RBR-Type E3 Ligases and the Ubiquitin-Conjugating Enzyme ... › content › plantphysiol › 182 › 4 › 1723.full.pdf · Sabrina Iñigo,b,c Lesia Rodriguez,a Eduardo Bueso,a Alain

RBR-Type E3 Ligases and the Ubiquitin-ConjugatingEnzyme UBC26 Regulate Abscisic Acid Receptor Levelsand Signaling1[OPEN]

Maria Angeles Fernandez ,a,2 Borja Belda-Palazon,a,2 Jose Julian,a Alberto Coego,a Jorge Lozano-Juste,a

Sabrina Iñigo,b,c Lesia Rodriguez,a Eduardo Bueso,a Alain Goossens,b,c and Pedro L. Rodrigueza,3,4

aInstituto de Biología Molecular y Celular de Plantas, Consejo Superior de InvestigacionesCientíficas-Uiversidad Politécnica de Valencia, 46022 Valencia, SpainbGhent University, Department of Plant Biotechnology and Bioinformatics, 9052 Ghent, BelgiumcVIB Center for Plant Systems Biology, 9052 Ghent, Belgium

ORCID IDs: 0000-0003-4926-1917 (B.B.-P.); 0000-0001-7034-566X (J.L.-J.); 0000-0002-7244-7237 (L.R.); 0000-0002-1599-551X (A.G.);0000-0002-5886-9425 (P.L.R.).

The turnover of abscisic acid (ABA) signaling core components modulates the plant’s response to ABA and is regulated byubiquitination. We show that Arabidopsis (Arabidopsis thaliana) RING Finger ABA-Related1 (RFA1) and RFA4 E3 ubiquitinligases, members of the RING between RING fingers (RBR)-type RSL1/RFA family, are key regulators of ABA receptor stabilityin root and leaf tissues, targeting ABA receptors for degradation in different subcellular locations. RFA1 is localized both in thenucleus and cytosol, whereas RFA4 shows specific nuclear localization and promotes nuclear degradation of ABA receptors.Therefore, members of the RSL1/RFA family interact with ABA receptors at plasma membrane, cytosol, and nucleus, targetingthem for degradation via the endosomal/vacuolar RSL1-dependent pathway or 26S proteasome. Additionally, we provideinsight into the physiological function of the relatively unexplored plant RBR-type E3 ligases, and through mutagenesisand biochemical assays we identified cysteine-361 in RFA4 as the putative active site cysteine, which is a distinctive featureof RBR-type E3 ligases. Endogenous levels of PYR1 and PYL4 ABA receptors were higher in the rfa1 rfa4 double mutant than inwild-type plants. UBC26 was identified as the cognate nuclear E2 enzyme that interacts with the RFA4 E3 ligase and formsUBC26-RFA4-receptor complexes in nuclear speckles. Loss-of-function ubc26 alleles and the rfa1 rfa4 double mutant showedenhanced sensitivity to ABA and accumulation of ABA receptors compared with the wild type. Together, our results reveal asophisticated mechanism by which ABA receptors are targeted by ubiquitin at different subcellular locations, in which thecomplexity of the ABA receptor family is mirrored in the partner RBR-type E3 ligases.

The plant hormone abscisic acid (ABA) regulatesmany key processes in plants, including seed germi-nation and development and various biotic and abioticstress responses (Cutler et al., 2010; Finkelstein, 2013;Rodriguez et al., 2019). The ABA signaling pathway isinitiated by ABA perception through the 14-memberPYRABACTINRESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS(RCAR) family of proteins (Ma et al., 2009; Park et al., 2009;Santiago et al., 2009b; Nishimura et al., 2010). Specificfunctions for some members of the PYR/PYL/RCARfamily have emerged in recent years (Antoni et al.,2013; Zhao et al., 2016; Belda-Palazon et al., 2018;Dittrich et al., 2019). ABA perception by ABA recep-tors leads to interaction with and inactivation of cladeA protein phosphatase type 2Cs (PP2Cs), such asABA INSENSITIVE1 (ABI1) and ABI2, HYPERSEN-SITIVE TO ABA1 (HAB1) and HAB2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 (PP2CA/AHG3), which are key negative regulators of ABAsignaling (Ma et al., 2009; Park et al., 2009; Rubio et al.,2009; Santiago et al., 2009b; Umezawa et al., 2009; Vladet al., 2009).

1This work was supported by the Ministerio de Ciencia, Innova-cion y Universidades(MICIU), Fondo Europeo de Desarrollo Re-gional, and Consejo Superior de Investigaciones Cientificas (grantsBIO2014-52537-R and BIO2017-82503-R to P.L.R.), by a Juan de laCierva contract from MICIU and by the Marie Skłodowska-CurieAction H2020-MSCA-IF-2015-707477 to J.L-J., by Programa VALi1dGVA APOSTD (2017/039 to B.B.-P.), by an FPI contract from MICIU(to J.J.) and by an FPU contract from MECD (to M.A.F.), and by theBelgian Science Policy Organization with postdoctoral fellowshipsto S.I.

2These authors contributed equally to the article.3Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Pedro L. Rodriguez ([email protected]).

M.A.F., B.B.-P., J.J., A.C., J.L.-J., S.I., L.R., E.B., A.G., and P.L.R.designed the experiments; M.A.F., B.B.-P., J.J., A.C., J.L.-J., S.I., L.R.,and E.B. performed the experiments; P.L.R. conceived the project andwrote the article; all authors analyzed the data, discussed the results,and revised the article.

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https://orcid.org/0000-0003-4926-1917

https://orcid.org/0000-0003-4926-1917

https://orcid.org/0000-0001-7034-566X

https://orcid.org/0000-0001-7034-566X

https://orcid.org/0000-0002-7244-7237

https://orcid.org/0000-0002-7244-7237

https://orcid.org/0000-0002-1599-551X

https://orcid.org/0000-0002-1599-551X

https://orcid.org/0000-0002-5886-9425

https://orcid.org/0000-0002-5886-9425

https://orcid.org/0000-0003-4926-1917

https://orcid.org/0000-0001-7034-566X

https://orcid.org/0000-0002-7244-7237

https://orcid.org/0000-0002-1599-551X

https://orcid.org/0000-0002-5886-9425

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Structural and biochemical studies have revealedthat PP2Cs are necessary ABA coreceptors able tomonitor the occupancy of the ABA-binding pocket toachieve nanomolar affinity for ABA binding (Ma et al.,2009; Melcher et al., 2009; Miyazono et al., 2009;Santiago et al., 2009a; Yin et al., 2009; Dupeux et al.,2011; Moreno-Alvero et al., 2017). Comparison of thestructure of ligand-bound crop ABA receptors in theabsence and presence of clade A PP2Cs reveals theirproactive role to form productive ternary complexes inwhich the phosphatase activity is efficiently inhibited(Moreno-Alvero et al., 2017). Hence, clade A PP2C-mediated inhibition on three ABA-activated SNF1-related protein kinases (SnRK2s; i.e. SnRK2.2/D, SnRK2.3/I, and SnRK2.6/E/OST1) is relieved (Umezawa et al.,2009; Vlad et al., 2009). These SnRK2s subsequentlyactivate downstream signaling by phosphorylation ofnumerous players, including ABA-responsive tran-scription factors (Fujii et al., 2009; Fujii and Zhu, 2009;Nakashima et al., 2009), the chromatin-remodelingATPase BRAHMA (Peirats-Llobet et al., 2016), ionand water channels (Geiger et al., 2009; Lee et al., 2009;Grondin et al., 2015), and other mediators/effectorsinvolved in ABA signaling and action (Umezawa et al.,2013; Wang et al., 2013). Whereas transcription ofsome PYR/PYL/RCARs is repressed in response toABA, that of PP2Cs is stimulated (Santiago et al.,2009b; Szostkiewicz et al., 2010), indicating that anegative feedback transcriptional mechanism is pre-sent to modulate ABA signaling by controlling tran-script levels of core elements. Recently, it has beendiscovered that degradation of PP2Cs is a comple-mentary mechanism to PYR/PYL/RCAR-mediatedinhibition of PP2C activity (Kong et al., 2015; Wu et al.,2016; Belda-Palazon et al., 2019; Julian et al., 2019).

The ubiquitin (Ub)-26S proteasome system (UPS)plays a crucial role in plant hormone signaling (Santnerand Estelle, 2009; Vierstra, 2009). Approximately 6% ofthe Arabidopsis (Arabidopsis thaliana) genome encodescomponents that can be connected to the UPS, repre-senting one of the most elaborate and prevalent regu-latory mechanisms in plants (Smalle and Vierstra, 2004;Vierstra, 2009). In addition to committing proteins todegradation by the 26S proteasome, ubiquitination(ubiquitylation) also serves proteasome-independentroles, including the endocytosis of membrane proteins(MacGurn et al., 2012; Kim et al., 2013; Gao et al., 2017;Yu and Xie, 2017). The turnover of ABA receptors fol-lows a double mechanism involving both the UPS andthe cellular pathway that comprises endocytosis, traf-ficking through the endosomal sorting complex requiredfor transport (ESCRT), and vacuolar degradation (Buesoet al., 2014; Irigoyen et al., 2014; Belda-Palazon et al.,2016; Yu et al., 2016; García-León et al., 2019). The rela-tive contribution of each pathway to regulate endoge-nous levels of ABA receptors remains to be investigated,particularly regarding degradation of ABA receptorsthrough the UPS.

PYR/PYL/RCAR ABA receptors are subjected toubiquitination, either at the nucleus through the

CULLIN4-RING E3 ubiquitin ligase (CRL4) complexformed by COP10-DET1-DDB1 and the substrateadapter DDB1-ASSOCIATED1 (DDA1; Irigoyen et al.,2014) or at the plasmamembrane through themonomericE3 Ub ligase RING FINGER OF SEED LONGEVITY(RSL1; Bueso et al., 2014; Belda-Palazon et al., 2016). RSL1bears a C-terminal transmembrane (TM) domain thattargets the E3 ligase to the plasma membrane, where theinteraction between RSL1 and ABA receptors occurs(Bueso et al., 2014; Belda-Palazon et al., 2016). The ubiq-uitination of PYL4 and PYR1 by RSL1 at the plasmamembrane targets the receptors to the vacuolar degra-dation pathway (Belda-Palazon et al., 2016; Yu et al.,2016). We found that FYVE1/FREE1, which is a keycomponent of the ESCRT-I machinery, interacts withRSL1-receptor complexes and recruits PYL4 to endosomalcompartments (Belda-Palazon et al., 2016). Recently, amoonlight function in the nucleus of FYVE1/FREE1 toattenuate ABA signaling has also been reported (Li et al.,2019). Additionally, the ESCRT-I component VPS23A,which is a Ub-conjugating enzyme variant lacking thecatalytic Cys, also recognizes ABA receptors for endo-somal degradation (Yu et al., 2016). Finally, both FYVE1and VPS23A interact with the ESCRT-III protein ALIX,which in turn interacts with ABA receptors and contrib-utes to regulation of receptor turnover and stomatal ap-erture (García-León et al., 2019). Thus, although theESCRT pathway had been assumed to be reserved forintegral membrane proteins, it also mediates the degra-dation of ABA receptors, which can be found associatedwith membranes through CAR proteins or RSL1 butwhich are not integral membrane proteins (Rodriguezet al., 2014; Diaz et al., 2016). ESCRT-mediated sortingof proteins ubiquitinated at the plasma membrane leadsto degradation in the vacuole after endosomal andmultivesicular body trafficking (MacGurn et al., 2012).By contrast, proteins polyubiquitinated in cytosol ornucleus are degraded via the 26S proteasome (Smalleand Vierstra, 2004).

