The N-Terminal UNDMotif of the Arabidopsis U-Box E3 LigasePUB18 Is Critical for the Negative Regulation of ABA-MediatedStomatal Movement and Determines Its UbiquitinationSpecificity for Exocyst Subunit Exo70B1

Dong Hye Seo, Min Yong Ahn, Ki Youl Park, Eun Yu Kim, and Woo Taek Kim1

Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea

ORCID IDs: 0000-0003-1243-4466 (D.H.S.); 0000-0001-5027-4622 (W.T.K.)

The Arabidopsis thaliana U-box E3 ligases PUB18/PUB19 and PUB22/PUB23 are negative regulators of drought stressresponses. PUB18/PUB19 regulate the drought stress response in an abscisic acid (ABA)-dependent manner, whereasPUB22/PUB23 regulate this response in an ABA-independent manner. A major structural difference between PUB18/PUB19and PUB22/PUB23 is the presence of the UND (U-box N-terminal domain). Here, we focused on elucidating the molecularmechanism that mediates the functional difference between PUB18 and PUB22 and found that the UNDPUB18 was criticallyinvolved in the negative regulation of ABA-mediated stomatal movements. Exo70B1, a subunit of the exocyst complex, wasidentified as a target of PUB18, whereas Exo70B2 was a substrate of PUB22. However, the ΔUND-PUB18 derivative failed toubiquitinate Exo70B1, but ubiquitinated Exo70B2. By contrast, the UNDPUB18-PUB22 chimeric protein ubiquitinated Exo70B1instead of Exo70B2, suggesting that the ubiquitination specificities of PUB18 and PUB22 to Exo70B1 and Exo70B2,respectively, are dependent on the presence or absence of the UNDPUB18 motif. The ABA-insensitive phenotypes of the pub18pub19 exo70b1 triple mutant were reminiscent of those of exo70b1 rather than pub18 pub19, indicating that Exo70B1functions downstream of PUB18. Overall, our results suggest that the UNDPUB18 motif is crucial for the negative regulation ofABA-dependent stomatal movement and for determination of its ubiquitination specificity to Exo70B1.

INTRODUCTION

As sessile organisms, terrestrial higher plants have developedintricate mechanisms to cope with adverse environmental con-ditions. Drought is one of the most critical factors that limit thegrowth, development, and productivity of agricultural crops(Chaves et al., 2002; Reynolds and Tuberosa, 2008; Osakabeet al., 2014). Under water-deficit conditions, the phytohormoneabscisic acid (ABA) acts as a major internal signal that regulatesstress-adaptive responses (Tuteja, 2007; Nakashima andYamaguchi-Shinozaki, 2013; Yu et al., 2016). However, recentstudies have revealed that an ABA-independent drought-tolerantpathway also plays an important role in Arabidopsis thaliana(Shinozaki andYamaguchi-Shinozaki, 2007; Yoshida et al., 2014).Therefore, understanding the comprehensive defense mecha-nisms requires deciphering of the crosstalk and demarcationbetween ABA-dependent and ABA-independent signaling path-ways against abiotic stress.

In eukaryotes, the ubiquitin (Ub)-proteasome system (UPS)plays an indispensable role in modulating various vital cellularprocesses, including hormone signaling, DNA repair, endocyto-sis, and biotic and abiotic stress responses (Welchman et al.,2005; Dreher and Callis, 2007; Vierstra, 2009; Lee and Kim, 2011;

Bartel andCitovsky, 2012; Sadanandomet al., 2012; Stone, 2014;Zhang et al., 2015; Yu et al., 2016). The ubiquitination cascaderequires the sequential action of three enzymes: E1 Ub-activatingenzymes, E2Ub-conjugating enzymes, andE3Ub ligases (Guerraand Callis, 2012; Berndsen and Wolberger, 2014). The expandeddiversity of E3 Ub ligases suggests that the selection of spe-cific substrates to strictly regulate different cellular processesis determined by E3s (Mazzucotelli et al., 2006; Vierstra, 2009;Sadanandom et al., 2012).The E3 ubiquitin ligases are classified into four different types:

really interesting new gene (RING), U-box, homology to E6-AP Cterminus (HECT), and Cullin (Cul)-RING ligases (CRLs) (Vierstra,2009; Yu et al., 2016). Themultisubunit E3 complexes, such as theanaphase-promoting complexes and CUL1-based SCF com-plexes, belong to the CRL-type E3 Ub ligases (Mazzucotelli et al.,2006; Spratt et al., 2014).The U-box E3 ligase, which has a modified RING finger motif,

was first found in yeasts. Based on sequence conservation, theU-box motif-containing proteins are widely present in eukaryoticorganisms. For example, Arabidopsis, a dicot model plant, con-tains 64 U-box E3 Ub ligases, whereas the monocot model croprice (Oryza sativa) contains 77 U-box containing proteins (PUBs).Recently, 125 putative PUBs were identified in soybean (Glycinemax; Wang et al., 2016). By contrast, yeast and humans possessa smaller number of U-box E3 Ub ligases than higher plants do(Mudgil et al., 2004; Wiborg et al., 2008; Yee and Goring, 2009;Lyzenga and Stone, 2012). The existence of a large number ofPUBs implies that the U-box E3 ligases have evolved to performplant-specific functions, such as plant immune responses (Yang

1Address correspondence to wtkim@yonsei.ac.kr.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Woo Taek Kim (wtkim@yonsei.ac.kr).www.plantcell.org/cgi/doi/10.1105/tpc.16.00347

The Plant Cell, Vol. 28: 2952–2973, December 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.

et al., 2006; Trujillo et al., 2008; Lu et al., 2011, 2012; Stegmannet al., 2012; Antignani et al., 2015; He et al., 2015), abiotic stressresponses (Yan et al., 2003; Cho et al., 2006, 2008; Bergler andHoth, 2011; Liu et al., 2011; Salt et al., 2011; Seo et al., 2012;Hwang et al., 2015), self-incompatibility (Liu et al., 2007; Samuelet al., 2008, 2009; Indriolo and Goring, 2014; Zhang et al., 2014),hormonal responses (Luo et al., 2006; Vogelmann et al., 2012; Huet al., 2013;Konget al., 2015), anddevelopment (Raabet al., 2009;Wang et al., 2013; Deb et al., 2014; Kinoshita et al., 2015).

Unlike other multigene families that are divided by their se-quence homology, PUBs are classified based on the presence offunctional domains in addition to the U-box (Azevedo et al., 2001;Wang et al., 2016). Among the 64 U-box E3 Ub ligases in Ara-bidopsis, 41 PUBs contain the armadillo (ARM) repeat, which isa protein-protein interacting domain, in their C-terminal regions(Azevedo et al., 2001; Mudgil et al., 2004; Zeng et al., 2008; Yeeand Goring, 2009). The PUB-ARM proteins are then divided intotwo subgroups based on the presence or absence of the U-boxN-terminal domain (UND). At least 17 members of the UND-containing PUB-ARM (UND-PUB-ARM) proteins are present inArabidopsis. According to recent reports, the UND motif is in-volved in the interaction of U-box E3 ligase and its target protein(Samuel et al., 2009; Indriolo et al., 2012; Indriolo and Goring,2014). Furthermore,UNDhasbeen reported tobenot essential forE3Ub ligaseenzymeactivity (Andersenetal., 2004).Nevertheless,detailed cellular functions of UND remain to be elucidated.

Ourpreviousstudiesshowedthat fourArabidopsisU-boxE3Ubligases, PUB18, PUB19, PUB22, and PUB23, play negative rolesin response to drought stress (Cho et al., 2008; Seo et al., 2012).The PUB18 and PUB19 homologs regulate the drought stressresponse in an ABA-dependent manner, whereas PUB22 andPUB23 function in an ABA-independent manner. PUB18 andPUB19 possess an N-terminal UND and C-terminal ARM repeatsin addition to theU-boxdomain in their central region.Conversely,PUB22 and PUB23 are composed of the U-box motif and theC-terminal ARM repeats. A major difference between PUB18/PUB19andPUB22/PUB23 is thepresenceof theN-terminalUND.These findings raised the possibility that the N-terminal UND inPUB18/PUB19 participates in the negative regulation of an ABA-mediateddrought stress response. In this study,we report that theN-terminal UND of PUB18 is indeed critical for the negativeregulation of ABA-mediated stomatal closure. We identifiedExo70B1, a subunit of the exocyst complex, as a target of PUB18.Ourdata furthersuggest that theN-terminalUNDmotifdeterminesthe binding and ubiquitination specificity of PUB18 to Exo70B1.

RESULTS

Deletion of the N-Terminal UND of PUB18 Impairs the ABA-Mediated Stomatal Movements, but Does Not AffectDrought-Induced Stomatal Closure in Arabidopsis

Seo et al. (2012) reported that four Arabidopsis U-box E3 Ub li-gases, i.e., PUB18, PUB19, PUB22, and PUB23, play negativeroles in response to drought stress. The modes of action of thesenegative regulators were divided into two distinct pathways:PUB18 and PUB19 regulate the drought stress response in an

ABA-dependent manner, whereas PUB22 and PUB23 work in anABA-independent manner (Cho et al., 2008; Seo et al., 2012). Thehomologs PUB18 and PUB19 possess N-terminal UND andC-terminal ARM repeats in addition to the U-box domain in theircentral region (Mudgil et al., 2004; Yee and Goring, 2009).However, PUB22 and PUB23 consist only of the U-box motif andthe C-terminal ARM repeats. A major difference between PUB18/PUB19 and PUB22/PUB23 is the presence of the N-terminalUND (Supplemental Figure 1). Thus, we considered the possibilitythat the N-terminal UND in PUB18/PUB19 participates in thenegative regulation of ABA-dependent drought stress response.To test this possibility, the DUND-PUB18 mutant protein, inwhich the N-terminal 269-amino-acid-long UND was deleted(Figure 1A), was characterized in termsof ABA-mediated stomatalmovements.Liuetal. (2011)usedwheat (Triticumaestivum) E1andhumanE2

(UBCh5b) to measure the in vitro E3 Ub ligase activity of PUB19.WeusedArabidopsis E1 (UBA1) andvariousE2s, includingUBC7,UBC8, UBC9, UBC10, and UBC13, as well as human E2, for anin vitro self-ubiquitination assay of PUB18, but failed to show E3Ub ligase activity of bacterially expressed MBP-PUB18. Thus, inthis study, a small amount of crude extract (10 mg total proteins)prepared fromABA-treatedpub18pub19pub22pub23quadruplemutant leaves was used as a source of E2 enzyme for in vitro self-ubiquitination assays of PUB18. A crude extract of pub18 pub19pub22 pub23 mutant leaves, rather than wild-type leaves, wasused as E2 to exclude a possible effect of endogenous PUB18present in the protein crude extract on the ubiquitination assay.Recombinant full-length MBP-PUB18 and MBP-OUND-PUB18proteinswere incubatedwithATPandFlag-taggedUb (Flag-Ub) inthe presence or absence of Arabidopsis UBA1 (E1) and a proteincrude extract (E2) at 30°C for 2 h. The reaction mixture was an-alyzed by immunoblotting using anti-MBP and anti-Flag anti-bodies. The results indicated that MBP-OUND-PUB18 exhibitedcomparable E3 Ub ligase activity to the full-length MBP-PUB18,as evidenced by similar levels of high molecular mass smearladders detected by both anti-MBP and anti-Flag antibodies(Figure1B).ExclusionofeitherUBA1 (E1)orcrudeextract (E2) fromthe incubation mixture abolished these high molecular massbands. In addition, a single amino acid substitution in both MBP-PUB18V305I and MBP-OUND-PUB18V36I abrogated E3 ligaseactivity even in the presence of Flag-Ub, UBA1 (E1), and crudeextract (E2) (Figure 1B). When a crude protein extract was in-activated by boiling, ubiquitinated products were not detected inthe presence of all reaction components (Supplemental Figure 2).These results suggest that the N-terminal UND of PUB18 is notessential for E3 Ub ligase activity, which is consistent with pre-vious results that the UNDmotif in PUBs is not required for E3 Ubligase enzyme activity (Andersen et al., 2004). For consistentexperimental conditions, a crude protein extract of pub18 pub19pub22 pub23 leaves pretreated with ABA was used as an E2source in all other E3 Ub ligase assays in this study.The possible role of UND in response to ABA was examined by

ectopically expressing HA-PUB18 and Flag-ΔUND-PUB18 inpub18 pub19 double mutant plants under the control of the 35SCaMV promoter (Figure 1C). Ectopic expression of HA-PUB18and Flag-ΔUND-PUB18 proteins was detected by anti-HA andanti-Flag antibodies, respectively, in two independent transgenic

Role of the UND Motif of Arabidopsis PUB18 2953

Figure 1. Deletion of the N-Terminal UND of PUB18 Impairs ABA-Mediated Stomatal Movements, but Does Not Affect the Drought-Induced StomatalClosure in Arabidopsis.

