, ROC1 (RBX1 homologue),DDB1, DET1 and COP1, a RING E3 ligase initially ident-ified in plants for its role in regulating photomorphogen-esis (Deng et al., 1991; Wertz et al., 2004). In arabidopsis,CUL4-L appears to associate more directly with the CDD



complex, composed of the UEV protein COP10, alongwith DDB1 and DET1, but lacking COP1 (Yanagawaet al., 2004) as indicated by yeast-two-hybrid, in vitro andplant-based co-immunoprecipitation experiments (Chenet al., 2006). However, the CDD complex shows signs ofassociating with COP1 in arabidopsis plants, and mayregulate COP1’s ubiquitylation activity. Although theDET1, COP1 and COP10 proteins all contribute to light-regulated signalling, the varying phenotypes of arabidopsisplants with reduced levels of CUL4 protein suggest that itmay participate in CRLs with other substrate adaptor unitsto regulate the degradation of additional proteins (Chenet al., 2006).

Interestingly, there was some early evidence that DDB1could bind either directly to a substrate (like a BTBprotein) or function as an adaptor (like SKP1), but recentwork favours the latter interpretation. Tandem affinitypurification followed by mass spectrometry has showninteraction between human DDB1 and a large family ofproteins termed DCAFs (DDB1-Cullin4-AssociatedFactors) (Angers et al., 2006; Jin et al., 2006). Themajority of these proteins contain WD40 repeats, includ-ing DDB2, CSA and COP1 previously shown to functionin CUL4-based E3 ligases (Wertz et al., 2004; Groismanet al., 2006; H. Wang et al., 2006). Mutational studiesdemonstrate that two conserved WDXR motifs in DCAFsare important for their binding to varying portions of adouble beta propeller fold in DDB1 (Jin et al., 2006). Alimited blastp search through the NCBI database usingseveral human DCAF proteins reveals the existence ofWD40-repeat-containing proteins with two or moreWDXR motifs in a number of plant species, including ara-bidopsis, rice (Oryza sativa) and Medicago truncatula,suggesting that plant homologues of DCAFs may exist(Madden et al., 1996). In addition to binding DDB1,DCAFs presumably recruit substrate proteins and bringthem into proximity with the CUL4-ROC1-tetheredUb-charged E2. For instance, the DCAF Cdt2 binds toSpd1 in fission yeast (Schizosaccharomyces pombe) andpromotes its degradation (Liu et al., 2005). As previouslyobserved for some F-box proteins (Zhou and Howley,1998) and BTB proteins (Wee et al., 2005), DCAF pro-teins may also become victims of their own CRL, as ubi-quitylation of the DCAF human DDB2 can be promotedby a CUL4A-DDB1-based E3 ligase (Matsuda et al.,2005). To date, it remains unknown whether DDB1 is theonly adaptor protein to function in CUL4-based E3ligases. Two human proteins, CPSF160 and SAP130,show structural similarity to DDB1 (Martinez et al., 2001;Li et al., 2006), suggesting that other CUL4-based ligasesmay exist. Defining the nature of CUL4 CRL complexes iscurrently an active area of investigation in plants and inother eukaryotes.

Humans also possess a CRL that uses the adaptor pro-teins elongin B/C instead of SKP to bring substrates tohuman CUL2 (not homologous to arabidopsis CUL2) andhuman CUL5 (Kamura et al., 2004; reviewed in Willemset al., 2004). One arabidopsis protein is reported to havesimilarity to elongin C (Risseeuw et al., 2003) but noclear homologues of the elongin B adaptor protein, nor

human CUL2/CUL5 orthologues have been discovered inplants to date (Shen et al., 2002).

Finally, the APC, among its 11 or more subunits,contains a cullin-like protein, APC2, and a small RING-domain-containing protein, APC11. This CRL, found inplants (Capron et al., 2003), most likely helps to controlthe stability of cell-cycle regulatory proteins (Fulop et al.,2005).

Regulation of CRL activity

Cullin-based ubiquitin ligases are subject to regulationby a cycle of covalent addition and removal of the76-amino-acid ubiquitin-like protein called RUB (Relatedto Ubiquitin; known as Nedd8 in fission yeast andanimals). RUBs/Nedd8 attach to a conserved lysyle-amino group near the C-terminus of all six human cullinfamily members (Hori et al., 1999). To date the samemodification has been directly demonstrated for arabidop-sis CUL1 (del Pozo and Estelle, 1999), CUL3a andCUL3b (Figueroa et al., 2005) and CUL4 is present intwo electrophoretic forms, including a slower migratingform that increases in abundance in COP9 signalosomemutants (see below for relevance), strongly suggesting thatit is also modified by RUB (Chen et al., 2006). RUBattachment, similar to ubiquitin attachment, occurs throughthe activities of E1-, E2- and E3-like enzymes. TheE1-like protein is heterodimeric, composed of AXR1 andECR1 subunits that activate RUB and pass it to the RCE1RUB E2 conjugating enzyme. Finally, RBX1 appearsto act as the RUB E3 ligase that facilitates RUB attach-ment to the cullin (Gray et al., 2002), although inCaenorhabditis elegans and budding yeast, an additionalprotein, DCN-1/Dcn1p, facilitates rubylation/neddylationin vitro and in vivo (Kurz et al., 2005).

Arabidopsis contains two closely related (RUB1 andRUB2) and one more divergent (RUB3) RUB familymembers (Rao-Naik et al., 1998); their precise functionalroles are currently under investigation. RUB conjugationappears to stimulate CRL-based activity in vitro (Readet al., 2000; Wu et al., 2000). Curiously, de-rubylationcatalysed by the COP9 signalosome (CSN) (Lyapina et al.,2001; Schwechheimer et al., 2001; Cope et al., 2002) alsoappears to be required for in vivo robust CRL activity inthe degradation of Aux/IAA (auxin/indole-3-acetic acid)proteins (Schwechheimer et al., 2001) and HY5 (longhypocotyl5) (Osterlund et al., 2000) in plants, Spd1 infission yeast (Liu et al., 2003), MEI-1 in C. elegans(Pintard et al., 2003a), and cyclin E in mice (Musmusculus) (Lykke-Andersen et al., 2003) and Drosophilamelanogaster (Doronkin et al., 2003). By contrast, in vitrowork with yeast and mammalian proteins demonstratesthat the CSN effect is inhibitory rather than stimulatory onSCF activity (Lyapina et al., 2001; Zhou et al., 2001;Yang et al., 2002; Groisman et al., 2003).

These seemingly contradictory results have led to thehypothesis that a dynamic cycling of RUB/Nedd8 attach-ment and removal is needed for robust CRL activity(Lyapina et al., 2001). Recently, one specific mechanismwas proposed to explain this observation. The CSN may



be required for CRL activity in vivo by preventing proteol-ysis of CRL components when substrates are not present(reviewed in Wu et al., 2006).

CRL activity is further impacted by the CAND1 proteinthat binds to both the N- and C-termini of cullin homol-ogues (Min et al., 2003; Goldenberg et al., 2004; Huet al., 2004). By such interactions it interferes with thebinding of substrate recruiting modules (J. Liu et al.,2002; Zheng et al., 2002; Min et al., 2003; Lo andHannink, 2006) and attachment of RUB/Nedd8, respecti-vely (Hwang et al., 2003; Goldenberg et al., 2004), thusfunctioning as a negative regulator of SCF activity invitro. Therefore, one would expect in vivo loss of CAND1to hyperactivate SCF activity, but the opposite is observed.Elimination of CAND1 from arabidopsis stabilizes thetranscriptional regulators HY5 (Feng et al., 2004), RGA(repressor of ga1-3) (Feng et al., 2004) and IAA7/AXR2(indole-3-acetic acid7/auxin resistant2) (Chuang et al.,2004) that are normally slated for proteasome-mediateddegradation, suggesting that CAND1 acts as a positiveregulator of ubiquitin-mediated degradation in vivothrough its impact on CRL activity. Therefore, the precisemolecular events involved in cycles of CAND1/cullin/RUB/SKP association and dissociation require furthercharacterization. For instance, in vitro, once humanCAND1 binds to unmodified human CUL1, even a supras-toichiometric amount of the Nedd8/RUB E2 enzyme inthe presence of the Nedd8/RUB E1 cannot attach Nedd8to CUL1 nor promote the displacement of CAND1(Goldenberg et al., 2004), leaving open the question ofhow a new round of CAND1 release and CRL activationcan be achieved. Additional recent in vitro studies demon-strate that the F-box protein Skp2 together with Skp1 canpromote CAND1 dissociation from CUL1, while a smallcomplex including the p27 substrate recognized by Skp2protein can inhibit deneddylation of CUL1 by the CSN(Bornstein et al., 2006). These two processes can bothpromote SCF activity and provide an attractive explanationfor increased p27 degradation as Skp2 levels rise near theend of G1. However, the authors point out that additionalfactors may be required for this process of CAND1 displa-cement and acknowledge that more work will be requiredto determine if these findings are broadly applicable toother CRLs (Bornstein et al., 2006).

While the biochemical characterization of CAND1 pro-ceeds, insights into the biological function of CAND1 inplants continue to arise from the identification and charac-terization of cand1 mutant alleles in arabidopsis. Aberrantphenotypes observed include altered venation patterns invegetative leaves and cotyledons (Alonso-Peral et al.,2006), modest auxin and ethylene resistance and increasedABA sensitivity compared with wild-type in roots (Chenget al., 2004; Chuang et al., 2004; Feng et al., 2004), dark-grown hookless hypocotyls, and enhanced sensitivity tolow-fluence red light in hypocotyl elongation (Chuanget al., 2004; Feng et al., 2004). Both a missense allele andT-DNA insertion alleles exhibit dwarfing as adult plantswith an increased number of rosette leaves with wrinklyblades (Chuang et al., 2004; Feng et al., 2004). T-DNAinsertion alleles are almost completely sterile with reduced

apical dominance and delayed flowering (Chuang et al.,2004; Feng et al., 2004). These diverse phenotypes are notsurprising given CAND1’s ability to bind to multiplecullins and consequently to regulate the activities of manyCRLs.

Proteasomal recognition and substrate degradation

Clearly, many proteins co-operate to accomplish theregulated attachment of ubiquitin to a substrate, but thestory does not end there. Once the protein has been polyu-biquitylated, it must be brought to the proteasome, recog-nized as a proteolytic substrate, deubiquitylated, threadedinto the 20S core particle and hydrolysed therein.Selective and regulated deubiquitylation of proteins mayplay a counteracting role prior to or coincident with pro-teasomal docking. For instance, deubiquitylation of thehuman p53 protein by HAUSP inhibits p53 degradation(Li et al., 2002). Although plants contain many putativeubiquitin hydrolases (reviewed in Smalle and Vierstra,2004), their in vivo roles remain largely uncharacterized.Mechanisms governing substrate recognition, deubiquityla-tion and degradation by the proteasome also provideimportant points of regulatory control that require furtherresearch.

Ongoing identification of the components of the UPSand characterization of their biochemical activitiesenhances our understanding of targeted protein degra-dation in plants. At the same time, a combination offorward genetic, reverse genetic and biochemical tech-niques enables us to examine the relationship between theUPS and various aspects of plant growth and development,including hormonal signalling and plant defence.

During functional characterization of hormone ordefence pathways (discussed in detail here relative toUPS), scientists often monitor the consequences of alteredlevels of specific UPS components in vivo or in vitro. Themolecular and phenotypic consequences depend on thenature of the UPS component (whether it promotes degra-dation or inhibits degradation) and on the function of thesubstrate in the pathway under investigation. When a par-ticular UPS component that promotes degradation, such asan E3 ligase, is down-regulated, e.g. through mutation ortranscriptional silencing, its substrate(s) should accumu-late. So, in the case of a transcriptional activator, thisshould lead to increased expression of downstream genes(Fig. 2A). However, if the E3 ligase under considerationnormally targets a transcriptional repressor for degra-dation, plants lacking that E3 ligase would have elevatedlevels of the repressor, and hence reduced transcription ofdownstream genes (Fig. 2A). The opposite effects wouldbe observed upon over-expression of the E3 ligase, i.e.reduced transcription if the substrate is a transcriptionalactivator, owing to increased degradation and hence lowersteady-state levels, but increased transcription if the sub-strate is a repressor (Fig. 2A).

Similar predictions of expected phenotypes can bedeveloped for other potential E3 ligase substrates such asa kinase, phosphatase, hormone biosynthetic enzyme orhormone catabolic enzyme (Fig. 2B). Additionally, the



effects of perturbations in the degradative components ofthe UPS can be examined at the level of the downstreampathway. For instance, a transcriptional activator can beeither a positive or a negative regulator of a signallingpathway (e.g. in response to hormones, light, drought,pathogen attack, etc.) depending on the types of genes itturns on. Similarly, kinases, phosphatases, receptors, trans-porters, GTPases, acetylases, ion pumps, etc., can all func-tion as either negative or positive regulators of pathways.If one of these proteins that acts as a positive regulator ofa pathway (such as the EIN3 transcription factor in ethyl-ene signalling – see below) is subject to UPS-mediateddegradation, then a plant lacking the E3 ligase responsiblefor degradation of the positive signalling component

should be hypersensitive to the stimulus (e.g. ethylene) orshould be more disease resistant if the protein positivelyregulates plant defence (Fig. 2B).

In searching for and analysing novel E3 ligase/substrateinteractions, these predictable phenotypes (Fig. 2) can beused to make and test hypotheses about the regulatoryroles of the UPS and the substrates in the signalling path-ways under investigation. Similar molecular and pathway-level predictions can be made for many components of theUPS. For example, altered levels of other proteins thatpromote degradation, such as 20S proteasome core sub-units, AXR1 and E2 ubiquitin-conjugating enzymes,would be expected to cause similar effects to altered levelsof E3 ligases. By contrast, changes in the levels of

If substrate is a. . .

If substrate is a. . .

Transcriptional activator

Positive regulator of pathway Negative regulator of pathway

Transcriptional repressor

E3 ligase:knock-out/

knock-down

E3 ligase:knock-out/

knock-down

E3 ligase:normal levels

E3 ligase:normal levels

PotentialE3 ligase

substrates:

E3 ligase:overexpression

E3 ligase:overexpression

TA

TA TA

TA

TA TR

TR

TR

TR

TRPredicted phenotypes

Predicted phenotypes

A

B

• Transcriptional activator • Transcriptional repressor• Phosphatase• Receptor• Ion pump• Glycosylase• Etc

• Transcriptional repressor• Phosphatase• Receptor• Ion pump• Glycosylase• Etc

• Kinase• Acetylase• GTPase• Transporter• Biosynthetic enzyme

• Hypersensitivity• Increased resistance

• Hypersensitivity• Increased resistance

• Insensitivity• Increased susceptibility

• Insensitivity• Increased susceptibility

• Normal sensitivity• Normal resistance

• Normal sensitivity• Normal resistance

• Transcriptional activator• Kinase• Acetylase• GTPase• Transporter• Catabolic enzyme

PositivePositive

Positive

Positive

Positive

Negative

Negative

Negative

Negative

Negative

FI G. 2. Predicted phenotypic consequences of altering the levels of E3 ubiquitin ligases at the molecular level (A) and from the perspectiveof an overall pathway (B). The figure displays the predicted effects of altering the quantity of the E3 ligase components, but its predictions can beextended to mutations that affect the activity of the E3 ligase as well. The effects of mutations that decrease the activity of the E3 ligase, for exampleby impairing its ability to bind to the substrate or to the E2 enzyme, should resemble the effects of reducing the levels of the E3 protein. And similaroutcomes might be expected when an E3 ligase is expressed at a higher level, or exhibits higher activity, for instance owing to an enhanced

affinity for its substrate.



components of the UPS that act to stabilize substrates,such as deubiquitylating enzymes, would most likelypromote opposite outcomes.