RSL1 belongs to a gene family composed of at leastnine additional members, which we have named RFA1(for RING finger ABA-related1) to RFA9 (Bueso et al.,2014). Initially, RSL1 was annotated as a RING-type E3ligase (Bueso et al., 2014); however, further inspectionof the gene family revealed that RSL1/RFAs are struc-turally characterized by the presence of three putativeRING domains in tandem, named as RING1-IN BE-TWEEN RING (IBR)-RING2, and accordingly they be-long to the RBR-type E3 ligase family (Marín, 2010;Callis, 2014). Molecular insights into the function ofRBR-type E3 Ub ligases have been obtained mostly inhumans from the study of Parkin, associated with au-tosomal recessive Parkinsonism, and Human Homologof Ariadne (HHARI), involved in the regulation oftranslation (Dove et al., 2016). Thus, studies in mam-mals have shown that RBR E3s combine properties ofboth RING- and HECT-type E3s (Wenzel et al., 2011;Wenzel and Klevit, 2012). Noncovalent interaction withE2-Ub occurs at the RING1 domain as in RING/U-boxproteins, but then the activated Ub is transferred to a

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conserved Cys residue in RING2. Finally, this cysteinylresidue of RING2 transfers Ub to target proteins in aHECT-type mechanism (Callis, 2014). Structural anal-yses of HHARI and Parkin have revealed that RING1 isthe only domain with a cross-brace zinc-coordinationtopology, whereas the zinc-liganding residues in IBRand RING2 domains are arranged in a sequentialfashion (Duda et al., 2013; Riley et al., 2013). Therefore,the RING nomenclature for IBR and RING2 does notreflect the canonical RING cross-brace structure that isonly present in RING1.Five members of the RSL1/RFA family (i.e. RSL1 and

RFA6–RFA9) contain a TM domain at the C terminus ofthe protein, which suggests that they are membranelocalized like RSL1 (Bueso et al., 2014; Belda-Palazonet al., 2016). In contrast, RFA1 to RFA5 lack theC-terminal TM domain, and their functional charac-terization, as well as their subcellular localization, havenot yet been investigated. In this work, we investi-gated RFA1 and RFA4 function in ABA signaling. Weidentified UBIQUITIN CONJUGATING ENZYME26(UBC26) as the cognate E2 enzyme for RFA4 and ob-served the formation of UBC26-ABA receptor-RFA4complexes in nuclear speckles. This represents an ad-ditional pathway to the DDA1-CRL4 complex to pro-mote the degradation of ABA receptors in the nucleus(Irigoyen et al., 2014). Altogether, our results reveal asophisticated targeting of ABA receptors at differentsubcellular locations, where the complexity of the ABAreceptor family is mirrored in the partner RBR-type E3ligases.

RESULTS

Subcellular Localization of RFA1 and RFA4

In this work, we focused on RFA1 and RFA4 to in-vestigate the branch of the RFA E3 family whosemembers lack the C-terminal TM domain, namelyRFA1 to RFA5. Overall, the mRNA levels of RFA1 andRFA4 are higher than RFA2/3 genes in different tissuesand developmental stages, but they are relatively sim-ilar to RFA5 (Supplemental Fig. S1A; http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi; Waese et al.,2017). The heat map viewer also shows a similar pro-file for the overall expression of RFA1/4/5 and PYR/PYLgenes (Supplemental Fig. S1A). Additionally, we useddata mining to analyze the expression of RFA1 andRFA4 in seedlings subjected to different abiotic stresses(Goda et al., 2008). As a result, we found that RFA4expression was up-regulated in aerial tissue in re-sponse to cold, osmotic, salt, drought, and heat stress,whereas RFA1 expression was less consistently af-fected (Supplemental Fig. S1B). In contrast, analysis ofmicroarray data in guard cells under different treat-ments (ABA, high CO2, darkness, and low humidity)did not reveal significant expression changes of RFA1and RFA4 (Gene Expression Omnibus database ac-cession nos. GSE41054 and GSE118520; Dittrich et al.,

2019). Expression of RFA4 peaked at earlier stages ofsilique development and embryo globular/heart/torpedo stage, whereas expression of RFA1 increasedat later stages, from embryo walking-stick to cotyle-don stage (Supplemental Fig. S1C). A similar doublepeak in the expression of some ABA receptors (e.g.PYR1, PYL4, and PYL5) could be observed during seedand silique development, which suggests some over-lapping between the expression of RFA1/4 and PYLgenes (Supplemental Fig. S1C).Alignment of RFA1 and RFA4 reveals the RING1-

IBR-RING2 domains in tandem (Bueso et al., 2014;Supplemental Fig. S2). In the case of RFA4, after RING2,there is a very acidic C-terminal domain that containsmore than 40 Asp residues (Supplemental Fig. S2). Incontrast, RFA1 lacks this domain or the C-terminal TMdomain of RSL1 (Supplemental Fig. S2). We generated35S:GFP-RFA1, 35S:RFA1-GFP, 35S:GFP-RFA4, and35S:GFP-RFA4 C-terminal deletion (RFA4DC, lackingamino acid residues 385–468) constructs and deliveredthem into leaf cells of Nicotiana benthamiana by agro-infiltration (Fig. 1A). Expression of the correspondingfusion proteins was verified by immunoblot analysisusing anti-GFP antibodies (Fig. 1A). Expression of GFP-RFA4 was consistently lower than that of other GFPfusion proteins, likely because of the large acidicC-terminal domain. We found that RFA1 is apparentlylocalized in cytosol and nucleus. The cytosolic locali-zation of RFA1 was confirmed by the lack of colocali-zation with the plasma membrane marker OFP-TM23,both for RFA1-GFP and GFP-RFA1 proteins (Fig. 1B).This marker was previously used to show the plasmamembrane localization of GFP-RSL1 (Bueso et al.,2014). In order to investigate whether the C-terminalacidic domain of RFA4 affects its subcellular location,we compared 35S:GFP-RFA4DC and 35S:GFP-RFA4localization (Fig. 1A). Both RFA4DC and RFA4 werelocalized in the nucleus of N. benthamiana leaf cells,which suggests that the acidic domain of RFA is dis-pensable for nuclear localization of the protein. Indeed,a nuclear localization signal (NLS; type SV40 T antigen)composed of four basic residues (KKRR) was identifiedat the N terminus of RFA4 (Supplemental Fig. S2).Deletion of the NLS led to a change in RFA4 subcellularlocalization, and GFP-RFA4DNLS was apparently lo-calized in cytosol and nucleus, similar to RFA1(Fig. 1A).

The Interaction of RFA1, RFA4, and RSL1 with ABAReceptors Covers Different Subcellular Localizations inPlant Cells

Comparison of RFA1, RFA4, and RSL1, excludingthe C-terminal TM domain of RSL1 and the acidicdomain of RFA4, reveals high sequence similarity(Supplemental Fig. S2), which suggests that theymight all interact with ABA receptors. Only the in-teraction of RSL1 with ABA receptors has been ana-lyzed previously (Bueso et al., 2014); therefore, we

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RFA1/RFA4 RBR-Type E3 Ligases Target ABA Receptors


http://www.plantphysiol.org/cgi/content/full/pp.19.00898/DC1

http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi

http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi











tested whether RFA1 and RFA4 interact with ABAreceptors using bimolecular fluorescence comple-mentation (BiFC) assays (Fig. 2). We coexpressedYFPN-RFA1 or YFPN-RFA4 with YFPC-PYR/PYLsusing agroinfiltration and found that RFA1 interactswith different ABA receptors both at cytosol and nu-cleus ofN. benthamiana leaf cells (Fig. 2A). On the otherhand, the interaction of RFA4 and PYR/PYLs was lo-calized exclusively in the nucleus, in agreement withthe nuclear localization of GFP-RFA4 (Fig. 2B). Coex-pression of YFPC-OST1D280, which is expressed innucleus and cytosol (Vlad et al., 2009), with eitherYFPN-RFA1 or YFPN-RFA4 did not produce fluores-cence reconstitution. We also investigated whether the

very acidic C-terminal domain of RFA4 affects theinteraction with ABA receptors. To this end, we testedthe interaction of YFPN-RFA4DCwith PYR/PYLs. Theinteraction of RFA4DC with PYR/PYLs did not differfrom that of wild-type RFA4, which suggests thatrecognition of the target by RFA4 is not dependent onthe C-terminal acidic domain (Supplemental Fig. S3A).We also verified that YFPN-RFA1, RFA4, and RFA4DC,aswell as YFPC-PYR/PYLs or YFPC-OST1D280 proteins,were correctly expressed (Supplemental Fig. S3B). Tovalidate the interactions observed using the BiFC assays,we performed a split-luciferase complementation assayin N. benthamiana leaves (Fig. 2C). In contrast to BiFCassays, the restoration of luciferase activity upon

Figure 1. Subcellular localization of RFA1 and RFA4. A, RFA1 localizes to nucleus and cytosol, whereas RFA4 localizes tonucleus, upon transient expression in N. benthamiana leaf cells. Confocal images show transiently transformed epidermal cellsexpressing GFP, RFA1-GFP, GFP-RFA1, GFP-RFA4, GFP-RFA4DC, or GFP-RFA4DNLS. The merge of the GFP channel and bright-field microscopy imaging is abbreviated as GFP1BF. Immunoblotting analysis (IB) using anti-GFP was used to verify the ex-pression of the corresponding fusion proteins. B, RFA1 does not localize to the plasma membrane. Confocal images showtransiently transformedN. benthamiana leaf cells coexpressing RFA1-GFP or GFP-RFA1 and the plasma membrane marker OFP-TM23. The degree of colocalization between the two fluorescent signals was analyzed using merged images and Zeiss software(ZEN Lite 2012).N. benthamiana leaf cells were plasmolyzed using a 500 mM NaCl treatment for 30 min. Lack of colocalizationwithOFP-TM23 is shown in relative intensity (x and y axes) scatterplots and values of Pearson (RP) and Spearman (RS) coefficients.Bars 5 30 mm.


Fernandez et al.





protein-protein interaction of the candidate proteins isreversible (Gehl et al., 2011). The coexpression of RFA1-nLUC and cLUC-PYR1, cLUC-PYL4, or cLUC-PYL8reconstituted luciferase activity, although in the case ofcLUC-PYL8 a weak signal was obtained (Fig. 2C, top).Luciferase activity was also reconstituted when RFA4-nLUC and cLUC-PYR1, cLUC-PYL4, or cLUC-PYL8were coexpressed (Fig. 2C, bottom). As a positive con-trol, we used the pGB0164 construct expressing full-length luciferase (Vazquez-Vilar et al., 2017), whereasexpression of RFA1-nLUC or RFA4-nLUC with cLUCserved as negative controls.