2954 The Plant Cell

lines (#1 and #2) (Figure 1D). These complementation transgenicplants (pub18 pub19/35S:HA-PUB18 and pub18 pub19/35S:Flag-ΔUND-PUB18) were subsequently used to analyze ABA-dependent stomatalmovements.Consistentwithprevious results(Seo et al., 2012), stomatal movement of the pub18 pub19mutantwas hypersensitive compared with that in the wild-type plant inresponse to ABA treatments (1 and 10 mM). After 10 mM ABAtreatment, the average stomatal apertures (the ratio of width tolength) of the wild type and pub18 pub19were 0.1086 0.003 and0.0776 0.003, respectively (Figure 1E). In addition, the degree ofABA sensitivity of pub18 pub19/35S:HA-PUB18 complemen-tation progeny was approximately the same as that of the wild-type plant, with an average stomatal aperture of 0.113 6 0.011(transgenic line #1) and 0.105 6 0.003 (transgenic line #2),corroborating the role of PUB18 as a negative regulator in ABA-mediated stomatal closure. By contrast, the constitutive ex-pression of Flag-ΔUND-PUB18 inpub18pub19 could not rescuethe hypersensitive phenotype of the mutant toward ABA (Figure1E). In the presence of 10 mM ABA, stomatal apertures of pub18pub19/35S:Flag-ΔUND-PUB18 were 0.074 6 0.006 (transgenicline #1) and 0.072 6 0.002 (transgenic line #2). These resultssuggest that the N-terminal UND in PUB18 is critical for thenegative regulation of ABA-dependent stomatal behaviors inArabidopsis.

Stomatal movements of wild-type, mutant (pub18 pub19), andcomplementation (pub18 pub19/35S:HA-PUB18 and pub18pub19/35S:Flag-ΔUND-PUB18) plants in response to mannitoltreatments (0.2 and 0.4 M) were measured. The results revealedthat the hypersensitive stomatal behavior of pub18 pub19 undermannitol treatmentwasefficientlyoffsetby theectopicexpressionof both HA-PUB18 and Flag-ΔUND-PUB18 (Figure 1F). Theseresults indicate that theN-terminalUND inPUB18 isnotnecessaryfor the negative regulation of mannitol-induced stomatal closure.

Taken together, our data suggest that the deletion of theN-terminal UND of PUB18 impairs the ABA-mediated stomatalmovements but does not affect the mannitol (drought)-inducedstomatal closure in Arabidopsis.

ABA-Responsive Stomatal Movement Was Restored byOverexpression of the N-Terminal UND in pub22 pub23Mutant Plants

To further investigate the biological role of UND in the ABA re-sponse and unravel the possible functional relationship betweenPUB18 and PUB22, the UNDPUB18-PUB22 chimeric protein, inwhich the N-terminal 269-amino acid residues of PUB18 werefused to the N terminus of PUB22, was constructed (Figure 2A).Whether the UNDPUB18-PUB22 protein has E3 Ub ligase activitywas determined by conducting an in vitro self-ubiquitinationassay. The results showed that purified MBP-PUB22 and MBP-UNDPUB18-PUB22possessedE3Ub ligaseactivitiesdetectedbyanti-MBP and anti-Flag antibodies, whereas MBP-PUB22V24I

and MBP-UNDPUB18-PUB22V293I mutant derivatives failed toshow E3 ligase activity (Figure 2B). This indicates that the at-tachment of UNDPUB18 to PUB22 did not affect E3 Ub ligaseactivity.The 35S:PUB22-GFP and 35S:Flag-UNDPUB18-PUB22 con-

structs were then introduced into pub22 pub23 double mutantplants. Ectopic expression of PUB22-GFP and Flag-UNDPUB18-PUB22weredetectedbyRT-PCR (Figure2C) and immunoblottingusing anti-GFPandanti-Flag-antibodies, respectively (Figure 2D).Subsequently, the stomatal behaviors of pub22 pub23 andcomplementation lines (pub22 pub23/35S:PUB22-GFP andpub22 pub23/35S:Flag-UNDPUB18-PUB22) in response to ABAwere monitored. Stomatal movement of pub22 pub23 after ABAtreatments (1 and 10 mM) was very similar to that of wild-type

Figure 1. (continued).

(A)Schematic structureof full-lengthPUB18cDNAandPUB18andΔUND-PUB18proteins. Solid lines indicate5ʹ- and3ʹ-untranslated regions. Thegraybarin the upper diagram represents the coding region. The N-terminal UND motif, U-box domain, and armadillo (ARM) repeats are indicated.(B) In vitro self-ubiquitination assay of PUB18 and ΔUND-PUB18. Recombinant MBP-PUB18, MBP-PUB18V305I, MBP-ΔUND-PUB18, and MBP-ΔUND-PUB18V36I proteins were incubated with Flag-tagged Ub (Flag-Ub) at 30°C for 2 h in the presence or absence of E1 (UBA1) and crude extract (10 mg totalproteins) prepared fromABA (100mM)-treated pub18 pub19 pub22 pub23 quadruple mutant leaves, as a source of E2 enzyme, along withMG132 (80mM)and protease inhibitor cocktail. Reaction products were resolved using SDS-PAGE and subjected to immunoblot analysis with anti-MBP and anti-Flagantibodies. The level of a-tubulin detected by anti-a-tubulin antibodywas used as an equal loading control of input crude extract. The presence of an equalamount of Flag-Ub in each lane was confirmed by anti-Flag antibody. Arrows indicate the migration of the unmodified PUB proteins.(C)RT-PCRanalysis ofwild-type,pub18pub19doublemutant, andpub18pub19/35S:HA-PUB18 (transgenic lines #1and#2) andpub18pub19/35S:Flag-ΔUND-PUB18 (transgenic lines #1 and #2) complementation plants. Each experiment was performed with three independent biological replicates. TheUbiquitin Conjugating Enzyme10 (UBC10) gene was used as a loading control.(D) Immunoblot analysis of pub18 pub19 double mutant and pub18 pub19/35S:HA-PUB18 (lines #1 and #2) and pub18 pub19/35S:Flag-ΔUND-PUB18(lines #1 and #2) complementation plants. Expression levels of HA-PUB18 and Flag-ΔUND-PUB18 proteins were determined using anti-HA and anti-Flagantibodies, respectively. The level of a-tubulin was an equal loading control.(E)Stomatalmovements ofwild-type,pub18pub19doublemutant, andpub18pub19/35S:HA-PUB18 (lines#1and#2) andpub18pub19/35S:Flag-ΔUND-PUB18 (lines #1and#2) complementationplants in response toABA. Light-grownmature rosette leaveswere immersed in stomatal opening solution for 2hand transferred to the solution containing different concentrations (0, 1, and 10mM) of ABA for 2 h. Stomata were imaged using bright-fieldmicroscopy. Atleast 30 stomatal apertures in each epidermal stripweremeasured per replicate. Three replicateswere performed for each experiment. Error bars represent6SE (n = 90; **P < 0.005, one-way ANOVA). Bars = 10 mm.(F)Stomatalmovements ofwild-type,pub18pub19doublemutant, andpub18pub19/35S:HA-PUB18 (lines#1and#2) andpub18pub19/35S:Flag-ΔUND-PUB18 (lines #1and#2) complementationplants in response to osmotic stress imposedby various concentrations (0, 0.2, and0.4M) ofmannitol. Error barsrepresent 6SE (n = 90; *P < 0.05, **P < 0.005, one-way ANOVA). Bars = 10 mm.

Role of the UND Motif of Arabidopsis PUB18 2955

Figure 2. ABA-Responsive Stomatal Movement Was Restored by the Overexpression of the N-Terminal UND in pub22 pub23 Mutant Plants.

(A) Schematic representation of full-length PUB22 cDNA and PUB22 and UNDPUB18-PUB22 proteins. Solid lines indicate 5ʹ- and 3ʹ-untranslated regions,and the gray bar represents the coding region. The UNDPUB18 motif, U-box domain, and armadillo (ARM) repeats are indicated.(B) In vitro self-ubiquitination of PUB22 and UNDPUB18-PUB22. The E3 Ub ligase activities of bacterially expressed MBP-PUB22 and MBP-UNDPUB18-PUB22 recombinant proteins and their single amino acid substitution variants (MBP-PUB22V24I and MBP-UNDPUB18-PUB22V293I) were examined with orwithout E1 (UBA1) and crude extract (10mg total proteins) of ABA (100mM)-treatedpub18pub19pub22pub23 leaves, as a source of E2 enzyme, alongwithFlag-tagged Ub (Flag-Ub), MG132 (80 mM), and protease inhibitor cocktail. Reaction products were analyzed by SDS-PAGE, followed by immunoblottingwith anti-MBP and anti-Flag antibodies. The level of a-tubulin detected by anti-a-tubulin antibody was used as an equal loading control of input crude

2956 The Plant Cell

plants. The average stomatal aperture in pub22 pub23 was;0.1036 0.004 at 10mMABA, whichwas about the same as thatof the wild-type plants (Figure 2E). This is consistent with theprevious reports that PUB22 regulates an ABA-independentdrought signaling pathway (Cho et al., 2008; Seo et al., 2012).Moreover, the overexpression of PUB22-GFP in pub22 pub23could not change the ABA-independent phenotype. By contrast,pub22 pub23/35S:Flag-UNDPUB18-PUB22 complementationlines exhibited markedly decreased sensitivity toward ABA(stomatal apertures = 0.148 6 0.007 for transgenic line #1 and0.15260.01 for line #2 at 10mMABA), indicating that the ectopicexpression of 35S:Flag-UNDPUB18-PUB22 in pub22 pub23 re-sulted in ABA-insensitive stomatal closure (Figure 2E). Theseresults strongly suggest that the UNDPUB18-PUB22 chimericprotein, in contrast to PUB22, plays a negative role in ABA-dependent stomatal movement. Based on the results presentedin Figures 1 and 2, we inferred that theN-terminal UNDof PUB18is negatively involved in ABA-regulated stomatal movement inArabidopsis.