Auxin and the UPS

In plants, the hormone auxin appears indispensable forplant survival. Often present as indole-3-acetic acid (IAA),auxin plays a critical role in regulating cell growth, div-ision and differentiation, and on a gross morphologicalscale, auxin clearly impacts apical dominance, rootelongation, lateral root formation and many otherprocesses (reviewed in Davies, 2004; Paciorek and Friml,2006).

Many, but not all, of auxin’s effects are linked to auxin-mediated alterations of the transcriptome and the UPSappears to be involved in allowing these transcriptionalchanges. Multiple members of the Aux/IAA family ofproteins act as repressors of auxin-mediated transcriptionin transient transfection assays using carrot (Daucuscarota) or arabidopsis protoplasts (Ulmasov et al., 1997;Tiwari et al., 2001, 2004). These proteins are subjected torapid degradation and stabilized versions disrupt normalplant growth and auxin signalling, indicating the biologicalsignificance of rapid Aux/IAA proteolysis (reviewed inReed, 2001). Although there is no published report of ubi-quitylated forms of these extremely short-lived proteins,evidence for their degradation via the UPS is compelling.The TIR1 F-box protein (Ruegger et al., 1998), and threeadditional related F-box proteins called AFB1, 2 and 3(Dharmasiri et al., 2005a, b) interact with Aux/IAAfamily members in vitro, suggesting that these proteins aresubstrates of a CUL1-based CRL, either an SCFTIR1 or anSCFAFBx. Two additional family members, AFB4 andAFB5, remain to be characterized biochemically, but therecent identification of afb5 mutants with reduced sensi-tivity to the structurally distinct picolinate class of syn-thetic auxins suggests that these proteins could act in arelated pathway (Gagne et al., 2002; Dharmasiri et al.,2005b; Walsh et al., 2006).

Evidence for Aux/IAA ubiquitylation also comesfrom the observations that in vivo degradation of Aux/IAA fusion proteins such as IAA1:LUC (firefly luciferase)(Ramos et al., 2001), AXR2(IAA7):GUS (b-glucuroni-dase) and AXR3(IAA17)NT:GUS (a fusion with theN-terminal 102 amino acids of IAA17) (Gray et al., 2001)is slowed by the proteasome inhibitor MG132 and bothtagged and endogenous Aux/IAA proteins are stabilizedby mutations in several components that affect CRLactivity in particular, including AXR1, CAND1, CUL1 andCSN component CSN5 (Gray et al., 2001; Schwechheimeret al., 2001; Zenser et al., 2003; Chuang et al., 2004;Quint et al., 2005).

How do auxin signalling and Aux/IAA proteolysisintersect? Surprisingly, increased levels of auxin promoteaccelerated degradation of these Aux/IAA transcriptionalrepressors (Gray et al., 2001; Zenser et al., 2001) byincreasing their interactions with TIR1 and related AFBs(Gray et al., 2001; Kepinski and Leyser, 2004; Dharmasiriet al., 2005b). Attempts to uncover the signalling steps

between auxin perception and accelerated degradation ledto the exciting discovery that auxin itself directly promotesthe interaction between TIR1 and tagged forms of Aux/IAA proteins and through this increased interaction prob-ably prompts accelerated proteolysis (Dharmasiri et al.,2005a; Kepinski and Leyser, 2005; and reviewed in Parryand Estelle, 2006). In all experiments, auxin bindingoccurs in the presence of both a TIR1/AFB protein and itsAux/IAA substrate, leaving open the possibility that bothproteins are required to bind this crucial hormone(Dharmasiri et al., 2005a; Kepinski and Leyser, 2005).This mechanism of proteolytic regulation represents a newparadigm for regulation of SCF activity and suggests thatsmall-molecule non-covalent modulation of CRL functionmay occur in other contexts.

A recent study shows that auxin directs the movementof IAA17:GFP into nuclear protein bodies (NPBs) alongwith components of SCFTIR1, the COP9 signalosome andthe 26S proteasome core (Tao et al., 2005). In arabidopsiscells, this relocalization depends on the RAC1 GTPase, asmall signalling protein. RAC1’s interactions with variousdownstream effectors is regulated by rounds of GTPbinding and hydrolysis. Expression of a dominant negativeform of AtRAC1 suppresses auxin-mediated formation ofthese NPBs and decreases loss of IAA17:GFP protein,whereas IAA17:GFP levels drop more in cells expressinga constitutively active form of RAC1 (Tao et al., 2005). Inaddition, work performed in tobacco (Nicotiana tabacum)demonstrates that induction of auxin-responsive genesoccurs while GFP-NtRac1 localizes predominantly to theplasma membrane, pointing to the existence of anunknown membrane-associated protein linking RACGTPases to auxin-mediated acceleration of Aux/IAAdegradation (Tao et al., 2002).

There is also evidence that mutation of the HOBBITCdc27-like component of the APC stabilizes the IAA17/AXR3 protein. This does not result from a global decelera-tion of proteolysis as the HY5 transcription factor that par-ticipates in light signalling is not similarly stabilized inhobbit mutants (Blilou et al., 2002). It is unclear if thestabilization of IAA17 in hobbit mutants results from anydirect interaction between the APC and Aux/IAA proteins,but suggests a link between cell cycle regulation and Aux/IAA degradation.

Although several experiments demonstrate that auxindoes not promote a global acceleration of proteolysis(Zenser et al., 2003; H. Li et al., 2004), its effects onproteolysis are not limited to the Aux/IAA proteins. Forexample, degradation of a phosphorylated E2FB transcrip-tion factor that helps regulate cell cycle progression isdramatically slowed in arabidopsis cultured cells incubatedwith exogenous auxin [(1-NAA)1-naphthalene-acetic acid](Magyar et al., 2005). By contrast, levels of theNAC1 transcription factor, a positive regulator of auxin-responsive lateral root formation (Xie et al., 2000), dropfollowing treatment with auxin even though its transcriptlevels are initially induced by auxin (Xie et al., 2000,2002). Post-translational modification must be the cause ofthe lowered protein levels because plants expressing35S:6�Myc:NAC1 show steady levels of transcript



following auxin treatment even though 6�Myc:NAC1protein levels drop. The authors found that the SINAT5RING E3 ligase is capable of ubiquitylating NAC1in vitro and affecting its accumulation in transgenicplants (Xie et al., 2002). SINAT5 is transcriptionallyup-regulated by auxin more slowly than NAC1, but priorto observed decreases in NAC1 protein levels, suggestingthat increased levels of SINAT5 cause increased degra-dation of NAC1 protein in the presence of auxin. It is notclear whether auxin has any additional role in promotingthe degradation of NAC1 (e.g. by stimulating post-translational modification that increases its affinity forSINAT5) (Xie et al., 2002).

The auxin signal transduction pathway demonstrates thepower of the UPS to control plant behaviour tightly inresponse to transient or changing signals. For instance, apulse of auxin will rapidly trigger increased Aux/IAAdegradation and turn on genes required for auxin signal-ling. In roots, this might lead to lateral root formation,mediated in part by the NAC1 protein. But to prevent anover-proliferation of lateral roots, auxin also promotesdegradation of the NAC1 protein through increased levelsof an E3 ligase that catalyses NAC1 ubiquitylation. Otherhormonal and defence signalling pathways could alsoemploy the UPS both to initiate and then to dampen a bio-logical response.

Like SINAT5, two other components of the UPSinvolved in lateral root development are also transcription-ally up-regulated by auxin, namely the RING E3 ligase,XBAT32 (Nodzon et al., 2004), and the F-box proteinCEGENDUO (Dong et al., 2006); however, no substratesfor these proteins have been identified. It is interesting tonote that the former is a positive regulator of lateral rootformation while the latter inhibits lateral root development,again suggesting a potential dual stimulatory and inhibi-tory role for the UPS in this developmental process.

Several years ago, scientists also uncovered a linkbetween the UPS and auxin transport. AtPIN2/EIR1, anauxin efflux carrier (Petrasek et al., 2006), does not appearto be transcriptionally regulated by auxin, but the levels ofa PIN2/EIR1:GUS fusion protein drop following treatmentwith the auxin 1-NAA or a protein synthesis inhibitor,suggesting that the protein is short-lived and its degra-dation is regulated by auxin (Sieberer et al., 2000).NAA-mediated loss of EIR1:GUS is slowed by the axr1-3mutation, intimating that degradation of this protein isregulated by a CRL (Sieberer et al., 2000). This could bethrough a direct mechanism, or the stability of PIN2/EIR1could be controlled by a component of the UPS underAXR1-mediated control.

A recent report verified and extended that study bydemonstrating that inhibition of proteasome activity resultsin accumulation of PIN2 protein and by visualizingubiquitylated forms of PIN2 (Abas et al., 2006).Gravistimulation leads to asymmetric distribution of bothendogenous PIN2 and a PIN2:GFP reporter protein thatcan be blocked by treatment with a proteasome inhibitor.Internalization of the PIN2 protein also appears to beaffected by a proteasome-dependent process (Abas et al.,2006). Thus, the UPS affects steady-state levels of PIN2 at

the plasma membrane and consequently influences theamount and direction of auxin transport within a plant, butthen auxin itself appears to affect the stability of PIN2.This may constitute an important feedback loop to localizeand then limit auxin in specific cells.

Gibberellins and the UPS

The gibberellins (GAs), a group of diterpenoid com-pounds with phytohormone activity, affect various stagesof plant development, including seed germination, stemelongation, root growth, flowering and pollen tubeelongation (reviewed in Davies, 2004; Swain and Singh,2005) and like auxin signalling, gibberellin-inducedchanges in transcription are an important part of theresponse pathway (Fleet and Sun, 2005). Several featuresof the GA/UPS-mediated signalling cascade resemblecharacteristics of the Aux/IAA branch of the auxin signal-ling pathway. The DELLA proteins, named for highly con-served amino acids at their N-termini, act, like Aux/IAAproteins, as negative regulators of GA signalling. Thereare five proteins in this family in arabidopsis [GAI (GAinsensitive), RGA (Repressor of ga1-3), RGL1(RGA-like), RGL2 and RGL3 (Dill et al., 2001; Peng andHarberd, 2002)]. SLENDER RICE1 in rice (SLR1) andSLENDER1 in barley (Hordeum vulgare) (SLN1) aresingle DELLA proteins in these plants (Ikeda et al., 2001)and various other species also have homologous proteins(Thomas and Sun, 2004). As for Aux/IAA proteins,DELLA proteins are subject to hormone-mediated accel-eration of degradation. Levels of both endogenous RGAand a GFP:RGA fusion protein drop following GA treat-ment of arabidopsis seedlings (Silverstone et al., 2001).Protein loss depends on an SCF complex containing theAtSLY1 (SLEEPY) F-box protein (Swain and Singh,2005). sly1 mutants contain elevated levels of RGA andcan no longer eliminate this protein following GA treat-ment (McGinnis et al., 2003). DELLA proteins (RGA andGAI) and SLY1 interact directly through bothyeast-two-hybrid assays and in vitro pull-down exper-iments using GST-SLY1 (Dill et al., 2004). A single arabi-dopsis homologue, SNEEZY (SNE), when overexpressed,can compensate for loss of SLY1 suggesting that it alsoacts as a substrate-recruiting protein for DELLA proteins(Strader et al., 2004). The rice DELLA protein SLR1 alsoappears to be ubiquitylated through the activity of an SCFcomplex using the GID2 (Gibberellin-Insensitive Dwarf2)F-box protein (Itoh et al., 2003) in a GA-dependentmanner, indicating a conserved function for rice GID2 andits arabidopsis orthologue SLY1.

How gibberellin promotes DELLA protein degradationis unclear. One possibility is that GA-induced post-translational modification of the DELLA proteins enhancestheir interactions with the F-box component of the SCFE3 ligase. The DELLA proteins do appear to becomephosphorylated in the presence of elevated levels of GA inrice (Itoh et al., 2003) and only phosphorylated SLR1 caninteract with the GID2 F-box protein in vitro (Gomi et al.,2004). In arabidopsis, there is also evidence for binding ofphosphorylated GAI by the F-box protein SLY1 (Fu et al.,



2004). But the steps connecting GA and DELLA phos-phorylation have not been elucidated.

Recent identification of the first bona fide gibberellinreceptors in rice (Ueguchi-Tanaka et al., 2005) and arabi-dopsis (Nakajima et al., 2006) indicates that the receptorplays a role in GA-induced DELLA degradation. Thesoluble GA receptors, called GID1 in rice and GID1a, band c in arabidopsis, bind bioactive GAs. gid1 ricemutants exhibit classic GA insensitivity phenotypes suchas dwarfed stature, dark green leaves and lack ofalpha-amylase induction in seeds. Sequence homologygives little insight into the molecular mechanisms thatunderlie GID1-mediated signalling. These proteinsresemble hormone-sensitive lipases (HSLs), but lack oneof the three conserved residues in the catalytic triad, andrice GID1 cannot hydrolyse an artificial HSL substrate(Ueguchi-Tanaka et al., 2005). However, it is clear thatGID1 can bind to the DELLA proteins in a GA-dependentmanner recapitulated in yeast and in vitro (Ueguchi-Tanaka et al., 2005; Nakajima et al., 2006). In the ricegid1 mutant, the SLR1 DELLA protein accumulates tohigher levels and SLR1 levels fail to drop following treat-ment with GA, demonstrating that GA-mediated SLR1degradation depends upon GID1 (Ueguchi-Tanaka et al.,2005). Interestingly, the DELLA proteins RGA and GAIcan actually enhance GID1c’s affinity for 16,17-dihydro-GA4, suggesting that this DELLA/GID1 two-protein complex may actually be the biological bindingtarget of the hormone (Nakajima et al., 2006). If thismodel is correct, formation of the GA-GID1-DELLAcomplex may be required for productive SCFSLY1/GID2-mediated ubiquitylation of the DELLA proteins, and it isunclear how phosphorylation affects this process(Nakajima et al., 2006).

In addition to contributing to GA-mediated regulationof germination, growth and flowering (Wen and Chang,2002; Macmillan et al., 2005; reviewed in Peng andHarberd, 2002), recent studies demonstrate the importanceof the DELLA signalling pathway for plant survival undersalt stress. Arabidopsis seedlings grown on 100 mM saltaccumulate lower levels of bioactive GA1 and GA4, whichshould lead to increased DELLA stability. Indeed, a 1-htreatment with 50 mM NaCl is sufficient to increase thelevels of a GFP:RGA reporter protein. Salt-induced stabil-ization of DELLA proteins can then inhibit plant growthto enhance stress tolerance (Achard et al., 2006).

