The E3 Ligase DROUGHT HYPERSENSITIVE Negatively Regulates ... · Cuticular wax plays crucial roles...

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The E3 Ligase DROUGHT HYPERSENSITIVE Negatively Regulates Cuticular Wax Biosynthesis by Promoting the Degradation of Transcription Factor ROC4 in Rice Zhenyu Wang, a Xiaojie Tian, a,b Qingzhen Zhao, c Zhiqi Liu, d Xiufeng Li, a Yuekun Ren, a,b Jiaqi Tang, a,b Jun Fang, a Qijiang Xu, d and Qingyun Bu a,1 a Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin 150081, China b Graduate University of Chinese Academy of Sciences, Beijing 100049, China c School of Life Sciences, Liaocheng University, Liaocheng 252000, China d School of Life Sciences, Northeast Forestry University, Harbin 150040, China ORCID IDs: 0000-0001-9040-5156 (X.T.); 0000-0003-3107-1525 (Z.L.); 0000-0002-5386-3577 (Q.B.) Cuticular wax plays crucial roles in protecting plants from environmental stresses, particularly drought stress. Many enzyme- encoding genes and transcription factors involved in wax biosynthesis have been identi fied, but the underlying posttranslational regulatory mechanisms are poorly understood. Here, we demonstrate that DROUGHT HYPERSENSITIVE (DHS), encoding a Really Interesting New Gene (RING)-type protein, is a critical regulator of wax biosynthesis in rice (Oryza sativa). The cuticular wax contents were significantly reduced in DHS overexpression plants but increased in dhs mutants compared with the wild type, which resulted in a response opposite that of drought stress. DHS exhibited E3 ubiquitin ligase activity and interacted with the homeodomain-leucine zipper IV protein ROC4. Analysis of ROC4 overexpression plants and roc4 mutants indicated that ROC4 positively regulates cuticular wax biosynthesis and the drought stress response. ROC4 is ubiquitinated in vivo and subjected to ubiquitin/26S proteasome-mediated degradation. ROC4 degradation was promoted by DHS but delayed in dhs mutants. ROC4 acts downstream of DHS, and Os-BDG is a direct downstream target of the DHS-ROC4 cascade. These results suggest a mechanism whereby DHS negatively regulates wax biosynthesis by promoting the degradation of ROC4, and they suggest that DHS and ROC4 are valuable targets for the engineering of drought-tolerant rice cultivars. INTRODUCTION Rice (Oryza sativa) is a staple food for more than half of the global population. However, its production is threatened by drought stress, and water resources have become one of the major limiting factors for rice production due to increased industrialization and water pollution (Kumar et al., 2014). Fortunately, rice has evolved various strategies to cope with drought stress. Among these strategies, cuticular wax provides an essential barrier for de- creasing nonstomatal water loss during drought stress and en- hancing cuticular wax contents, thereby markedly increasing drought tolerance in rice (Wang et al., 2012; Zhu and Xiong, 2013). Cuticular wax is mainly composed of very-long-chain fatty acids (VLCFAs) and their derivatives (including aldehydes, alcohols, alkanes, ketones, and wax esters) with chain lengths ranging from C20 to C34 (Haslam and Kunst, 2013). Over the past few decades, increasing numbers of genes controlling cuticular wax bio- synthesis have been identified via the characterization of ecer- iferum (cer) mutants in Arabidopsis thaliana and reverse genetics approaches (McNevin et al., 1993; Greer et al., 2007), and the associated biosynthetic processes have been uncovered (Yeats and Rose, 2013; Borisjuk et al., 2014). Briefly, wax biosynthesis begins with a de novo C16 or C18 fatty acid, which is converted to C16 or C18 acyl-CoA by a long-chain acyl-CoA synthase and is then used as a substrate for the fatty acid elongase complex, including b-ketoacyl-CoA synthase, b-ketoacyl-CoA reductase, b-hydroxy acyl-CoA dehydratase, and enoyl-CoA reductase. Through a series of enzyme catalysis steps, two carbons per cycle are successively added to produce VLCFAs-CoA. Finally, the resulting VLCFAs-CoAs are further modified to yield various de- rivatives, such as primary alcohols, aldehydes, and alkanes (Yeats and Rose, 2013). Several wax biosynthesis genes have been identified in rice through characterizing wax crystal-sparse leaf mutants. Mutations in these genes result in markedly reduced cuticular wax contents and drought-sensitive phenotypes (J.Y. Zhang et al., 2005; Yu et al., 2008; Mao et al., 2012; Wang et al., 2012; Zhu and Xiong, 2013; Gan et al., 2016; Wang et al., 2017). Some transcription factors have also been shown to control wax biosynthesis by regulating the expression of downstream wax biosynthesis genes (Borisjuk et al., 2014). For example, APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/EFR) family members, including WAX INDUCER1 (WIN1)/SHINE1 in Arabi- dopsis, rice wax biosynthesis regulatory proteins Os-WR1 to Os-WR4, and Medicago truncatula WAX PRODUCTION1 positively regulate wax biosynthesis by directly promoting the expression of wax and cutin biosynthesis genes (Broun et al., 2004; J.Y. Zhang et al., 2005; Wang et al., 2012; Zhou et al., 2014). In contrast, another AP2 protein in Arabidopsis, DECREASE WAX BIOSYNTHESIS, 1 Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Qingyun Bu (buqingyun@ iga.ac.cn). www.plantcell.org/cgi/doi/10.1105/tpc.17.00823 The Plant Cell, Vol. 30: 228–244, January 2018, www.plantcell.org ã 2018 ASPB.

Transcript of The E3 Ligase DROUGHT HYPERSENSITIVE Negatively Regulates ... · Cuticular wax plays crucial roles...

  • The E3 Ligase DROUGHT HYPERSENSITIVE NegativelyRegulates Cuticular Wax Biosynthesis by Promoting theDegradation of Transcription Factor ROC4 in Rice

    Zhenyu Wang,a Xiaojie Tian,a,b Qingzhen Zhao,c Zhiqi Liu,d Xiufeng Li,a Yuekun Ren,a,b Jiaqi Tang,a,b Jun Fang,a

    Qijiang Xu,d and Qingyun Bua,1

    a Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy ofSciences, Harbin 150081, ChinabGraduate University of Chinese Academy of Sciences, Beijing 100049, Chinac School of Life Sciences, Liaocheng University, Liaocheng 252000, Chinad School of Life Sciences, Northeast Forestry University, Harbin 150040, China

    ORCID IDs: 0000-0001-9040-5156 (X.T.); 0000-0003-3107-1525 (Z.L.); 0000-0002-5386-3577 (Q.B.)

    Cuticular wax plays crucial roles in protecting plants from environmental stresses, particularly drought stress. Many enzyme-encoding genes and transcription factors involved in wax biosynthesis have been identified, but the underlyingposttranslational regulatory mechanisms are poorly understood. Here, we demonstrate that DROUGHT HYPERSENSITIVE(DHS), encoding a Really Interesting New Gene (RING)-type protein, is a critical regulator of wax biosynthesis in rice (Oryzasativa). The cuticular wax contents were significantly reduced in DHS overexpression plants but increased in dhs mutantscompared with the wild type, which resulted in a response opposite that of drought stress. DHS exhibited E3 ubiquitin ligaseactivity and interacted with the homeodomain-leucine zipper IV protein ROC4. Analysis of ROC4 overexpression plants androc4 mutants indicated that ROC4 positively regulates cuticular wax biosynthesis and the drought stress response. ROC4 isubiquitinated in vivo and subjected to ubiquitin/26S proteasome-mediated degradation. ROC4 degradation was promoted byDHS but delayed in dhs mutants. ROC4 acts downstream of DHS, and Os-BDG is a direct downstream target of the DHS-ROC4cascade. These results suggest a mechanism whereby DHS negatively regulates wax biosynthesis by promoting the degradationof ROC4, and they suggest that DHS and ROC4 are valuable targets for the engineering of drought-tolerant rice cultivars.