PUB18 Interacts with Exo70B1

The specificity of E3 Ub ligases to their target proteins is a crucialfactor for the UPS. Identification of target proteins is thus anessential step in elucidating the cellular roles of E3 ligases.Therefore, we performed a yeast two-hybrid screen to identifythe target protein of PUB18. Based on this screen, we selectedExo70B1 as a putative interacting partner of PUB18. Exo70B1is an Arabidopsis paralog of mammalian Exo70 protein, whichis a subunit of the exocyst involved in vesicle tethering dur-ing exocytosis (Li et al., 2010; Wang et al., 2010; Kulich et al.,2013). Interaction of PUB18 and Exo70B1 appeared to bespecific, since PUB18 was not associated with Exo70B2,a homologof Exo70B1, in yeast cells (Figure 3A). To corroboratethe interaction between PUB18 and Exo70B1, we conductedan in vitro pull-down assay. In this experiment, purified MBP-PUB18 recombinant protein was coincubated with His-Myc-Exo70B1 or His-Myc-Exo70B2 in the presence of an Excelloseresin, and bound proteins were eluted with maltose (10 mM),followed by immunoblot analysis with anti-Myc and anti-MBPantibodies. His-Myc-Exo70B1 was pulled down from the Ex-cellose affinity matrix by MBP-PUB18 (Figure 3B). This in-dicates a physical interaction between PUB18 and Exo70B1.

By contrast, MBP-PUB18 and His-Myc-Exo70B2 did notinteract with each other in vitro (Figure 3B). In addition toPUB18, PUB19 had a binding activity to Exo70B1 in yeastcells and in vitro pull-down assay (Supplemental Figures3 and 4).Previous subcellular localization studies showed that Arabi-

dopsis Exo70B1 localized to small spherical bodies and/or dis-crete punctae (Wang et al., 2010; Kulich et al., 2013). When the35S:PUB18-GFP construct was transiently expressed in Ara-bidopsis mesophyll protoplasts by means of the polyethyleneglycol (PEG)-mediated transformation, the fluorescent signal forPUB18-GFPdisplayedapunctate pattern (Figure 3C),whichwashighly similar to that of Exo70B1-DsRED. These punctate lo-calization signals for PUB18-GFP and Exo70B1-DsRED weremerged in tobacco (Nicotiana benthamiana) leaf epidermal cells(Figure 3D), suggesting cosubcellular localization of PUB18 andExo70B1.The interaction between PUB18 and Exo70B1 was further

examined by a bimolecular fluorescence complementation (BiFC)assay. The yellow fluorescent protein (YFP) was spliced into theN-terminal region (YFPN) and the C-terminal region (YFPC). YFPN

and YFPC were fused to the C termini of PUB18 and Exo70B1,respectively. When PUB18-YFPN + Exo70B1-YFPC were coex-pressed in tobacco leaf epidermal cells, punctate fluorescentsignals were detected (Figure 3E). However, coexpression ofPUB18-YFPN + Exo70B2-YFPC did not give rise to reconstitutedfluorescent signal (Figure 3E; Supplemental Figure 5A). Thus, itappeared that PUB18 interacted with Exo70B1, but not withExo70B2, in tobacco cells.The35S:PUB18-GFPand35S:Flag-Exo70B1 fusionconstructs

were infiltrated into tobacco leaves. The total proteins were thenisolated and fractionated into soluble and membrane compo-nents. As shown in Figure 3F, both PUB18-GFP and Flag-Exo70B1 were exclusively found in the membrane fractiondetected by anti-GFP and anti-Flag antibodies, respectively. ThePUB18-GFPandFlag-Exo70B1proteinswerepartially solubilizedby 1 M NaCl and 0.1 M Na2CO3. These results suggest thatPUB18-GFP and Flag-Exo70B1 are membrane-associated pro-teins. Taken together, yeast two-hybrid, in vitro pull-down, sub-cellular localization, BiFC, and protein fractionation experimentsindicated that PUB18 interacted with Exo70B1. This suggestedthat Exo70B1 might be a target protein recognized by PUB18 E3Ub ligase.

Figure 2. (continued).

extract. Thepresenceof anequal amount of Flag-Ub ineach lanewasconfirmedbyanti-Flag antibody. Arrows indicate themigration of theunmodifiedPUBproteins.(C) RT-PCR analysis of wild-type, pub22 pub23 double mutant, and pub22 pub23/35S:PUB22-GFP (transgenic lines #1 and #2) and pub22 pub23/35S:Flag-UNDPUB18-PUB22 (transgenic lines #1 and #2) complementation plants. Four independent biological replicate were performed. TheUBC10 genewasused as a loading control.(D) Immunoblot analysis of pub22 pub23 double mutant and pub22 pub23/35S:PUB22-GFP (lines #1 and #2) and pub22 pub23/35S:Flag-UNDPUB18-PUB22 (lines #1 and #2) complementation plants. Ectopic expression of PUB22-GFP and Flag-UNDPUB18-PUB22 proteins was detected using anti-GFPand anti-Flag antibodies, respectively. The level of a-tubulin was used as an equal loading control.(E) Stomatal movements of wild-type, pub22 pub23 double mutant, and pub22 pub23/35S:PUB22-GFP (lines #1 and #2) and pub22 pub23/35S:Flag-UNDPUB18-PUB22 (lines #1 and #2) complementation plants in response to different concentrations (0, 1, and 10mM) of ABA. Thirty stomatal apertures pertreatmentweremeasured, and three replicateswere performed for each experiment. Error bars represent6SE (n= 90; **P < 0.005, one-wayANOVA). Bars =10 mm.

Role of the UND Motif of Arabidopsis PUB18 2957

Figure 3. PUB18 Interacts with Exo70B1.

(A) Yeast two-hybrid analysis. The full-length coding region of PUB18 was cloned into the pGBKT7 vector, and Exo70B1 and Exo70B2 were cloned intopGADT7. Different combinations of bait and prey plasmids were cotransformed as indicated into the yeast strain AH109. The protein-protein interactionswere examined by plating yeast cells on SD/-Leu/-Trp/-His solid medium containing 2 mM 3-amino-1,2,4,-triazole (3-AT). p53 + T-antigen was used asa positive control. Lambda + T-antigen and empty pGADT7 (ø) plasmid were used as negative controls.(B) In vitropull-downassay.PurifiedMBP-PUB18+His-Myc-Exo70B1,MBP-PUB18+His-Myc-Exo70B2,MBP+His-Myc-Exo70B1,andMBP+His-Myc-Exo70B2 recombinant proteins were coincubated with an Excellose resin, and bound proteins were eluted by maltose (10 mM), followed by immunoblotanalysis with anti-Myc and anti-MBP antibodies. Arrows indicate MBP-PUB18 and MBP. MBP was used as a negative control.

2958 The Plant Cell

The UNDPUB18 Motif Determines the Binding Specificities ofPUB18 and PUB22 toward Exo70B1 andExo70B2, Respectively

The aforementioned results provide evidence that PUB18 inter-acts with Exo70B1. On the other hand, Stegmann et al. (2012)reported thatPUB22bindsandubiquitinatesExo70B2,ahomologof Exo70B1, to regulate the negative response to pathogen in-fection in Arabidopsis. Because the presence of the N-terminalUND in PUB18 is a noteworthy distinction of PUB18 fromPUB22,we speculated that the binding activities of PUB18 and PUB22 toExo70B1 and Exo70B2, respectively, are stimulated by thepresence or absence of the UNDPUB18 motif. To test this hy-pothesis, we estimated the binding activities of ΔUND-PUB18 toExo70B1 and Exo70B2 by yeast two-hybrid assay. Interestingly,ΔUND-PUB18 failed to interactwithExo70B1 in yeast cells (Figure4A). This suggests that the UNDPUB18 motif is required for theinteraction of PUB18 with Exo70B1. By contrast, ΔUND-PUB18was able to bind to Exo70B2.

Interaction of ΔUND-PUB18 to Exo70B2 was reinforced by anin vitro pull-down assay. MBP-fused ΔUND-PUB18 recombinantprotein was coincubated with His-Myc-Exo70B1 or His-Myc-Exo70B2 in the presence of an Excellose resin. Bound proteinswere thenelutedbymaltose (10mM)andsubjected to immunoblotanalysis with anti-Myc and anti-MBP antibodies. As shown inFigure 4B, His-Myc-Exo70B2 was pulled down by MBP-ΔUND-PUB18, indicating that ΔUND-PUB18 directly interacts withExo70B2 in vitro. By contrast, MBP-ΔUND-PUB18 did not bind toHis-Myc-Exo70B1 in vitro (Figure 4B). These results were con-firmed by an in vivo coimmunoprecipitation (co-IP) assay. Toeliminate possible effects of self-ubiquitination activity of ΔUND-PUB18, a single amino acid substitution derivative ΔUND-PUB18V36I was used. The 35S:Flag-ΔUND-PUB18V36I constructwas transiently expressed with 35S:Myc-Exo70B1 or 35S:Myc-Exo70B2 in tobacco leaf cells by means of Agrobacteriumtumefaciens-mediated infiltration. Total leaf proteins (200 mg)were immunoprecipitated with an anti-Flag affinity gel matrix. Theboundproteinswere elutedandsubjected to immunoblot analysiswith anti-Flag and anti-Myc antibodies. The results showedthat Flag-ΔUND-PUB18V36I and Myc-Exo70B2 were coimmu-noprecipitated by anti-Flag antibody (Figure 4C). Thus, ΔUND-PUB18V36I interactedwithExo70B2 in tobaccocells.However, the

interaction between Flag-ΔUND-PUB18V36I and Myc-Exo70B1was very weak and barely detectable in tobacco cells (Figure 4C).Therefore, the deletion of the N-terminal UND from PUB18 re-sulted in a change in its binding property from Exo70B1 toExo70B2.To further corroborate the role of UNDPUB18, we determined the

binding activities of the UNDPUB18-PUB22 chimeric protein toExo70B1 and Exo70B2 using yeast two-hybrid, in vitro pull-downanalysis, and an in vivo co-IP assay. Notably, UNDPUB18-PUB22interacted with Exo70B1 in yeast cells (Figure 4D). In addition, theinteraction between UNDPUB18-PUB22 and Exo70B2 was negli-gible. These results were in sharp contrast to those suggestingthat PUB22 does not bind to Exo70B1 (Figure 4D), but binds toExo70B2 (Figure4D;Stegmannet al., 2012). In vitropull-downandin vivo co-IP experiments confirmed the interaction betweenUNDPUB18-PUB22 and Exo70B1 (Figures 4E and 4F). Thus, itseems highly likely that the addition of the UNDPUB18 domain toPUB22 altered its binding specificity from Exo70B2 to Exo70B1,indicating that the N-terminal UND is a critical factor for the in-teraction of UNDPUB18-PUB22 with Exo70B1. Collectively, thedomain-swapping results presented in Figure 4 strongly suggestthat the binding specificities of PUB18 and PUB22 towardExo70B1 and Exo70B2, respectively, depend on the presence orabsence of the N-terminal UNDPUB18 motif.