Interestingly, three other hormones have been implicatedin regulation of DELLA stability. Depletion of auxin fromarabidopsis seedlings through decapitation leads toreduced GA-mediated loss of GFP:RGA from root nucleiwhereas application of auxin to the shoot restores rapidGA-induced reduction in GFP:RGA levels (Fu andHarberd, 2003). Ethylene exerts an opposite effect; seed-lings grown in ethylene-rich air show slower loss ofGFP:RGA from nuclei following GA treatment (Achardet al., 2003). This implies that a ctr1-1 mutant exhibiting aconstitutive ethylene response phenotype should accumu-late more DELLA proteins capable of promoting stresstolerance. In keeping with this model, ctr1-1 mutantsdisplay enhanced survival under high salt conditions. This

improved salt tolerance depends at least partially onDELLA proteins as a triple mutant ctr1-1 gai-t6 rga-24that lacks two DELLA proteins is more susceptible to saltstress than ctr1-1 (Achard et al., 2006). Abscisic acid(ABA) levels also affect DELLA accumulation. Rootstreated with ABA show enhanced accumulation of aGFP:RGA fusion protein, both under basal conditions andfollowing treatment with exogenous GA. But, plantsmutant for abi1, a serine/threonine phosphatase that posi-tively regulates ABA signalling, no longer accumulateGFP:RGA following ABA treatment (Achard et al., 2006).Intriguingly, this regulation may be cell-type-, organism-or family member-specific as SLN1 protein levels are notaffected by ABA in barley aleurone cells (Gubler et al.,2002). It is unclear how these changes in auxin, ethyleneand ABA levels cause differential effects on DELLAprotein stability, but it does provide an intriguing exampleof hormonal cross-talk in which multiple pathways canconverge to influence the stability of a single family oftranscriptional regulators.

GA and ABA also play well-characterized antagonisticroles in regulation of hydrolase production in the aleuronelayer of germinated cereal seeds through their effects onanother transcription factor, GAMYB. This positive regu-lator of alpha-amylase expression is transcriptionallyup-regulated by GA (Gubler et al., 1995) and repressed byABA (Gomez-Cadenas et al., 2001). The SLN1 DELLAprotein is implicated in the GA-mediated transcriptionalregulation of GAMYB (Gomez-Cadenas et al., 2001;Gubler et al., 2002), but a more recent yeast-two-hybridassay pulled out another potential regulator of GAMYB inbarley, named GMPOZ (Woodger et al., 2004). ThisGAMYB interacting protein derives part of its name fromits POZ domain, also known as a BTB domain. Thisprovides a potential link between GAMYB and aCUL3-based CRL. Knock-down of GMPOZ in aleuronecells decreases GA-mediated transcription of an alpha-amylase reporter construct, suggesting that GMPOZ nor-mally positively regulates GA signalling. At the sametime, the cells show enhanced ABA-mediated stimulationof a dehydrin reporter, arguing that GMPOZ normallynegatively regulates ABA signalling in the barley aleuronelayer. As GAMYB protein levels were not assayed, it isunclear how GMPOZ directly affects transcription of GA-and ABA-responsive genes. But, based on the observedfunctions of BTB proteins in other organisms, it couldpotentially interact directly with elements of the basaltranscription machinery, as seen for BAB1 and BAB2 inDrosophila (Pointud et al., 2001), regulate the stability ofGAMYB and/or other transcription factors as seen inKeap1-mediated destabilization of the Nrf2 transcriptionfactor in humans (Furukawa and Xiong, 2005), or even bedegraded itself by a CUL3-based CRL as observed forMEL-26 in C. elegans (Pintard et al., 2003b).

Genetic evidence also suggests that a HECT-type E3ubiquitin ligase, UPL3, influences the GA-regulatedprocess of trichome development as upl3-1 and upl3-2mutants possess trichomes with elevated numbers ofbranches, similar to some GA-signalling mutants. upl3-1and upl3-2 mutants are moderately hypersensitive to GA



in a hypocotyl elongation assay, suggesting that somepositive potentiator of GA signalling is more abundant inthe absence of the UPL3 protein (Downes et al., 2003).

PHOR1, a predicted E3 ligase initially identified inpotato (Solanum tuberosum), also appears to participate inGA signalling (Amador et al., 2001). This protein containsa U-box domain (Hatakeyama et al., 2001; Monte et al.,2003), and a series of ARM repeats. An ARM repeat, firstidentified in the Drosophila protein ARMadillo, isapproximately 40 amino acids in length and is typicallypresent in a variable number of tandem copies and func-tions in protein–protein interactions (reviewed in Hatzfeld,1999). Plants expressing lowered levels of PHOR1 haveshorter, GA-insensitive stems, whereas plants constitu-tively over-expressing PHOR1 develop longer stems thatare hypersensitive to treatment with low levels of GA.Both sets of experiments argue that PHOR1 is normally apositive regulator of GA-stimulated growth. Increasedlevels of bioactive (GA20) and inactive (GA8, GA29) gib-berellins accumulate in PHOR1 antisense lines consistentwith a role for PHOR1 in regulation of GA metabolism,and indicative of a lack of normal feedback inhibition inthis genetic background. Although a PHOR1:GFP fusionprotein can be found in the cytoplasm or nucleus oftobacco BY cells following transient transfection, themajority of PHOR1:GFP signal is localized to the nucleusfollowing treatment with exogenous GA (Amador et al.,2001). Three arabidopsis proteins, AtPUB27, AtPUB28and AtPUB29, are more than 50% identical to PHOR1and may link the UPS to GA signalling in arabidopsisthrough degradation of unidentified substrate(s) (Monteet al., 2003).

Although there is no evidence for UPS-mediated regu-lation of GA transport, a recent paper presents an excitinglink between light signalling, targeted protein degradationand regulation of GA biosynthesis prior to seed germina-tion (Oh et al., 2006). For many years it has been knownthat light signalling through the phytochrome family ofphotoreceptors can promote seed germination (Shinomuraet al., 1994; Hennig et al., 2002), and more recently it wasdiscovered that this occurs through light-induced transcrip-tional up-regulation of GA biosynthetic enzymes(Yamaguchi et al., 1998). The PIL5 (PIF3-Like 5) tran-scription factor represses seed germination by regulatingthe abundance of transcripts for GA biosynthetic and cata-bolic enzymes to decrease GA levels in seeds kept in thedark. Consistent with this finding, over-expression of PIL5blocks up-regulation of the GA biosynthetic enzymesGA3ox1 and GA3ox2 and simultaneously promotesexpression of the GA2ox2 oxidase that inactivates GA4

and other biologically active GAs (Oh et al., 2006). Nowthere is evidence that PhyA and PhyB-mediated signallingcan prompt degradation of Myc-tagged PIL5 to increaseGA levels and promote germination in response to light.Higher levels of the PIL5 fusion protein accumulate inphyA mutants than in wild-type plants following a pulse ofred light, and when examined over a longer period, phyBmutants also show elevated levels of Myc-tagged PIL5relative to wild-type seedlings following a pulse of red orfar-red light. The proteasome inhibitor MG132 blocks

light-stimulated loss of Myc-PIL5 following a red orfar-red light pulse, arguing that the UPS is required for thenormal reduction of PIL5 protein levels in plants exposedto light (Oh et al., 2006). As PIL5 is degraded, GA bio-synthetic genes are up-regulated while a GA catabolicgene is repressed, leading to the overall accumulation ofGA required for seed germination.

Abscisic acid and the UPS

The hormone abscisic acid (ABA) is another prominentregulator of seed germination that also enables plants torespond to abiotic stresses such as drought. ABA candirectly affect ion transport in guard cells to alter stomatalaperture rapidly in response to changing water availability(reviewed in Roelfsema et al., 2004), but other slowerABA responses require changes in transcription. And, asobserved for other phytohormones, the UPS is implicatedin regulation of ABA-responsive transcription.

The first evidence of ABA-mediated changes in proteinstability was observed for the ABI5 (abscisic acid insensi-tive5) protein in arabidopsis. Unlike the Aux/IAA andDELLA proteins, this basic leucine zipper transcriptionfactor acts as a positive regulator of ABA responses.Therefore, it might not seem surprising that rather thanpromoting degradation of ABI5, abscisic acid is requiredfor the maintenance of high levels of the ABI5 protein.This was discovered using transgenic arabidopsis plantsexpressing an HA:ABI5 fusion protein under the controlof the constitutive 35S promoter. Although the levels oftranscript for this fusion protein remain unaltered follow-ing ABA treatment, HA:ABI5 protein levels increase sub-stantially. ABA treatment also blocks loss of the ABI5protein from cycloheximide-treated seedlings incapable ofperforming de novo protein synthesis (Lopez-Molinaet al., 2001), and treatment of seedlings with proteasomeinhibitors leads to the accumulation of ubiquitylatedHA:ABI5 (Lopez-Molina et al., 2003), implicating theUPS in ABI5 degradation. This hypothesis is strengthenedby the observation that ABI5 is stabilized in the rpn10-1mutant that has a defect in one of the nine RPN(Regulatory Particle, Non-ATPase) subunits of the 26Sproteasome lid. Interestingly, two other proteasome sub-strates, PhyA and HY5, are not stabilized in this mutantbackground, suggesting that RPN10 may promote degra-dation of a subset of proteasome substrates includingABI5 (Smalle et al., 2003). It is unclear how ABA stabil-izes ABI5, but several experiments demonstrate that theABI5-interacting protein (AFP) plays a role in down-regulating ABI5 protein levels in the absence of ABA(Lopez-Molina et al., 2003). Upon removal of ABA fromthe media, seedlings that are over-expressing AFP show amuch more rapid loss of ABI5 than wild-type seedlings orafp mutants. ABI5 and AFP were initially shown to inter-act in a yeast-two-hybrid assay and this association wasconfirmed in planta through co-immunoprecipitationexperiments. Fluorescently tagged AFP and ABI5 proteinsalso co-localize in nuclear protein bodies in transgenic ara-bidopsis seedlings treated with ABA. These nuclearprotein bodies also include the COP1 RING E3 ligase



when all three proteins are bombarded into onion epider-mal cells (Lopez-Molina et al., 2003). However, to datethere is no evidence that COP1 ubiquitylates ABI5, so thesearch is still on for the E3 ligase that acts upon ABI5,and for the abscisic acid receptor and/or other signallingcomponents that link altered ABA levels to changes inABI5 proteolysis.

The degradation pathway of another ABA-responsivetranscription factor, ABI3, has also been studied. ThisB3-domain-containing transcription factor plays a promi-nent role in ABA-signalling. Like ABI5, an ABI3:6Mycfusion protein was recently found to be relatively short-lived under basal conditions, but to be long-lived in thepresence of proteasome inhibitors (Zhang et al., 2005). Itappears that ABI3 degradation is mediated, at least in part,by the AIP2 (ABI3-Interacting Protein2) RING-type E3ubiquitin ligase. ABI3 and AIP2 interact in a yeast-two-hybrid assay and tagged versions of these proteinsco-immunoprecipitate in arabidopsis extracts. Moreover,ABI3 is a substrate of AIP2-mediated ubiquitylation in anin vitro assay. Not surprisingly, over-expression ofAIP2:3�HA reduces the accumulation of ABI3 in arabi-dopsis seedlings, whereas levels of the endogenous ABI3protein increase in aip2-1 mutants. ABA regulates thestability of ABI3, but unlike for ABI5, elevated levels ofABA reduce ABI3 accumulation. Similar to the NAC1/SINAT5 signalling system in the auxin pathway, it seemsthat ABA-mediated transcriptional up-regulation of theAIP2 RING-E3 ligase allows it to degrade more ABI3,providing a partial mechanistic explanation for the linkbetween ABA levels and ABI3 proteolysis. Nevertheless,Zhang et al., (2005) conclude that AIP2 might target otherproteins for ubiquitin-mediated degradation, and that ABI3may also be ubiquitylated by other E3 ligases.

Yet another positive regulator of ABA transcription, theABF2 (ABRE Binding Factor2) transcription factor, alsointeracts with a potential component of an E3 ligase,ARIA (ARM protein Repeat Interacting with ABF2), inboth yeast-two-hybrid assays and in vitro pull-down exper-iments (Kim et al., 2004). Over-expression or knock-outof ARIA affects a subset of ABA-regulated processes andindicates that ARIA is a positive regulator of ABA signal-ling. For example, aria mutants show reduced sensitivityto ABA in root elongation assays. In addition to its ARMrepeats, ARIA possesses a BTB domain. Although aGST:ARIA fusion protein can pull down ABF2 in vitro(Kim et al., 2004), it could also potentially mediateinteractions with a CUL3 protein and thereby linkABF2-regulated processes to the UPS. As in the case ofthe GMPOZ BTB-protein involved in negative regulationof ABA signalling in barley (see above), there is noevidence of whether ARIA or its potential substratesundergo UPS-mediated degradation.

Several other sets of experiments highlight potentialrelationships between ABA signalling and the UPS. First,a characterization of the ATL [Arabidopsis Toxico paraLevadura (toxic to fungi)] family of RING E3 ligasesincluded a phenotypic analysis of lines with T-DNA inser-tions in various ATL genes. A mutation in ATL43 rendersseedlings insensitive to ABA in a seed germination assay

(Serrano et al., 2006). The U-box E3 ligase AtCHIP mayalso be involved in ABA signalling. Over-expression ofthe protein makes plants hypersensitive to the effects ofABA in a germination assay and in a stomatal apertureassay. AtCHIP seems capable of attaching a single ubiqui-tin moiety to the A subunit of protein phosphatase 2A, butit is unclear whether this is functionally related to theABA signalling pathway (Luo et al., 2006).

The possible involvement of an SCF-complex in ABAsignalling was uncovered through characterization of TLP9(Lai et al., 2004). This member of the TUBBY-LIKEfamily of genes in arabidopsis contains a C-terminal tubbydomain [named for a protein identified in obese mutantmice (Kleyn et al., 1996)] and an N-terminal F-boxdomain. Yeast-two-hybrid assays confirm that this F-boxprotein can interact with arabidopsis ASK1. Phenotypicanalyses of two tlp9 insertional mutants indicate that thisprotein is required for normal ABA signalling. The mutantplants show reduced sensitivity to ABA in seedling germi-nation assays whereas plants over-expressing TLP9 underthe control of the 35S promoter exhibit ABA hypersensi-tivity and lower rates of germination. These phenotypeslead to the prediction that TLP9’s substrate(s) are negativeregulators of ABA signalling or levels (Fig. 2B), but nonehas been identified to date. It is interesting to note that inthe abi1 ABA-insensitive mutants, TLP9 transcript levelsare reduced (Lai et al., 2004).

Finally, there is new evidence that the XERICO RINGE3 ligase contributes to regulation of ABA levels in arabi-dopsis, potentially in response to osmotic and salt stress.Transcription levels of this gene rise in response to highsalt and osmotic stress – conditions that normally increaseABA levels (Ko et al., 2006; reviewed in Zhu, 2002).Over-expression of XERICO prompts an increase in ABAlevels in transgenic plants relative to wild-type plantswhen both are grown in well-watered conditions; droughtconditions further increase ABA accumulation even in the35S:XERICO plants. Transcriptional profiling demon-strates an up-regulation of the NCED ABA biosyntheticgene in both wild-type and transgenic plants followingtreatment with ABA, but the level of induction isgreater in the 35S:XERICO plants. XERICO interactswith the E2 ubiquitin-conjugating enzyme, UBC8, inyeast-two-hybrid assays. Unexpectedly, the TLP9 F-boxprotein also interacts with XERICO in this experiment.Although both appear to function in ABA signalling andshow a high degree of co-expression at the transcriptionallevel, the functional significance of this novel RING/F-box protein interaction has not been discerned (Koet al., 2006).