    INTRODUCTION

    Rice (Oryza sativa) is a staple food for more than half of the globalpopulation. However, its production is threatened by droughtstress, andwater resourceshavebecomeoneof themajor limitingfactors for rice production due to increased industrialization andwater pollution (Kumar et al., 2014). Fortunately, rice has evolvedvarious strategies to cope with drought stress. Among thesestrategies, cuticular wax provides an essential barrier for de-creasing nonstomatal water loss during drought stress and en-hancing cuticular wax contents, thereby markedly increasingdrought tolerance in rice (Wang et al., 2012; Zhu and Xiong, 2013).

    Cuticularwax ismainlycomposedof very-long-chain fattyacids(VLCFAs) and their derivatives (including aldehydes, alcohols,alkanes, ketones, andwax esters) with chain lengths ranging fromC20 toC34 (HaslamandKunst, 2013). Over the past fewdecades,increasing numbers of genes controlling cuticular wax bio-synthesis have been identified via the characterization of ecer-iferum (cer) mutants in Arabidopsis thaliana and reverse geneticsapproaches (McNevin et al., 1993; Greer et al., 2007), and theassociated biosynthetic processes have been uncovered (Yeats

    and Rose, 2013; Borisjuk et al., 2014). Briefly, wax biosynthesisbeginswith a de novoC16 or C18 fatty acid, which is converted toC16 or C18 acyl-CoA by a long-chain acyl-CoA synthase and isthen used as a substrate for the fatty acid elongase complex,including b-ketoacyl-CoA synthase, b-ketoacyl-CoA reductase,b-hydroxy acyl-CoA dehydratase, and enoyl-CoA reductase.Througha series of enzymecatalysis steps, twocarbonsper cycleare successively added to produce VLCFAs-CoA. Finally, theresulting VLCFAs-CoAs are further modified to yield various de-rivatives, suchasprimary alcohols, aldehydes, andalkanes (Yeatsand Rose, 2013). Several wax biosynthesis genes have beenidentified in rice through characterizing wax crystal-sparse leafmutants. Mutations in these genes result in markedly reducedcuticular wax contents and drought-sensitive phenotypes (J.Y.Zhang et al., 2005; Yu et al., 2008; Mao et al., 2012; Wang et al.,2012; Zhu and Xiong, 2013; Gan et al., 2016; Wang et al., 2017).Some transcription factors have also been shown to control

    wax biosynthesis by regulating the expression of downstreamwax biosynthesis genes (Borisjuk et al., 2014). For example,APETALA2/ETHYLENE-RESPONSIVEFACTOR (AP2/EFR) familymembers, including WAX INDUCER1 (WIN1)/SHINE1 in Arabi-dopsis, rice wax biosynthesis regulatory proteins Os-WR1 toOs-WR4, andMedicago truncatulaWAXPRODUCTION1positivelyregulate wax biosynthesis by directly promoting the expression ofwax and cutin biosynthesis genes (Broun et al., 2004; J.Y. Zhanget al., 2005;Wanget al., 2012;Zhouetal., 2014). In contrast, anotherAP2 protein in Arabidopsis, DECREASE WAX BIOSYNTHESIS,

    1 Address correspondence to [email protected] 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: Qingyun Bu ([email protected]).www.plantcell.org/cgi/doi/10.1105/tpc.17.00823

    The Plant Cell, Vol. 30: 228–244, January 2018, www.plantcell.org ã 2018 ASPB.

    http://orcid.org/0000-0001-9040-5156http://orcid.org/0000-0001-9040-5156http://orcid.org/0000-0001-9040-5156http://orcid.org/0000-0001-9040-5156http://orcid.org/0000-0001-9040-5156http://orcid.org/0000-0003-3107-1525http://orcid.org/0000-0003-3107-1525http://orcid.org/0000-0003-3107-1525http://orcid.org/0000-0002-5386-3577http://orcid.org/0000-0002-5386-3577http://orcid.org/0000-0002-5386-3577http://orcid.org/0000-0002-5386-3577http://orcid.org/0000-0001-9040-5156http://orcid.org/0000-0003-3107-1525http://orcid.org/0000-0002-5386-3577http://crossmark.crossref.org/dialog/?doi=10.1105/tpc.17.00823&domain=pdf&date_stamp=2018-01-26mailto:[email protected]://www.plantcell.orgmailto:[email protected]:[email protected]://www.plantcell.org/cgi/doi/10.1105/tpc.17.00823http://www.plantcell.org

  • functions as a transcriptional repressor of wax biosynthesis andnegatively regulateswax loads (Go et al., 2014). SeveralMYB familyproteins are also involved in cuticular wax biosynthesis and de-position via different mechanisms. MYB16 and MYB106 regulatecutinbiosynthesis inasimilarmanner toWIN1,whereasMYB30andMYB96 mediate pathogen and drought-induced wax biosynthesis(Raffaele et al., 2008; Seo et al., 2011; Oshima et al., 2013).

    Another important cuticularwax regulator is the homeodomain-leucine zipper IV (HD-ZIP IV) family of transcription factors, whichcontain a conserved homeodomain (HD) associated with a Leuzipper domain (ZIP), a steroidogenic acute regulatory-related lipidtransfer domain (START), and an HD-START-associated domain(Nakamura et al., 2006; Ariel et al., 2007). Arabidopsis HDG1,tomato (Solanum lycopersicum) CUTIN DEFICIENT2 (CD2), andmaize (Zea mays) OUTER CELL LAYER1 (OCL1) are highly ho-mologous HD-ZIP IVmembers. These proteins regulate cutin andwaxbiosynthesisbydirectingbinding to theconservedL1boxcis-element in the promoters of downstream target genes, includingwax biosynthesis genes BODYGUARD (BDG) and FIDDLEHEAD,LIPID TRANSPORTER (LTP), and ATP BINDING CASSETTEtransporters involved in the transport ofwax (Isaacsonet al., 2009;Javelle et al., 2010; Wu et al., 2011). However, the mechanismby which HD-ZIP IV proteins are involved in cuticular wax bio-synthesis is not clear. In addition, the posttranslational regulationof the HD-ZIP IV protein remains unknown.

    The ubiquitin/26S proteasome (UPS) pathway degradesubiquitinated substrate proteins and is extensively involved invarious cellular processes. This pathway plays key roles in diverseaspects of plant growth anddevelopment (Vierstra, 2009; SantnerandEstelle, 2010). The ubiquitination process is achieved throughthe sequential action of ubiquitin-activating enzymeE1, ubiquitin-conjugating enzymeE2, and ubiquitin ligase E3. Really InterestingNew Gene (RING) finger proteins, featuring eight conserved CysandHis residues, areone themost important typesofE3 ligases.Awealth of studies have shown that RING finger proteins play keyroles in plant hormone signaling pathways, defense responses,and development processes (Bu et al., 2009; Ryu et al., 2010;W. Li et al., 2011; Liu and Stone, 2011; Park et al., 2012; Kim andKim, 2013; Zhang et al., 2015). Arabidopsis CER9, encodinga RING-variant domain-containing protein, negatively regulateswax loads,although theE3 ligaseactivityandmechanismhavenotyet been determined (Lü et al., 2012). However, the roles offunctionalRING-typeE3 ligasesother thanCER9 in regulatingwaxbiosynthesis are currently unclear.