Turnover of Exo70B1 Is Regulated by PUB18 ina Proteasome-Dependent Manner

The specific interaction between PUB18 and EXO70B1 (Figure 4)suggests that the cellular level of Exo70B1 is regulated via theUPS. To investigate this possibility, we conducted a cell-freedegradation assay. Bacterially expressed His-Myc-Exo70B1 re-combinant protein was incubated with a crude extract (100 mgtotal proteins) prepared from wild-type and pub18 pub19 doublemutant leaves pretreated with 100 mM ABA and subjected toimmunoblot analysis with anti-Myc antibody. The level of His-Myc-Exo70B1was rapidly reducedover time inwild-type cell-freeextracts. As shown in Figure 5A, 49.3% 6 3.7% of His-Myc-Exo70B1 was degraded after 3.0 h of incubation. By contrast, theHis-Myc-Exo70B1 protein remained stable in the presence ofMG132, an inhibitor of the proteasome complex. After 3 h of in-cubationwith50mMMG132,19.2%61.2%ofHis-Myc-Exo70B1

Figure 3. (continued).

(C)Subcellular localization ofPUB18 inArabidopsis protoplasts. The35S:GFP and35S:PUB18-GFP constructswere transfected intoprotoplasts obtainedfrom Arabidopsis leaf tissues using PEG-mediated transformation. The GFP signals were visualized by fluorescence microscopy under dark-field con-ditions. Bars = 10 mm.(D) Subcellular localization of PUB18 and Exo70B1 in tobacco leaf epidermal cells. The 35S:PUB18-GFP and 35S:Exo70B1-DsRED constructs wereintroduced into tobacco leaf epidermal cells using an Agrobacterium-mediated infiltration method. The localization signals of PUB18-GFP and Exo70B1-DsRED were detected by confocal microscopy under dark-field conditions. Bars = 10 mm.(E)BiFCanalysis for in vivo interaction betweenPUB18andExo70B1. The full-length coding regionofPUB18was fused to theN-terminal region (YFPN; 1 to155 aminoacids)ofYFP.Exo70B1andExo70B2were fused to theC-terminal region (YFPC; 156 to239amino-acids)ofYFP. ThePUB18-YFPN+YFPC, YFPN+Exo70B1-YFPC, PUB18-YFPN + Exo70B1-YFPC, and PUB18-YFPN + Exo70B2-YFPC proteins were transiently coexpressed in tobacco leaf epidermalcells. Reconstituted fluorescent signals were detected via confocal microscopy. Bars = 20 mm.(F)Membrane association assay of PUB18 and Exo70B1. The 35S:PUB18-GFP and 35S:Flag-Exo70B1 constructs were transiently expressed in tobaccoleaves.Supernatant (S) andpellet (P) fractionsof total leafproteinextractswere treatedwith1MNaClor0.1MNa2CO3andsubjected to immunoblot analysiswith anti-GFP and anti-Flag antibodies.

Role of the UND Motif of Arabidopsis PUB18 2959

Figure 4. The UNDPUB18 Domain Determines the Binding Specificities of PUB18 and PUB22 toward Exo70B1 and Exo70B2, Respectively.

(A) ΔUND-PUB18 and Exo70B2 interact in yeast cells. The ΔUND-PUB18 coding region was cloned into the pGBKT7 vector, and Exo70B1 and Exo70B2were cloned intopGADT7. TheΔUND-PUB18-pGBKT7+Exo70B1-pGADT7 andΔUND-PUB18-pGBKT7+Exo70B2-pGADT7plasmidswere transformedintoyeastAH109cells. Theyeast cellswereplatedonSD/-Leu/-Trp/-His solidmedium in thepresenceof2mM3-AT.p53+T-antigenandPUB18+Exo70B1were used as positive controls. Lambda + T-antigen and empty pGADT7 (ø) were used as negative controls.(B) In vitro interaction ofΔUND-PUB18 andExo70B2. PurifiedMBP-ΔUND-PUB18 +His-Myc-Exo70B1,MBP-ΔUND-PUB18 +His-Myc-Exo70B2,MBP+His-Myc-Exo70B1, andMBP +His-Myc-Exo70B2 recombinant proteins were coincubated in the presence of an Excellose resin. After extensive washing,bound proteins were eluted withmaltose (10mM). Eluted proteins were subjected to immunoblot analysis with anti-Myc and anti-MBP antibodies. Arrowsindicate MBP-ΔUND-PUB18 and MBP. MBP was used as a negative control.

2960 The Plant Cell

was degraded (Figure 5A), suggesting that Exo70B1 is degradedin a proteasome-dependent manner with wild-type cell-free ex-tracts. The results further show that His-Myc-Exo70B1 was de-graded more slowly in pub18 pub19 cell-free extracts than in thewild-type extracts. After a 3.0-h incubation, 81.7%66.4%ofHis-Myc-Exo70B1was still detected in thepub18pub19extracts, andthis slower degradation was almost completely impeded byMG132 (Figure 5A). The degradation of REPRESSOR OF GA1-3,which is regulated by the UPS (Dill et al., 2004; Lee et al., 2010),exhibited similar degradation patterns in wild-type and pub18pub19 crude extracts, confirming the specific role of PUB18 inExo70B1 degradation (Figure 5A). The amount of Rubisco largesubunit (rbcL) remained unchanged in both wild-type and pub18pub19 extracts.

The PUB18-dependent turnover of Exo70B1 was further in-vestigated using a protoplast transient expression system. The35S:Myc-Exo70B1 and 35S:sGFP constructs were transfectedintoprotoplastsprepared fromwild-typeandpub18pub19doublemutant leaves using PEG-mediated transformation (Kim et al.,2016). After overnight transfection, protoplasts were incubatedwith cycloheximide (CHX; 250 mM), an inhibitor of protein syn-thesis, for 2 h with or without MG132 (50 mM) and ABA (100 mM).Total proteins were extracted and examined by immunoblotanalysis with anti-Myc and anti-GFP antibodies. The results re-vealed that the Myc-Exo70B1 level was rapidly decreased up to51.3% 6 8.8% in the presence of ABA in wild-type protoplasts(Figure 5B). However, His-Myc-Exo70B1 was more stable inpub18 pub19 protoplasts and 93.8% 6 2.1% of the protein wasdetected in the presence of ABA. Degradation of Myc-Exo70B1was inhibited byMG132 in bothwild-type andmutant protoplasts(Figure 5B). Theamount ofMyc-Exo70B1wasslightly increased inthe presence of MG132, suggesting that degradation of Myc-Exo70B1 is subject to control of 26S proteasome. The level ofGFP, which was used to gauge the protoplast transfection effi-ciency and as an equal loading control, remained unchangedregardless of thepresenceof ABAandMG132.Overall, the resultsof in vitro and in vivo protein degradation assays suggest that theturnover of Exo70B1 is regulated, at least in part, by PUB18 ina proteasome-dependent fashion.

The UNDPUB18 Motif Is Critical for the Ubiquitination ofExo70B1 and Exo70B2 by PUB18 and PUB22, Respectively

Next, an in vitro ubiquitination assaywasperformed (SupplementalFigure 6; Figure 6). His-Myc-PUB18 and His-Myc-ΔUND-PUB18were coincubated with MBP-Exo70B1 or MBP-Exo70B2, alongwith Flag-Ub, E1, and ATP, in the presence or absence of a proteincrude extract (as a sourceof E2) prepared fromABA-treatedpub18pub19 pub22 pub23mutant leaves. The reactionmixture was thensubjected to immunoblot analysis with anti-Flag, anti-MBP, anti-Myc, and anti-a-tubulin antibodies. Ubiquitinated bands weredetected by anti-Flag antibody. Equal loading of each lane wasconfirmed by anti-MBP and anti-Myc antibodies. The level ofa-tubulinwasusedasa loadingcontrolofcrudeextract.The resultsshowed that coincubation of His-Myc-PUB18 and MBP-Exo70B1yielded high molecular mass smear bands detected by anti-Flagantibody (left panel in Figure 6A). The produced smear bands werenotbecauseof theendogenousPUB18present in theproteincrudeextract, since thiswasclearly detectedwitha crudeextract ofABA-pretreated pub18pub19pub22pub23mutant leaves. This result inconjunction with those of yeast two-hybrid and pull-down assays(Figure4) indicates thatExo70B1 isasubstrateofPUB18.However,coincubation of His-Myc-ΔUND-PUB18withMBP-Exo70B1 failedto produce any detectable highmolecular mass smear ladders (leftpanel in Figure 6A), suggesting that Exo70B1was ubiquitinated byPUB18, but not by ΔUND-PUB18. Conversely, His-Myc-ΔUND-PUB18 gave rise to highmolecular mass smear bandswhen it wascoincubated with MBP-Exo70B2 (right panel in Figure 6A). Theseresults indicate that deletion of the N-terminal UND of PUB18 re-sulted in the alteration of ubiquitination specificity of PUB18 fromExo70B1 to Exo70B2.An identical set of in vitro ubiquitination experiments was

performedwith His-Myc-PUB22 andHis-Myc-UNDPUB18-PUB22E3 ligases. Consistent with previous findings (Stegmann et al.,2012), His-Myc-PUB22 effectively ubiquitinated MBP-Exo70B2(left panel in Figure 6B). By contrast, His-Myc-UNDPUB18-PUB22was unable to ubiquitinate MBP-Exo70B2, suggesting thatthe addition of UNDPUB18 to PUB22 hampered its ubiquiti-nation activity toward Exo70B2 (left panel in Figure 6B). Although

Figure 4. (continued).

(C) In vivo co-IP assay of ΔUND-PUB18 and Exo70B2. The 35S:Flag-ΔUND-PUB18V36I construct was transiently coexpressed with 35S:Myc-Exo70B1 or35S:Myc-Exo70B2 in tobacco leaf epidermal cells by an Agrobacterium-mediated infiltration method. Total leaf proteins (200 mg) were extracted andimmunoprecipitatedwith anti-Flag affinity gelmatrix. The boundproteinswere elutedbyboilingwith 43SDSsample buffer. Elutedproteinswere separatedby SDS-PAGE and subjected to immunoblot analysis with anti-Flag and anti-Myc antibodies.(D) UNDPUB18-PUB22 and Exo70B1 interact in yeast cells. The UNDPUB18-PUB22-pGBKT7 + Exo70B1-pGADT7 and UNDPUB18-PUB22-pGBKT7 +Exo70B2-pGADT7 constructs were introduced into yeast cells. The yeast cells were plated on SD/-Leu/-Trp/-His solid medium in the presence of 2 mM3-AT. p53 + T-antigen and PUB22 + Exo70B2 and were used as positive controls. Lambda + T-antigen and empty pGADT7 (ø) plasmid were negativecontrols.(E) In vitro interaction of UNDPUB18-PUB22 and Exo70B1. Purified MBP-UNDPUB18-PUB22 + His-Myc-Exo70B1, MBP-UNDPUB18-PUB22 + His-Myc-Exo70B2, MBP + His-Myc-Exo70B1, and MBP + His-Myc-Exo70B2 recombinant proteins were coincubated in the presence of an Excellose resin. Afterextensive washing, bound proteins were eluted with maltose (10 mM). Eluted proteins were detected by immunoblot analysis with anti-Myc and anti-MBPantibodies. Arrows indicate MBP-UNDPUB18-PUB22 and MBP. MBP was used as a negative control.(F) In vivo co-IP assay of UNDPUB18-PUB22 and Exo70B1. The 35S:Flag-UNDPUB18-PUB22V293I construct was transiently coexpressed with 35S:Myc-Exo70B1or 35S:Myc-Exo70B2 in tobacco leaf epidermal cells. Total leaf proteins (200mg)were extracted and immunoprecipitatedwith an anti-Flag affinitygel matrix. The bound proteins were eluted by boiling with 43 SDS sample buffer and detected by immunoblot analysis with anti-Flag and anti-Mycantibodies.