Since RSL1 interacts with ABA receptors at theplasma membrane (Bueso et al., 2014), the above-mentioned results indicate that other members of theRSL1/RFA family target PYR/PYLs at different sub-cellular locations. This was further investigated usingmulticolor BiFC by cloning PYR1 or PYL4 intop(MAS)-SCYCE(R), RSL1 into pDEST-SCYNE(R), andRFA4 into pDEST-VYNE(R) vectors, as describedpreviously (Gehl et al., 2009). We coexpressedSCFPC-PYR1, SCFPN-RSL1, and VENUSN-RFA4 orSCFPC-PYL4, SCFPN-RSL1, and VENUSN-RFA4 in N.benthamiana cells. We observed the simultaneous

Figure 2. RFA1 and RFA4 interact withABA receptors. A, RFA1 and PYL pro-teins interact in nucleus (asterisks) andcytosol of N. benthamiana leaf cells.Confocal images show transiently trans-formed epidermal cells coexpressingYFPN-RFA1 and YFPC-PYR/PYL proteins.Interaction with PYR1 is observed in thenuclear envelope but excluded from thenucleus. The reconstituted YFP signalwas quantified from 10 randomly cho-sen regions of interest of infiltratedleaves by ImageJ software, and the ar-bitrary units of fluorescence are indi-cated in numbers. The GFP channelshows the subcellular localization of theinteraction, whereas bright-field (BF)microscopy imaging served to identifythe nuclei. B, Nuclear interaction ofRFA4 and ABA receptors. Confocal im-ages show transiently transformed N.benthamiana leaf cells coexpressingYFPN-RFA4 and YFPC-PYR/PYL proteins.The reconstituted YFP signal was quan-tified from 10 randomly chosen regionsof interest of infiltrated leaves by ImageJsoftware, and the arbitrary units of fluo-rescence are indicated in numbers. Bars5 30 mm. C, Split-luciferase comple-mentation assay reveals interaction ofRFA1 and RFA4 with PYR1, PYL4, andPYL8. The indicated construct pairswerecoexpressed in N. benthamiana leaves(shown in gray) by A. tumefaciens-mediated infiltration, and 50 mM MG132was applied into the infiltrated region 6 hbefore measurement of luciferase activity,which was performed 72 h after infiltra-tion. The 35S:LUCFL construct served as apositive control. Luciferase activity wasmeasured by applying 1 mM D-luciferinand imaging with a CCD imaging system.Luciferase signal was converted to falsecolors with ImageJ. The color scale rep-resents luciferase activity. Three inde-pendent experiments were performed,and images correspond to representativeleaves (n 5 5 per experiment).





formation of receptor-RSL1 and receptor-RFA4 com-plexes in plasma membrane and nucleus, respectively,of N. benthamiana cells (Supplemental Fig. S4). Thissuggests that the turnover of ABA receptors can beregulated differently by RSL1 and RFA4, targetingABA receptors for degradation via the vacuolar or 26Sproteasome pathway, respectively.

RFA1 and RFA4 Promote Ubiquitination of PYR1 in Vitro

We generated maltose-binding protein (MBP)-RFA1and MBP-RFA4DC fusion proteins to carry out pull-down and ubiquitination assays of ABA receptors.The C-terminal acidic domain of full-length RFA4 pre-cluded efficient bacterial expression and purification ofrecombinant protein. Since RFA4DC could interact withABA receptors in BiFC assays (Supplemental Fig. S3A),we used the MBP-RFA4DC fusion protein. Using apull-down assay, we found that both MBP-RFA1 andMBP-RFA4DC could interact with PYR1 and PYL4receptors, whereas MBP did not (Fig. 3A). In order totest whether RFA1 and RFA4DC catalyze the transferof Ub to PYR1, we performed an enzymatic reaction incolumn using HA-PYR1 immunoprecipitated fromArabidopsis transgenic lines. The enzymatic reactionwith immunoprecipitated HA-PYR1, biotinylated-Ub,ATP, human activating enzyme E1, Arabidopsis E2UBC8, and either MBP-RFA1 or MBP-RFA4DC wasperformed as described previously (Bueso et al., 2014).We used the generic E2 UBC8 because in vitro activitywas detected using this E2 together with the RBR-typeenzymes RSL1 or ARI8, and UBC8 is the most com-monly used E2, displaying high and promiscuous ac-tivity with different E3s (Kraft et al., 2005; Bueso et al.,2014; Kowarschik et al., 2018). The incorporation ofbiotinylated Ub in the reaction was monitored usingstreptavidin-horseradish peroxidase (St-HRP), and theincorporation of Ub into the substrate was confirmedusing anti-HA antibody. RFA1 and RFA4 were able tocatalyze the transfer of Ub to the PYR1 ABA receptor,as revealed using anti-HA immunoblotting (Fig. 3B).Using St-HRP, we could detect other proteins thatwere ubiquitinated during the reaction, whichaccording to the expected molecular mass likelyrepresent ubiquitinated components of the ubiquiti-nation cascade (i.e. E1, E2, MBP-RFA1, or MBP-RFA4DC; Fig. 3B). In addition to in vitro biochemicalassays, we performed coimmunoprecipitation ex-periments in plant extracts to further validate theobserved interactions. To this end, we mixed Arabi-dopsis MG132-treated protein extracts obtained fromlines expressing either HA-RFA1 or HA-RFA4 withN. benthamiana protein extracts that contain eitherGFP or GFP-PYL4 proteins (Fig. 3C). We immuno-precipitated GFP or GFP-PYL4 using paramagneticbeads coupled to anti-GFP antibody. Following anti-HA immunoblotting analysis, we could detect coim-munoprecipitation of HA-RFA1 or HA-RFA4 withGFP-PYL4 but not with the GFP control (Fig. 3C).

RFA1 and RFA4 Are Ubiquitinated in Vivo

Studies done in mammals on the mechanism of ac-tion of RBR-type E3s have shown that their cognate E2enzymes transfer the activated Ub to a conserved Cysresidue of the E3 in RING2, which in turn transfers theUb from this cysteinyl residue (active site Cys) to targetLys residues (Dove et al., 2016). Therefore, RBR-typeE3s are ubiquitinated by the E2 during this processand subsequently RBRs ubiquitinate their targetsthrough a mechanism similar to HECT-type E3 ligases(Callis, 2004). In order to investigate whether RFA1 andRFA4 are ubiquitinated in vivo, we constructed Ara-bidopsis lines expressing HA-tagged RFA1 and RFA4from a 35S promoter (Fig. 4A; Supplemental Fig. S5).Interestingly, these lines showed reduced sensitivity toABA-mediated inhibition of root growth, which is incommon with the phenotype of RSL1-overexpressinglines that enhance degradation of ABA receptors (Buesoet al., 2014; Supplemental Fig. S5A). Protein extracts fromArabidopsis RFA1- and RFA4-overexpressing lines wereprepared and immunoblotting analysis revealed that bothRFA1 and RFA4 proteins reached higher levels uponMG132 treatment, which suggests that they are them-selves subjected to ubiquitination and 26S proteasomedegradation (Fig. 4A; Supplemental Fig. S5B). High-mo-lecular-mass forms of RFA1 and RFA4 were detected insamples treated withMG132, which might correspondto ubiquitinated forms of the E3 ligases (Fig. 4B). Toverify this, we immunoprecipitated HA-RFA1 andHA-RFA4 from MG132-treated samples, and immu-noblot analysis using anti-Ub antibodies confirmedthat both RFA1 and RFA4 are ubiquitinated in vivo(Fig. 4B).

The Cys-361 Residue of RFA4 Affects Its OwnUbiquitination and Capability to Ubiquitinate PYL4

In order to identify the putative catalytic cysteinylresidue of RFA4 involved in the transfer of Ub totarget Lys residues, we performed a multiple se-quence alignment of RFA4 RING2, HHARI RING2,and Parkin RING2 (Supplemental Fig. S6A). As aresult, we identified Cys-361 in RFA4 as the putativeactive site Cys, which corresponds to the well-knownactive site Cys-431 in Parkin and Cys-357 in HHARI(Wenzel and Klevit, 2012; Duda et al., 2013; Rileyet al., 2013), and we introduced a Cys-361Ala muta-tion in RFA4 that did not affect nuclear localization ofthe protein (Supplemental Fig. S6B). Additionally, wegenerated HA-tagged RFA4DC and RFA4Cys-361AlaDCconstructs, which were used to obtain stable lines inArabidopsis (Fig. 4C) and that were delivered into leafcells of N. benthamiana by agroinfiltration (Fig. 4D).Expression of HA-RFA4DC was lower than that ofHA-RFA4Cys-361AlaDC both in mock- and MG-treatedsamples obtained in Arabidopsis (Fig. 4C; SupplementalFig. S5B). Upon immunoprecipitation with anti-HAantibodies and subsequent analysis with anti-Ub


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antibodies, we could detect more ubiquitination ofHA-RFA4DC compared with HA-RFA4Cys-361AlaDC(Fig. 4C). Similar results were obtained after tran-sient expression in N. benthamiana cells, where

expression of HA-RFA4DCwas lower than that of HA-RFA4Cys-361AlaDC (Fig. 4D). However, high-molecular-mass forms of HA-RFA4DC were more abundantthan those of HA-RFA4Cys-361AlaDC (Fig. 4D). In order

Figure 3. RFA1 and RFA4 pull down ABA receptors and show E3 Ub ligase activity with PYR1. A, Interaction among PYR1/PYL4with RFA1 or RFA4DC in a pull-down assay. Purified MBP, MBP-RFA1, or MBP-RFA4DC (5 mg each) and 5 mg of 6His-PYR1 orPYL4 protein were incubated for 1 h at 4°C with constant rocking in 0.5 mL of binding buffer (20 mM Tris-HCl, pH 7.5, 200 mM

NaCl, 1 mM EDTA, and 0.5% [v/v] Tween 20). Next, MBP, MBP-RFA1, or MBP-RFA4DC protein was purified using amyloseaffinity chromatography, eluted, and analyzed by immunoblotting using anti-His and Ponceau staining. B, MBP-RFA1 or MBP-RFA4DC (2 mg) was assayed for E3 ligase activity in the presence of 100 ng of human E1, 250 ng of purified 6His-AtUBC8 (E2),3HA-PYR1 immunoprecipitated from an Arabidopsis transgenic line, and 1 mg of biotinylated Ub. After incubation at 30°C for 2h, the mixture was subjected to SDS-PAGE/blotting followed by detection using either St-HRP or anti-HA-HRP to detect eitherubiquitylated or 3HA-tagged proteins, respectively. The components of the ubiquitination cascade (i.e. E1-Ub, E2-Ub, ubiq-uitinated MBP-RFA1, or MBP-RFA4DC) are indicated by arrows. C, PYL4 coimmunoprecipitates with RFA1 and RFA4. GFP orGFP-PYL4 proteins were expressed inN. benthamiana, and each protein extract was combined with MG132-treated Arabidopsisprotein extracts obtained from lines expressing either HA-RFA1 or HA-RFA4. Proteins were immunoprecipitated using anti-GFPand next immunoblotted with anti-HA to detect coimmunoprecipitation of either HA-RFA1 or HA-RFA4.





to analyze the ubiquitination of HA-RFA4DC andHA-RFA4Cys-361AlaDC, protein extracts were sub-jected to immunoprecipitation using anti-HA anti-bodies, and the immunoprecipitates were analyzedusing anti-Ub antibodies. This revealed an ;50% re-duction of the ubiquitination in the Cys-361Ala

mutant compared with the wild type (Fig. 4D). Takentogether, the results obtained in Arabidopsis and N.benthamiana suggest that Cys-361 is involved in theubiquitination of RFA4, although it is possible thatother Lys or Cys residues of RFA4 could also beubiquitinated.