Brassinosteroids and the UPS

Brassinosteroids (BRs), a class of polyhydroxylatedsteroid hormones, also have pleiotropic effects on plantdevelopment and regulate processes such as cell division,cell elongation, photosynthesis, vascular differentiationand stem elongation (reviewed in Bishop and Koncz,2002).



In the BR signal transduction pathway, the first hint thatthe UPS is involved in BR-mediated transcriptionalresponses came from analyses of the bes1-D(bri1-EMS-suppressor 1 Dominant) and bzr1-1D(brassinazole-resistant 1-1Dominant) mutants that displayBR hypersensitivity (Wang et al., 2002; Yin et al., 2002).These dominant mutants harbour identical proline toleucine missense substitutions in two related proteins thatare transcription factors. Analysis of bzr1-1D:CFP andBZR1:CFP fusion proteins in arabidopsis seedlingsdemonstrates that the mutant protein accumulates to sub-stantially higher levels, indicating that it is stabilized rela-tive to its wild-type counterpart. This suggests that thesepositive regulators of BR-mediated transcriptional changesmust normally be degraded to prevent constitutive BR sig-nalling. Subsequent work identified additional regulatorycomponents that led to the development of a model of BRsignalling with some similarity to the Wnt/GSK3/b-catenin signalling pathway found in metazoans(reviewed in Vert et al., 2005; Cadigan and Liu, 2006).New findings, such as those regarding subcellular localiz-ation of BR signalling components, highlight the potentialfor significant differences between these signal transduc-tion cascades (reviewed in Vert et al., 2005; Vert andChory, 2006), but some similarities may exist. Forinstance, in the absence of BR, the BIN2 (Brassinosteroidinsensitive2) GSK3-like kinase is hypothesized to phos-phorylate and thereby destabilize BES1 and BZR1. BIN2can interact directly with BZR1 in a yeast-two-hybridassay and can phosphorylate it in vitro (He et al., 2002).Treatment with proteasome inhibitors stabilizes the BZR1protein, suggesting that it is degraded by the UPS. Inaddition, the phosphorylated form of BZR1 appears to bethe substrate for proteolysis as its abundance is affectedmore significantly by the proteasome inhibitor treatmentthan the hypo/un-phosphorylated form of the protein (Heet al., 2002). But when BR levels rise, the membrane-bound BRI1 (Brassinosteroid insensitive1) and BAK1(BRI1-associated receptor kinase) leucine-rich repeatreceptor kinases somehow cause an accumulation of hypo-phosphorylated BES1 and BZR1 (He et al., 2002; Yinet al., 2002). Two mechanisms may contribute to thisoutcome. First, the receptors might signal to BIN2 todecrease its phosphorylation activity toward BES1 andBZR1. Second, the activity of the nuclear-localized BSU1(bri1 Suppressor1) phosphatase may also increase. BSU1can dephosphorylate BES1 in vitro and an activation-tagged line that expresses elevated levels of the BSU1protein shows an accumulation of the hypophosphorylatedform of BES1, demonstrating that this phosphatase alsoacts on BES1 in vivo (Mora-Garcia et al., 2004). As thelevels of dephosphorylated BZR1 and BES1 proteins risewithin the nucleus, they can turn on BR-responsive genes(reviewed in Vert et al., 2005). Although their phosphoryl-ation status was initially suggested to be a prime regulatorof BES1 and BZR1 protein accumulation, recent evidencesuggests that phosphorylation has a much more dramaticeffect on BES1’s ability to interact with other transcriptionfactors and to bind to and activate transcriptionfrom BR-sensitive promoters (Vert and Chory, 2006).

So, although BES1 and BZR1 may be relatively short-lived proteins, it does not seem that BR-mediated stabiliz-ation of these proteins is required for BR signalling.Nevertheless, identification of the E3 ubiquitin ligasesresponsible for BES1 and BZR1 degradation will contrib-ute to a fuller understanding of the BR signal transductioncascade.

Although the stability of the BIN2 kinase (Vert andChory, 2006) and BSU1 phosphatase (Mora-Garcia et al.,2004) are reported to be unaffected by brassinosteroidlevels, other components of the BR pathway may beregulated by the UPS. As seen in the auxin and abscisicacid signalling pathways, BR appears to regulate transcrip-tionally a RING E3 ubiquitin ligase. The BRH1(Brassinosteroid-responsive RING-H2) transcript is down-regulated by addition of brassinolide in a BRI1-dependentmanner. No putative substrates have been identified forthis E3 ligase, but it is interesting to note that it is tran-scriptionally up-regulated by the elicitor chitin, suggestinga potential and novel antagonistic link between BR andpathogen signalling that involves the UPS (Molnar et al.,2002).

Ethylene and the UPS

The small gaseous hormone ethylene regulates severalaspects of the plant life cycle including seed germination,flower development, flower senescence and fruit ripening.Ethylene is also produced in response to several types ofabiotic and biotic stresses, including pathogen attack(reviewed in Klee, 2004; Chen et al., 2005; van Loonet al., 2006). Thus, ethylene is a regulator both ofendogenous developmental programmes, and externallystimulated stress adaptations and plant defences. The UPSappears to function in all of these ethylene-mediatedprocesses.

Transcriptional changes resulting from altered ethylenelevels play a vital role in the ethylene signal transductionpathway. The EIN3 (Ethylene-insensitive 3) transcriptionfactor and its homologues act as positive regulators ofethylene responses by turning on downstream transcrip-tional regulators such as the ERF1 (Ethylene ResponseFactor) gene (Solano et al., 1998). Ethylene-dependentstabilization of EIN3 thus initiates an important transcrip-tional cascade (reviewed in Alonso and Stepanova, 2004).EIN3 abundance is controlled by an SCF E3 ubiquitinligase containing one of two related F-box substrate speci-ficity factors: EBF1 or EBF2 (EIN3-Binding F-box)(Potuschak et al., 2003; Gagne et al., 2004; Guo andEcker, 2004). These proteins interact with EIN3 and therelated EIL1 protein in a yeast-two-hybrid experiment, andEIN3 is pulled down by both GST:EBF1 and GST:EBF2in vitro (Potuschak et al., 2003). The ethylene precursorACC increases the stability of an EIN3:FLAG fusionprotein (Yanagisawa et al., 2003) and endogenous EIN3(Guo and Ecker, 2003) in arabidopsis seedlings, but it isunclear exactly how ethylene regulates this process.

Several upstream components in the ethylene signallingpathway, including the ETR/ERS receptor, and EIN2,EIN5 and EIN6 all need to be functional for EIN3 to



accumulate. In addition, the CTR1 kinase also contributesto EIN3 proteolytic regulation, because constitutivelyactive ctr1 mutants show elevated levels of EIN3. ButCTR1 cannot be the sole regulator of EIN3 stability, asaddition of exogenous ethylene still promotes EIN3accumulation even in a ctr1-1 mutant (Guo and Ecker,2003). One possible mode of regulation would be at thelevel of EBF abundance. This model would predict adecreased level of EBF protein as ethylene levels rise.But, interestingly, activation of the ethylene signallingcascade, through EIN3, actually induces EBF2, but notEBF1, transcription, perhaps to promote a negative feed-back loop to prevent excessive ethylene response(Potuschak et al., 2003). This differential transcriptionalregulation of EBF1 and EBF2 fits with the hypothesis thatEBF1 and EBF2 play slightly different roles in regulatingEIN3 stability. Analysis of mutant phenotypes suggeststhat EBF1 may be sensitive to lower levels of ethyleneand act as a constitutive damper on the ethylene pathway,whereas EBF2 may play a more significant role at higherethylene levels, and when the plant wants to shut down anacute ethylene response (Gagne et al., 2004). As the abun-dance of the EBFs does not seem to be inversely corre-lated with EIN3 stability, post-translational modification ofone or the other could influence their binding abilities.Given that these proteins interact both in yeast-two-hybridassays and in vitro, post-translational modification of EIN3or the F-boxes is unlikely to be a requirement for the inter-actions. However, it is possible that ethylene-mediatedmodification of EIN3 or the EBFs could disrupt theirbinding and thereby slow EIN3 degradation.

Although there is much interest in characterizing ethy-lene’s ability to modulate EIN3 stability, efforts are alsounderway to ascertain the relationship between sugar signal-ling and EIN3 proteolysis. EIN3 stability is subject to regu-lation by glucose through the hexokinase signallingpathway. Because ethylene and glucose often have antago-nistic effects on plant growth, it makes sense that glucoseactually promotes proteolysis of EIN3, which was demon-strated using Flag-, Myc- and GFP- tagged EIN3 proteinsin protoplasts and transgenic arabidopsis seedlings(Yanagisawa et al., 2003). Although glucose-mediated lossof EIN3 is severely impaired by treatment with proteasomeinhibitors, the specific components of the UPS that regulateEIN3 stability in response to glucose have not been iden-tified. Glucose could act through the EBF1/2-based system,or through a completely novel ubiquitin ligase.

EIN3 may not be the only transcription factor involvedin the ethylene response that is regulated at the level ofproteolysis. The ERF3 gene of tobacco encodes a trans-cription factor capable of repressing ethylene responses(Koyama et al., 2003). The mRNA level of its homologuein Nicotiana sylvestris, NsERF3, is increased by ethylene(Kitajima et al., 2000). Yeast-two-hybrid experimentsdemonstrate an interaction between the ERF3 protein andan E2 ubiquitin-conjugating enzyme NtUBC2 thatdepends on the region found between the conserved ERFdomain and the EAR-motif-containing repressor domainin ERF3. Interestingly, two ERFs from different subfam-ilies that possess transcriptional activation, as opposed to

repressive, capabilities do not interact with the UBC. Theyeast-two-hybrid truncated NtUBC2 clone capable ofinteracting with ERF3 was used to identify full-lengthNtUBC2 and a closely related protein designated NtUBC1.There is some evidence that NtUBC2 might regulateERF3. Transient co-expression of ERF3 and a putativedominant-negative form of NtUBC2 in tobacco cellsincrease ERF3’s ability to repress transcription, presum-ably owing to stabilization of ERF3 by the mutantNtUBC2 (Koyama et al., 2003). To date, it is unknownwhether this process is affected by ethylene or whether anE3 is required to bring UBC2 and ERF3 together in vivo.

Although there is evidence that ethylene is capable ofmodulating the degradation of components of its own sig-nalling pathway, the identification of the hss1 mutantdemonstrated that ethylene can also regulate the stabilityof proteins functioning in the auxin signalling pathway,providing another example of cross-talk occurring throughthe UPS. Both ethylene and auxin play roles in regulatingapical hook development in dark-grown seedlings.One protein required for this developmental programmeis HOOKLESS1 (HLS1). HLS1 encodes a putativeN-acetyltransferase that is transcriptionally up-regulated byethylene and is required for apical hook maintenance(Lehman et al., 1996). It also appears to mediate cross-talkwith the auxin signalling pathway as a mutation in ARF2(Auxin response factor2) can suppress many aspects of thehls1 phenotype. ARF2 is a member of a family of tran-scriptional regulators that bind to auxin response elements(AuxREs) in gene promoters (reviewed in Guilfoyle et al.,1998; Okushima et al., 2005), and recent studies posit thatARF2 is a negative regulator of cell division and organgrowth (Schruff et al., 2006). Although ARF2 transcriptlevels do not change in the hls1 mutants, the level ofthe ARF2 protein rises, suggesting that HLS1 normallynegatively regulates ARF2 accumulation. Consequently,increased levels of ethylene sufficient to increase HLS1levels cause a concomitant drop in ARF2 protein levels(H. Li et al., 2004). Ethylene’s ability to modulate ARF2levels depends on the presence of the HLS1 protein, andon a functional proteasome as treatment with the MG132proteasome inhibitor blocks ACC-mediated loss of ARF2.

Light also affects the accumulation of HLS1 and thusARF2. Even in the presence of exogenous ACC, light candown-regulate the levels of the HLS1 protein, which iscorrelated with a subsequent rise in ARF2 protein levels.It is unclear both how light affects HLS1 protein abun-dance, and how HLS1 lowers the stability of ARF2. Thetransfer of an acetyl group to a substrate by HLS1 couldaffect ARF2 proteolysis, but based on several experiments,ARF2 does not appear to be its substrate (H. Li et al.,2004). However, other components of the degradationmachinery could be regulated by this post-translationalmodification and their identification should lend insightinto UPS-mediated integration of light, ethylene and auxinsignalling in apical hook maintenance.

Rising ethylene levels certainly lead to UPS-mediatedchanges in transcription, but several lines of evidencedemonstrate that ethylene production itself is governed inpart by the UPS. Evidence for CRL-based regulation of



ethylene production arose from two distinct lines ofresearch. From the UPS side of the story, post-transcriptional gene silencing of the closely related RUB1and RUB2 proteins, which modify CRLs (see above) andregulate their activity, causes overproduction of ethylenein dark-grown arabidopsis seedlings (Bostick et al., 2004).And mutation of the RUB-conjugating enzyme RCE1also increases ethylene production in dark-grownseedlings (Larsen and Cancel, 2004), again suggesting thatRUB conjugation to one or more CRLs negatively regu-lates some aspect of ethylene production. Despite theexistence of hundreds of unique CRLs in arabidopsis thatcould be affected by the rubylation pathway, one candidateemerged from independent studies of plants with defectsin regulation of ethylene synthesis, namely EthyleneOverproduction (ETO) mutants. The eto1-1 mutationdisrupts a protein with a BTB domain capable of bindingto CUL3 and acting as the substrate recognition subunit ina CUL3-based E3 ubiquitin ligase (Wang et al., 2004).

ACC synthase (ACS) isozymes are probable substratesfor this CUL3ETO1 E3 ligase. The abundance of ACS bio-synthetic enzymes largely governs ethylene productionlevels (reviewed in Kende, 1993; Chae et al., 2003).Changes in ACS transcript levels have been shown toaffect ethylene levels (Bleecker and Kende, 2000), butpost-translational regulation is also evident for at leastsome ACS family members found in tomato (Solanumlycopersicum) and other plants (Kim and Yang, 1992).ETO1 and related family members ETO-LIKE1 and 2(EOL1, 2) can bind to ACS5 in yeast-two-hybrid assays.Two other ethylene overproduction mutants, eto2 andeto3, bear mutations in the ACS genes ACS5 and ACS9,respectively (Vogel et al., 1998; Chae et al., 2003). Botheto2-1/acs5 and eto3-1/acs9 mutants possess mutationswithin their C-termini. Mutant eto2-1/acs5 protein islonger-lived than wild-type ACS5. This increased stabilitydepends upon the ETO1 protein (Chae et al., 2003).Whereas wild-type HA:ACS5 can be pulled down fromplants by wild-type RGS:His-tagged ETO1, a mutant formof HA:acs5/eto2-1 does not interact with this protein,suggesting that this mutant protein remains stable becauseit escapes detection and ubiquitylation mediated by ETO1.In addition, ETO1 interaction with ACS5 inhibits ACSactivity, suggesting an additional mode of regulation(Wang et al., 2004).

ACS5 appears to be another point of convergence fortwo hormone signalling pathways because cytokinin treat-ment of arabidopsis etiolated seedlings leads to ethyleneoverproduction and to an increase in the half-life of anMyc:ACS5 fusion protein (Chae et al., 2003). These find-ings allow the development of a general model in which aCUL3-based ubiquitin ligase containing the ETO1 adaptorprotein binds to the C-terminus of ACS5 and targets it forubiquitylation and degradation. However, at least twomajor questions remain to be answered.