Role of the UND Motif of Arabidopsis PUB18 2961

His-Myc-PUB22 failed to ubiquitinate MBP-Exo70B1, His-Myc-UNDPUB18-PUB22wasable toubiquitinateMBP-Myc-Exo70B1, asevidenced by the formation of high molecular weight ladders de-tected by anti-Flag antibody (right panel in Figure 6B). Taken to-gether, the results of in vitro ubiquitination assays indicate that thedeletion of the N-terminal UND motif from PUB18 changes thetarget specificity from Exo70B1 to Exo70B2. By contrast, additionof UNDPUB18 to PUB22 resulted in the change in its ubiquitinationactivity from Exo70B2 to Exo70B1. Therefore, the ubiquitinationspecificities of PUB18 and PUB22 to Exo70B1 and Exo70B2, re-spectively, aremost likelydependenton thepresenceorabsenceofthe N-terminal UNDPUB18 motif.

Exo70B1 and Exo70B2 Are Positive Regulators of theResponse to Mannitol (Drought) Treatments in ABA-Dependent and ABA-Independent Manners, Respectively

Our results indicate that ABA responsiveness and target specif-icities of PUB18 and PUB22 were dependent on the presenceor absence of the N-terminal UNDPUB18 motif (Figures 1 to 4).Exo70B1 is a substrate of PUB18 that contains the N-terminalUND, whereas Exo70B2 is a target protein of PUB22, in which theUNDPUB18motif is absent (Figures 5 and 6; Stegmann et al., 2012).Based on these results, we presumed that Exo70B1 playsa positive role in the ABA response and, by contrast, the cellularrole of Exo70B2 is ABA independent. To elucidate this possibility,

Figure 5. In Vitro and in Vivo Degradation Assays of Exo70B1.

(A) Cell-free degradation assay of Exo70B1. Bacterially expressed His-Myc-Exo70B1 was incubated with a cell-free crude extract (100 mg total proteins)prepared from ABA (100 mM)-treated mature rosette leaves of wild-type or pub18 pub19mutant plants in the presence or absence of 50 mMMG132. Thetime-dependent changes of protein level were monitored by immunoblotting with anti-Myc antibody. The RGA1-2xFlag protein was used as a positivecontrol for the 26S proteasome-dependent degradation. Equal loading of cell-free extracts was confirmed with Rubisco and visualized by Coomassiestaining. Theprotein levelswerequantifiedusing ImageJsoftware.Results arepresented asanaverage valueof five independentbiological replicates. Errorbars represent 6SE (n = 5; *P < 0.05, **P < 0.005, one-way ANOVA).(B) In vivo degradation assayof Exo70B1 inwild-type andpub18pub19protoplasts. The 35S:Myc-Exo70B1 and35S:GFP constructswere transfected intoprotoplasts freshly isolated fromwild-typeandpub18pub19 leaf tissuesusingPEG-mediated transformation.After overnight transfection,protoplastswereincubatedwithCHX (250mM) for 2hwith orwithoutMG132 (50mM)andABA (100mM). Total proteinswere extracted andexaminedby immunoblot analysiswith anti-Mycandanti-GFPantibodies.The levelsofGFPareshownas transfectionefficiencyofprotoplast andequal loadingcontrol. Results arepresentedas an average value of three independent biological replicates. Error bars represent 6SE (n = 6; *P < 0.05, **P < 0.005, one-way ANOVA).

2962 The Plant Cell

we obtained the loss-of-function T-DNA inserted knockout mu-tants ofExo70B1 andExo70B2. Theexo70b1 (SALK_202386) andexo70b2 (SALK_091877) mutant lines contained a single T-DNAinsertion in the exon at nucleotide 734 and in the intron at nu-cleotide 539, respectively (Figure 7A). In addition, transgenicArabidopsis plants that constitutively expressed Exo70B1 andExo70B2 under the control of the 35S CaMV promoter wereconstructed. Disruption and ectopic expression of Exo70B1and Exo70B2 were verified by genotyping PCR (SupplementalFigure 7) and RT-PCR (Figure 7B), respectively. As shown inFigure 7C, the exo70b1 mutant line displayed retarded growthwith wrinkled rosette leaves under long-day growth conditions(16 h light/8 h dark) when compared with growth of wild-typeplants. On the other hand, 35S:Exo70B1, exo70b2, and 35S:Exo70B2progenyweremorphologically normal under the samegrowth conditions.

Next, ABA-mediated stomatal behaviors of these mutant andoverexpressing linesweremonitored. After 10mMABA treatment,the average stomatal apertures of wild-type, exo70b1, and 35S:Exo70B1 were 0.1006 0.004, 0.1546 0.010, and 0.0726 0.003(line #1) to 0.068 6 0.005 (line #2), respectively (Figure 7D).Thus, ABA-mediated stomatal closurewas inhibited in exo70b1mutant plants and, by contrast, highly enhanced in Exo70B1-overexpressingplants, implying thatExo70B1playsapositive rolein ABA-promoted stomatal closure. To further examine the role ofExo70B1, the second mutant allele (exo70b1-1, GK-114C03;Hong et al., 2016) of Exo70B1 was obtained and its response toABA was tested (Supplemental Figure 8). Recently, Hong et al.(2016) reported that exo70b1-1 showed similar stomatal move-ments to wild-type plants in response to ABA treatment (1 mM for0.5 to 1 h). Although phenotypes of exo70b1-1 were less severethan exo70b1, the exo70b1-1 progeny exhibited retarded growth

Figure 6. The UNDPUB18 Domain Is Critical for the Ubiquitination of Exo70B1 and Exo70B2 by PUB18 and PUB22, Respectively.

(A) In vitro ubiquitination assays of Exo70B1andExo70B2byPUB18andΔUND-PUB18, respectively. RecombinantHis-Myc-PUB18andHis-Myc-ΔUND-PUB18E3Ub ligaseswere incubatedwithMBP-Exo70B1 (left panel) orMBP-Exo70B2 (rightpanel)withorwithoutcrudeextract (10mg total proteins) ofABA(100 mM)-treated pub18 pub19 pub22 pub23 quadruple mutant leaves, along with Flag-tagged Ub (Flag-Ub), E1 (UBA1), MG132 (80 mM), and proteaseinhibitor cocktail. The reaction mixtures were subjected to immunoblot analysis with anti-Flag, anti-MBP, anti-Myc, and anti-a-tubulin antibodies.Ubiquitinated bands were detected by anti-Flag antibody. Equal loading of each lane was confirmed by anti-MBP and anti-Myc antibodies. The level ofa-tubulin was used as a loading control of crude extract. Arrows indicate the migration of the unmodified PUB proteins.(B) In vitro ubiquitination assays of Exo70B2 and Exo70B1 by PUB22 and UNDPUB18-PUB22, respectively. His-Myc-PUB22 and His-Myc-UNDPUB18-PUB22E3Ub ligaseswere incubatedwithMBP-Exo70B2 (left panel) orMBP-Exo70B1 (rightpanel)withorwithoutcrudeextract (10mg total proteins) ofABA(100mM)-treatedpub18pub19pub22pub23quadruplemutant leaves, alongwith Flag-Ub, E1 (UBA1),MG132 (80mM), andprotease inhibitor cocktail. Thesamples were resolved by SDS-PAGE and immunoblotted with anti-Flag, anti-MBP, anti-Myc, and anti-a-tubulin antibodies. Ubiquitinated bands weredetectedbyanti-Flagantibody.Equal loadingof each lanewasconfirmedbyanti-MBPandanti-Mycantibodies. The level ofa-tubulinwasusedasa loadingcontrol of crude extract. Arrows indicate the migration of the unmodified PUB proteins.

Role of the UND Motif of Arabidopsis PUB18 2963

Figure 7. Exo70B1 and Exo70B2 Are Positive Regulators of the Response to Mannitol Treatments in ABA-Dependent and ABA-Independent Manners,Respectively

2964 The Plant Cell

under long-day conditions andwere hyposensitive toABA (10mMfor 2 h) in stomatal closure compared with wild-type plants(Supplemental Figure 8). By contrast, stomatal apertures of wild-type, exo70b2mutant, and 35S:Exo70B2 transgenic plants wereindistinguishable in response to ABA treatment, indicating thatExo70B2 is not involved in theABA-induced stomatalmovements(Figure 7D).

However, in response to mannitol treatment (0.4 M), bothexo70b1 and exo70b2mutants displayed insensitive phenotypesin stomatal closure (Figure 7D). Furthermore, stomatal move-ments in Exo70B1 and Exo70B2 overexpressors showed similarlevels of hypersensitivity in response to mannitol-derived waterdeficit. These results suggest that Exo70B1, a target protein ofPUB18, is a positive regulator of both ABA-promoted andmannitol (drought)-promoted stomatal closure. Conversely,Exo70B2, a substrate of PUB22, is positively involved in mannitol-induced stomatal closure, but not in the ABA-mediated sto-matal movement.

PUB18 Is Epistatic to Exo70B1 in the ABA-Mediated DroughtStress Responses

Exo70B1 is a target protein of PUB18 and plays a positive rolein the mannitol (drought)-promoted stomatal closure in an ABA-dependent manner (Figure 7). To define whether Exo70B1 actsdownstream of PUB18, we generated the pub18 pub19 exo70b1triple knockout mutant line (Figure 8A), and its phenotypicproperties were compared with those of wild-type, pub18 pub19,and exo70b1 plants. Real-time RT-qPCR revealed that the ex-pressions ofPUB18 andPUB19weremarkedly higher in exo70b1knockout mutant than in wild-type plants (Figure 8A). In addition,theexpressionofEXO70B1washigher in thepub18pub19doublemutant line than in wild-type plants. The reason for these higherexpression levels ofPUB18/PUB19andEXO70B1 inexo70b1andpub18 pub19 progeny, respectively, is unknown.

As indicated in Figures 7B and 8B, the exo70b1 single mutantshowed smaller leaves with partial lesions and a twisted mor-phology compared with the leaves of wild-type and pub18 pub19double mutant plants under long-day growth conditions. Thesemorphological abnormalities were also evident in pub18 pub19exo70b1 triple knockout mutant plants (Figure 8B), suggestingthat Exo70B1 acts downstream of PUB18/PUB19. To further

examine the epistatic interaction of Exo70B1 and PUB18, theABA- and mannitol-mediated stomatal behaviors of wild-type,exo70b1 single, pub18 pub19 double, and pub18 pub19 exo70b1triple mutant plants were compared. In response to 10 mM ABAand0.4Mmannitol treatments, stomatalmovements of thepub18pub19 exo70b1 triple mutant exhibited insensitive phenotypes,which were reminiscent of those of the exo70b1 single mutantrather than the pub18 pub19 double mutant (Figure 8C). Thesemorphological and stomatal movement studies suggest thatPUB18 is epistatic to Exo70B1 in the ABA-mediated droughtstress responses. Taken together, we concluded that Exo70B1 isa target protein of the U-box E3 ligase PUB18 and exerts itspositive effects on the ABA signaling pathway downstream ofPUB18.