    Here, we report that DHS (DROUGHT HYPERSENSITIVE),a RING-type E3 ligase, regulates rice wax biosynthesis by con-trolling the protein stability of ROC4 (anHD-ZIP IV familymember)via the UPS and consequently influences the drought stress re-sponse. The overexpression ofDHS resulted inmarkedly reducedwax loads and strikingly drought-hypersensitive phenotypes,whereas dhs mutants showed increased wax contents and en-hanced drought tolerance. In addition, we found that DHS pos-sesses E3 ligase activity, interacts with ROC4, and promotes thedegradation of ROC4. Moreover, analysis of ROC4 over-expression plants and roc4 mutants indicated that ROC4 posi-tively regulates the wax biosynthesis and drought stressresponse.More importantly,wediscovered thatROC4geneticallyacts downstream of DHS. Furthermore, Os-BDG, which might

    control wax biosynthesis, was identified as the direct target of theDHS-ROC4 cascade. Collectively, these findings demonstratethat the E3 ligase DHS cooperates with its putative ubiquitinationsubstrate, ROC4, to fine-tune wax biosynthesis and the droughtstress response in rice, thereby providing valuable targets forbreeding drought-tolerant rice cultivars.

    RESULTS

    Overexpression of DHS in Rice ConfersDrought Hypersensitivity

    To identify the critical genes involved in the drought stressresponse in rice, we screened a rice mutant library in whichvarious transcription factor genes were overexpressed. Onemutant carrying LOC_Os02g45780 exhibited a strikingly drought-hypersensitive phenotype and thus was selected for furtheranalysis. LOC_Os02g45780 was thus named DHS (DROUGHTHYPERSENSITIVE ).DHS overexpression (DHSOE) plantlets grewmore slowly than

    thewild type (transformedwith empty vector). After 30 d growth inregeneration culture medium, DHS OE was significantly smallerthan the wild type, exhibiting a dwarfed plant height and shorterleaves (Supplemental Figures 1A to 1C). The leaves of DHS OEwere lightergreen than thewild type (SupplementalFigure2A).Themost obvious phenotype of DHS OE was that its leaves wiltedrapidly (Figures 1A and 1B). Once DHS OE seedlings were re-moved from culture bottles and transplanted into the soil, theleaves of DHS began to roll within an hour, the leaf tips turnedyellow in around 3 d, and the seedlings stopped growing and diedgradually within 1 month (Figures 1A and 1B), which was incontrast to the normal growth of the wild type. To confirm thestrikingly drought-hypersensitive phenotype of DHS OE, we an-alyzed thewater loss ratesofdetached leavesanddiscovered thatDHS OE lost water much more rapidly than the wild type (Figure1C). As most of the independent DHSOE plants displayed similarphenotypes, and the transcript level of LOC_Os02g45780 wasindeed markedly increased in DHS OE plants (SupplementalFigure 1D), we hypothesized that the overexpression of DHSconfers drought hypersensitivity in rice.Plants lose water mainly via their stomata (Schroeder et al.,

    2001; Nilson and Assmann, 2007). However, a preliminary ex-amination of the stomatal density showed that the average sto-mataldensitywascomparablebetween thewild typeandDHSOE,which implied that stomatal water lossmight be normal inDHSOE(Supplemental Figure 1E).Cuticular wax is the outermost membrane that protects plants

    from water loss, and the content and structure of the waxes areclosely associated with water loss rate (Lü et al., 2012; Zhu andXiong, 2013). The light-green color of DHS OE is also reminiscentof some rice cuticular wax-deficient mutants (SupplementalFigure 2A) (Yu et al., 2008; Mao et al., 2012; Wang et al., 2017). Toexamine whether the cuticular wax in DHS OE was defective, weperformed several assays. First, we soaked the rice seedlings inwater and then removed them.Comparedwith thewild type, therewere more water drops on the leaves of DHSOE, suggesting thatthere may be less cuticular wax in DHS OE than in the wild type

    DHS Regulates Cuticular Wax and Drought Response 229

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  • (Supplemental Figure 2A). Second, measurement of the mem-brane permeability demonstrated that the chlorophyll-leachingrate inDHSOEwas faster than that in thewild type (SupplementalFigure 2B). Third, scanning electronmicroscopy analysis revealedthat the platelet-like wax crystals on the leaf surface of DHS OEwere sparser than those on the wild type (Figures 1D and 1E).Fourth, ultrastructural analysis by transmission electron micro-copy (TEM) showed that the cuticle membranes in wild-typeleaves were smooth and contracted, in contrast to the loose, ir-regular, and vague membranes in DHS OE leaves (SupplementalFigures 2C and 2D). Fifth, the compositions and contents ofcuticular waxes were analyzed by gas chromatography-mass

    spectrometry (GC-MS).Comparedwith thewild type, the totalwaxloads inDHSOEwere severely reduced (Figure 1F; SupplementalFigure 2E). Together, these data suggest that the overexpressionof DHS disrupts the biosynthesis and development of cuticularwax, which results in drought hypersensitivity in DHS OE.To verify this notion, we generated dhs mutants via CRISPR/

    Cas9-mediated genome editing and characterized three in-dependentmutant alleles,dhs-1,dhs-2, anddhs-3 (SupplementalFigure 3). Unlike DHS OE plants, the growth and development ofthe dhsmutantswere similar to thewild type, and they showed novisible phenotypes (Figure 2B). However, data from the GC-MSanalysis indicated that the cuticular wax contents in dhs were

    Figure 1. Overexpression of DHS Leads to Drought Hypersensitive Phenotypes and Disrupts Cuticular Wax Structure and Composition.

    (A) and (B) Phenotypes of three independentDHSOE lines and the wild type at 5min (A) and 60min (B) after the seedlings were transplanted into soil fromculture bottles.(C) DHS OE plants lose water more rapidly than the wild type. Leaves of DHS OE and the wild type at the same developmental stages were excised andweighedat various timepointsafter detachment.Water loss is representedas thepercentageof initial freshweight at each timepoint. Valuesaremeans6 SEof three individual plants per genotype.(D) and (E) Scanning electronmicroscopy images of cuticular wax crystal patterns on the surfaces of leaf blades in the wild type (D) andDHSOE (E). Bar =1 mm.(F) Cuticular wax composition on the leaf surfaces of wild-type and DHS OE plants analyzed by GC-MS. Wax constituents are grouped by carbon chainlength and chemical class. Data are means 6 SE of three biological replicates using independent seedling samples grown under the same condition.Asterisks denote significant differences from the wild type (*P < 0.05 and **P < 0.01) determined by Student’s t test.

    230 The Plant Cell

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  • increased compared with the wild type (Figure 2A; SupplementalFigure4D). Inaddition, tosomeextent, thecuticularwaxcrystals indhsweredenser than those in thewild type (Supplemental Figures4A and 4B). In agreement with these results, the chlorophyll-leaching rate of dhs was slower than that of the wild type(Supplemental Figure 4C). Together, these data suggest that themutation of DHS leads to increased cuticular wax biosynthesis.Compared with the wild type, the drought tolerance of dhs wasconsistently significantly enhanced (Figures 2B to 2D), alongwitha higher recovery rate following dehydration treatment (Figure2E) and a slower water loss rate (Figure 2F), which furthersupports the notion that DHS plays negative roles in controllingwax biosynthesis and consequently affects the drought stressresponse.