Figure 4. Analysis of RFA1 and RFA4 ubiquitination and the role of the Cys-361 residue of RFA4 in the ubiquitination and activityof the E3 ligase. A, Both RFA1 and RFA4 are themselves subjected to degradation via the 26S proteasome. Arabidopsis transgeniclines expressing HA-tagged RFA1 or RFA4 accumulate higher levels of these proteins after 50 mM MG132 treatment for 24 h.Values are averages6 SD obtained from three independent experiments (n5 5 plants in each experiment). *, P, 0.05 (Student’st test) with respect to the correspondingmock-treated sample. B, Both RFA1 andRFA4 are ubiquitinated in vivo. TheHA-RFA1 andHA-RFA4 proteins were immunoprecipitated fromMG132-treated samples using anti-HA antibodies and subsequently analyzedby immunoblotting using anti-HA or anti-Ub (P4D1) antibody. C, The Cys-361Ala mutation markedly reduces the ubiquitinationof RFA4 and stabilizes the protein compared with the wild type. Arabidopsis transgenic lines expressing HA-tagged RFA4DC orRFA4Cys-361AlaDC accumulate higher levels of these proteins after 50 mM MG132 treatment for 24 h. Protein extracts wereimmunoprecipitated using anti-HA antibodies and subsequently analyzed by immunoblotting using anti-HA or anti-Ub (P4D1)antibodies. Values are averages 6 SD obtained from three independent experiments (n 5 5 plants in each experiment), andRFA4DC protein levels in mock-treated plants were taken as 100%. *, P, 0.05 (Student’s t test) comparedwith the correspondingmock-treated sample. D, Transient expression in N. benthamiana of HA-tagged RFA4DC or RFA4Cys-361AlaDC reveals reducedubiquitination of RFA4Cys-361AlaDC compared with the wild type. *, P, 0.05 (Student’s t test) compared with the wild type. E, Invivo ubiquitination of PYL4 by RFA4DC in N. benthamiana leaf cells. Agrobacteria encoding GFP-PYL4 were coinfiltrated in N.benthamiana cells with agrobacteria encoding either HA-RFA4DC or RFA4Cys-361AlaDC. Protein extracts were immunoprecipi-tated using anti-GFP and subsequently immunoblotted with anti-GFP, anti-PYL4, anti-Ub, and anti-HA antibodies. *, P , 0.05(Student’s t test) comparedwith thewild type. For D and E, values are averages6 SD obtained from three independent experiments(n 5 5 leaves in each experiment).


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Next, we examined whether the Cys-361Ala muta-tion affects the capability of RFA4 to ubiquitinate thePYL4 target. We set up an in vivo ubiquitination assayby coexpressing GFP-PYL4 in N. benthamiana togetherwith HA-RFA4DC or HA-RFA4Cys-361AlaDC (Fig. 4E).Two days after agroinfiltration, protein extracts fromdisc samples were prepared, immunoprecipitated us-ing anti-GFP, immunoblotted, and analyzed with fourdifferent antibodies (Fig. 4E). In samples where GFP-PYL4 was coexpressed with HA-RFA4DC, the use ofanti-GFP, anti-PYL4, and anti-Ub antibodies revealedmore ubiquitinated PYL4 comparedwith sampleswhereGFP-PYL4 was coexpressed with HA-RFA4Cys-361AlaDC(Fig. 4E). These results indicate that Cys-361 is an im-portant residue for the activity of RFA4. Interestingly,coimmunoprecipitation of HA-RFA4Cys-361AlaDC withGFP-PYL4was probably enhanced by the higher proteinlevel of the Cys-361Ala mutant (Fig. 4E, a-HA).

RFA1 and RFA4 Promote Degradation of ABA Receptorsin Vivo

Once we confirmed the capability of RFA1 and RFA4to interact with and ubiquitinate PYR1 and PYL4 ABAreceptors, coinfiltration experiments in N. benthamianawere performed to test whether RFA1/RFA4 promotedegradation of their targets in vivo (Zhao et al., 2013).Increasing amounts of the agrobacterium (Agrobacteriumtumefaciens) that drives expression of the E3 ligase werecoinfiltrated with different agrobacteria encoding con-structs to express ABA receptors. Sampleswere collectedfor the detection of both protein and RNA levels (Fig. 5,A and B). Increasing amounts of RFA1 led to decreasedlevels of PYR1 and PYL4, whereas the internal GFPcontrol was not significantly affected by increasing theamount of RFA1 (Fig. 5A). Similarly, increasing amountsof RFA4 led to degradation of PYR1 and PYL4, whereasthe internal RFP control remained stable (Fig. 5B).We also analyzed whether RFA1 promoted PYR1 and

PYL4 degradation using a semi-in vivo degradation as-say (Liu et al., 2010). RFA1 and each PYR/PYL wereexpressed separately via different agroinfiltrations.Then, we mixed together the N. benthamiana extractscontaining RFA1 and each PYR/PYL and monitoredprotein levels after 1 h of incubation at 4°C (Fig. 5C). Theaddition of RFA1 enhanced the degradation of PYR/PYLs comparedwith the samplewith nonagroinfiltratedextract (mock). When the MG-132 proteasome inhibitorwas included, the level of PYR/PYL increased 2- to 3-fold with respect to the sample lacking the inhibitor(Fig. 5C), which indicates that RFA1 promotes the deg-radation of PYR1 and PYL4 through the 26S proteasome.

Identification of UBC26 as the Cognate Nuclear E2 EnzymeThat Interacts with the RFA4 E3 Ligase

Attachment of Ub to substrate proteins is catalyzedby the concerted action of E1, E2, and E3 enzymes. E3

ligases mediate substrate specificity of the ubiquitina-tion machinery; however, E2s also play a key role inmediating Ub chain assembly on the target (Callis,2014; Kowarschik et al., 2018). In the case of U-box/RING-type E3 ligases, concerted action of the E2/E3pair regulates substrate specificity and the type of Ubmodification on the substrate proteins (Zhao et al.,2013). In the case of mammalian HECT- and RBR-type E3s, residues in the active site Cys determine thetype of ubiquitination, although little is known for plantenzymes (Kim and Huibregtse, 2009; Dove et al., 2016).In plants, the cohort of E2s that function together withRBR-type ligases is unknown, as are the features andconsequences of this interaction for the ubiquitinationof the target. The Arabidopsis genome encodes 48 UBCdomain-containing proteins; however, only 37 carry theactive site Cys required for E2 Ub conjugase activity(Callis, 2014). We were particularly interested in theidentification of nuclear E2s that could interact withRFA4 to form a nuclear E2-E3 complex; therefore, weconducted a yeast two-hybrid (Y2H) screen to identifycognate E2 enzymes for RFA4. To this end, either GAD-RFA4 or GAD-RFA4DCwas used as a bait to find preysin a yeast pGBD library containing 30 E2s. As a result,we found that both UBC24 andUBC26, which belong tofamily XI of Arabidopsis E2 proteins, interacted withboth RFA4 and RFA4DC (Fig. 6A; Supplemental Fig.S7). In contrast, the other E2s analyzed did not interactwith RFA4 (Supplemental Fig. S7). We pursued furtherwork with UBC26 because it contains an NLS(KKKTRKR) at the C terminus (Supplemental Fig. S8).Indeed, GFP-UBC26 is localized in the nucleus (Fig. 6B),whereas UBC24 has been reported to be mostly colo-calized with endoplasmic reticulum and Golgi markers(Liu et al., 2012; Fig. 6B). In addition to RFA4, UBC26also interacts with RFA1 in the Y2H assay; however,since RFA1 also shows cytosolic localization, it is likelythat other unidentified UBCs interact with RFA1(Fig. 6A). Additionally, using the Y2H assay, we alsoconfirmed that RFA1 and RFA4DC could interact withABA receptors (Fig. 6B; Supplemental Fig. S9A), whichindicates that these E3s can interact in Y2H assays withboth the E2 and the substrate.To investigate the interaction in planta of RFA1

and RFA4 with UBC26, we performed BiFC assays(Fig. 6C). We coexpressed YFPN-RFA4, YFPN-RFA4DC,or YFPN-RFA1 with YFPC-UBC26 and found that theyinteract in the nucleus of N. benthamiana cells. In con-trast, the closely related RFA5 protein did not interactwith UBC26, even though it was expressed to similarlevels to other RFAs (Fig. 6C; Supplemental Fig. S3B).The nuclear speckles formed by RFA4-UBC26 weredifferent from those formed by RFA4DC-UBC26, whichare more similar to RFA1-UBC26 speckles. These re-sults suggest that although the C-terminal acidic do-main of RFA4 is dispensable for interaction with thesubstrate and the E2, it determines the location of theinteraction in a different nuclear territory. Finally, weperformed a multicolor BiFC assay (Gehl et al., 2009) toanalyze the possible formation of nuclear receptor-E3-E2











complexes. RFA4 interacted with both the E2 and ABAreceptors when coexpressed in N. benthamiana cells(Fig. 6D).

UBC26 has been reported to be recalcitrant to ex-pression in Escherichia coli (Zhao et al., 2013). Indeed,some E2s are not induced or are insoluble whenexpressed in bacteria, and in these cases, E2 activity canbe recovered after expression in plants (Zhao et al.,2013). Since we had successfully expressed GFP-UBC26 by agroinfiltration in N. benthamiana cells andnuclear PYL4-RFA4-UBC26 complexes could be detec-ted (Fig. 6D), we set up an in vivo ubiquitination assayinvolving GFP-PYL4, GFP-UBC26, and GFP-RFA4(Fig. 6E). The GFP fusion proteins were immunopreci-pitated and the ubiquitination of PYL4 was analyzed

using anti-PYL4 and anti-Ub antibodies (Fig. 6E). High-molecular-mass forms of PYL4, recognized both byanti-PYL4 and anti-Ub antibodies, were notably en-hanced when the E2/E3 proteins were coexpressedwith PYL4, which suggests that ubiquitination of PYL4was markedly enhanced by the action of UBC26 andRFA4 (Fig. 6E). Some ubiquitination of PYL4 in N.benthamiana cells could be detected in the absence ofE2/E3 coexpression, which had been also previouslyobserved in Arabidopsis lines expressing HA-PYL4(Bueso et al., 2014) and might be explained by the en-dogenous endowment of E2/E3s. Finally, the expres-sion of GFP-PYL4 plus only the E2 or E3 did notsignificantly enhance the basal ubiquitination of PYL4(Supplemental Fig. S9B).