DISCUSSION

A large number of plant PUBs, arising from extensive gene pro-liferation and diversification, compared with those in yeasts andmammals, provides the possibility that U-box E3 Ub ligases playcrucial roles in various cellular processes in higher plants. Of the64 U-box E3 ligases in Arabidopsis, the largest group of PUBs iscomposed of 41 PUB-ARM proteins, among which 17 proteins(UND-PUB-ARM) possess the UND motif in their N termini. Al-though the UND motifs were first identified based on theirN-terminal positional conservation in a subset of PUBs (Mudgilet al., 2004), a high level of amino acid sequence diversity of UNDimplies that functional specificities of UND-PUB-ARM proteinsare dependent, at least in part, on the UND motifs (SupplementalFigure 9; Samuel et al., 2009; Antignani et al., 2015). However, thedetailed cellular functions of UND are largely unknown in higherplants.Arabidopsis U-box E3 Ub ligases PUB18, PUB19, PUB22, and

PUB23actasnegative regulatorsofdroughtstress responses,butthey exhibit distinct modes of action: PUB18/PUB19 regulate thedrought stress response in an ABA-dependent manner, whereasthe roleofPUB22/PUB23wasABA-independent (Choet al., 2008;Seo et al., 2012). In addition, PUB22/PUB23 along with PUB24regulate thenegative response topathogen infection (Trujillo et al.,2008). In this study, we focused on elucidating the molecularmechanismthatmediates this functional separationofPUB18and

Figure 7. (continued).

(A) Schematic structure of exo70b1 and exo70b2 T-DNA inserted loss-of-function mutant alleles. Inverted triangles indicate T-DNA insertion sites. Blackbars, open bars, and solid line represent coding regions, untranslated regions, and intron, respectively. Gene-specific and T-DNA-specific primers used inRT-PCR are shown with arrows.(B)RT-PCRanalysis todetect the transcript levelsofExo70B1andExo70B2 inwild-type,exo70b1,35S:Exo70B1 (transgenic lines #1and#2),exo70b2, and35S:Exo70B2 (transgenic lines #1 and #2) plants. Each experiment was performed with three independent biological replicates. UBC10 was used asa loading control.(C) Phenotypes of wild-type, exo70b1, 35S:Exo70B1, exo70b2, and 35S:Exo70B2 plants under normal growth conditions. Seedlings were grown in half-strengthMurashige and Skoogmedium containing 1% sucrose and 0.7% phytoagar (pH 5.7) for 9 d, transferred to soil, and grown for 4 weeks in a growthchamber at 22°C under long-day conditions (16 h light/8 h darkness).(D) ABA- and mannitol-induced stomatal closure in wild-type, exo70b1, 35S:Exo70B1 (transgenic lines #1 and #2), exo70b2, and 35S:Exo70B2 (transgeniclines#1and#2) plants. Light-grownmature rosette leaveswere immersed in stomatal openingsolution for 2h and transferred tosolution that contained10mMABAor0.4Mmannitol for 2 h. Stomatawere capturedusingbright-fieldmicroscopy. At least 30 stomatal apertures in each epidermal stripweremeasured perreplicate. Three replicates were performed for each experiment. Error bars represent 6SE (n = 90, **P < 0.005, one-way ANOVA). Bars = 10 mm.

Role of the UND Motif of Arabidopsis PUB18 2965

PUB22 in relation to ABA responses. We found an apparent dif-ference between PUB18/PUB19 and PUB22/PUB23: the presenceof the N-terminal UND motif in the former. According to the phy-logenetic tree of 41 PUB-ARM proteins constructed on the basis ofthe alignment of U-box sequences, UND-PUB-ARM proteins weredivided into three distinct clusters, indicating that the UND motifswere independently obtained multiple times for specialized func-tions during evolution (Mudgil et al., 2004; Ishizaki et al., 2013;SupplementalFigure10andSupplementalDataSet1). Interestingly,PUB18/PUB19 and PUB22/PUB23 fall into the same cluster.

Our results indicate that theUNDPUB18motif is critically involvedin the negative regulation of ABA-dependent stomatal behaviors

(Figures 1E and 2E). Because the mannitol-induced drought re-sponse was not affected by UNDPUB18 (Figure 1F), the UNDPUB18

motif is not likely to be a general negative factor for stress re-sponses, but is specific to the ABA-mediated drought response.Thus, the functional differentiation of PUB18 and PUB22 in re-sponse toABAmight result from thepresenceor absenceofUND.Liu et al. (2016) recently reported that the N-terminal domain ofBrassica oleracea ARC1, the closest homolog of ArabidopsisPUB17, confers a binding specificity of ARC1 toward Exo70A1.Exo70B1 was identified as an interaction partner of PUB18

(Figures 3A and 3B). The Exo70 (exocyst component of 70 kD)protein is a core component of an evolutionarily conserved

Figure 8. PUB18 Is Epistatic to Exo70B1 in the ABA-Mediated Drought Stress Responses.

(A)Real-time qRT-PCRanalysis to detect the transcripts levels ofPUB18,PUB19, andExo70B1 inwild-type andpub18pub19double, exo70b1 single, andpub18 pub19 exo70b1 triple mutant plants. ACT8 was used as an endogenous control gene to normalize the expression fold of PUB18, PUB19, andExo70B1. Error bars represent 6SE from three independent experiments.(B) Growth morphologies of wild-type and pub18 pub19, exo70b1, and pub18 pub19 exo70b1 mutant plants under normal conditions. Seedlings weregrown on half-strengthMurashige and Skoog agar plates for 9 d, transplanted to individual pots, and grown for 4weeks in a growth chamber at 22°C underlong-day conditions (16 h light/8 h darkness).(C)Stomatalmovementsofwild-type andpub18pub19, exo70b1, andpub18pub19 exo70b1mutant plants in response toABA (10mM)andmannitol (0.4M).ABA-andmannitol-mediatedstomatalbehaviorsweremonitoredasdescribed inFigure7D. Imagesofstomatawerecapturedusingbright-fieldmicroscopy.Atleast30stomatal apertures ineachepidermalstripweremeasuredper replicate.Three replicateswereperformed foreachexperiment.Errorbars represent6SE

(n = 90, **P < 0.005, one-way ANOVA). Bars = 10 mm.

2966 The Plant Cell

exocyst vesicle-tethering complex, which consisted of eightsubunits (Novicket al., 1980).Among these,Exo70hasundergonethe most remarkable expansion in plants. Therefore, there are23 paralogs of Exo70, including Exo70B1, in Arabidopsis. Thisexpansion implies that plants have developed a functionally dif-ferentiatedExo70gene family tomediateplant-specificprocessesat the evolutionary point of view (Chong et al., 2010; Li et al., 2010;Cvr�cková et al., 2012; Zárský et al., 2013; Indriolo et al., 2014; Linet al., 2015). Punctate subcellular localization and membraneassociation patterns of PUB18 were highly similar to those ofExo70B1, further supporting their physical interactions as bindingpartners (Figures 3C to 3F). Furthermore, Exo70B1 is degradedbya 26S proteasome complex in a PUB18-depenent fashion (Figure5). Exo70B2 was shown to be a target substrate of PUB22 E3 Ubligase (Stegmann et al., 2012). Among the 23 Exo70 paralogousproteins in Arabidopsis, Exo70B2 is the closest homolog ofExo70B1 (54% identity) (Li et al., 2010; Stegmann et al., 2012;Supplemental Figure 11).With this inmind,we speculated that theUNDPUB18 motif affects the binding activities of PUB18 andPUB22 to Exo70B1 and Exo70B2, respectively.

Itwaspreviously shown that theGFP-PUB18 fusionproteinwaslocalized to the cytosol and nucleus, rather than to punctatebodies, in Arabidopsis protoplasts (Supplemental Figure 1 inDrechsel et al., 2011). The reason for this discrepancy betweenthose and our results is currently unknown. It is possible that theN-terminally fused GFP to PUB18 may cause a subtle confor-mational change in the UND domain, which resulted in differentsubcellular localizationofGFP-PUB18.However, thispossibility isunlikely because DUND-PUB18-GFP also displayed a punctatesubcellular localization (Supplemental Figure 5B). Alternatively,proper trafficking of transiently expressed proteins to differentsubcellular organelles (e.g., cytosol and punctate exocyst bodies)in the protoplastsmay need differential temporal processes; thus,more detailed time-course localization studies of GFP-PUB18after protoplast transformation appear to be necessary to answerthese questions.

Thedomain-exchangeanalysis indicated that theubiquitinationspecificities, as well as binding activities, of PUB18 and PUB22 toExo70B1 and Exo70B2, respectively, are dependent on thepresence or absence of the UNDPUB18 motif (Figures 4 and 6).Because ΔUND-PUB18 colocalized to punctate bodies withExo70B1 (Supplemental Figure 5B), the UNDPUB18 motif may notbe involved in the subcellular localization of PUB18. Consistently,UNDPUB18 was able to interact with Exo70B1 in yeast cells(Supplemental Figure 12). Exo70B1 plays a positive role in bothABA- and mannitol-induced stomatal closure downstream ofPUB18, whereas Exo70B2 positively regulated mannitol-inducedstomatal closure, but in an ABA-independent manner (Figures 7Dand 8C). Taken together, these results strongly suggest thatExo70B1 is regulated by PUB18 in ABA-mediated stomatalmovement and that the N-terminal UNDPUB18 is critically involvedin this negative regulation of ABA-dependent drought responses.

Although PUB18 was identified as a functional U-box E3 Ubligase (Mudgil et al., 2004; Bergler and Hoth, 2011; Seo et al.,2012), we failed to detect in vitro E3 self-ubiquitination activity ofbacterially expressed MBP-PUB18 using Arabidopsis E1 (UBA1)and various E2 enzymes. This was in sharp contrast to the resultsthat in vitro E3Ub ligase activities of recombinant PUB19, PUB22,