    DHS Has E3 Ubiquitin Ligase Activity

    DHS contains 165 amino acid residues as well as a predictedtransmembrane segment in theN terminus and a conservedRINGdomain in the C terminus of the protein (Supplemental Figure 5).RING-type proteins always function as E3 ubiquitin ligases (Stoneet al., 2005; Bu et al., 2009; H. Li et al., 2011). To examine whetherDHS has E3 ligase activity, DHS fused with the maltose bindingprotein (MBP) was expressed and used for an in vitro autoubi-quitination assay. In the presence of E1, E2, and the His-ubiquitinprotein, the MBP-DHS protein, similar to the MBP-tagged ubiq-uitin ligase positive control MBP-CIP8 (Hardtke et al., 2002),showedclear autoubiquitination, suggesting that DHSexhibits E3ligase activity in vitro (Figure 3A). In contrast, when E1 or E2 wereomitted, we did not detect any ubiquitination (Figure 3A). In ad-dition, an ubiquitination assay inEscherichia coli also showed thatDHS has E3 ligase activity (Supplemental Figure 6). ConservedCys and His residues in the RING domain are critical for E3 ligaseactivity (Buetal., 2009;H.Li etal., 2011). Thus, themutatedproteinDHSC95S (Cys-95 in theRINGdomainwasmutated toSer-95) wasexpressed and its activity was examined (Supplemental Figure7A). We did not detect any ubiquitination in DHSC95S (Figure 3A),suggesting that the conserved RING domain is indispensable fortheE3 ligase activity ofDHS. Furthermore,wegeneratedDHSC95S

    overexpression plants (DHSC95S OE). Unlike DHS-OE, DHSC95S

    OEwas similar to thewild type in terms of growth speed and plantarchitecture (Supplemental Figure 7B). In addition, the cuticularwax contents and drought stress response of DHSC95S OE werealso comparable with the wild type (Supplemental Figures 7C to7F). Taken together, these results indicate thatDHS is anactiveE3ligase and that its E3 ligase activity is necessary for its biologicalfunction.

    DHS Physically Interacts with ROC4

    In general, RING-type E3 ligases function by ubiquitinating targetproteins and triggering their degradation via the 26S proteasome(X. Zhang et al., 2005; Dong et al., 2006; Qin et al., 2008). To revealthe role of DHS in the regulation of wax biosynthesis, we at-tempted to identify the ubiquitination target protein of DHS. Asdescribed previously, DHS functions as a negative regulator ofwax biosynthesis (Figures 1 and 2), and we speculated that theubiquitinated target of DHS might be a positive regulator of the

    cuticular wax pathway. Several transcription factors thus far havebeen shown toplaypositive roles in regulatingwaxbiosynthesis inArabidopsis, rice, tomato, and maize (Zea mays), including theHD-ZIP IV family (HDG1, OCL1, andCD2), AP2/EFR family (WIN1,Os-WR1, and Os-WR2), and MYB family (MYB16, MYB106,MYB30, and MYB96; Broun et al., 2004; Raffaele et al., 2008;Javelle et al., 2010; Seo et al., 2011; Wang et al., 2012). To pre-liminarily screen for the possible interaction partner of DHS, weused the protein sequences of the abovementioned transcriptionfactors as queries for BLAST analysis against the rice proteindatabase, and we chose the corresponding rice homologs forfurther analysis (Supplemental Figure 8). First, we performeda yeast two-hybrid assay to examine the possible interactionbetween DHS and the homologs in rice. We discovered that theHD-ZIP IV family member ROC4 interacted with DHS (Figure 3B),whereas the homologs of the AP2/EFR and MYB familymembers did not (Supplemental Figure 8). Second, we con-firmed the physical interaction between DHS and ROC4 usingan in vitro pull-down assay (Figure 3C). Third, an in plantaluciferase complementation imaging assay also showed thatthe coexpression of DHS with ROC4 generated strong lumines-cence signals that were not detected in the control pairs (Figure3D). Collectively, these results suggest that DHS physically in-teracts with ROC4.

    ROC4 Positively Regulates Wax Development

    ROC4, containing 813 amino acid residues, contains a typicalhomeobox domain and a SMART domain and belongs to theHD-ZIP IV family (Supplemental Figure 9). As the homologs ofROC4 in Arabidopsis, maize, and tomato have been shown to beinvolved in cuticular wax development (Isaacson et al., 2009;Javelle et al., 2010; Wu et al., 2011), we investigated whetherROC4 regulates wax biosynthesis in rice. For this purpose, wegenerated roc4mutants (Supplemental Figure 10) and ROC4 OEplants (overexpressing GFP-fused ROC4) (Supplemental Figures11A and 11B). GC-MS analysis showed that there were morecuticular waxes in ROC4 OE plants, but fewer in the roc4 mutantcompared with the wild type (Figure 4A; Supplemental Figure11G). In addition, scanning electron microscopy analysis showedthat the wax crystals were dense in ROC4 OE, but they wereobviously sparser in the roc4mutant compared with the wild type(Supplemental Figures 11C to 11E). Accordingly, the chlorophyll-leaching rate was slower in ROC4 OE but faster in roc4(Supplemental Figure11F). Inagreementwith thesefinding,ROC4OE exhibited drought tolerance, whereas roc4 was droughtsensitive compared with the wild type (Figures 4B to 4E), and thisresult was further supported by the data from thewater loss assay(Figure 4F). Thesedata thusstrongly suggest thatROC4positivelyregulateswax biosynthesis and the corresponding drought stressresponse.

    ROC4 Is Subjected to UPS-Dependent Degradation

    As described earlier, DHS and ROC4 physically interact with eachother and play opposite roles in the control of wax biosynthesis. Inaddition, DHS exhibits E3 ligase activity. These findings suggestthat ROC4 might be a ubiquitination target of DHS. If this is true,

    DHS Regulates Cuticular Wax and Drought Response 231

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  • ROC4 protein might be unstable and modified by ubiquitination.To test this hypothesis, we examined the protein stability ofROC4 in a cell-free degradation assay,which indicated that ROC4protein was unstable and degraded rapidly (Figures 5A and 5B). Inaddition, we treated ROC4 OE callus with the protein synthesis

    inhibitor cycloheximide (CHX) or the proteasome inhibitor MG132for 4 h and assessed the level of ROC4 protein by immunoblotanalysis. The level of ROC4 protein decreased markedly whentreated with CHX, whereas the addition of MG132 efficientlyblocked ROC4 degradation (Figures 5C and 5D). To confirm this

    Figure 2. Characterization of the Wax Composition and Drought Tolerance Phenotypes of dhs Mutants.

    (A) Cuticular wax composition on the leaf surfaces of the wild type and dhsmutants analyzed by GC-MS. Wax constituents are grouped by carbon chainlength and chemical class. Data are means6 SE of three biological replicates using independent seedling samples grown at the same condition. Asterisksdenote significant differences from the wild type (*P < 0.05 and **P < 0.01) determined by Student’s t test.(B) to (D)Phenotypesofdhsandthewild typeundernormalconditions (B),withholdingwater for3d (C), andrehydration for3dafterwithholdingwater for7d (D).(E)Recovery rate ofdhsand thewild type in (D). Data aremeans6 SEof threebiological replicateswith30seedlingsused for each replicate. Asterisksdenotesignificant differences from the wild type (**P < 0.01) determined by Student’s t test.(F)Water loss rate in detached leaves from dhs and thewild type. Leaves at the same developmental stageswere excised andweighed at various time points afterdetachment.Thewater lossrate is representedasthepercentageof initial freshweightateachtimepoint.Valuesaremeans6SEof three individualplantspergenotype.