Figure 5. Analysis of RFA1- and RFA4-promoted degradation of PYR1 and PYL4 by in vivo and semi-in vivo assays. A and B, Invivo degradation of either PYR1 or PYL4 was observed in coinfiltration experiments with increasing amounts of RFA1 or RFA4.The ratio of the relative concentration of agrobacteria used in the different coinfiltrations is indicated by numbers (top). In A, cellextracts were analyzed using anti-HA to detect HA-tagged PYR1/PYL4 and anti-GFP to detect GFP-RFA1, whereas in B, anti-HAwas used to detect both HA-tagged PYR1/PYL4 and HA-RFA4. Anti-GFP or anti-RFP antibodies were used to detect the internalcontrol of GFP or RFP, respectively. The mRNA expression levels of the target PYR1 and PYL4 genes and ACTIN8 (ACT8) wereanalyzed by reverse transcription-semiquantitative PCR. Molecular masses of marker proteins are indicated in kilodaltons. C,Cell-free experiments show HA-tagged PYR1/PYL4 degradation promoted by RFA1. Degradation of either HA-PYL4 or HA-PYR1was performed by mixingN. benthamiana cell extracts from separated agroinfiltrations. HA-PYL4 or HA-PYR1 extract was mixedwith RFA1-GFP extract or mock control extract and incubated at 4°C during 1 h either in the absence or presence of MG132.Proteinswere detected by immunoblot as described above. Ponceau staining of the Rubisco protein is shown as a loading control.Graphs at right show the quantification of the experiment. Values are averages6 SD obtained from three independent experiments(n 5 3 leaf extracts in each experiment). *, P , 0.05 (Student’s t test) compared with the corresponding mock-treated sample.


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Figure 6. UBC26 and the UBC26-RFA4-ABA receptor complex are localized in the nucleus. A, Y2H interactions of RFA1 andRFA4. Transformed yeast cultures were grown overnight in liquid synthetic defined (SD) medium lacking Leu and Trp, and adilution (1021) of these cultures was dropped on either control medium lacking Leu and Trp (SD–Leu–Trp1His) or selectivemedium additionally lackingHis (SD–Leu–Trp–His). Empty vectorswere used as negative controls, and yeast was allowed to growfor 3 d at 30°C before interaction was scored. B, GFP-UBC26 localizes in the nucleus upon transient expression in N. ben-thamiana, whereas GFP-UBC24 seems to be membrane associated. Immunoblotting analysis using anti-GFP was used to verifythe expression of GFP-UBC26. C, BiFC interaction of RFA4 and UBC26 in the nucleus of N. benthamiana epidermal cells.Confocal images show transiently transformed leaf cells coexpressing YFPC-UBC26 and YFPN-RFA4, RFA4DC, RFA1, or RFA5.Note that the E2-E3 interaction decorates nuclear speckles, whereas the PYL4-E3 interaction is absent in the nucleolus (Fig. 2A).Deletion of the acidic domain of RFA4 changes the nuclear pattern of the E2-E3 interaction comparedwith RFA4FL. YFPC-UBC26does not interact with YFPN-RFA5, which is another member of the RFA family. Quantification of YFP signal was analyzed byImageJ software, and the arbitrary units of fluorescence are indicated in numbers. D, Multicolor BiFC reveals the formation of E2-E3-PYL4/8 nuclear complexes. Confocal images show transiently transformed N. benthamiana epidermal cells coexpressingSCFPC-RFA4, SCFPN-PYL4/8, and VENUSN-UBC26. The RFA4-PYL4/8 interaction was visualized through reconstitution of theSCFP, whereas the RFA4-UBC26 interaction gave rise to SCFPC-VENUSN fluorescent protein. SCFPC-RFA4 coexpressed withSCFPN or VENUSN did not reconstitute fluorescent protein (Supplemental Fig. S3B). Constructs were delivered into N. ben-thamiana epidermal leaves through A. tumefaciens-mediated transfection. Leaves were examined using confocal laser scanningmicroscopy 48 to 72 h after infiltration. Bars 5 30 mm in B to D. E, In vivo ubiquitination of PYL4 by UBC26-RFA4 in N.






To provide genetic support to the possible role ofUBC26 in ABA signaling, we generated CRISPR/Cas9 lines specifically impaired in UBC26 function(Supplemental Fig. S10). The ubc26-c1 and ubc26-c2alleles contain one-base insertions at position 1,215(counting from the ATG), which generates a frame-shift mutation and creates a stop codon at position1,246. As a result, the Ub-conjugating E2 domain ofUBC26 is disrupted (Supplemental Fig. S10, A and B).We analyzed ABA sensitivity of the ubc26-c1 andubc26-c2 alleles in germination and root growthassays. We found that both alleles showed enhancedsensitivity to ABA-mediated inhibition of seedlingestablishment and root growth (Fig. 6, F and G;Supplemental Fig. S10C). We examined protein levelsof PYL4, using the specific antibodies described by Yuet al. (2016), and found higher PYL4 levels in bothubc26-c1 and ubc26-c2 alleles compared with the wildtype (Fig. 6H). This result suggests that UBC26 medi-ates the nuclear degradation of PYL4, likely throughits interaction with RFA4.

The rfa1 rfa4 Double Mutant Accumulates More PYR1 andPYL4 Receptors

We screened the Arabidopsis Biological ResourceCenter (ABRC)/Nottingham Arabidopsis Stock Cen-tre collection to identify T-DNA insertion rfa1 andrfa4 mutants (Supplemental Fig. S11A). The T-DNAinsertion strongly impairs the expression of RFA1and RFA4; however, single rfa1 and rfa4 mutantsshow wild-type sensitivity to ABA, which likely re-flects a certain functional redundancy observed inmultigene families (Supplemental Fig. S11, A–C).Therefore, we generated an rfa1 rfa4 double mutantand analyzed its sensitivity to ABA (Fig. 7A). As acontrol for this experiment, we used the ABA-hypersensitive RNA interference (RNAi) 7.16.2 line,where expression of at least three members of theRSL1/RFA1 family was impaired (i.e. RSL1, RFA1,and RFA3; Bueso et al., 2014). The rfa1 rfa4 double

mutant was impaired in germination and showedenhanced sensitivity to ABA-mediated inhibition ofseedling establishment and root growth comparedwith the wild type (Fig. 7, A and B; Supplemental Fig.S11, D and E). We also performed water-loss experi-ments, and detached leaves of rfa1 rfa4 showed nosignificant difference in water-loss assays comparedwith the wild type (Fig. 7C), which is in agreementwith the low expression of RFA1 and RFA4 in guardcells (accession nos. GSE41054 and GSE118520, GeneExpression Omnibus database). Using infrared ther-mography of leaves to monitor transpiration, we didnot observe significant differences in rfa1 rfa4 com-pared with the wild type; however, the RNAi 7.16.2line showed a significant increase of leaf temperature(Fig. 7D). This suggests that ubiquitination and en-docytosis of ABA receptors triggered by RSL1 mightregulate stomatal aperture, as observed for the com-ponents of the ESCRT pathway FYVE1/FREE1 andALIX (Belda-Palazon et al., 2016; García-León et al.,2019). Finally, we examined protein levels of two re-ceptors, PYR1 and PYL4, that play a major role inABA signaling and for which there are specific anti-bodies available (Fig. 7E; Supplemental Fig. S12). Wefound that rfa1 rfa4 accumulates higher levels of bothPYR1 and PYL4 compared with the wild type, whichwas also observed in the RNAi 7.16.2 line. This resultconfirms that the RSL1/RFA family of E3 ligasesregulates endogenous protein levels of ABA recep-tors. Additionally, we performed a time-course studyof the PYL4 protein in the presence of cycloheximide(CHX) and observed that degradation of PYL4 wasslower in rfa1 rfa4 compared with the wild type(Fig. 7F). Finally, we analyzed the effect of ABAtreatment on PYL4 both in leaves and roots and foundthat ABA promotes the degradation of PYL4 bothin the wild type and rfa1 rfa4. This result suggeststhat ABA-induced down-regulation of PYL4 can oc-cur in the absence of RFA1 and RFA4, likely throughtranscriptional down-regulation of PYL4 expres-sion in response to ABA (Goda et al., 2008; Santiagoet al., 2009b) and protein degradation through the

Figure 6. (Continued.)benthamiana leaf cells. Agrobacteria encodingGFP-PYL4were coinfiltrated inN. benthamiana cells with agrobacteria lacking orencoding GFP-UBC26/GFP-RFA4 proteins (2/1 E2E3). Protein extracts were immunoprecipitated using anti-GFP and nextimmunoblotted with anti-GFP, anti-PYL4, and anti-Ub to detect ubiquitination of GFP-PYL4 in the absence or presence ofGFP-UBC26/GFP-RFA4 proteins. Extraction of GFP-RFA4 for SDS-PAGE analysis seems to be inefficient because of the largeC-terminal acidic domain. F, Enhanced sensitivity to ABA-mediated inhibition of seedling establishment. Approximately100 seeds of each genotype (three independent experiments) were sown on Murashige and Skoog (MS) plates lacking orsupplemented with 0.5 mM ABA and scored for the presence of both green cotyledons and the first pair of true leaves after 7d.Values are averages 6 SD. *, P , 0.01 (Student’s t test) with respect to wild-type Columbia-0 (Col-0) assayed in the sameconditions. G, Enhanced sensitivity to ABA-mediated inhibition of root growth. Quantification is shown for ABA-mediatedinhibition of root growth in the indicated genotypes comparedwith the wild type. *, P, 0.01 (Student’s t test) with respect towild-type Col-0 assayed in the same conditions. H, Accumulation of endogenous PYL4 protein in ubc26-c1 and ubc26-c2alleles compared with the wild type. Seedlings of the indicated genotypes were grown for 7 d in MS medium, and totalproteins were extracted and subjected to immunoblot analysis using the indicated antibodies. Actin was analyzed asa protein-loading control, and relative PYL4 protein levels were quantified. Values are averages 6 SD obtained fromthree independent experiments (n. 20 seedlings in each experiment). *, P, 0.05 (Student’s t test) compared with wild-typeCol-0.