UNDPUB18-PUB22, andPUB23proteinswere routinely detectable(Cho et al., 2008; Trujillo et al., 2008; Liu et al., 2011; Stegmannetal., 2012;Supplemental Figure13). Thus, theE3 ligaseactivityofPUB18 was speculated to be mediated by another specific E2protein or to require an as yet unidentified cellular factor. Thein vitro E3 activity of PUB18 was clearly detected when a smallamount of crude extract (10mg total proteins) prepared fromABA-treated leaves was included as a source of E2 in the reactionmixture (Figures 1B and 6A). Because an ABA-treated crudeextract was essential for the detection of E3 activity of PUB18, weare tempted to propose that the activation of E3 Ub ligase activityof PUB18 is subject to control byABA-mediated cellular processessuch as protein modifications. This hypothesis is supported byrecent results that an Ub ligase enzyme activity of RING-type E3RZFP34/CHYR1 was enhanced by SnRK2.6-mediated phos-phorylation, a key regulator of ABA signal transduction in Arabi-dopsis (Ding et al., 2015). In addition, the phosphorylation of theC-terminal ARM repeats of OsPUB15, a rice UND-PUB-ARMprotein, triggered its E3 Ub ligase activity (Wang et al., 2015).Exocytosis is a fundamental cellular mechanism that regulates

the transport of various compounds to the plasmamembrane andextracellularmatrix (TerBush et al., 1996;Hsu et al., 2004;Orlandoand Guo, 2009; Liu and Guo, 2012). In higher plants, exocytosisplays various roles in cell growth, pollen incompatibility, and theresponse to pathogen invasion (Wen et al., 2005; Samuel et al.,2009; Sup Yun et al., 2013; Kissoudis et al., 2014). The Exo70B1subunit contributes to the regulation of autophagy-relatedtransport, vesicle trafficking, and pathogen-induced immune re-sponses (Kulich et al., 2013; Stegmann et al., 2013; Teh andHofius, 2014; Zhao et al., 2015), whereas Exo70B2 participates inthe process of pathogen-associated molecular pattern (PAMP)-triggered defense responses (Pecenková et al., 2011; Stegmannet al., 2012). Stegmann et al. (2012) proposed that the detailedmechanisms by which Exo70B1 and Exo70B2 regulate PAMP-induced immune signaling are different. The exo70b1 knockoutmutant plants displayed morphological abnormalities, such assmaller and twisted leaves with partial lesions, whereas theexo70b2 progeny were phenotypically normal (Figure 7C). Theseresults further indicate that Exo70B1 and Exo70B2 paralogsperform distinct cellular roles.Under adverse growth conditions, many stress-related genes

are activated systematically and simultaneously (Cramer et al.,2011; Lv et al., 2014). Because stress-responsive proteins oc-casionally resulted in multiple destructive traits, they functiontransiently and then should be subsequently degraded (Gilmouret al., 2000; Xiong et al., 2007; Flick and Kaiser, 2012). However,how these transiently induced proteins are effectively removedhas not yet been determined. Recently, physiological relevancebetween autophagy and ABA and abiotic stress responses hasattracted considerable interest (Xiong et al., 2007; Slavikova et al.,2008; Liu et al., 2009; Han et al., 2011). Notably, Vanhee andBatoko (2011) proposed the existence of a selective autophagysystemto remove theABA-inducedAt-TSPO.Furthermore,NBR1was reported to be an important regulator of selective autophagy-mediated antiproteotoxic pathways in abiotic stress responses(Zhou et al., 2014). Tzfadia and Galili (2013) suggested that21 paralogs of Arabidopsis EXO70 exocyst subunits, includingExo70B1 and Exo70B2, contain an ATG8-interacting motif (strict

Role of the UND Motif of Arabidopsis PUB18 2967

consensus W/YxxL/V/I), implying that the functions of Exo70paralogs could be related to autophagy regulation. Moreover,Exo70B1 was found to participate in the autophagy-relatedtransport of exocysts to the vacuole (Kulich et al., 2013). Inter-estingly, PUB22 ubiquitinates 26S proteasome subunits RPN6and RPN12 (Cho et al., 2008, 2015). Ubiquitination of RPN12 byPUB22 resulted in the partial dissociation of the 26S proteasomecomplex, whereas the ubiquitinated RPN6 subunit was rapidlydegraded, both of which might decrease the proteasomal proteindegradation activity. The changes in the proteasome activity bydrought-induced PUB22 might affect cellular autophagy activityto maintain continuous elimination of toxic or unnecessary pro-teins to fine-tune the plant responses to drought stress. Thus,further studies are warranted to elucidate the possible regulatorylinksbetweenPUB18andPUB22U-boxE3Ub ligasesandExo70-related autophagy in ABA-mediated as well as ABA-independentdrought stress responses.

Inconclusion, our results suggest that theN-terminalUNDmotifof the U-box E3 ubiquitin ligase PUB18 is critical for the negativeregulationofABA-mediatedstomatalmovements anddeterminesits binding and ubiquitination specificity to Exo70B1, a subunit ofthe exocyst complex in Arabidopsis.

METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col-0) was used asthewild type in this study.Mutant alleles used to produce thepub18pub19and pub22 pub23 double mutants have been previously described (Choet al., 2008;Seoet al., 2012). TheT-DNA insertion exo70b1 (SALK_202386)and exo70b2 (SALK_091877)mutantswere obtained from theABRCat theOhio State University (http://www.arabidopsis.org). The pub18 pub19exo70b1 tripleknockoutmutant linewasgenerated throughagenetic crossbetween pub18 pub19 and exo70b1 homozygous mutant plants.

Chimeric transgenes were constructed by obtaining the N-terminalUND region of PUB18 by using PCR and ligating it to the N terminus of thefull-length coding sequence ofPUB22. TheUNDPUB18-PUB22 and ΔUND-PUB18 constructs, as well as a full-length PUB18, were inserted into themodifiedpENTRSD/D topovector (Invitrogen) and transferred to thebinaryvector pEarleyGate202 (ABRC stock no. CD3-688) using a Gatewaycloning kit (Invitrogen). The full-length coding sequence of PUB22 wasintroduced into a GFP-fused pENTR SD/D topo vector and transferred tothe pEarlyGate100 destination vector (ABRC stock no. CD3-724). TheExo70B2 cDNA clone (ABRC stock no. CD4-36) was acquired from theABRC. The full-length Exo70B1 and Exo70B2 coding sequences wereamplified using PrimeSTAR polymerase (Takara) and incorporateddownstream of the CaMV 35S promoter in the pEarleyGate202 vectorcontaining the Flag-tag sequence.

All transgenic plants used in this study were generated via floral-diptransformation. Methods for seed surface sterilization and growth con-ditions of wild-type and transgenic Arabidopsis plants were described byRyu et al. (2010) andwere applied with slight modifications. All plants weregrown at 22°C under continuous spectrum light. Primer sequences used inRT-qPCR, genotyping PCR, and plasmid constructions are listed inSupplemental Tables 1 and 2.

Stomatal Aperture Measurements

Light-grown fully expanded rosette leaves of 4- to 6-week-old plantswere detached and submerged in stomatal opening solution (30mMKCl,

100 mM CaCl2, and 10 mM MES, pH 6.15) for 2 h at 25°C as describedpreviously (Kwaketal., 2001). Theadaxial surfaceof the leaveswasappliedto label tape (Bel-Art Products) to peel off the abaxial epidermal layers.Epidermal strips were floated on stomatal opening solution containingvarious concentrations of mannitol (0, 0.2, and 0.4 M) or ABA (0, 1, and10mM) for 2 h inwhite light (100mmolm22 s21). After incubation, leaf stripswere mounted on glass slides. Stomata images were observed andphotographed using an Olympus CX41 microscope equipped with aJUJAK560CCDcamera (DixiOptics).At least30stomatalapertures ineachepidermal peel were measured and analyzed using Photoshop CS6. Exceldata files of stomatal apertures are provided in Supplemental Data Set 2.

In Vitro Self-Ubiquitination and Substrate Ubiquitination Analyses

Recombinant MBP-PUB18, MBP-ΔUND-PUB18, MBP-PUB22, MBP-UNDPUB18-PUB22, and their single amino acid substitution mutant variantproteins (MBP-PUB18V305I,MBP-ΔUND-PUB18V36I,MBP-PUB22V24I, andMBP-UNDPUB18-PUB22V293I) were expressed in Escherichia coli andpurified by affinity chromatography using MBP Excellose resin (Takara).Purified MBP-tagged proteins were immobilized on the resin withoutelution andapplied for in vitro self-ubiquitination analyses. Each reaction of60mL final volumecontainedubiquitination buffer (50mMTris-HCl, pH7.5,5mMMgCl2, 2mMDTT, 4mMATP, 15mg ubiquitin, 400mMPMSF, 80mMMG132 [AG Scientific], and 13 protease inhibitor cocktail) and 1 mg ofbacterially expressed E3s. The reaction was conducted at 30°C for 2 h inthe presence or absence of E1 (Arabidopsis UBA1) (50 ng) and a crudeextract (10mg total proteins), as a sourceof E2enzyme, prepared fromABA(100mM)-pretreated pub18 pub19 pub22 pub23quadruplemutant leaves.The pub18 pub19 pub22 pub23 quadruple mutant leaves, instead of wild-type leaves, were used as E2 to avoid the possible effects of endogenousPUB18 and PUB22 present in the protein crude extract on the ubiquiti-nation assays. The resin-bound proteinswerewashed five timeswithMBPcolumnbuffer (20mMTris-HCl, pH7.4, 200mMNaCl, and1mMEDTA) andeluted with 10 mMmaltose. The eluted samples were mixed with 43 SDSsample buffer and heated at 90°C for 5 min. The reaction products wereseparated on a 6% (w/v) SDS-PAGE gel, followed by immunoblot analysiswith anti-MBP antibody (rabbit, 1:5000; Applied Biological Materials; catno. G079; lot no. AP4779).

For in vitro substrate ubiquitination assays, Ni-NTA resin (Qiagen)-bound His-Myc-Exo70B1 and His-Myc-Exo70B2 were used as targetsubstrates. The reactionwasperformed in80mLof totalmixture containingubiquitination buffer, 50 ng of E1, and 1mgof target protein in the presenceor absenceof anABA-pretreatedpub18pub19pub22pub23 crudeextract(10mg total proteins) and200ngof E3s at 30°C for 3 h. After incubation, theresin-bound substrate proteins were washed five times with Ni-NTAwashing buffer (13 PBS, 300 mM NaCl, and 20 mM imidazole) and elutedusing43SDSsamplebufferwithboiling for 5min. Theelutedproteinsweresubjected to immunoblot analysis with anti-Myc antibody (rabbit, 1:5000;Applied Biological Materials; cat no. G077; lot no. 0512)

A protein crude extract was prepared from the pub18 pub19 pub22pub23 quadruple mutant plants. Fully expanded mutant rosette leavespretreated with 100 mMABA for 3 h were ground under liquid nitrogen andsuspended in an extraction buffer containing 50 mM Tris-HCl, pH 7.4,10mMMgCl2, 1mMEDTA, 1MNaCl, 80mMMG132, 1mMPMSF, and 13protease inhibitor cocktail. The suspensions were vortexed gently at 4°Cfor 30 min. After centrifugation (13,200 rpm) at 4°C for 10 min, the su-pernatant was used as a source of E2 for ubiquitination assays.

Yeast Two-Hybrid Assays

The PUB18, ΔUND-PUB18, PUB22, and UNDPUB18-PUB22 constructswere cloned in the pGBKT7 vector (Clontech) as described previously (Leeet al., 2009). The full-length coding sequences of Exo70B1 and Exo70B2

2968 The Plant Cell

were amplified and introduced into the pGADT7 vector (Clontech). Variouscombinations of bait and prey plasmidswere cotransformed into the yeaststrain AH109. Yeast two-hybrid analyses were performed according to themanufacturer’s instructions of theMatchmaker Yeast Two-Hybrid System(Clontech). Interactions between two proteins were confirmed by growthonSD/-Leu/-Trp/-Hismediaplates, including2mM3-amino1,2,4,-triazole.

In Vitro Pull-Down Assay

The MBP-PUB18 and MBP-ΔUND-PUB18 recombinant proteins were im-mobilized on theMBPExcellose resin (Takara) and incubatedwith His-Myc-Exo70B1 and His-Myc-Exo70B2, respectively, in immunoprecipitationbuffer at 30°C for 1 h. In addition, the resin-bound MBP-UNDPUB18-PUB22chimeric protein was coincubated with His-Myc-Exo70B1 under the sameconditions. The affinity beads were extensively washed five times withMBPcolumn buffer, and bound proteins were eluted with 10 mM maltose. Theeluted proteins were separated by SDS-PAGE and analyzed by immuno-blotting using anti-MBP (rabbit, 1:5000; Applied BiologicalMaterials; cat no.G079; lot no. AP4779) and anti-Myc (rabbit, 1:5000; Applied BiologicalMaterials; cat no. G077; lot no. 0512) antibodies.