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  • result, we used 2-week-old ROC4 OE seedlings to observe theGFP fluorescence. Confocal microscopy observation showedthatROC4was localized to thenucleus, and theGFPfluorescencewas enhanced by MG132 treatment but reduced by CHX treat-ment (Figure 5E), indicating that the degradation of ROC4 occursvia the UPS. Additionally, ROC4 protein was immunoprecipitatedfrom ROC4 OE callus using an anti-ROC4 antibody and wasprobed with antiubiquitin and anti-ROC4. The higher molecularweight smear bands in the protein gel blot indicated that ROC4is indeed polyubiquitinated in vivo (Figure 5F). Together, thesedata suggest that ROC4 is subjected to proteasome-mediateddegradation.

    DHS Promotes UPS-Dependent Degradation of ROC4

    We further investigated whether DHS promotes UPS-mediateddegradation of ROC4. First, we examined the degradation speedof ROC4 in a cell-free degradation assay. For this experiment, weused calli from wild-type and DHS OE plants at the same growthstage. Crude ROC4 protein extracted from ROC4 OE callus wasdivided into several aliquots, and each aliquot was mixed withequal amountsofwild-typeandDHSOEproteinextract. Followingincubationat roomtemperature for the indicated time, the reactionwas stopped and ROC4 protein levels were examined by proteingel blot analysis. This assay indicated that ROC4 degraded over

    Figure 3. DHS Has E3 Ubiquitin Ligase Activity and Interacts with ROC4.

    (A) E3 ubiquitin ligase activity of DHS. MBP-DHS fusion proteins were assayed for E3 ubiquitin ligase activity in the presence of El, E2, and His-ubiquitin(His-Ub). Ubiquitinated proteins were detected by immunoblottingwith anti-His and anti-MBP antibodies, respectively. The slowlymigrating bands are theubiquitinated forms of DHS. DHSC95S is the mutated form of DHS. AtCIP8 was used as a positive control.(B) Interaction between DHS and ROC4 in a yeast two-hybrid assay. The DHS protein was fused with the GAL4 binding domain to generate BD-DHS andROC4 with the GAL4 activation domain to form AD-ROC4. Clones that grew on SD/-Trp-His-Ade medium indicate protein interaction in yeast cells. Theempty AD/BD-DHS combination was used as a negative control.(C)Pull-downassay showing the interaction betweenDHSandROC4.GST-ROC4waspulled downbyMBP-DHS immobilized onamylose resin beads andanalyzed by immunoblotting using anti-GST antibody. The inputwas immunoblotted using anti-MBPandanti-GST antibodies. The uncut picture is showedin Supplemental File 1.(D) Luciferase complementation imaging assays showing the interaction betweenDHS andROC4 inNicotiana benthamiana leaf epidermal cells. DHSwasfused with nLUC (N terminus of LUC) and ROC4 with cLUC (C terminus of LUC). The LUC signals were observed at 48 h after infiltration.

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  • time, and importantly, the degradation speed of ROC4 combinedwith DHS OE was faster than that combined with the wild type(Figure 6A). In contrast, a similar assay showed that the degra-dation speed of ROC4 combined with dhs was slower than thatcombined with the wild type (Figure 6A). Quantification and

    statistical analysis of ROC4 degradation speed from three in-dependent experiments demonstrated that the degradation ofROC4 was promoted in DHS OE but was delayed in dhs (Figure6B). Second, we transiently coexpressed ROC4 with DHS orDHSC95S in a rice protoplast system. Protein gel blot analysis

    Figure 4. ROC4 Positively Regulates Wax Loads and Drought Stress Responses.

    (A)Cuticular wax composition on the leaf surfaces ofROC4OE, roc4, and thewild type analyzedbyGC-MS.Waxconstituents are groupedby carbonchainlength and chemical class. Data are means6 SE of three biological replicates using independent seedling samples grown at the same condition. Asterisksdenote significant differences from the wild type (*P < 0.05) determined by Student’s t test.(B) to (D)PhenotypesofROC4OE, roc4, and thewild typeunder normal conditions (B),withholdingwater for 3d (C), and rehydration for 3dafterwithholdingwater for 7 d (D).(E) Recovery rate of ROC4 OE, roc4, and the wild type in (D). Data are means6 SE of three biological replicates with 30 seedlings used for each replicate.Asterisks denote significant differences from the wild type (**P < 0.01) determined by Student’s t test.(F)Water loss rate in ROC4 OE, roc4, and the wild type. Leaves at the same developmental stages were excised and weighed at various time points afterdetachment. Thewater loss rate is representedas thepercentageof initial freshweightateach timepoint. Valuesaremeans6 SEof three individualplantspergenotype.

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  • showed that the protein level of ROC4 in the presence of DHSwasmuch lower than that in theabsenceofDHS (Figures6Cand6D). Incontrast, the coexpression of DHSC95S did not significantly affectROC4 protein level (Figures 6C and 6D). Third, we examined theprotein level of ROC4 in different dhs allelicmutants andwe found

    that the dhs mutants accumulated more ROC4 protein than thewild type (Figure 6E). As a control, ROC4 transcript levels weresimilar between the dhs mutants and the wild type (Figure 6F).Collectively, thesedata suggest thatDHSpromotes thedegradationof ROC4, suggesting that ROC4 is the ubiquitination target of DHS.

    Figure 5. ROC4 Is Unstable and Is Degraded via the 26S Proteasome System.

    (A) Degradation of ROC4 in a cell-free system. ROC4 protein was extracted from ROC4 OE callus, and ROC4 levels at the indicated time points wereexamined by protein gel blot analysis using anti-ROC4 antibody. HSP contents detectedwith anti-HSP antibodywere used as a loading control. The uncutphoto is showed in Supplemental File 1.(B) Quantification of ROC4 degradation kinetics in (A). The relative ratio of the signal intensity between ROC4 and HSP was quantified by Image J. HSPwas used for normalization. The ratio at the starting point was set to 1. Error bars represent the SE of three independentmeasurements shown in SupplementalFile 1.(C)The in vivo stability ofROC4 is enhancedbyMG132.ROC4OEcalluswas treatedwithCHXorMG132 for 4 h, andROC4 levelswere examinedbyproteingel blot analysis with anti-ROC4 antibody. HSP contents detected with anti-HSP antibody were used as a loading control. The uncut picture is showed inSupplemental File 1.(D) Quantification of ROC4 levels in (C). The relative ratio of the signal intensity between ROC4 and HSP was quantified by Image J. HSP was used fornormalization.Dataaremeans6 SEwith three independentmeasurementsshown inSupplemental File1.Asterisksdenotesignificantdifferences frommock(**P < 0.01) determined by Student’s t test.(E)The in vivo stability of ROC4 is enhancedbyMG132. Two-week-oldROC4OE seedlingswere treatedwithCHXorMG132, andnucleus-localizedROC4-GFP fluorescence was observed in roots by confocal microscopy.(F)ROC4 isubiquitinated invivo.ROC4was immunoprecipitated fromprotein extractsofOsROC4OEandwild-type seedlings, and the immunoprecipitatedsamples were separated on SDS-PAGE gels and probed with anti-ROC4 and antiubiquitin antibodies. The slowly migrating bands are the ubiquitinatedforms of ROC4. IgG was used as an internal control.