First, ethylene levels change during many developmen-tal processes and in response to external stimuli. Thisimplies that ETO1-based ACS5 degradation should beslower under conditions that promote ethylene synthesis,but there is no definitive explanation for this phenomenon.

Post-translational modification of ACS5 may regulate itsinteractions with ETO1 or affect components of the UPS.A tomato ACC synthase, ACS2 (Tatsuki and Mori, 2001),undergoes phosphorylation in its C-terminus by awound-inducible calcium-dependent protein kinase. If thisdisrupted interactions between the ACS and ETO1,thereby stabilizing the enzyme, it could contribute towound-induced ethylene production (Tatsuki and Mori,2001). Although neither tomato ACS2 nor arabidopsisACS5 have predicted consensus CDPK phosphorylationsites, experiments performed using synthetic peptidesdemonstrate that they both contain a non-canonical CDPKtarget site in their C-termini. Furthermore, a comparisonof peptides derived from the C-terminus of arabidopsisACS5/9 (which are identical in this region) containing thewild-type sequence or the equivalent of an eto3 mutationshowed that the latter is a better substrate for CDPKI frommaize (Zea mays) (Hernandez Sebastia et al., 2004), pro-viding some support for a model in which increasedCDPK-dependent phosphorylation of acs mutants blocksETO1 binding, and hence ubiquitylation. However, thereis no definitive evidence of ACS5 or ACS9 phosphory-lation in arabidopsis to date.

There is evidence that other kinases can play a role inregulating the stability of different ACS family members.SIPK, a tobacco MAPK that is induced by numerousstimuli including drought, osmotic stress and UVirradiation, also increases ethylene signalling by increasingACS activity (Kim et al., 2003). Examination of AtMPK6,the SIPK orthologue, indicates that this MAPK contributesto increased ethylene production in arabidopsis, as well.No putative MAPK phosphorylation sites exist in ACS5 orACS9, although they are found in the C-terminus of ACS2and ACS6. Accordingly, plant-derived MPK6 can phos-phorylate recombinant ACS2 and ACS6, but not ACS5.Conditions that activate MPK6 increase the levels ofFLAG-tagged ACS6 in arabidopsis seedlings, and a phos-phomimic ACS6 mutant (with serine to aspartate substi-tutions) accumulates even in the absence of elevatedMPK6 activity (Liu and Zhang, 2004). It will be interest-ing to determine whether specific kinase/ACS combi-nations that regulate ethylene production all go throughETO1 and its homologues, or if other specific E3 ligasecomponents are involved.

Another outstanding question concerns cytokinin’sinfluence on ACS5 stability. Although the C-termini of theACSs appear to be hot spots for regulatory modifications,cytokinin-mediated stabilization of ACS5 cannot be solelyexplained by post-translational modification of theC-terminus. The mutant eto2 protein lacks the proper final12 amino acids of the protein, including the conservedserine phosphorylated by CDPK (Chae et al., 2003).Nevertheless, eto2 is still stabilized by cytokinin, prompt-ing a search for some novel mode of cytokinin-mediatedchanges in ACS5 accumulation. One of the first prioritiesmight be to determine whether an ETO1-based E3 ligaseor other components of the UPS system are involved incyokinin’s control of ACS5 longevity.

Once a plant produces an appropriate amount of ethy-lene based on its developmental context and ambient



conditions, the hormone needs to be perceived to regulatedownstream signalling steps, such as gene transcription(see above). In arabidopsis, there is a family of five recep-tors that contribute to ethylene perception (reviewed inAlonso and Stepanova, 2004). Analysis of several mutantforms of the ethylene receptors suggested that their stab-ility might also be regulated in a post-transcriptionalmanner (Zhao et al., 2002). For example, four dominantmutations in ETR1 that confer ethylene insensitivity causehigher accumulation of the ETR1 receptor in dark-grownseedlings. Further analysis of etr1-1 revealed that itsmRNA levels are not elevated relative to wild-type ETR1mRNA, indicating that the elevated levels of etr1-1 proteinresult from a post-transcriptional change. ETR1 proteinlevels are not augmented in ein2 or ein3 mutants,suggesting that reduced ethylene sensitivity per se is notthe causative agent of ETR1 stabilization. Intriguingly,addition of silver nitrate, which renders the receptorsinsensitive to ethylene, also increases the accumulation ofthe wild-type ETR1 protein, but does not further increasethe stability of the ethylene-insensitive forms of ETR(Zhao et al., 2002). Given that some animal hormonereceptors show accelerated degradation in the presenceof their binding hormone, it is possible that theethylene-insensitive forms of ETR1 that cannot bind thehormone have a lower rate of degradation.

Jasmonates and the UPS

Jasmonates (JAs), derived from linolenic acid (reviewedin Turner et al., 2002), like ethylene, function in normaldevelopmental pathways, but also play a crucial role inallowing plants to mount a defence to biotic challenges.JA affects processes such as pollen development and fruitripening, and also promotes resistance to insects andpathogens (reviewed in Creelman and Mullet, 1997).

The first indication of UPS involvement in jasmonatesignalling came with the identification of the coronatineinsensitive1 (coi1) mutant. coi1 plants are insensitive tothe JA-like compound coronatine, as well as to methyljasmonate (MeJA) (Feys et al., 1994). COI1 encodes anF-box protein and both tagged and native forms of COI1can interact with ASK1, ASK2, RBX1 and CUL1 inplanta, suggesting that COI1 assembles into an SCF-typeE3 ubiquitin ligase (Devoto et al., 2002; Xu et al., 2002).Four subunits of the CRL-modifying COP9 signalosomecan also be co-immunoprecipitated with FLAG-taggedCOI1 (Feng et al., 2003). JA-related phenotypes observedin plants with mutations in UPS components bolster theclaim of its involvement in JA signalling. For instance,RNAi-mediated knock-down of RBX1 reducesMeJA-dependent induction of genes such as Allene OxideSynthase (AOS) (Xu et al., 2002), and a mutation in AXR1impairs MeJA-dependent inhibition of root growth(Tiryaki and Staswick, 2002). In addition, the jai4 mutantobtained through a screen for JA-insensitive mutants in anein3 background (Lorenzo et al., 2004) has a mutation inthe SGT1b protein (Lorenzo and Solano, 2005). As homo-logues of SGT1 in barley (Azevedo et al., 2002) andtobacco (Y Liu et al., 2002) interact with components of

the SCF and the COP9 signalosome, the loss of JA sensi-tivity in a jai4/sgt1b mutant further links JA signalling toSCF-mediated processes.

Despite the demonstrated importance of COI1 andUPS-related proteins in JA signalling, no substrates of theSCF-COI1 E3 ligase have been conclusively identified todate. The RPD3b/HDAC6 histone deacetylase interactswith COI1 in yeast-two-hybrid assays and in planta,suggesting that it could be a substrate for COI1-mediatedubiquitylation and degradation (Devoto et al., 2002),although further work will be required to substantiate thishypothesis. Over-expression of the related HDAC19 inarabidopsis enhances expression of the JA-responsive PR(Pathogenesis-Related) genes Basic Chitinase andBeta-1,3-glucanase (Zhou et al., 2005). This would implythat if HDAC6 and/or HDAC19 are substrates ofCOI1-mediated proteolysis, then JA should reduce theinteraction between COI1 and the deacetylases to promoteJA-mediated chromatin modification and induce PR geneexpression. Another potential substrate forCOI1-dependent ubiquitylation, the small subunit 1b ofRubisco, was also pulled out of a yeast-two-hybrid screenfor COI1-interactors (Devoto et al., 2002). Previousstudies demonstrated MeJA-stimulated degradation ofRubisco, and indicated that this might be part ofMeJA-induced leaf senescence (reviewed in Parthier,1990), but no further work has been done to determinewhether the Rubisco subunit is subject to COI1-mediateddegradation.

Plant defence and the UPS

Plants consistently face challenges from a wide array ofbiotic stresses, including insect herbivoury, and fungal,bacterial and viral attacks. Both specific and general, andoften overlapping defences are mounted in response toeach type of biological onslaught, and increasing evidencepoints to the participation of the UPS in these variedforms of defence.

Regulated proteolysis of endogenous or pathogen-produced proteins can contribute to multiple levels ofplant defence. First, the ‘basal defence’ system allowsplants to recognize and respond to a broad range of poten-tially pathogenic (non-host) organisms based on thepathogen-associated molecular patterns (PAMPs) theypresent to the plant. For instance, the fungal cell wall com-ponent chitin and bacterial flagellin proteins both stimulateseveral protective measures, such as callose deposition(Gomez-Gomez et al., 1999) and oxidative bursts (Felixet al., 1999) in many plant species (for references seeM.G. Kim et al., 2005). However, plants also have heigh-tened abilities to respond to particular (host) pathogensthrough the gene-for-gene, or R-mediated resistance path-ways. In this system, a pathogen may evolve a factor, oftena protein or peptide, that specially enhances its patho-genesis on specific ‘host’ plants. If a plant protein evolvesto counteract this pathogenic factor, the plant will be ableto prevent pathogenesis of this specific invader by initiat-ing resistance responses often accompanied by localizedcell death through the hypersensitive response (HR).



In this case, the pathogenic factor is called an avirulence(Avr) factor and the plant defence protein is termed an R(resistance) protein. If a plant targeted by the pathogenwith a particular Avr lacks the cognate R gene, the plantwill be susceptible to attack.

In addition to the immediate and local consequences ofbasal and R-mediated defence signalling, pathogen attackalso prompts the development of systemic acquired resis-tance (SAR), which renders a plant less susceptible tosubsequent attacks by a variety of organisms, includingfungi, bacteria and viruses (reviewed in Durrant and Dong,2004). Typically, tissue damage caused by necrotrophicpathogens or the induction of an HR triggers increases inthe levels of salicylic acid (SA). This prompts local anddistal signalling events, largely through the activity of theNPR1 (non-expresser of PR genes) regulatory protein, as itstimulates expression of many Pathogenesis-Related (PR)genes. A similar NPR1-dependent but SA-independentsuite of responses, termed induced systemic resistance(ISR), is stimulated by non-pathogenic root-colonizingbacteria, but also heightens a plant’s ability to fend offfuture attacks from fungi and bacteria (reviewed in Feysand Parker, 2000; Durrant and Dong, 2004).

Ongoing studies of plant defence continue to uncovercomplex interactions between basal defence, R-mediateddefence and systemic defence responses. Some recentexperiments have specifically led to interesting modifi-cations of the original gene-for-gene (R-protein-based)model and highlight new links between it and basaldefence. First, the ‘guard’ hypothesis (Van Der Biezenand Jones, 1998; Dangl and Jones, 2001) posits thatR-mediated resistance is not necessarily initiated through adirect interaction between an Avr factor and its cognate Rprotein. Rather, R proteins may survey the status of thedirect targets of the Avr factor. For example,Pseudomonas syringae directly secretes the AvrRpm1protein into plant cells, where AvrRpm1 induces phos-phorylation of the RIN4 protein. The plant RPM1R-protein functions as the guard. It does not directly inter-act with AvrRpm1, but rather recognizes RIN4 phos-phorylation as a sign of P. syringae attack and stimulatesthe hypersensitive response (Mackey et al., 2002). Second,mounting evidence points to cross-talk and shared signal-ling between R-mediated and basal defences. For example,several studies highlight the ability of pathogens to try tosubvert basal defence mechanisms (Jakobek et al., 1993;Hauck et al., 2003; Keshavarzi et al., 2004) and some Rproteins may recognize and fight back against this tactic(e.g. M.G. Kim et al., 2005). For basal, R-mediated andsystemic defence mechanisms, there is a growing recog-nition that UPS-mediated events may be important factorsboth in plant defence and in pathogen virulence pro-grammes (reviewed in Zeng et al., 2006).

Convergence of hormone signalling, plantdefence, and the UPS

As previously mentioned, both the ethylene and thejasmonic acid signalling pathways contribute to plantdefence. As highlighted above, the UPS appears to be

required for various steps in these signal transduction cas-cades that impact plant defence signalling. For example,ethylene synthesis increases in response to several types ofbiotic challenges [e.g. bacteria, fungi (reviewed inBroekaert et al., 2006), and insects (Kahl et al., 2000)].It is interesting to note that ethylene does not alwaysimprove disease resistance and actually seems to becorrelated with enhanced disease progression in manycircumstances (reviewed in Broekaert et al., 2006; vanLoon et al., 2006), but in either case, the level of ethyleneproduced can affect plant susceptibility, and ethylenebiosynthesis is subject to UPS-mediated regulation.

Furthermore, ethylene affects the transcription ofnumerous defence-related genes. For instance, in arabi-dopsis, ethylene up-regulates many genes encodingcell-wall-modifying enzymes or protein components, oxi-dative burst regulators, and PR proteins such as b-1, 3-glucanase and osmotin (Zhong and Burns, 2003). Allknown ethylene signalling to date has been shown toproceed through the EIN3 family of transcription factors(van Loon et al., 2006) and as these are substrates of theUPS, proteolytic regulation must affect transcription ofthese defence-related genes. Interestingly, many of thesegenes, especially those in the PR class, are also regulatedby JA (reviewed in van Loon et al., 2006) as both JA andethylene can modulate the transcription of ERF/AP2 tran-scription factors that function downstream of the EIN3family (reviewed in Guo and Ecker, 2004). Initial identifi-cation of this co-ordinated regulation of ERF1 in arabi-dopsis demonstrated that over-expression of ERF1 couldrestore defence responses in a coi1 (F-box protein) mutant,indicating that perturbation of UPS function in the JAsignalling pathway could also compromise defence geneexpression (Lorenzo et al., 2003). The observation thatplants with reduced signalosome levels ( fus6/csn1-11)lose JA-stimulated induction of PR genes such as PDF1.2further substantiates the link between JA, the UPS anddefence-related gene expression (Feng et al., 2003).

In tomato, additional evidence of the requirements forCOI1 in defence against herbivores arose from the identifi-cation of the jai1/coi1 mutant. Methyl jasmonate (MeJA)is no longer able to induce expression of the anti-herbivoryserine proteinase inhibitor (PI-II) in jai1/coi1 plants, butgene induction is restored by introduction of a 35S:SlCOI1transgene. Glandular trichome development and monoter-pene production are also compromised in the jai1/coi1mutants and most likely contribute to the severely reducedresistance to spider mites. Fecundity of female spidermites, elevated on jai1/coi1 leaves, is also restored towild-type levels in 35S:COI1-complemented mutants(L. Li et al., 2004).