Subcellular Localization

The full-length PUB18 coding sequence was inserted into the 326-GFPexpression vector provided by Inhwan Hwang at POSTECH (Pohang,Korea). The PUB18-GFP plasmid was transformed into protoplasts pre-pared from wild-type Arabidopsis rosette leaves using the PEG method(Ryu et al., 2010). For an Agrobacterium tumefaciens-mediated transientexpression assay, the 35S:PUB18-GFP, 35S:GFP, 35S:Exo70B1-DsRED,and 34S:DsRED constructs were introduced into the binary vector pEar-lyGate100 and transformed into Agrobacterium strain GV3101 (Kim andKim, 2013). Agrobacteriumcells harboring each plasmidwere incubated inthe infiltration medium (10 mM MgCl2, 10 mM MES, pH 5.7, and 500 mMacetosyringone) containing 40mMMG132 for 30min and infiltrated into theabaxial side of Nicotiana benthamiana leaves. The expression of PUB18-GFPandExo70B1-DsREDwasenhancedbycoexpressingaviral-encodedsilencing suppressor P19 that enables high levels of transient expression(Hellens et al., 2005). Transiently expressed GFP signals were obtainedusing a fluorescence microscope (BX51; Olympus) and a cooled CCDcamera (PCO) or by confocal microscopy (LSM510META; Carl Zeiss). Thelaser signals weremeasured using a sliding 0.313-mmdetectionwindow ina confocal microscope. GFP and YFPwere excited by a 488-nm laser, andDsRED was excited by 550-nm laser.

BiFC Assay

PUB18 and ΔUND-PUB18 were cloned in the pSPYNE-35S vector thatcontained theN-terminal region (YFPN;1 to155aminoacids)ofYFP (Walteret al., 2004). The full-length coding sequences of Exo70B1 and Exo70B2were amplified and introduced into the pSPYCE-35S vector containing theC-terminal region (YFPC; 156 to 239 amino acids) of YFP (Walter et al.,2004). Various combinations of Agrobacterium strain GV3101 harboringthe YFPN- and YFPC-fused constructs were coinfiltrated into the abaxialside of tobacco leaves. After 3 d, reconstituted fluorescent signals inepidermal cells of infiltrated leaves were examined by confocal micros-copy.

Protein Membrane Association Analysis

The 35S:PUB18-GFP and 35S:Flag-Exo70B1 fusion genes were tran-siently coexpressed with 35S:P19 using Agrobacterium-mediated in-filtration in N. benthamiana as described by Kim et al. (2016). Tobaccoleaves were ground in liquid nitrogen, suspended in extraction buffer

(50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM EDTA, and 1 M NaCl), andcentrifuged at 28,000g for 10 min at 4°C. The pellet was resuspended inextraction buffer with or without NaCl (1 M) and Na2CO3 (100 mM) andvigorously vortexed at 4°C for 15 min. Protein samples were then centri-fuged at 28,000g for 30 min at 4°C. The supernatant and pellet fractionswere analyzed using immunoblotting with anti-GFP (mouse, 1:5000;Clontech; cat no. 632381; lot no. A5033481) and anti-Flag (mouse, 1:5000;Sigma-Aldrich; cat no. A8592; batch no. SLBD9930) antibodies.

In Vivo Co-IP Assay

The 35S:Flag-ΔUND-PUB18V36I and 35S:Flag-UNDPUB18-PUB22V293I

constructswere introduced into thebinary vector pEarlyGate202. The35S:Myc-Exo70B1 and 35S:Myc-Exo70B2 constructs were inserted intothe modified pENTR SD/D topo vector (Invitrogen) and transferred to thebinary vector pEarleyGate100. 35S:Flag-ΔUND-PUB18V36I and 35S:Flag-UNDPUB18-PUB22V293I were transiently coexpressed with 35S:Myc-Exo70B1or35S:Myc-Exo70B2 in thepresenceof35S:P19 inN.benthamianaleaves using Agrobacterium-mediated infiltration as described by Kim et al.(2016). After 2.5 d, harvested tobacco leaves were ground in liquid nitrogen,suspended in the extraction buffer (50 mM Tris-HCl, pH 7.4, 10 mMMgCl2,1 mM EDTA, and 1MNaCl), and vigorously vortexed at 4°C for 15 min. Totalleaf proteins (200 mg) were incubated for 1 h at 4°C with anti-Flag affinity gelmatrix. The precipitated samples were extensively washed three times withTBSbufferandelutedbyboilingwith43SDSsamplebuffer.Eachsamplewasseparated bySDS-PAGEandanalyzedby immunoblottingwith anti-Flag andanti-Myc antibodies.

In Vitro and in Vivo Protein Degradation Assay

Light-grown mature rosette leaves of wild-type and pub18 pub19 mutantplants were treated with ABA (100mM) for 2 h and rapidly frozen with liquidnitrogen. Frozen powder of sample leaves was suspended in extractionbuffer and vigorously vortexed at 4°C for 1 h. After the mixture wascentrifuged at 28,000g at 4°C for 10 min, the supernatant was collected.The His-Myc-Exo70B1 and RGA1-2xFlag recombinant proteins wereincubated with a cell-free crude extract (100 mg total proteins) for 1.5and 3 h in the presence or absence of 50 mM MG132 (AG Scientific).The reactions were terminated by adding 23 SDS sample buffer. Theprotein degradation patterns were analyzed by immunoblotting withanti-Myc (Applied Biological Materials) and anti-Flag (Sigma-Aldrich)antibodies.

For the in vivo protein degradation assay, the full-length Exo70B1coding sequence was inserted into the 326-GFP expression vector thatwas provided by Inhwan Hwang. The Exo70B1-GFP and GFP only plas-mids were transfected into protoplasts prepared from Arabidopsis rosetteleaves of wild-type and pub18 pub19 double mutant plants using the PEGmethod (Ryu et al., 2010). After incubation for 12 h, the protoplasts weretreated with MG132 (50 mM) or DMSO for 3 h. Subsequently, protoplastswere incubatedwithCHX (250mM) for2hwithorwithoutABA (100mM).Thereactions were terminated by adding 43 SDS sample buffer. The patternsof protein degradation were analyzed by immunoblot analysis with anti-Myc and anti-GFPantibodies. The levels ofGFP anda-tubulin were shownas transfection efficiency control of protoplasts and equal loading control,respectively. The protein levels were quantified using ImageJ software(NIH; http://imagej.nih.gov/ij/).

Sequence Alignment and Phylogenetic Analysis

The U-box domain sequences of Arabidopsis PUB-ARM proteins werealigned using ClustalW2 with default parameters. The multiple sequencealignment was generated and the phylogenetic tree was constructed. Theevolutionary history was inferred using the maximum likelihood method

Role of the UND Motif of Arabidopsis PUB18 2969

based on the Poisson correction model. The bootstrap consensus treeinferred from 5000 replicates is taken to represent the evolutionary historyof the taxa analyzed (Felsenstein, 1985). The initial tree(s) for the heuristicsearchwasobtainedautomaticallybyapplyingneighbor-joiningandBioNJalgorithms to a matrix of pairwise distances estimated using a JTT modeland then selecting the topology with superior log likelihood value. Thephylogenetic dendrogram was constructed in MEGA5 software (Tamuraet al., 2011).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under the following accession numbers: PUB18 (At1g10560),PUB19 (At1g60190), PUB22 (At3g52450), PUB23 (At2g35930), Exo70B1(At5g58430),Exo70B2 (At1g07000),UBA1 (At2g30110),UBC7 (At5g59300),UBC8 (At5g41700), UBC9 (At4g27960), UBC10 (At5g53300), and UBC13(At3g46460).

Supplemental Data

Supplemental Figure 1. Multiple Alignment of Amino Acid Sequencesof PUB18, PUB19, PUB22, and PUB23 Arabidopsis U-box E3 UbLigases.

Supplemental Figure 2. In Vitro Self-Ubiquitination Assay of PUB18,PUB18V305I, PUB22, and PUB22V24I with or without Heat-InactivatedLeaf Crude Protein Extract.

Supplemental Figure 3. Arabidopsis PUB19 Interacts with Exo70B1in Yeast Cells.

Supplemental Figure 4. The in Vitro Pull-Down Assay between MBP-PUB19 and His-Myc-Exo70B1.

Supplemental Figure 5. BiFC Assay and Subcellular Localization ofGFP, DsRED, ΔUND-PUB18-GFP, and Exo70B1-DsRED.

Supplemental Figure 6. In Vitro Ubiquitination Assays of Exo70B1 andExo70B2 by PUB18, ΔUND-PUB18, PUB22, and UNDPUB18-PUB22.

Supplemental Figure 7. Molecular Characterization of exo70b1 andexo70b2 T-DNA-Inserted Homozygous Knockout Mutant Plants.

Supplemental Figure 8. Characterization of the Second Mutant Allele(exo70b1-1) of Exo70B1.

Supplemental Figure 9. Multiple Alignments of Amino Acid Sequen-ces of Arabidopsis UND Motifs.

Supplemental Figure 10. Molecular Phylogenetic Analysis of Ara-bidopsis PUB-ARM Proteins Using the Maximum LikelihoodMethod.

Supplemental Figure 11. Amino Acid Sequence Alignment of Arabi-dopsis Exo70B1 and Exo70B2.

Supplemental Figure 12. Yeast Two-hybrid Assay of the UNDPUB18

Motif and Exo70B1.

Supplemental Figure 13. In Vitro Self-Ubiquitination Assay ofUNDPUB18-PUB22.

Supplemental Table 1. Primer Sequences Used for Genotyping PCR,qRT-PCR, and RT-PCR.

Supplemental Table 2. Primer Sequences Used for Gene Construc-tions and Mutagenesis.

Supplemental Data Set 1. Alignment used to generate the phylogenypresented in Supplemental Figure 10.

Supplemental Data Set 2. Excel Data Files of Stomatal ApertureAnalyses.

ACKNOWLEDGMENTS

Thisworkwassupportedbygrants from theWooJangChunSpecial Project(PJ009106) funded by the Rural Development Administration and fromthe National Research Foundation, Project No. 2014R1A2A2A01003891,Republic of Korea, to W.T.K.

AUTHOR CONTRIBUTIONS

D.H.S., M.Y.A., K.Y.P., and E.Y.K performed the experiments. D.H.S.,M.Y.A., and W.T.K. analyzed the data. D.H.S. and W.T.K. planned theproject and drafted the manuscript. W.T.K. supervised the project andcomplemented the writing.

Received April 27, 2016; revised November 18, 2016; acceptedDecember9, 2016; published December 12, 2016.

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Role of the UND Motif of Arabidopsis PUB18 2973

DOI 10.1105/tpc.16.00347; originally published online December 12, 2016; 2016;28;2952-2973Plant Cell

Dong Hye Seo, Min Yong Ahn, Ki Youl Park, Eun Yu Kim and Woo Taek KimSpecificity for Exocyst Subunit Exo70B1

Negative Regulation of ABA-Mediated Stomatal Movement and Determines Its Ubiquitination The N-Terminal UND Motif of the Arabidopsis U-Box E3 Ligase PUB18 Is Critical for the

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