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  • ROC4 Genetically Acts Downstream of DHS

    To examine whether ROC4 is the ubiquitinated target of DHSgenetically, wegenerated thedhs roc4doublemutant by crossingdhs-1 with roc4-1 and subjected it to phenotypic analysis.Scanning electron microscopy analysis showed that the cuticularwax in dhs roc4 was sparse, as observed in roc4 (SupplementalFigures 12A to 12D). In addition, GC-MS analysis showed that thewax contents in dhs roc4 were lower than in dhs-1 (Figure 7A;Supplemental Figure 12F). Accordingly, the slower chlorophyll-leaching rate in dhs-1 also was suppressed in dhs roc4(Supplemental Figure 12E). Furthermore, the results of both thewater loss assay and drought stress assay demonstrated that thedrought tolerance of dhs roc4was similar to that of the roc4 singlemutant andwassignificantlyweaker than that ofdhs-1 (Figures7Bto 7F). Collectively, these data clearly demonstrate that the ac-cumulationofROC4 is required for thedhsmutant phenotype, andthey support the notion that ROC4 acts downstream of DHS incontrolling wax biosynthesis and the corresponding droughtstress response.

    Os-BDG Is a Direct Target of the DHS-ROC4 Cascade

    Asdescribedabove,ROC4actsdownstreamofDHS incontrollingwax biosynthesis. We investigated how the DHS-ROC4 cascadeis involved in thispathway. InArabidopsis,BDGplaysan importantrole in cuticular development, and HDG regulates the expressionof BDG by direct binding to the L1 box region in its promoter(Kurdyukov et al., 2006; Wu et al., 2011). Sequence alignmentdemonstrated that there are three homologs of Arabidopsis BDGin rice (Supplemental Figure 13), and there are two conserved L1boxes in the promoter region of Os-BDG (LOC_Os06g04169)(Figure 8A). To investigate whether the Os-BDG is direct target ofROC4, we conducted an electrophoretic mobility shift assay(EMSA). ROC4 protein fused to His tag (His-ROC4) was found tobind to thebiotin-labeledOs-BDGpromoter inanL1box-dependentmanner (Figure 8B). To verify this result in vivo, we performeda chromatin immunoprecipitation (ChIP) assay using ROC4 OEplants. ROC4-bound fragments enriched by immunoprecipitationwith anti-ROC4 antibody were used for quantitative PCR. Wefound that the L1 box-containing fragment in the Os-BDG pro-moter was significantly enriched in ROC4 OE plants (Figure 8C).Moreover, the transient expression assay in rice protoplastsshowed that ROC4markedly activated the expression ofOs-BDG(Figure 8D). More important, the expression level of Os-BDG wassignificantly reducedwhenROC4wascoexpressedwithDHS,butnot when coexpressed with DHSC95S (Figure 8D). Furthermore,analysis of the expression of Os-BDG in ROC4 OE and roc4showed that Os-BDG expression was enhanced inROC4OE andreduced in roc4 (Figure 8E). Together, these results indicate thatOs-BDG is a direct target of ROC4. Becausewe showed that DHSacts upstream of ROC4 genetically and negatively regulatesROC4 protein stability, we also analyzed the expression ofOs-BDG in dhs. As shown in Figure 8E, the expression ofOs-BDGwas higher in dhs than in the wild type. More importantly, theincreasedexpressionofOs-BDG indhswassuppressed in thedhsroc4 double mutant (Figure 8E). The differential expression ofOs-BDG in dhs, roc4, and the dhs roc4 double mutant suggested

    that the DHS-ROC4 cascade regulates the wax biosynthesispathway by controlling the expression of Os-BDG. In conclusion,we propose a working model in which DHS negatively regulateswax biosynthesis and the drought stress response by promotingthe degradation of ROC4, which directly regulates the expressionof the downstream target gene, Os-BDG (Figure 8F).

    DISCUSSION

    Cuticular wax plays crucial roles in protecting plants from envi-ronmental stresses. In particular, increasing cuticular wax con-tents can reduce nonstomatal water loss in plants, therebyimproving drought tolerance. Many catalytic enzyme-encodinggenes and transcription factors involved in the wax biosynthesispathway have been characterized in plants (Broun et al., 2004;Raffaele et al., 2008; Yu et al., 2008; Javelle et al., 2010; Seo et al.,2011;Maoet al., 2012;Nadakuduti et al., 2012;HaslamandKunst,2013; Oshima et al., 2013; Borisjuk et al., 2014; Zhou et al., 2014;Gan et al., 2016; Wang et al., 2017). In contrast, there are fewreports regarding the posttranslational regulation of wax bio-synthesis-related transcription factors.In this study, we characterized the RING-type E3 ligase DHS as

    a negative regulator of cuticular wax biosynthesis and droughttolerance (Figures 1 and 2). We also revealed that DHS interactswith the HD-ZIP IV transcription factor ROC4 (Figure 3;Supplemental File 1). We found that ROC4, in contrast to DHS,positively regulates wax deposition and drought tolerance (Figure4). In addition, ROC4 was found to be an unstable protein, andDHS promoted the UPS-mediated degradation of ROC4 (Figures5 and 6). Moreover, we demonstrated that ROC4 acts geneticallydownstream of DHS in controlling wax biosynthesis and thedrought stress response (Figure 7). Furthermore, we identifiedOs-BDG as a direct downstream target of ROC4 and proposedthat the DHS-ROC4 cascade regulates the wax biosynthesispathway by controlling the expression of Os-BDG (Figure 8). Al-though these data do not demonstrate that DHS directly ubiq-uitinates ROC4, the combined physiological, biochemical, andgenetic data presented here strongly suggest that DHS is in-volved in the ubiquitination and subsequent degradation ofROC4, and the data establish that the DHS-ROC4 moduleregulates the drought stress response by controlling wax bio-synthesis in rice.The protein structure of DHS is simple, consisting of 165 amino

    acid residues and a transmembrane domain in theN terminus anda RINGdomain in the C terminus (Supplemental Figure 5A). Usingthe DHS as a query for BLAST analysis in the Arabidopsis proteindatabase, we discovered a subgroup of RING-type proteins (in-cludingRHA2a, RHA2b, and XERICO) that displayed homology toDHS to various extents (Supplemental Figures 5B and 5C andSupplemental File 2). These RING proteins play common andcritical roles in the drought stress response and abscisic acidsignaling (Koetal., 2006;Buetal., 2009;H.Li etal., 2011), althoughthe direct targets and mechanisms are not well known. Previousand current results suggest that these simple-structured RING-typeproteinsmight playmajor roles in the abiotic stress response.It would be interesting to examine whether DHS is involved in theabscisic acid signaling response in the future.

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  • In this study, we characterized DHS as a negative regulator ofcuticular wax biosynthesis. Similarly, the Arabidopsis RING-typeprotein CER9 also functions in controlling wax biosynthesis (Lüet al., 2012). The underlyingmechanisms of CER9 andDHSmightdiffer, however. In thecer9mutant,C22-C26VLCFAscontentsareelevated,whichcontributes to theelevated totalwaxcontents andenhanced drought tolerance, although the contents of VLCFAs

    derivatives (aldehyde, alcohol, and alkanes) are reduced (Lü et al.,2012). By contrast, nearly all wax composition contents werereduced in DHS-OE plants, but they were increased in dhscompared with the wild type (Figures 1F and 2A). In addition, thelower transpiration rate and improved water use efficiency in thecer9 mutant also contributes to improved drought tolerance (Lüet al., 2012).Moreover, theCER9 sequence is highly similar to that

    Figure 6. DHS Promotes the Degradation of ROC4.