General UPS components in plant defence

Clues as to the importance of targeted protein degra-dation in defence mechanisms can be gleaned from ananalysis of general UPS components that display alteredexpression levels in response to biotic stresses promptedboth by non-host and by host-specific pathogens.However, thorough explanations of underlying



mechanisms and causal relationships are lacking. At avery gross level, the finding that tomato mosaic virus(ToMV), tobacco mosaic virus (TMV), wounding, ACC(ethylene precursor), JA and SA all up-regulate twoubiquitin-activating enzymes (NtUBA1 and NtUBA2) intobacco suggests that plants increase their ubiquitylationcapacity when faced with an attack (Takizawa et al.,2005). Up-regulation of additional general components ofthe UPS has been observed in other plant/pathogen inter-actions. For instance, sunflower (Helianthus annuum)hypocotyls accumulate higher levels of ubiquitin tran-scripts following challenge with an incompatible (aviru-lent) strain of powdery mildew, but not with a compatiblestrain (Mazeyrat et al., 1999). A glucan fungal elicitorfrom Phytophthora megasperma also causes up-regulationof ubiquitin transcripts in soybean cells (Levine et al.,1994). A ubiquitin-conjugating enzyme OsUBC5b (but notits homologue OsUBC5a) also shows elevated levels oftranscripts following treatment of rice cells with the fungalelicitor N-acetylchitoheptaose (Takai et al., 2002).Transcript levels for two UBCs are also up-regulated inNicotiana attenuata during herbivorous attack by theinsect Manduca sexta (Hui et al., 2003). And, at the pro-teolytic end of the UPS pathway, three specific 20S protea-some subunits are transcriptionally induced by theproteinaceous fungal elicitor cryptogein in tobacco (Dahanet al., 2001). Although changes in levels of ubiquitin, E2enzymes and proteasome subunits might be predicted tohave broad effects on a plant cell’s level of ubiquitin-mediated proteolysis, scientists seek to identify how thesechanges affect specific host–pathogen interactions.

Specific E3 ligase components in plant defence

Efforts to find pathogen-responsive genes have unco-vered several E3 ligases with mRNA levels affected by anumber of different general elicitors and avirulence factors(reviewed in Zeng et al., 2006) and genes encoding UPScomponents have surfaced in screens for mutants withaberrant pathogenesis-related phenotypes. Both lines ofresearch support a role for the UPS in the convergenceof both basal and specific defence strategies, as well assystemic responses.

CRL components. NPR1 emerged from a screen formutants lacking SA-responsive induction of a PR reportergene (Cao et al., 1994). The initial npr1 mutant failed toachieve SAR following treatment with SA, or an analogue,INA (2,6-dichloroisonicotinic acid), and remained highlysusceptible to P. syringae pv. maculicola. This npr1mutant could still mount an HR following treatment withan avirulent strain of P. syringae but showed defects inavirulent pathogen-mediated induction of SAR as well(Cao et al., 1994). Additional alleles of npr1/nim1/sai1,as well as further molecular and physiological experimentsusing NPR1/NIM1/SAI1, demonstrate the crucial roleplayed by this protein in SAR, ISR and other defencepathways (reviewed in Dong, 2004). The NPR1 proteincontains ankyrin repeats, implicated in protein–proteininteractions (reviewed in Michaely and Bennett, 1992), as

well as a BTB/POZ domain (Aravind and Koonin, 1999),implying that it could potentially interact with aCUL3-based CRL as well as other proteins, such as sub-strates for ubiquitylation, simultaneously. Yeast-two-hybrid assays do not point to strong binding betweenNPR1 and CUL3a or CUL3b (Dieterle et al., 2005), butsome NPR1 interactions observed in planta cannot berecapitulated through this experimental technique (Despreset al., 2003). Several studies focusing on NPR1’s effect onpathogen-responsive transcription suggest that in responseto SA, and redox-mediated conformational changes, NPR1moves into the nucleus to interact with TGA transcriptionfactors to turn on PR genes (reviewed in Dong, 2004). Ananalysis of several TGA transcription factors indicates thatthey undergo differential, proteasome-dependent proteol-ysis (Pontier et al., 2002). There is some precedent forubiquitin-mediated activation, followed by degradation oftranscription factors in other systems (Salghetti et al.,2001), and it will be interesting to determine if NPR1participates in a similar process or affects SAR and plantdefence in any other UPS-dependent manner, for exampleby degrading repressors of SAR induction.

SAR-related investigations have also identified theSON1 F-box protein through a mutant screen for supp-ressors of nim1/npr1-related susceptibility in arabidopsis(Kim and Delaney, 2002). SON1 is hypothesized to act asa negative regulator of plant defence responses, becauseson1 nim1-1 mutants are less susceptible to inoculationwith the Peronospora parasitica oomycete (downymildew) or P. syringae than nim1-1 alone, and becausesusceptibility, as measured by downy mildew sporulation,can be restored by constitutive expression of SON1.Interestingly, the increased resistance conferred bymutation of son1 in an npr1/nim1-1 background is notachieved through restoration of PR protein induction, nordoes it depend on salicylic acid. And it also does notresult from up-regulation of the JA-responsive defensingene, PDF1.2. However, son1 mutants express constitu-tively several PR transcripts in the presence of the wild-type NPR1/NIM1 protein. This gives rise to the hypo-thesis that the SON1 F-box protein normally suppressesNPR/NIM1-mediated induction of PR genes in theabsence of an elicitor, but that it may also take part insome sort of SA-independent plant defence response (Kimand Delaney, 2002). SON1 transcript levels do not appearto change in response to pathogenic attack nor after treat-ment with a salicylic acid analogue, but it is possible thatpost-translational modifications to SON1 or its substratesreduce SON1’s activity following a biotic challenge,thereby enabling the plant to mount a successful defence(Kim and Delaney, 2002).

In an effort to understand R-mediated/gene-for-generesistance pathways better, up-regulated mRNA sequenceswere identified in tobacco cells carrying the Cf9 resistance(R) gene after inoculation with the Avr9 elicitor of the bio-trophic fungus Cladosporum fulvum. cDNA clones forseveral of these ACRE (Avr/Cf9 Rapidly Elicited) geneswere used for further analysis (Durrant et al., 2000).ACRE189 encodes an F-box protein, providing evidencethat SCFCOI1 and SCFEBF1/EBF2 may not be the only the



SCF complexes involved in pathogen defence (Rowlandet al., 2005). Viral-induced gene silencing of ACRE189diminishes the ability of tobacco leaves to generate a Cf4-and a Cf9-mediated hypersensitive response. These datasuggest that SCFACRE189 may act downstream of at leasttwo different R proteins during incompatible interactions(Rowland et al., 2005).

Additional components of multiple CRLs, or factorsthat modulate CRL activity, have surfaced as contributorsto plant defence. Increased transcription of SKP1 andSGT1, two potential components of many SCF-type E3ligases (see above), occurs in pepper (Capsicum annuum‘Bukang’) following inoculation with an incompatiblestrain of Xanthomonas bacteria (Chung et al., 2006). Infact, sgt1b mutants were found based on their compro-mised disease resistance (Austin et al., 2002; Tor et al.,2002). Arabidopsis plants with two different mutations inaxr1, which functions to activate many SCFs and perhapsall CRLs in vivo (see above), show reduced resistance tothe opportunistic fungus Pythium irregulare (Tiryaki andStaswick, 2002). All these data together reinforce themodel that CRLs contribute to plant defence.

Interestingly, the F-box proteins that function in auxinsignalling are also subject to transcriptional regulationduring pathogen attack via induction of a microRNA(Navarro et al., 2006). The P. syringae general flagellinelicitor induces accumulation of the miR393a transcript inarabidopsis. This microRNA then promotes cleavage ofTIR1, AFB2 and AFB3 transcripts, causing reduced levelsof these F-box proteins. An AXR3NT:GUS reporterprotein is stabilized under these conditions, and theaccumulation of Aux/IAA repressors following flg22inoculation presumably contributes to the reduced induc-tion of several auxin-responsive genes. This inhibition ofauxin signalling appears to enhance resistance to virulentpathogens as overexpression of AFB1:Myc, which shouldnot be cleaved by the microRNA, can increase suscep-tibility of arabidopsis plants to a virulent strain ofP. syringae. But the auxin response probably does notimpact R-mediated signalling, as overexpression ofAFB1:Myc does not influence interactions with an aviru-lent strain of P. syringae carrying the AvrRpt2 gene. Thesestudies reveal that bacterially induced suppression ofTIR1/AFB-mediated degradation and auxin signallingincreases plant resistance to virulent P. syringae by modu-lating the abundance of specific host UPS components(Navarro et al., 2006).

Disruptions in the auxin signalling pathway also appearto affect arabidopsis resistance to TMV infection(Padmanabhan et al., 2005, 2006). IAA26 and IAA27, butnot several other Aux/IAA family members, interact withthe TMV replicase protein in yeast-two-hybrid assays(Padmanabhan et al., 2006), and this interaction wasconfirmed in vitro for IAA26 (Padmanabhan et al., 2005).Wild-type TMV can induce symptoms on arabidopsis,ecotype Shahdara, but the severity of specific phenotypesis reduced when the plants are inoculated with a mutantversion of TMV (TMV-V1087I). This has a mutantreplicase that spreads and accumulates normally withinarabidopsis but shows diminished interaction with IAA26

in yeast-two-hybrid assays, suggesting that binding of thereplicase to IAA26 is important for symptom development(Padmanabhan et al., 2005). Interestingly, co-transfectionof IAA26:GFP and wild-type TMV results in a reducedpercentage of cells with visible levels of fluorescence,suggesting that TMV could potentially destabilize IAA26(Padmanabhan et al., 2005) and thus stimulate auxinsignalling. In fact, a microarray analysis of TMV-infectedplants shows that approx. 30 % of the transcriptionallyaltered genes lie downstream of auxin response elements(Golem and Culver, 2003; Padmanabhan et al., 2005).However, there is additional evidence that interactionswith the replicase prompt fluorescently tagged forms ofIAA26 and IAA27 to relocalize from the nucleus to thecytoplasm (Golem and Culver, 2003; Padmanabhan et al.,2005, 2006). This could also compromise their ability toperform their normal transcriptional regulatory roles in thenucleus, and could reduce the visibility of GFP- andDsRed-tagged versions of the proteins as they diffuse inthe cytoplasm. So although there is evidence that TMVpromotes movements of IAA26 and IAA27 within thecell, the question of whether TMV also induces theirdegradation during the infection process remains open. Ifthis is the case, additional work will be required toascertain if the well-characterized SCFTIR1/AFBx complexor other E3 ubiquitin ligases contribute to this proteolyticprocess.

Non-CRL RING proteins. Although many RING domain-containing proteins exist in plants, only a small subset hasbeen shown to be transcriptionally up-regulated by bioticstress to date. Nevertheless, initial functional characteriz-ation of some of these putative E3 ligases suggests thatthey do play an important role in plant defence (reviewedin Zeng et al., 2006). For example, levels of ATL2 andATL6 mRNAs rapidly rise in arabidopsis in response tochitin, a basal defence elicitor associated with fungal cellwalls and insect exoskeletons. These genes encode tworelated RING E3 ligases (Salinas-Mondragon et al., 1999).The BRH1 RING protein, as mentioned above, also showsincreased transcription following treatment with a chitinelicitor (Molnar et al., 2002). In a separate study, twoforms of chitin spur mRNA accumulation of the arabi-dopsis At2g35000 gene (Ramonell et al., 2005), whichencodes a RING domain-containing protein active in invitro E3 ligase assays (Stone et al., 2005). Subsequentpowdery mildew inoculation causes increased diseasesymptoms in three independent lines with a T-DNA inser-tion in At2g35000, demonstrating the importance of thisgene in plant immune responses (Ramonell et al., 2005).The rice EL5 (elicitor-induced) RING E3 ligase isup-regulated by a different fungal elicitor. Functionalanalysis of this protein indicates that it possesses ubiquity-lation activity in vitro when coupled with the OsUBC5a orOsUBC5b E2 conjugating enzymes. Interestingly, asdescribed above, the latter E2 is up-regulated by the sameelicitor, suggesting that these might function in the samespecific response pathway, but their substrate is not known(Takai et al., 2002). The general bacterial flagellin peptideelicitor (flg22), known to stimulate basal defence



responses (Felix et al., 1999), prompts greater than a2.5-fold change in the levels of over 250 transcripts (outof a population of 8200 examined) in arabidopsis cellcultures and seedlings. Among those, ten RING fingerputative E3 ligases are up-regulated, including RHA3b,RHA1b, RMA1 and ATL6 (Navarro et al., 2004).

RING domain-containing proteins have also surfaced inscreens for gene-for-gene resistance-initiated transcrip-tional responses. For instance, the screen for Avr9-respon-sive transcripts that uncovered the F-box proteinACRE189 also identified the RING E3 ligase ACRE132.This protein is most closely related to the ATL2 protein ofarabidopsis, implying functional conservation in fungalresponse pathways (Durrant et al., 2000).

Curiously, one protein with potential RING- and/orSCF-related function appears to affect plant resistance.Studies have also shown that SIP (Siah-InteractingProtein) from arabidopsis is transcriptionally induced byinoculation with P. syringae, as well as after woundingand hydrogen peroxide application (Kim et al., 2006). SIPappears to play a positive, though relatively minor, role inplant defence given that sip mutants show slightlyincreased susceptibility to this pathogen, whereas SIPoverexpressers are somewhat more resistant to P. syringae.SIP is 21 % identical to SGT1b, but it is also 32 % identi-cal to the human SIP protein. Human SIP functions in anatypical E3 ligase (Matsuzawa and Reed, 2001). It canbind to human Skp1, but it can also bind to the Siah-1 andSiah-2 RING-based E3 ligases (similar to SINAT5 inarabidopsis). In humans, beta-catenin can be degradedwhen it binds to the Ebi F-box protein. Ebi1 binds toSkp1, but then, rather than connecting to CUL1, links tothe Siah E3 ubiquitin ligases through SIP. AtSIP does notinteract with SINAT5 in yeast-two-hybrid assays, but notests have yet been done to ascertain if it interacts withany of the four other arabidopsis SINAT family membersor the ASK proteins (Kim et al., 2006). Therefore, theproteolytic function of arabidopsis SIP remains obscure,but its noted impact on plant defence leaves it well-poisedto link further the UPS to plant resistance pathways.

U-box proteins. Recent results also reveal a prominentrole for ARM-repeat-containing U-box E3 ligases in plantdefence. In arabidopsis, 41 proteins contain these twodomains (Mudgil et al., 2004) including PHOR1, impli-cated in GA signalling (see above). In these proteins, theU-box functions as the E2 interaction domain and theARM repeat protein–protein interaction domain mightcontribute directly or indirectly to substrate recognition.

At least two U-box/ARM repeat E3 ligases were pulledout of the screen for ACRE genes (Durrant et al., 2000).ACRE276, found in tobacco and tomato, appears to berequired for Cf4- and Cf9-stimulated hypersensitiveresponse because after virus-induced gene silencing ofACRE276, tomatoes have increased susceptibility toC. fulvum leaf mould (Yang et al., 2006). Loss of thisprotein also compromises the plants’ ability to generatethe N-mediated hypersensitive response following treat-ment with the p50 elicitor of TMV. In this gene-for-geneinteraction, the N TIR-NBS-LRR protein acts as a

resistance factor against the Avr-like p50 elicitor fromTMV. A GST:ACRE276 U-box protein does possess E3ligase activity in vitro, as does a close homologue fromarabidopsis, PUB17, and this activity seems to be requiredfor the hypersensitive response based on experimentsperformed with mutant versions of the PUB17 proteinexpressed in tobacco (Yang et al., 2006).

Several other U-box/ARM-repeat-containing proteinsare induced by elicitors in other plants and appear toparticipate in the plant defence response in both basal andR-mediated pathways. SPOTTED LEAF11 (Spl11) isinduced in both a susceptible and a resistant cultivar ofrice following inoculation with rice blight and may func-tion in basal defence. It encodes a functional E3 ligasebased on in vitro assays. As the plants with mutated spl11exhibit a lesion mimic phenotype, it is hypothesized thatSPL11 normally degrades a positive regulator of thehypersensitive response and programmed cell death (Zenget al., 2004) (Fig. 2B).