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Figure 7. The rfa1 rfa4 doublemutant shows enhanced sensitivity to ABA and accumulation of the PYR1 and PYL4 ABA receptorsas compared with the wild type. A, Enhanced sensitivity to ABA-mediated inhibition of seedling establishment. Approximately100 seeds of each genotype were sown on MS plates lacking or supplemented with 0.5 mM ABA and scored for the presence ofboth green cotyledons and the first pair of true leaves after 7 d. For A and B, values are averages 6 SD obtained from three in-dependent experiments (n . 100 seeds in A; n . 20 seedlings in B). *, P , 0.01 (Student’s t test) with respect to the wild typeassayed in the same conditions. B, Enhanced sensitivity to ABA-mediated inhibition of root growth. Quantification of ABA-mediated inhibition of root growth is shown in the indicated genotypes compared with the wild type. The photographs showrepresentative seedlings 10 d after the transfer of 4-d-old seedlings to MS plates lacking or supplemented with 10 mM ABA. Bars51 cm. C,Water loss in detached leaves of the indicated genotypes. Loss of fresh weight was measured in detached leaves from 15-d-old plants submitted to the drying atmosphere of a laminar flow hood. Values are averages 6 SD obtained from three inde-pendent experiments (n 5 5 plants in each experiment), and ns indicates nonsignificant difference when comparing (usingStudent’s t test) data of the wild type and rfa1 rfa4. D, Leaf temperature of the indicated genotypes was quantified by infraredthermography. Data are means 6 SD (n 5 5, approximately 1,000 measurements of square pixels from several leaves of eachplant). *, P , 0.05 (Student’s t test) with respect to wild-type Col-0. E, Accumulation of endogenous PYR1 and PYL4 proteins inrfa1 rfa4 and RNAi 7.16.2 line compared with the wild type. Seedlings of the indicated genotypes were grown for 7 d in MSmedium, and total proteins were extracted and subjected to immunoblot analysis using the indicated antibodies. Actin wasanalyzed as a protein-loading control, and PYR1 or PYL4 protein levels were quantified relative to Col-0 (taken as 100%). Valuesare averages6 SD obtained from three independent experiments (n5 10 seedlings per genotype in each experiment). *, P, 0.05(Student’s t test) with respect to wild-type Col-0. F, Degradation of PYL4 is delayed in the rfa1 rfa4 mutant compared with wild-type Col-0. Seedlings of Col-0 or rfa1 rfa4were grown in liquidMSmedium for 10 d and then 50mMCHX treated for the indicatedtime periods. Actin was analyzed as a loading control. Histograms show the quantification of PYL4 protein levels in CHX-treatedsamples relative to time 0, whose value was taken as 100% for Col-0 or rfa1 rfa4 samples. Values are averages6 SD obtained fromthree independent experiments (n5 10 seedlings per genotype in each experiment). *, P, 0.05 (Student’s t test) with respect toCol-0 at the same time point. G, ABA treatment leads to down-regulation of the PYL4 protein. Seedlings of Col-0 or rfa1 rfa4weregrown in liquid MS medium for 10 d and then were mock or 50 mM ABA treated for 6 h. Leaf and root tissues were separated, andprotein extracts were analyzed by immunoblotting using anti-PYL4 and anti-ACT antibodies. Arrowheads indicate the bandcorresponding to PYL4. A new batch of the anti-PYL4 antibody recognized a second band below PYL4.





ESCRT/vacuolar pathway (Belda-Palazon et al.,2016; García-León et al., 2019).

DISCUSSION

In this work, we gained insight into the biologicalfunctions of some RBR-type E3 ligases, which arepoorly characterized enzymes in plants (Callis, 2014).There are 42 genes encoding RBR-type proteins inArabidopsis, which are classified into four subfam-ilies: Plant II (22 members), Plant I/helicase (threemembers), ARA54 (one member), and ARIADNE(16 members; Marín, 2010). Compared with otherfamilies of E3 ligases, very little is known regardingthe biological function of RBRs. The ARI12 and ARI14members of the ARIADNE family have been impli-cated in UV-B signaling and fertilization, respectively(Ron et al., 2010; Lang-Mladek et al., 2012), but the restof the family is hardly characterized. RSL1/RFAs be-long to clade A of the Plant II subfamily, and it wasdemonstrated that RSL1 targets ABA receptors inplasma membrane (Bueso et al., 2014; Belda-Palazonet al., 2016). Since 14 ABA receptors exist in Arabi-dopsis, we hypothesized that their interacting E3smight also belong to multigene families (Bueso et al.,2014). We demonstrated that ABA receptors can bealso targeted by RFA1 and RFA4, which are membersof the RSL1/RFA family not associated with plasmamembranes. We suggest that specific features ofRSL1/RFA members, such as their particular subcel-lular localization or differential expression pattern,could notably increase the capability to recognize andregulate the stability of the whole family of ABA re-ceptors. Interestingly, RFA4 shows specific nuclearlocalization, whereas RFA1 is localized both in nu-cleus and cytosol. Moreover, we demonstrated usingmulticolor BiFC that it is possible to detect the simul-taneous formation of receptor-RSL1 and receptor-RFA4 complexes in different subcellular locations.Thus, the concerted action of several members of theRSL1/RFA family enables fine regulation of ABA re-ceptor half-life in different subcellular compartments.Additionally, whereas RSL1-mediated ubiquitinationpromotes endosome-mediated vacuolar degradationof ABA receptors, RFA1 and RFA4 act likely throughthe 26S proteasome degradation pathway (Fig. 8).Therefore, our results further confirm that both 26Sproteasome and non-26S Ub-dependent degradationpathways regulate the stability of key components ofABA signaling (Irigoyen et al., 2014; Belda-Palazonet al., 2016; Yu et al., 2016; Yu and Xie, 2017). Thus,the regulation of the turnover of ABA receptors can beachieved both in plasma membrane and nucleus,which are two key places of the cell to regulate ABAsignaling either affecting ion/water transport ortranscriptional response to ABA, respectively (Cutleret al., 2010).

The interaction of RFA1/RFA4 with PYR1 and PYL4ABA receptors was confirmed both in vitro and in vivo.

Using agroinfiltration experiments, we demonstratedthat increasing amounts of RFA1 or RFA4 correlatedwith enhanced degradation of PYR1 and PYL4. More-over, using specific antibodies for PYR1 and PYL4, wecould demonstrate that endogenous levels of these re-ceptors were increased in the rfa1 rfa4 double mutant(Fig. 7). This finding strongly supports that endogenousABA receptors are bona fide targets of RFA1 and RFA4.Additionally, we provided evidence that a previouslydescribed ABA-hypersensitive RNAi line that targetsRSL1 and at least RFA1 and RFA3 also shows higherPYR1 and PYL4 levels than wild-type plants (Fig. 7).Endogenous PYL4 levels were also higher in the ubc26mutant (Fig. 6H), which correlated with enhancedsensitivity to ABA in establishment and root growthassays (Fig. 6, F and G). This result, together with in-teraction and ubiquitination assays, suggests thatUBC26 is the cognate E2 that forms ternary complexeswith RFA4 and ABA receptors in the nucleus of plantcells. Interestingly, only family XI members of Arabi-dopsis E2 proteins were found to interact with RFA4 inyeast cells, and for UBC26, we confirmed that this in-teraction occurs in the nucleus of plant cells. UBC26, aswell as other family XI E2 members, in addition to theC-terminal catalytic core domain present in all E2 en-zymes, contains a large N-terminal domain that mightbe involved in the interaction with E3 (SupplementalFig. S8). Future biochemical studies should furtherunravel the contribution of UBC26 to the ubiquitina-tion properties of RFA4.

In addition to regulating the half-lives of their targets,some E3s have been reported to be ubiquitinatedin vivo to regulate their own stability (Liu and Stone,2010). For instance, the KEEP ON GOING (KEG) E3ligase is autoubiquitinated in response to ABA, leadingto ABA-induced degradation of KEG and accumulationof ABI5 (Liu and Stone, 2010). Specific antibodies arenot available yet for RFAs; however, in epitope-taggedRFA1 and RFA4 lines, we observed that RFA1 andRFA4 are ubiquitinated and subjected to proteolyticdegradation by the 26S proteasome (Fig. 4, A and B).Autocatalytic ubiquitination of RSL1, RFA1, and RFA4has been observed in vitro (Bueso et al., 2014; Fig. 3B),which agrees with themechanism of action of RBR-typeE3s. In these enzymes, Ub is passed from the active siteCys of the E2 to that of the E3, which in turn catalyti-cally transfers the activated Ub from its RING2 domainto Lys target residues. A prediction of thismechanism isthat ubiquitinated forms of plant RBR-type E3s shouldbe detected in vivo, which was confirmed both forRFA1 and RFA4 (Fig. 4, A–C). Additionally, the Cys-361Ala mutation impaired the capability of RFA4 toubiquitinate the PYL4 target, which is in agreementwith its predicted role as active site Cys (Fig. 4E).

Since degradation of ABA receptors can be ach-ieved via the 26S proteasome and vacuolar pathways,it is expected that different types of Ub-linked chainsand E2/E3 ligases are involved in these processes. Forinstance, K48-linked polyubiquitination promotes26S proteasome degradation, monoubiquitination


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promotes internalization of plasma membrane pro-teins, and K63-linked polyubiquitination can pro-mote subsequent endosome trafficking (Yu and Xie,2017; Romero-Barrios and Vert, 2018). Indeed, bothK48- and K63-linked polyubiquitination has beendetected for PYL4 (Yu et al., 2016), which indicatesthat degradation of PYL4 occurs both through theUPS and vacuolar pathways. Degradation of ABAreceptors can be differentially regulated by differentmembers of the multigene RSL1/RFA family (Buesoet al., 2014; this work) or by different E3 ligases thathave been reported to promote the degradation ofABA receptors (Irigoyen et al., 2014; Li et al., 2016, 2018;Zhao et al., 2017). However, in these latter studies, theprotein levels of endogenous ABA receptors were notanalyzed using specific antibodies. Now, the availabilityof specific antibodies against ABA receptors makes itpossible to further analyze the contribution of thereported single- and multiple-subunit E3 ligases in reg-ulating endogenous ABA receptor levels.Recently, cooperation by E3-E3 pairs has been de-

scribed in humans to regulate substrate ubiquitination(Scott et al., 2016). The CULLIN4-RING E3 ubiqui-tin ligase (CRL4) complex formed by COP10-DET1-DDB1 and the substrate adapter DDA1 are involvedin nuclear degradation of ABA receptors; however,CRL4DDA1-mediated degradation of PYL8 was coun-teracted by ABA, which suggests that the CRL4DDA1

complex onlyworks at basal ABA levels (Irigoyen et al.,2014). Contrary to PYL8, which is stabilized by ligandbinding (Belda-Palazon et al., 2018), ABA prompts de-stabilization of PYL4 through the ESCRT/vacuolarpathway, since vp23a and alix1 mutants accumulatemore PYL4 than Col-0 after ABA treatment (Yu et al.,2016; García-León et al., 2019). Recent studies in hu-mans reveal that CRL- and RBR-type E3 ligases canwork in unison to regulate substrate ubiquitination

(Kelsall et al., 2013; Scott et al., 2016). For instance, it hasbeen shown that the association of neddylated-CRLswiththe RBR-type E3 ARIH1 leads to monoubiquitination ofCRL client substrates by ARIH1 and a reciprocal role ofRBRs as regulators of distinct CRLs (Kelsall et al., 2013;Scott et al., 2016). As a result, during the study of humanE3 ligases, the concept of E3-E3 team tagging hasemerged, which implies that different types of E3s can actsuccessively on a common target (Scott et al., 2016). Thus,exquisite regulation of substrate ubiquitination can beachieved via team tagging. Future studies should addressthe possible cooperation of E3 ligases that target ABAreceptors in the same cell compartment to jointly regulatesubstrate ubiquitination.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) plants were grown as described by Pizzioet al. (2013). The rfa4 and rfa1 T-DNA insertion lines (SALK_208771C andSALK_005363, respectively) were obtained from the Nottingham ArabidopsisStock Centre (http://nasc.nott.ac.uk). To confirm and identify homozygousT-DNA individuals, seedlings of each insertion line were grown individuallyand DNA from each plant was extracted and submitted to PCR-mediatedgenotyping using the primers described in Supplemental Table S1. The rfa1rfa4 double mutant was generated by crossing and genotyping of F2 individ-uals. The pAlligator2-35S:HA-RFA1 and pAlligator2-35S:HA-RFA4 constructswere transferred to Agrobacterium tumefaciens C58C1 (pGV2260; Deblaere et al.,1985) by electroporation and used to transform Col-0 wild-type plants by thefloral dip method (Clough and Bent, 1998). T1 transgenic seeds were selectedbased on seed GFP fluorescence and sown in soil to obtain the T2 generation.Homozygous T3 progeny were used for further studies, and the expression ofHA-tagged protein was verified by immunoblot analysis using anti-HA-HRP.