    (A)Degradation of ROC4 in the wild type,DHSOE, and dhs in a cell-free system. ROC4 protein was extracted from ROC4OE callus and divided into equalparts; eachpart was incubatedwith equal amounts of crudeprotein extract fromwild-type,DHSOE, anddhs callus. The degradation of ROC4was stoppedat the indicated time points and examined by immunoblotting with anti-ROC4 and anti-HSP antibodies. The experiment was repeated three times withsimilar results shown in Supplemental File 1.(B)Quantification of ROC4degradation kinetics in the wild type,DHSOE, and dhs (A). The relative ratio of the signal intensity between ROC4 andHSPwasquantified by Image J. HSP was used for normalization. The ratio at the starting point was set to 1. Error bars represent the SE of three independentmeasurements shown in Supplemental File 1.(C) DHS promotes the degradation of ROC4 in a transient expression assay. Different combinations of ROC4 and DHS or DHSC95S were transientlyexpressed in rice protoplasts, and ROC4 levels were examined by probing with anti-ROC4 and anti-HSP antibodies. CK indicates empty pRT107 vectorsused as control. The experiment was repeated three times with similar results shown in Supplemental File 1.(D) Quantification of ROC4 in (C). The relative ratios of the signal intensity between ROC4 and HSP were quantified by Image J. HSP was used fornormalization. Error bars represent the SE of three independent measurements shown in Supplemental File 1. Asterisks denote significant differences fromCK (**P < 0.01) determined by Student’s t test. CK indicates empty pRT107 vectors used as control.(E) dhs mutants accumulate more ROC4 protein than the wild type. Native ROC4 protein levels in wild-type and different dhs alleles were examined byimmunoblottingwith anti-ROC4 antibody. The numbers below the image are the relative ratios of the signal intensity betweenROC4 andHSPquantified byImage J; the ratio in the wild type was set to 1. The uncut picture is showed in Supplemental File 1.(F)ROC4 expression levels inwild-type anddhs allelesmeasured byRT-qPCR. The expression level in thewild typewas set to 1.0, and error bars representthe SE of three independent experiments.

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  • ofDoa10, anERAD (ER-associateddegradation) component. It isthought that CER9 might be involved in ERAD, by which manywax biosynthesis enzymes are degraded (Lü et al., 2012). Futurework should examine whether DHS is involved in the ERADprocess.

    Compared with dhs, roc4, and ROC4-OE, DHS-OE showedmore severe changes in wax loads and more strikingly drought-

    hypersensitive phenotypes. Because DHS exhibits E3 ligaseactivity, we speculated that DHS might have multiple ubiquiti-nation targets in addition to ROC4 and that these targets mightplay redundant or distinct roles, consequently contributing to thesevere phenotypes of DHS-OE. Supporting this notion, we dis-covered that DHS also interacts with ROC5, which shares thehighest sequence similarity with ROC4 (Supplemental Figures 8

    Figure 7. ROC4 Genetically Acts Downstream of DHS in Controlling Wax Biosynthesis and Drought Stress Response.

    (A)Cuticular wax composition on the leaf surfaces in the wild type, dhs, roc4, and dhs roc4 analyzed by GC-MS. Wax constituents are grouped by carbonchain length and chemical class. Data are means 6 SE of three biological replicates using independent seedling samples grown at the same condition.Asterisks denote significant differences from the wild type (*P < 0.05 and **P < 0.01) determined by Student’s t test.(B) to (D) Phenotypes of the wild type, dhs, roc4, and dhs roc4 under normal conditions (B), withholding water for 3 d (C), and rehydration for 3 d afterwithholding water for 7 d (D).(E)Recovery rate of thewild type,dhs, roc4, anddhs roc4 in (D). Data aremeans6 SE of three biological replicateswith 30 seedlings used for each replicate.Asterisks denote significant differences between two genotypes (*P < 0.05 and **P < 0.01) determined by Student’s t test.(F)Water loss rate in thewild type,dhs, roc4 and dhs roc4. Leaves at the samedevelopmental stageswere excised andweighed at various time points afterdetachment. The percentage of initial fresh weight at each time point is shown. Values are means 6 SE of three individual plants per genotype.

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  • and9 andSupplemental File 3). Further investigation is required toexamine whether DHS promotes the ubiquitination and degra-dationofROC5andwhetherROC5 is involved inwaxbiosynthesisand the relativedroughtstress response. Inaddition,we found thatROC4was still degraded far more slowly in the dhsmutant than in

    the wild type (Figures 6A and 6B), which implies that ROC4mightbe ubiquitinated by other E3 ligases as well.ROC4 belongs to rice HD-ZIP IV gene family, which contains

    nine members (ROC1 to ROC9), with five members specificallyexpressed in the epidermis (Ito et al., 2003). ROC5 controls leaf

    Figure 8. Os-BDG Is a Direct Target of the DHS-ROC4 Cascade in Controlling Wax Biosynthesis.

    (A)Schematic diagramof theOs-BDGgene.Black squares indicate L1boxes. Theprobewasused forEMSA, andDNA fragments (F1, F2, andF3)wereusedfor ChIP-qPCR.(B)ROC4binds to the conserve L1-box of theOs-BDGpromoter. His-GFPproteinwas used as a negative control. Mu indicatesmutated probe inwhich the59-TAAATGAA-39 motif was replaced by 59-TACGCGAA-39. Unlabeled probes were used as competitors.(C) ChIP assays showing that ROC4 binds to the promoter of Os-BDG in vivo. Immunoprecipitation was performed with anti-ROC4 antibody. Im-munoprecipitated chromatin was analyzed by qPCR. Enrichment was calculated by normalizing to ACTIN and to the total input of each sample. Data areshown as means 6 SE (n = 3). P values were calculated by Student’s t test (**P < 0.01).(D)ROC4activates theexpressionofOs-BDG in a riceprotoplast transient assay.Relative expression levels ofOs-BDG in rice protoplasts transformedwithvariouscombinationsofvectorswereanalyzedbyRT-qPCR.Theexpressionlevel inprotoplaststransformedwithPRT107emptyvectorwasusedasCKandset to1; data are shown as means 6 SE (n = 3). Asterisks denote significant differences between two genotypes (**P < 0.01) determined by Student’s t test.(E) The relative expression of Os-BDG in various plants was analyzed by RT-qPCR. The expression level in the wild type was set to 1; data are shown asmeans 6 SE (n = 3).(F) Schematic diagram of a working model for the roles of DHS and ROC4. DHS negatively regulates ROC4 protein level by promoting its UPS-mediateddegradation. ROC4 directly binds to and activates the expression of Os-BDG, which positively regulates wax biosynthesis, thereby affecting droughttolerance responses.