In parsley (Petroselinum crispum) the Pep25 fungalpeptide elicitor leads to rapid rises in the levels of thePcCMPG1/ELI17 (Cys-Met-Pro-Gly1/Elicitor-activatedgene) transcript, which encodes a U-box/ARM protein.And when elements from the PcCMPG1 promoter areplaced upstream of GUS and transformed into arabidopsisplants, GUS expression is increased at the site of infectionwhen plants are inoculated using both fungal and bacterialgeneral (flg22) and specific elicitors. Wounding alone,however, is insufficient to prompt GUS expression (Kirschet al., 2001). Two arabidopsis homologues of this protein,AtCMPG1/PUB20 and AtCMPG2, display similarly rapidtranscriptional induction by a fungal pathogen elicitor andby P. syringae (Heise et al., 2002). Further analysis of twohomologues of PcCMPG1 from tobacco (NtCMPG1/ACRE74) and tomato (SlCMPG1) affirmed that theseU-box/ARM proteins are transcriptionally up-regulated inresponse to a pathogen, and that NtCMPG1 possesses E3ubiquitin ligase activity in vitro. However, unlike SPL11,the CMPG1 protein appears to function as a positive regu-lator of the hypersensitive response, as tomato and tobaccoplants with reduced levels of CMPG1 display reducedhypersensitive responses following treatment with severalelicitors (Gonzalez-Lamothe et al., 2006). Two additionalU-box/ARM putative E3 ligases in arabidopsis, PUB12and PUB5, are also induced by the flagellin (flg22) elicitor(Navarro et al., 2004).

Overall, these studies suggest that plants increase theirtranscription of numerous E3 components when chal-lenged by pathogens, and highlight that among all of thevarious kinds of ubiquitin ligases, U-box/ARM E3 ligasesare over-represented in screens for pathogen-responsivetranscripts relative to their total number. In addition, theseexperiments support the idea that multiple pathogen per-ception systems converge on common downstream UPSelements, since, in many cases, both general elicitors andAvr-type specific elicitors up-regulate the same genes. Forinstance, a formal analysis identified homologues of thetomato Avr9-responsive (ACRE) genes in arabidopsis, andthen determined that many of the homologues, such asPUB17, are also up-regulated in response to the general



flagellin bacterial elicitor of basal defences (Navarro et al.,2004).

Potential substrates of the UPS involved in plant defence

There is ample evidence of E3 ligases responding topathogen attack and contributing to plant defence, butwhat are their biological substrates that need to bedegraded as part of the signalling process during plant/pathogen interactions? There are a few documented casesof plant defence proteins undergoing regulated proteolysisduring pathogenic interactions. RPM1 specifically helpsplants to defend themselves against P. syringae harbouringthe avrRpm1 and avrB genes (Grant et al., 1995). Thismembrane-associated protein is one of a large class ofR proteins that share predicted nucleotide binding sites(NBSs) and leucine-rich-repeats (LRRs). Rapid degra-dation of RPM1 coincident with the onset of the HRprovided one of the first clues that proteolysis contributesto R-mediated gene-for-gene defence (Boyes et al., 1998).Loss of RPM1:Myc from leaf extracts only occurs in ara-bidopsis plants inoculated with P. syringae carrying theavrRpm1, avrB, avrRps4 or avrRpt2 genes, but not withcomparable virulent strains. Importantly, several markerproteins do not show similarly rapid disappearance follow-ing bacterial inoculation, indicating that universal accelera-tion of protein degradation cannot explain RPM1 loss. Themode of RPM1 degradation remains unclear but theauthors speculate that degradation of this specificR-protein in response to several different Avr proteinsrepresents a general mechanism for limiting the spread ofthe HR (Boyes et al., 1998).

Recently, two proteins, RIN2 and RIN3, each with aRING domain and a ubiquitin binding domain called CUEwere shown to interact with RPM1 (Kawasaki et al.,2005). GST-RIN2 and RIN3 fusion proteins possess E3ubiquitin ligase activity in vitro. Analysis of the rin2 rin3double mutant indicates that these two proteins contributeto pathogen-elicited RPM1-dependent ion leakage. Itwould be tempting to speculate that these enzymescatalyse RPM1 ubiquitylation and degradation coincidentwith the HR. But neither GST-tagged RIN2 nor RIN3 canubiquitylate RPM1 in vitro, and there is no difference inpathogen-stimulated RPM1 disappearance in wild-typeversus rin2 rin3 plants. RIN2 and RIN3 may require otherproteins for in vitro activity and act redundantly in vivowith yet to be characterized E3s. Alternatively, they mayact on a protein or proteins downstream in the signallingpathway (Kawasaki et al., 2005).

There is evidence for three additional proteins affectingRPM1 accumulation. rar1/pbs2/lra1 mutants were pulledout of multiple pathogenesis-related screens in barley andarabidopsis (Freialdenhoven et al., 1994; Shirasu et al.,1999; Warren et al., 1999; Tornero et al., 2002). By anunknown mechanism, RAR1 contributes to the accumu-lation of a subset of R proteins, including RPM1, asRPM1:Myc levels drop drastically in an rar1 mutant back-ground (Tornero et al., 2002). Subsequent analysisrevealed that RAR1 is required for defence responsesdownstream of several different R genes (Muskett et al.,

2002), and not surprisingly has been shown to affect thestability of a number of R proteins including RPS5:HA inarabidopsis (Holt et al., 2005), MLA6:HA and MLA1:HAin barley, and Rx:HA from potato expressed in tobacco(Bieri et al., 2004).

Heat shock protein 90 (HSP90), a chaperonin requiredfor several R-mediated defence pathways, also contributesto R-protein accumulation. For instance, viral-inducedgene silencing of HSP90 in Nicotiana benthamianareduces the levels of an Rx:HA fusion protein (Lu et al.,2003) and an hsp90.2 mutant possesses reduced levels ofRPM1:Myc (Hubert et al., 2003). Intriguingly, in otherorganisms, HSP90 can contribute to both protein complexassembly and to ubiquitin-mediated degradation via theCHIP U-box E3 ubiquitin ligase (reviewed in Hohfeldet al., 2001).

Finally, the SGT1b protein can bind to both RAR1 andHSP90 (Azevedo et al., 2002; Takahashi et al., 2003) andSGT1b and SGT1a from arabidopsis and other plants func-tion in several R-mediated pathways (Austin et al., 2002;Tor et al., 2002; Leister et al., 2005; Azevedo et al.,2006). As previously mentioned, SGT1b can associatewith SCF E3 ligases (Azevedo et al., 2002; Y. Liu et al.,2002; Gray et al., 2003), and it appears to promoteSCF-mediated degradation of E3 ligase substrates inbudding yeast (Kitagawa et al., 1999) and in arabidopsis(Gray et al., 2003), potentially implicating an SCF in mul-tiple R-gene-dependent pathways, but it remains unclearwhether SGT1b enhances or suppresses substrate degra-dation in plant defence pathways. For instance, SGT1bappears to oppose RPS5:HA accumulation in arabidopsisplants (Holt et al., 2005) whereas a reduction in SGT1 inN. benthamiana causes Rx:HA levels to fall precipitously(Azevedo et al., 2006). Plants have a vested interest intightly controlling the abundance of each R protein asthere is evidence of dose-dependent resistance (Bieriet al., 2004) and increasing RPS5:HA levels correspondwith increased speed of HR onset (Holt et al., 2005). TheUPS could certainly contribute to this important process,but further exploration of this potential connection is war-ranted. On the one hand, treatment with proteasome inhibi-tors does not affect RPS5:HA stability in two differentassays (Holt et al., 2005) and most experiments focus ontotal R protein accumulation without determining whetherthe actual rate of R protein degradation changes inresponse to pathogen attack or altered RAR1, HSP90 orSGT1 levels. But, on the other hand, both HSP90 andSGT1 provide tantalizing links to the UPS. And evidencethat N-mediated gene-for-gene defence against TMV iscompromised by suppression of SKP1, SGT1 or COP9signalosome components (Y. Liu et al., 2002) givesadditional impetus to a thorough investigation of the roleof cullin-based E3 ligases in R-protein stability andsignalling.

Another protein that associates with the RPM1 andRPS2 R proteins, RIN4, can also undergo regulated degra-dation during pathogen attack (M.G. Kim et al., 2005) andthere is better experimental support for the involvement ofthe UPS in this process. The AvrRpt2 protein of the bac-terial P. syringae pathogen functions as a cysteine protease



capable of cleaving two distinct sites within membrane-bound RIN4 protein. One cleavage near the C-terminusleaves a small C-terminal anchor embedded in the plasmamembrane, but releases the remainder of the RIN4 protein.As the N-terminal portion of the protein can only beobserved in soluble fractions treated with proteasomeinhibitors, it appears that the RIN4 released from themembrane is rapidly degraded in an uncharacterizedproteasome-dependent manner (H.S. Kim et al., 2005).AvrRpt2-mediated cleavage at the second recognition sitein RIN4 unmasks a tertiary destabilizing residue (aspara-gine) that can potentially be recognized by a specific UPSpathway called the N-end rule. Ubiquitylation via N-endrule (reviewed in Varshavsky, 2003) depends on the natureof the N-terminal residue, and certain amino acids at thisposition can be recognized directly by an E3 ligase(primary residues), recognized after addition of an arginylgroup [secondary residues (aspartate and glutamate)] orrecognized after removal of an amino group followed byaddition of an arginyl group [tertiary residues (asparagineand glutamine)]. Presumably, presence of asparagine at theN-terminus of cleaved RIN4 leads to modification of theresidue, recognition by an N-end rule E3 ligase, ubiquity-lation and degradation of the RIN4 protein, given thatreplacing the asparagine with a residue not recognized bythe N-end pathway (glycine) stabilizes an N-terminalRIN4-30 N(N11G):GFP reporter protein. Several RIN4-like proteins possess similar AvrRpt2 cleavage sites,suggesting this mechanism may not be restricted to RIN4(Takemoto and Jones, 2005).

Degradation of the RIN4 protein has varying conse-quences for disease resistance depending on the suite ofAvr and R proteins expressed by P. syringae and arabidop-sis, respectively. RIN4 is required for defence against bac-teria harbouring the AvrRpm1 or AvrB avirulence factors(Mackey et al., 2002). Thus, AvrRpt2-mediated loss ofRIN4 blocks this defence mechanism. However, the RPS2R protein ‘guards’ against pathogen-mediated loss ofRIN4, so arabidopsis plants expressing RPS2 initiate anHR following RIN4 degradation (Axtell and Staskawicz,2003; Mackey et al., 2003). RIN4 also plays a role in reg-ulating basal defences so perturbations of RIN4 levels canpotentially influence these basal reactions to a broad rangeof pathogens (M.G. Kim et al., 2005).

Although very few examples of ubiquitylated host pro-teins have been implicated in plant defence pathways todate, a number of experiments suggest that viral proteinsmay be substrates of the host UPS. A general linkagebetween ubiquitin and viral/host interactions was revealedin tobacco plants expressing a mutant variant of ubiquitinwith lysine48 changed to arginine that exhibit spontaneouslesion development (Bachmair et al., 1990) and displayseveral alterations in TMV susceptibility and symptomprogression (Becker et al., 1993). Analysis of several plantvirus particles demonstrates the existence of ubiquitin-conjugated proteins (Hazelwood and Zaitlin, 1990),although the length of the ubiquitin chains and theirfunctional importance has not been determined. Treatmentof virally infected tissue with proteasome inhibitorsleads to the accumulation and stabilization of

high-molecular-weight forms of the TMV movementprotein, suggesting that this protein is normally subject toUPS-mediated degradation, as part of either the pathogen-esis or the defence process (Reichel and Beachy, 2000).Furthermore, a 66-kDa protein from the Turnip YellowMosaic Virus (TYMV), believed to function as an RNA-dependent RNA polymerase (RdRp), can be phosphory-lated at several serine and threonine residues within itsPEST domain when expressed in insect cells (Hericourtet al., 2000). PEST domains appear to act as phospho-sensitive destabilization motifs (‘degrons’) in severaleukaryotic substrates of the UPS (reviewed in Rechsteinerand Rogers, 1996), and further characterization of the66-kDa RdRp protein indicates that it undergoes ubiquity-lation in an insect expression system (Hericourt et al.,2000). It is possible that this RdRp is ubiquitylated in itsnatural plant hosts, but RdRp does not appear to beremarkably unstable when expressed in a proteolyticallyactive rabbit reticulocyte system (Drugeon and Jupin,2002). This could reflect a lack of proper modifyingenzymes, or indicate that the protein is naturally degradedat a modest rate. However, under the same conditions, the69-kDa movement protein of TYMV is degraded quiterapidly in an ATP- and proteasome-dependent manner.Furthermore, substantial evidence points to the accumu-lation of ubiquitylated forms of the 69-kDa movementprotein in proteolytically compromised rabbit reticulocytelysates (Drugeon and Jupin, 2002). To date, there is noconclusive evidence of whether these proteins are ubiqui-tylated in their hosts and, if so, what effect this has onTYMV pathogenicity.

In future characterizations of plant defence and theUPS, it will be interesting to see if there are any relation-ships between the set of UPS components shown to beinduced by, or required for, plant responses to pathogens(e.g. the ARM/U-box E3 ligases) and the R-related pro-teins that are degraded in response to specific biotic chal-lenges, or the viral proteins that may be ubiquitylated inplant cells, and to learn how all of these factors allowplants to mount integrated responses to an array of viral,bacterial, fungal and herbivore attacks.

Pathogenic usurpation of the host UPS

Given the demonstrated importance of the UPS forbiotic defence and general plant growth and development,it makes sense that several plant pathogens hijack or inter-fere with components of the UPS to promote their ownsurvival. For instance, the Polerovirus P0 protein possessesa minimal F-box motif that enables it to bind to the hostASK1/SKP1 and ASK2 proteins. This interaction allowsthe virus to suppress the plant’s ability to defend itself viapost-transcriptional silencing of viral RNAs presumablyby promoting degradation of a component of the plant’sRNA silencing machinery (Pazhouhandeh et al., 2006).The faba bean necrotic yellows virus (FBNYV) encodes asmall protein, Clink, that can bind both to SKP1 and tothe cell cycle protein pRB in vitro, in yeast and in planta(Aronson et al., 2000, 2002). Although mutations in theF-box portion of Clink do not interfere with replication of



the FBNYV DNA, it is possible that Clink/SKP-mediatedubiquitylation of host or viral proteins could influenceother aspects of FBNYV pathogenesis (Aronson et al.,2000). In addition, it was recently shown that Clink is notrequired for the induction of viral symptoms on Vicia faba(faba bean) plants in controlled experiments, but theredoes seem to be selective pressure to maintain this gene inthe FBNYV genome given that the viral DNA from 98 %of the symptomatic plants tested retain the Clink gene. Inaddition, the authors postulate that Clink might be requiredfor FBNYV infection of the wild relatives of the cultivatedfaba bean (TimchenKo et al., 2006) where itsSKP-binding capabilities could possibly connect it to tar-geted protein degradation.