Generation of CRISPR/Cas9 Mutants

The single guide RNA (sgRNA) targeting UBC26 (At1g53025) was designedusing the online tool CRISPR-PLANT (http://www.genome.arizona.edu/crispr/CRISPRsearch.html). A 19-bp sequence followed by the GGG PAM

Figure 8. A working model depicting the tar-geting of ABA receptors by RSL1, RFA1, orRFA4 at different subcellular locations. Ubiq-uitination of ABA receptors at the plasmamembrane by RSL1 triggers clathrin-mediatedendocytosis, transit through the trans-Golginetwork/early endosomes (TGN/EE), sortingof ubiquitinated cargo through the ESCRTpathway (involving FYVE1, VPS23A, and ALIXcomponents), delivery to multivesicular bod-ies/late endosomes (MVB/LE), and finally tothe vacuole for degradation. Ubiquitination ofreceptors at cytosol or nucleus leads to deg-radation by the 26S proteasome. The UBC26-RFA4-receptor complex regulates the half-lifeof ABA receptors in the nucleus. The cartoonrepresents the RING1-IBR-RING2 modularstructure of RBR-type E3 ligases and the lo-calization of the active site Cys-SH residue inRING2.




http://nasc.nott.ac.uk


http://www.genome.arizona.edu/crispr/CRISPRsearch.html

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sequence was selected. A modified pDONR207 vector (GenBank accession no.MG917725.1) was used to clone the sgRNA, which contains the AtU6-26 pro-moter followed by the sgRNA scaffold and the RNA Polymerase III terminator.We introduced the sgRNA sequence by site-directed, ligase-independentmutagenesis-PCR as described previously by Chiu et al. (2004). After verify-ing the cloning by sequencing, a fragment including the AtU6-26 promoter, thesgRNA, the scaffold RNA, and the terminator was amplified by PCR, withprimers containingHindIII and SpeI restriction sites (P1b-HindIII and P4b-SpeI).This fragment was cloned into the pHEE2E-TRI vector, which expresses Cas9driven by the egg cell-specific EC1.2 promoter (Wang et al., 2015). The resultingvector pHEE2E-TRI, containing Cas9 and the sgRNA, was transferred toA. tumefaciens C58C1 (pGV2260; Deblaere et al., 1985) by electroporation andused to transform Col-0 wild-type plants by the floral dip method (Clough andBent, 1998). Transformants were selected in medium supplemented with 20 mgmL21 hygromycin. To analyze the mutations caused by CRISPR/Cas9,genomic DNA was extracted from each transgenic plant using the cetyl-tri-methyl-ammonium bromide method. A 590-bp fragment containing theCRISPR target site was amplified using specific primers, cleaned with ExoSAP-IT (Affymetrix), and sequenced. T2 homozygous mutant plants were obtained,and their offspring were used in the indicated experiments.

Constructs

The OFP-TMD construct was obtained from Dr. Joerg Kudla (University ofMünster), and the pENTR221-RFA4 clone was obtained from the ABRC. Theopen reading frame (ORF) of RFA1was amplified by reverse transcription-PCRand cloned into pCR8/GW/TOPO. The pENTR221-RFA4 or pCR8-RFA1constructs were recombined by LR reaction into pAlligator2 to generate35S:HA-RFA4 or 35S:HA-RFA1 constructs, into pMDC43 to generate 35S:GFP-RFA4 or 35S:GFP-RFA1 constructs, or into pGADT7-GW to generate pGAD-RFA4 or pGAD-RFA1 constructs for Y2H assays. Additionally, we amplifiedthe ORF of RFA1 lacking the stop codon and cloned it into pCR8/GW/TOPO.This construct was recombined by LR reaction into pMDC83 to generate the35S:RFA1-GFP construct. The pENTR221-RFA4 and pCR8-RFA1 constructswere recombined by LR reaction into pYFPN43 for BIFC assays. The ORFs ofRFA4DC, lacking residues 385 to 468, and RFA4DNLS, lacking residues 1 to 14,were amplified using the primers indicated in Supplemental Table S1 andcloned into pCR8/GW/TOPO. We introduced the Cys-361Ala mutation intothe sequence of RFA4DCby site-directed, ligase-independentmutagenesis-PCRas described previously by Chiu et al. (2004).

Transient Protein Expression in Nicotiana benthamiana

A. tumefaciens infiltration of N. benthamiana leaves was performed basicallyas described by Saez et al. (2008). To investigate the interaction of RFA1 andRFA with ABA receptors in planta, we used the pYFPN43 and pYFPC43 vectorsfor BiFC assays (Belda-Palazón et al., 2012). To perform multicolor BiFC, wecloned PYR1/PYL4 into p(MAS)-SCYCE(R), RSL1 into pDEST-SCYNE(R), andRFA4 into pDEST-VYNE(R) vectors, as described by Gehl et al. (2009). In thecase of RFA4 interactions with UBC26 and ABA receptors, we cloned RFA4 intop(MAS)-SCYCE(R), UBC26 into pDEST-SCYNE(R), and ABA receptors intopDEST-VYNE(R) vectors. The different binary vectors were introduced into A.tumefaciens C58C1 (pGV2260) by electroporation, and transformed cells wereselected on Luria-Bertani plates supplemented with kanamycin (50 mg mL21).Then they were grown in liquid Luria-Bertani medium to late exponentialphase, and cells were harvested by centrifugation and resuspended in 10 mM

MES-KOH, pH 5.6, containing 10 mM MgCl2 and 150 mM acetosyringone to anOD600 of 1. These cells were mixed with an equal volume of A. tumefaciensC58C1 (pCH32 35S:p19) expressing the silencing suppressor p19 of Tomatobushy stunt virus so that the final density of A. tumefaciens solution was about1 (final concentration OD600 5 0.5, 0.33, or 0.25 each for bi, tri, or tetra infil-trations). Bacteria were incubated for 3 h at room temperature and then injectedinto young fully expanded leaves of 4-week-old N. benthamiana plants. Leaveswere examined 48 to 72 h after infiltration using confocal laser scanningmicroscopy.

Split-Luciferase Complementation Assay

The coding sequences of RFA1 and RFA4 lacking the stop codon wereamplifiedbyPCRandcloned intopCR8/GW.Then theywere recombinedbyLRreaction into pDEST-GWnLUC. For ABA receptors, coding sequences of PYR1,

PYL4, and PYL8 were recombined by LR reaction into pDEST-cLUCGW. Split-luciferase complementation assay was performed by transient expression inleaves of N. benthamiana by A. tumefaciens-mediated infiltration as describedabove. MG132 (50 mM) was applied into the infiltrated region 6 h before in-spection, which was performed 72 h after infiltration. To this end, leavescoexpressing different constructs were examined for luciferase activity by ap-plying 1 mM D-luciferin and placed in the dark for 5 min before imaging. Lu-ciferase complementationwas observedwith a CCD imaging system (LAS3000;Fujifilm) using 10-min exposures.

In Vivo and Semi-in Vivo Protein Degradation Assays

Protein degradation assays were performed as described by Liu et al. (2010)and Zhao et al. (2013) with small modifications. For in vivo protein degradationexperiments, A. tumefaciens cultures containing constructs that express the in-dicated E3 ligase, 3HA-PYR1 or 3HA-PYL4, the indicated GFP or RFP internalcontrol, and the silencing suppressor p19 were coinfiltrated at different ratios inN. benthamiana leaves. Three days after infiltration, samples were collected,ground in liquid nitrogen, and immediately placed in lysis buffer (50 mM

Tris-HCl, pH 8, 150 mM NaCl, 1% [v/v] Triton X-100, 1 mM phenyl-methylsulfonyl fluoride, and protease inhibitor cocktail) on ice for proteinextraction. Homogenates were cleared by centrifugation at 12,000g at 4°Cfor 15 min, and supernatants were used for protein immunoblot analysis.Samples were also collected for Actin and PYR1/PYL4 mRNA analyses toensure that equal amounts of PYR1/PYL4 transcripts were expressed indifferent coinfiltrations.

For semi-in vivo protein degradation experiments, we expressed RFA1-GFP,3HA-PYR1, and 3HA-PYL4 separately via different agroinfiltrations of N.benthamiana leaves. Samples were separately harvested, and proteins wereextracted in native extraction buffer (50 mM Tris-HCl, pH 8, 0.5 M Suc, 1 mM

MgCl2, 10 mM EDTA, 5 mM DTT, and protease inhibitor cocktail). Extracts wereclarified by centrifugation as mentioned above, and a final concentration of25 mM ATP was added to preserve the function of the 26S proteasome. Thenequal amounts of HA-PYR1 or 3HA-PYL4 extracts were mixed with N. ben-thamiana protein extracts lacking (mock) or containing RFA1-GFP, and theywere incubated at 4°C with rotation for 1 h in the presence or absence of 50 mM

MG132. Samples were mixed with 53 Laemmli buffer and subjected to im-munoblot analysis.

Protein Extraction, Analysis, Immunodetection,and Coimmunoprecipitation

Antibodies used in this work are listed in Supplemental Table S1. Proteinextracts for immunodetection experiments were prepared from Arabidopsistransgenic lines expressing HA-tagged RFA1 or RFA4. Material (;100 mg) fordirect western-blot analysis was extracted in 23 Laemmli buffer (125 mM Tris-HCl, pH 6.8, 4% [w/v] SDS, 20% [v/v] glycerol, 2% [v/v]mercaptoethanol, and0.001% [w/v] bromophenol blue), and proteins were run on a 10% SDS-PAGEgel and analyzed by immunoblotting. Proteins were transferred ontoImmobilon-P membranes (Millipore) and probed with anti-HA-peroxidase(Roche). Immunodetection of ubiquitylated proteins was performed using anti-Ub antibody (Ub P4D1:sc-8017; Santa Cruz Biotechnology). Antibodies wereused at a 1:1,000 dilution. Detection was performed using the ECL AdvanceWestern Blotting Chemiluminiscent Detection Kit (GE Healthcare). Imagecapture was done using the image analyzer LAS3000, and quantification of theprotein signal was done using Image Guache V4.0 software.

Coimmunoprecipitation experiments were performed by mixing proteinextracts in lysis buffer fromagroinfiltratedN. benthamiana leaves expressingGFPor GFP-PYL4 proteins with protein extracts from Arabidopsis transgenic linesexpressing HA-tagged RFA1 or RFA4. GFP or GFP-PYL4 proteins wereimmunoprecipitated using superparamagnetic micro-MACS beads coupled tomonoclonal anti-GFP antibody according to the manufacturer’s instructions (Mil-tenyi Biotec). Purified immunocomplexes were eluted in Laemmli buffer, boiled,and run on a 10% SDS-PAGE gel. Proteins immunoprecipitated with anti-GFP an-tibodywere transferred onto Immobilon-Pmembranes (Millipore) and probedwithanti-HA-peroxidase to detect coimmunoprecipitation of HA-tagged RFA1 or RFA4.

Pull-Down Assays

The construction, expression in bacteria, and purification of MBP fused toRFA1 or RFA4 lacking the C-terminal acidic domain was performed as


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described by Bueso et al. (2014). Purification of 6His-PYR1 was described bySantiago et al. (2009a). For pull-down assays, 5 mg of MBP-RFA1, MBP-RFA4DC, or MBP and 5 mg of either 6His-PYR1 or 6His-PYL4 were incubated1 h at 4°C with constant rocking in 0.5 mL of binding buffer (20 mM Tris-HCl,pH 7.5, 200mMNaCl, 1mM EDTA, and 0.5% [v/v] Tween 20). Afterward, MBPswere purified using amylose affinity chromatography, eluted, and analyzed bySDS-PAGE, followed by western blotting and immunodetection using anti-Histag monoclonal antibodies (Roche).

In Vitro Ub Assay

Enzymatic reactions were performed on the column by using immunopre-cipitated 3HA-PYR1 as a substrate, which was bound to superparamagneticmicro-MACS beads coupled to monoclonal anti-HA antibody. Immunopre-cipitation of 3HA-PYR1 was performed according to the manufacturer’s in-structions (Miltenyi Biotec) using Arabidopsis extracts of HA-tagged PYR1lines containing 600 mg of total protein in 50 mM Tris-HCl, pH 8, 0.5 M Suc, 1 mM

MgCl2, 10 mM EDTA, 5 mM DTT, 10 mM MG132, and protease inhibitor cocktail.The immobilized substrate was incubated at 30°C for 2 h in Ubi buffer (50 mM

Tris, pH 7.5, 10mMMgCl2, 0.2mMDTT, 50mMZnCl2, and 5mMATP) containing100 ng of human E1 (BostonBiochem), 250 ng of 6His-AtUBC8 (E2), 2 mg ofeither MBP-RFA1 or MBP-RFA4DC, and 1 mg of biotinylated Ub (Enzo LifeSciences). Reaction was stopped by washing the column with Ubi buffer fol-lowed by elution with Laemmli buffer. The eluted substrate was then subjectedto SDS-PAGE/blotting followed by detection using either St-HRP or anti-HA-HRP to detect either ubiquitinated or 3HA-tagged proteins, respectively.

In Vivo Ubiquitination Assay in N. benthamiana

A. tumefaciens cultures containing constructs that express GFP-PYL4, GFP-RFA4 (diluted 1:4 with respect to the other cultures), GFP-UBC26, and the si-lencing suppressor p19 were coinfiltrated in N. benthamiana leaves. Controlsamples lacked GFP-RFA4 and GFP-UBC26. Three days after infiltration,samples were collected, ground in liquid nitrogen, and immediately placed inlysis buffer supplementedwith 50mMMG132 and inhibitors of deubiquitinases:10 nM Ub aldehyde and 10 mM N-ethylmaleimide. GFP-tagged proteins wereimmunoprecipitated using superparamagnetic micro-MACS beads coupled tomonoclonal anti-GFP antibody according to the manufacturer’s instructions(Miltenyi Biotec). Purified immunocomplexes were eluted in Laemmli buffer,boiled, and run on a 10% SDS-PAGE gel. Proteins immunoprecipitated withanti-GFP antibody were transferred onto Immobilon-P membranes (Millipore)and probed with anti-GFP, anti-PYL4, and anti-Ub antibodies (SupplementalTable S1).

Y2H Assays

Protocols were similar to those described previously (Saez et al., 2008). Thescreening to identify cognate E2s of RFA4 was performed as described previ-ously (Cuéllar et al., 2013). In brief, the Saccharomyces cerevisiae PJ69-4A strainwas cotransformed with pGADT7 and pGBKT7 vectors containing the bait andprey, respectively. Three individually transformed cultures were grown over-night in liquid SD medium lacking Leu and Trp, and a 10-fold dilution of thesecultures was dropped on either control medium lacking Leu and Trp or selec-tive medium additionally lacking His. Empty vectors were used as negativecontrols, and yeast was allowed to grow for 3 d at 30°C before interaction wasscored.

Confocal Laser Scanning Microscopy

Confocal imaging was performed using a Zeiss LSM 780 AxioObserver.Z1laser scanning microscope with a C-Apochromat 403/1.20W corrective water-immersion objective. The following fluorophores, which were excited and flu-orescence emission detected by frame switching in the single or multitrackingmode at the indicated wavelengths, were used inN. benthamiana leaf infiltrationexperiments: SCFP (405 nm/450–485 nm), GFP (488 nm/500–530 nm), YFP orSCFPC1VENUSN (488 nm/529–550 nm), OFP (561 nm/575–600 nm), andchlorophyll (561 nm/685–760 nm). For BiFC experiments involving the detec-tion of two fluorescent proteins, sequential imaging of the fluorescent proteinswas performed using the sequential channel acquisition mode. Pinholes wereadjusted to 1 air unit for each wavelength. Postacquisition image processing

was performed using ZEN (Zeiss Efficient Navigation) Lite 2012 imagingsoftware and ImageJ (http://rsb.info.gov/ij/).

Seed Germination and Seedling Establishment Assays

After surface sterilization of the seeds, stratification was conducted in thedark at 4°C for 3 d.Approximately 100 seeds of each genotypewere sown onMSplates supplemented with different ABA concentrations per experiment. Toscore seed germination, radicle emergence was analyzed at 72 h after sowing.Seedling establishment was scored as the percentage of seeds that developedgreen expanded cotyledons and the first pair of true leaves at 5 or 7 d.

Root Growth Assay

Seedlingswere grownonvertically orientedMSplates for 4 to 5d.Afterward,20 plantswere transferred to newMSplates lackingor supplementedwith 10mM

ABA. The plates were scanned on a flatbed scanner after 10 d to produce imagefiles suitable for quantitative analysis of root growth using the NIH Imagesoftware ImageJ.

Water-Loss Experiments

For water-loss analysis, plants were grown under greenhouse conditions(40% to 50% room humidity) and standard watering for 21 d. At least 10 similarleaves per individual genotype (n 5 5) were excised and submitted to thedrying atmosphere of a laminar flow hood. Gravimetric analysis of water losswas performed and represented as the percentage of fresh weight loss at eachscored time point.

Infrared Thermography

Plants were grown in a controlled-environment growth chamber at 22°Cunder a 12-h-light/12-h-dark photoperiod at 100 mE m22 s21 and 40% to 50%room humidity. Philips bulbs were used (TL-D Super 8036W, white light 840,4000K light code). Infrared thermography images of rosette leaves were ac-quired from 6-week-old plants with a thermal camera FLIR E95 equipped witha 42° lens. Images were processed and quantified with the FLIR tools software.For quantification, the average temperature of 15 different sections corre-sponding to four leaves per plant was calculated. Five plants per genotype wereanalyzed in each experiment. The mean temperature 6 SD of all the plants foreach genotype was reported. Statistical comparisons among genotypes wereperformed by pairwise Student’s t tests.

Statistics and Colocalization Analyses

Student’s t test was performed for single comparisons. Values are averagesobtained from three independent experiments6 SD. Fluorescence colocalizationanalysis was performed using the PSC colocalization plug-in of ImageJ (Frenchet al., 2008). Pearson’s and Spearman’s correlation coefficients in the range 0.4 to1 indicate colocalization, whereas lower values indicate lack of colocalization.

Accession Numbers

The Arabidopsis Genome Initiative locus identifiers are as follows: RSL1,At2G26130; RFA1, At3G43750; RFA2, At3G45570; RFA3, At3G45580; RFA4,At2G21420; RFA5, At5G37560; RFA6, At3G45480; RFA7, At3G45460; RFA8,At3G45540; RFA9, At2g26135; UBC26, At1g53020; and UBC24, At2G33770.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Expression data of RFA genes compared withPYR/PYL genes.

Supplemental Figure S2. Amino acid sequence alignment of RSL1(At2G26130), RFA1 (At3G43750), and RFA4 (At2G21420).

Supplemental Figure S3. Nuclear interaction of RFA4DC and ABAreceptors.






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Supplemental Figure S4. Simultaneous formation of receptor-RSL1 andreceptor-RFA4 complexes in different subcellular locations revealed bymulticolor BiFC assays.

Supplemental Figure S5. Analysis of Arabidopsis lines overexpressingdifferent RSL1/RFA genes.

Supplemental Figure S6. Identification of Cys-361 as the putative activesite of RFA4.

Supplemental Figure S7. Y2H assay between RFA4 or RFA4DC and apanel of 30 Arabidopsis E2 Ub-conjugating enzymes.

Supplemental Figure S8. Amino acid sequence alignment of UBC23,UBC24, UBC25, and UBC26 group XI UBCs.

Supplemental Figure S9. Y2H assay between RFA1 or RFA4DC and sev-eral ABA receptors.

Supplemental Figure S10. Generation and phenotype of CRISPR/CAS9-mediated knockout alleles of UBC26.

Supplemental Figure S11. The rfa1 and rfa4 single mutants show wild-typesensitivity to ABA.

Supplemental Figure S12. Specificity of a-PYR1 and a-PYL4 antibodies.

Supplemental Table S1. List of primers and antibodies used in this work.

ACKNOWLEDGMENTS

We thank the ABRC and Nottingham Arabidopsis Stock Centre for provid-ing DNA constructs and seeds. pHEE2E-TRI was a gift from Qi-Jun Chen(https://www.addgene.org/, plasmid #71288). We thank Marc Nishimura forsharing the unpublished vector GenBank accession number MG917725.

Received July 19, 2019; accepted October 21, 2019; published November 7, 2019.

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