Another plant virus, Lettuce Mosaic Virus (LMV),influences the behaviour of the 20S proteasome (Ballutet al., 2005). Viral inoculation results in the formation oflarger proteasome-containing complexes, as assayed by gelfiltration. The viral protein HcPro can associate with puri-fied 20S proteasomes and might be part of these protea-somal aggregates. Functionally, HcPro appears to have aslight stimulatory effect on the chymotrypsin- and trypsin-like enzymatic activities of the 20S proteasome in in vitroassays. Intriguingly, proteasomes purified from sunfloweralso possess endonuclease activity capable of cleavingTMV and LMV RNA (Ballut et al., 2003), and pre-incubation of purified 20S proteasomes with HcPro signifi-cantly inhibits degradation of a TMV RNA substrate. Thispoints to a possible role for HcPro in ubiquitin-dependentand ubiquitin-independent proteasome-mediated processesin host/LMV interactions (Ballut et al., 2005).

In the realm of bacterial pathogens, the AvrPtoB proteinof P. syringae (which does not have an endogenous UPS)is a ubiquitin ligase that can presumably work downstreamof a host E1 and E2 enzyme to promote ubiquitylation ofitself and/or unidentified host substrates. The level of E3ligase activity exhibited by wild-type and mutant forms ofAvrPtoB appears correlated with AvrPtoB’s ability tosuppress HR-based programmed cell death in susceptibletomato plants and N. benthamiana (Abramovitch et al.,2006; Janjusevic et al., 2006). Meanwhile, Agrobacteriumtumefaciens introduces an F-box protein (VirF) into hostcells and utilizes host components to form a functionalSCF complex required for degradation of VirE2 andhost VIP1 as these proteins must be eliminated to allowintegration of the Agrobacterium’s T-DNA into the hostgenome (Tzfira et al., 2004).

Nematodes may also co-opt components of the hostUPS for their own advantage, as evidenced by the pro-duction of cDNAs for hexaubiquitin, a novel ubiquitinextension protein, a RING-H2 protein and an SKP1-likeprotein specifically within the dorsal esophageal glandcells of the nematode Heterodera glycines. These proteinsbear a signal peptide to direct them for secretion into thehost (Gao et al., 2003). An analysis of the secretion profileof the parasitic cyst nematode Heterodera schachtii led tothe discovery of another member of the new class of ubi-quitin extension proteins that is loaded into the host by thenematode (Tytgat et al., 2004). Plant-produced ubiquitinhydrolases most likely act on this protein to cleave

ubiquitin and release the C-terminal portion to performsome sort of function that enhances nematode feeding.

In conclusion, pathogenic and symbiotic interactions ofplants with their biosphere involve UPS components andoften result in the promotion or inhibition of selectiveproteolysis. Similarly, as a plant reacts to alterations inits abiotic environment or focuses inward to respondto internal signals, the UPS plays a central role.Undoubtedly, future research will reveal the functions ofthe other UPS components that are currently onlydescribed by in silico predictions.

ACKNOWLEDGEMENTS

We thank Dr Sophia Stone for the ubiquitin pathwaydiagram, Sara Hotton, Mandy Hsia, Dr Jemma Jowett,Edward Kraft and Lucy Stewart for helpful comments onthe manuscript, and Drs Ken Kaplan and RichardMichelmore for acronymic assistance. Research in theCallis lab is supported in part by grants from the NationalScience Foundation MCB-0519970 and IBN-0212659 andby the Paul K. and Ruth R. Stumpf Endowed Chair inPlant Biochemistry. K.D. was supported in part byNational Institutes of Health Predoctoral Training GrantGM-0007377–27.

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APPENDIX 1

FIG. 2.

Abbreviation* Full name/description

35S Promoter from the cauliflower mosaic virus thatmakes a 35S RNA transcript

ABA Abscisic acidABF ABRE binding factorABI Abscisic acid insensitiveABRE ABA response elementACC 1-amino-1-cyclopropane-carboxylic acid (ethylene

precursor)ACRE Avr/Cf9 rapidly elicitedACS ACC synthaseAFB Auxin signalling F-box proteinAFP ABI5 binding proteinAIP ABI3 interacting proteinAOS Allene oxide synthaseAP ApetalaAPC Anaphase promoting complexARF Auxin response factorARIA Arm protein repeat interacting with ABF2ARM Armadillo, a protein interaction domainASK Arabidopsis SKP1-likeAt Arabidopsis thaliana (arabidopsis)ATL Arabidopsis toxico para levadura (toxic to fungi)Aux/IAA Auxin/indole-3-acetic-acidAuxRE Auxin response elementAvr factor Avirulence factorAvrPtoB Avirulence – Pseudomonas syringae pv. tomatoAvrRpm Avirulence – resistance to Pseudomonas syringae

pv. maculicolaavrRpt Avirulence – resistance to Pseudomonas syringae

pv. tomatoAXR Auxin resistantAXR3NT:GUS Auxin resistant 3 N-terminus:beta-glucuronidase

protein fusionBAB Bric-a-brac proteinBAK BRI1 associated receptor kinaseBARD BRCA1 associated RING domainbes1-D bri1-EMS-suppressor 1 dominantBIN Brassinosteroid insensitiveblastp Basic local alignment search tool – proteinBRCA Breast cancer (BRCA1 is an E3)BRH Brassinosteroid-responsive RING-H2BRI Brassinosteroid insensitiveBSU bri1 suppressorBTB Domain first found in the Drosophila proteins:

broad complex, tramtrack and bric-a-bracBY Bright yellowbzr1-1D Brassinazole-resistant 1–1dominantC. fulvum Cladosporum fulvumCAND Cullin associated and neddylation-dissociatedCdc Cell-division cycleCDD COP10, DET1, DDB1 complexCDPK Calcium-dependent protein kinaseCdt Cdc10-dependent transcript 2C. elegans Caenorhabditis elegansCf Required for resistance to Cladosporum fulvumCFP Cyan fluorescent proteinCHIP C-terminal HSP interacting protein

Continued

Abbreviations used in the text


c-Jun Gene for this protein cloned from avian sarcomaisolate #17; ju-nana is 17 in japanese

Clink Cell cycle linkCMPG Cysteine-methionine-proline-glycine (first 4 strictly

conserved amino acids within domain)COI Coronitine insensitiveCOP Constitutive photomorphogenicCRL Cullin RING ligaseCSN COP9 signalosomeCtf Chromosome transmission fidelityCTR Constitutive triple responseCUE Ubiquitin-binding domain identified in yeast Cue1pCUL CullinDCAF DDB1-cullin4-associated factorDCN Defective in cullin neddylationDDB Damaged DNA bindingDET De-etiolatedDsRed Discosoma sp. ‘red’ (fluorescent protein)E1 Enzyme 1 (same as UBA, ubiquitin activating

enzyme)E2 Enzyme 2 (same as UBC, ubiquitin conjugating

enzyme)E2F E2 promoter binding factorE3 Enzyme 3 (same as ubiquitin protein ligase)EAR ERF-associated amphiphilic repressionEBF EIN3-binding F-boxEbi Shrimp (in Japanese)ECR E1 C-terminal relatedEID Empfindlicher im dunkelroten licht (sensitive to

far-red light – German)EIL EIN3-likeEIN Ethylene insensitiveEIR Ethylene insensitive rootEL5 Elicitor inducedELI Elicitor-activated geneEOL ETO-likeERF Ethylene response factorERS Ethylene response sensorETO Ethylene overproductionETR Ethylene receptorFBNYV Faba bean necrotic yellow virusFLAG Synthetic epitope tagflg22 Flagellin (22 amino acids from N-terminal part of

protein)FUS FuscaGA Gibberellic acid/gibberellinsGA2ox GA 2-oxidaseGA3ox GA 3-oxidaseGAI GA (gibberellin) insensitiveGAMYB Gibberellin-inducible Myb (identified from an

oncogene in avian myeblastosis virus)GFP Green fluorescent proteinGID Gibberellin insensitive dwarfGMPOZ GAMYB associated BTB/POZ proteinGSK Glycogen synthase kinaseGST Glutathione-S-transferaseGTPase Guanosine triphosphataseGUS Beta-glucuronidaseHA Peptide sequence from influenza haemagglutinin

protein (epitope tag)HAUSP Herpes-associated ubiquitin-specific proteaseHcPro Helper component proteaseHDAC Histone deacetylaseHECT Homologous to E6-AP COOH terminusHLS HooklessHR Hypersensitive responseHSL Hormone-sensitive lipaseHSP90 Heat shock protein 90 kDa

Continued




HSS Hookless1 suppressorHv Hordeum vulgare (barley)HY Long hypocotylIAA Indole-3-acetic acidINA 2,6-dichloroisonicotinic acid (synthetic inducer of

SAR)ISR Induced systemic resistanceJA Jasmonic acidJAI Jasmonate insensitiveKeap Kelch-like ECH-associated proteinLMV Lettuce mosaic virusLRA Loss of recognition of avrRpm1LRR Leucine-rich repeatLUC Photinus pyralis luciferase (firefly)MAPK Mitogen-activated protein kinaseMATH Meprin and TRAF homologyMEI MeioticMeJA Methyl jasmonateMEL Maternal effect lethalityMG132 Carboxybenzyl-leucyl-leucyl-leucinal (proteasome

inhibitor)Mla Mildew resistance locus AMPK Mitogen-activated protein kinaseMyc Epitope tag from c-Myc protein (originally ident-

ified from a chicken with myelocytomatosis)1-NAA 1-naphthaleneacetic acid (synthetic auxin)NAC NAM, ATAF, and CUC transcription factorsNBS Nucleotide binding siteNCBI National Center for Biotechnology InformationNCED Nine-cis-epoxycarotenoid dioxygenaseNEDD Neural precursor cell expressed, developmentally

down-regulated geneNIM Non-inducible immunityNPB Nuclear protein bodyNPR Non-expressor of pathogenesis-related genesNrf NF-E2-related factorNs Nicotiana sylvestrisNt Nicotiana tabacum (tobacco)Os Orzya sativa (rice)P. syringae Pseudomonas syringaePAMP Pathogen associated molecular patternPBS avrPphB susceptiblePc Petroselinum crispum (parsley)PDF Plant defensinPep25 25 amino acids from the 42-kDa Phytophthora

sojae glycoprotein elicitorPEST domain Enriched in proline, glutamate, serine, threoninePHOR Photoperiod-responsivePhy PhytochromePI-II Proteinase inhibitor-IIPIL PIF3-likePIN Pin-formedPOZ Pox virus and zinc fingerPR Pathogenesis-relatedpRB Phosphorylated retinoblastoma tumour suppressor

proteinPUB Plant U-boxR gene/protein Resistance gene/proteinRAC ras-related C3 botulinum toxin substrateRAR Required for MlA12 resistanceRBX RING-box protein, same as ROC1 and Hrt1pRCE RUB conjugating enzymeRcy RecyclingRdRp RNA-dependent RNA polymeraseRGA Repressor of ga1-3RGL RGA-likeRGS Epitope tag with arginine-glycine-serine upstream

of a polyhistidine tract

Continued


RHA RING-H2 group ARIN RPM1-interacting proteinRING Really interesting new gene protein domainRMA RING finger protein with membrane anchorRNAi RNA interferenceROC Regulator of cullinsRPD Reduced potassium dependencyRPM Resistance to Pseudomonas syringae pv.

maculicolaRPN Regulatory particle non-ATPaseRPS Resistance to Pseudomonas syringae pv. tomatoRUB Related to ubiquitinRubisco Ribulose 1,5-bisphosphate carboxylase/oxygenaseRx R protein conferring resistance to potato virus XSA Salicylic acidSAI Salicylic acid insensitiveSAR Systemic acquired resistanceSCF Skp1-Cullin1-F-boxSGT Suppressor of G2 allele of skp1Siah Seven in absentia homologueSINA Seven in absentiaSINAT SINA of Arabidopsis thalianaSIP Siah-interacting proteinSIPK Salicylic-acid induced protein kinaseSKP S phase kinase-associated proteinSl Solanum lycopersicum (tomato)SLN SlenderSLR Slender riceSLY SleepySNE SneezySON Suppressor of nim1-1Spd S-phase delayedSpl Spotted leafTGA Bind to DNA element with core sequence TGACGTIR1 Transport inhibitor response1TIR-NBS-LRR Toll Interleukin1 Receptor–Nucleotide Binding

Site–Leucine-Rich RepeatTLP TUBBY-like proteinTMV Tobacco mosaic virusToMV Tomato mosaic virusTYMV Turnip yellow mosaic virusUb UbiquitinUBA Ubiquitin activating enzymeUBC Ubiquitin conjugating enzymeUbDHFR Ubiquitin fused to the N-terminus of dihydrofolate

reductaseUbKn Ubiquitin lysine n, where n indicates specific lysine

residueU-box UFD2-homology domainUEV Ubiquitin-conjugating E2 enzyme variantUFO Unusual floral organsUPL Ubiquitin protein ligaseUPS Ubiquitin proteasome systemVIP Vir-E2 interacting proteinVirE2 Virulence E2VirF Virulence FWD40 �40-amino-acid residue domain with tryptophan

and aspartate residues in conserved positionsWDXR Motif with the sequence tryptophan-aspartate-any

amino acid-arginine.Wnt Wingless and int proteinsXBAT XB3 orthologue 2 in Arabidopsis thalianaXB3 Rice Xanthomonas oryzae pv. oryzae resistance 21

binding protein

* Numbers at the end of abbreviations have been removed when theydo not convey functional information. All genes/proteins found in thetext with the same abbreviations (but different numbers) have the samenames.



APPENDIX 2

Diagrams of (A) a generic, and (B) specific Cullin RINGligases. The binding of the ubiquitin-charged E2 (ubiquitinconjugating enzyme) to the RING protein RBX is depictedonly for the generic CRL. Many of the CRLs have alter-nate names (see Petroski and Deshaies, 2005). Questionmarks denote proteins with less well-characterized func-tions in CRLs. Recent experiments suggest that CRLs con-taining CUL1, CUL3 or CUL4a may homooligomerize inmammalian cells (Chew et al., 2007). Cullins associatedwith each class of CRL are listed below each diagram.

The identity of putative DCAFs and other proteins partici-pating in CUL4-based CRLs are also mentioned. TheAPC, which has a cullin-like subunit, is not shown. ECS:elongin BC, CUL2/5, SOCS/BC box; SOCS: suppressor ofcytokine signalling.

[Chew E-H, Poobalasingama T, Hawkeya CJ, ThiloHagen T. 2007. Characterization of cullin-based E3ubiquitin ligases in intact mammalian cells — evidencefor cullin dimerization. Cellular Signalling. In press,doi: 10.1016/j.cellsig.2006.12.002]

Generic CRL(Cullin RING ligase)

SCF

Arabidopsis- CUL1- CUL2 (?)

CUL3/BTB

CUL4 CRL

DCAFs(?): DDB2, DET1,WDXR proteins (?)

ArabidopsisCDD complex?(DDB1, DET1, COP10)+ COP1?

Recognition/AdaptorProteins

CULLIN

E2RBX

subiquitin

RING protein

Substrate

RUB

Arabidopsis- ?

Arabidopsis- CUL3a- CUL3b

Human- CUL1- CUL7(?)

Human- CUL3

ECS

Human- CUL2- CUL5

Arabidopsis- CUL4

Human- CUL4A

Specific CRLs

A

B

SGT1 (?)

Substrate

CULLIN

RBX

F-box

SKP/ASK

RUB

RUB

Substrate

CULLIN

RBXDDB1, ?

DCAF?

RUB

Substrate

CULLIN

RBX

SOCS/BC

Elongin C

Elongin B

RUB

CULLIN

RBXBTB

Substrate



Ubiquitin, Hormones and Biotic Stress in Plants

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