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  • rolling by regulating bulliform cell number and size (Zou et al.,2011), whereas the functions of other ROC members remainunknown. In this study, we discovered that ROC4 positivelyregulates cuticular wax biosynthesis, thereby influencing therelative drought stress response. These data point to divergentfunctions among ROC members, which is not unexpected. Forexample, the expression patterns of the majority of maize OCLgenes encoding HD-ZIP IV proteins are restricted to the epi-dermal and subepidermal layers of various organs (Ingram et al.,2000). OCL4, however, controls anther and trichome de-velopment (Vernoud et al., 2009), whereas OCL1 is involved inroot and kernel development (Khaled et al., 2005). Subsequently,through the identification and analysis of OCL1 targets, it wasshown that OCL1 also positively regulates cuticular wax bio-synthesis by directly modulating the expression of wax and lipidtransporter genes (WBC11a and LtpII.12) and the wax bio-synthesis gene FAR1 (Javelle et al., 2010). Another possible roleof ROC4 in wax biosynthesis was obtained through the charac-terization of CFL1 (CURLY FLAG LEAF1), which controls bothleaf rolling and cuticular wax development (Wu et al., 2011). InCFL1 overexpression plants, cuticular wax contents and cutincompositions are severely affected. In addition, HDG1, a ho-molog of ROC4,was identified as the interaction partner of CFL1and was found to be required for the functioning of CFL1 (Wuet al., 2011). These findings suggest that HD-ZIP IV proteinsplay multiple roles in various aspects of plant development andthat different members function in different developmentalprocesses.

    Rice plants have high water requirements, and drought hasbecome a major limiting factor for rice production due to watershortages. There is anurgent demand for thebreeding of drought-tolerant rice cultivars (Kumar et al., 2014). The overexpression ofMt-WXP1, At-CER1, Os-WR1, and Os-WR2 results in elevatedtotalwax loads, reducedwater loss, and less chlorophyll leaching,and consequently, improved drought adaptability (J.Y. Zhanget al., 2005; Bourdenx et al., 2011; Wang et al., 2012; Zhou et al.,2014). DWA1 (DROUGHT-INDUCED WAX ACCUMULATION1) isa critical enzyme that positively controls drought-induced waxproduction, and plants overexpressing DWA1 and dwa1mutantsexhibit clear changes in wax contents and opposite drought re-sponses (Zhu andXiong, 2013). These studies clearly indicate thatdrought tolerance could be improved by enhancing cuticular waxdeposition. In this study, we demonstrated that DHS and itsputative ubiquitination target, ROC4, are critical regulators of waxbiosynthesis. More important, dhs and ROC4 OE plants showedsignificantly enhanced drought tolerance, suggesting that thesegenes are valuable targets for engineering drought-tolerant ricecultivars.

    METHODS

    Plant Materials and Growth Conditions

    Rice cultivar Longjing 11 (Oryza sativa ssp japonica) was used to generatethe DHS and ROC4 overexpression plants and knockout mutants. Theseedlings were grown in a growth chamber (white fluorescent tubes,200–300 mmol m22 s21) at 30°C for 14 h (day) and 24°C for 10 h (night) at70% humidity or in the field (natural long-day conditions).

    Generation of Transgenic Plants and Mutants

    The coding sequences of DHS and ROC4 were cloned from Nipponbare(O. sativa ssp japonica) cDNA using a standard RT-PCR protocol. The full-length coding region of DHS was cloned into the binary vector pCAMBIA1300-221-HA to generate aDHS overexpression vector in whichDHSwasdriven by the CaMV35S promoter. To produce the ROC4 overexpressionconstruct, the full-lengthcoding regionofROC4wascloned intopENTR/D-TOPO (Invitrogen) and subcloned into the binary vector pH7WGF2 by LRreaction to generate 35Spro:GFP-ROC4. To generate the dhs and roc4mutants, twoandonesingle-guideRNAs (sgRNAs)weredesigned to targetDHS and ROC4, respectively. The sgRNA cassettes were sequentiallyligated into theCRISPR/Cas9binary vectorspYLCRISPR/Cas9Pubi-H (Maetal., 2015).All primersused for theseconstructsare listed inSupplementalTable 1. The constructs were introduced into Agrobacterium tumefaciensstrain EHA105, and rice cultivar Longjing11 was used as the recipient forAgrobacterium-mediated transformation as described previously (Tianet al., 2015). Homozygous T2 transgenic rice seedlings were used forphenotype analysis.

    Total RNA Isolation and RT-qPCR Analysis

    Total RNAwasextracted usingTRIzol (Invitrogen) and treatedwithDNaseI.cDNA was synthesized from 2 mg of total RNA using Superscript II reversetranscriptase (Invitrogen).RT-qPCRwasperformedwithSYBRGreenPCRmaster mix (Takara). Data were collected using a Bio-Rad Chromo 4 real-time PCR detector. All expression levels were normalized against theACTINgene (Os03g0718100). The primers used are listed at SupplementalTable 1.

    Water Loss Assay

    The water loss assay was performed as previously described with somemodifications (Tian et al., 2015) using 4-week-old rice seedlings grown inclimate chambers. Leaves at the same growth stages were detached fromthe plants, left on a laboratory bench, and weighed at the indicated timepoints. Time-course analysis ofwater losswasperformedand representedas the percentage of initial fresh weight at each time point.

    Chlorophyll Leaching Assays

    Chlorophyll leaching assays were used to measure the epidermal per-meability of rice leaves as described previously (Mao et al., 2012). The thirdleaf from the topwas sampled from4-week-old seedlings. The leaf was cutinto segments (;2 cm) and immersed in 30 mL 80% ethanol at roomtemperature. Aliquots of 0.5 mL were removed for chlorophyll quantifi-cation and returned to the same tube at the indicated time point. Thechlorophyll concentration was quantified using a Thermo BIOMATE3spectrophotometeratwavelengthsof664and647nm.Chlorophyll effluxateach time point was expressed as a percentage of total chlorophyll ex-tracted after 24 h of immersion.

    Scanning and Transmission Electron Microscopy

    Scanning electron microscopy was performed as previously described(Mao et al., 2012). Leaf blades excised from 4-week-old plants were usedfor scanning electron microscopy analysis. Samples were prefixed with2.5% glutaraldehyde-sodium phosphate buffer (0.1 M) at room temper-ature and postfixed in 1% OsO4 at 4°C. The samples were dehydratedthrough an ethanol series and dried with a critical point dryer. The driedsamples were coated with platinum using sputtering equipment andexamined by scanning electron microscopy (S-4800; Hitachi) at anaccelerating voltage of 10 kV. For TEM, the samples were processed asdescribed previously (Mao et al., 2012). Mature expanded leaves were

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  • cut into 3 3 1-mm segments between the midvein and the leaf margin.Ultrathin sections (80 nm) were cut using an Ultracut E Ultramicrotome(Leica) and mounted on copper grids. The sections were stained withuranyl acetate and lead citrate solution and observed by TEM (H-7650;Hitachi).

    Cuticular Wax Analysis

    Cuticular wax was extracted and measured as described previously (Maoet al., 2012). Briefly, leaves of 8-week-old seedlings were immersed in30 mL n-hexane at 67°C for 30 s, with 50 mg n-tetracosane as an internalstandard. The n-hexane was then evaporated under gaseous N2 andthe residue was derivatized with 100 mL of bis-N,N-(trimethylsilyl) tri-fluoroacetamide (Sigma-Aldrich) and 100mL of pyridine for 60min at 70°C.All wax samples were analyzed with an Agilent 7000C GC-MS/MS deviceon a 30 m HP-1MS column. The column was operated with helium as thecarrier gas and splitless injection at 250°C. The oven temperature wasincreased from 50°C to 200°C at 20°C min21, held for 2 min at 200°C,increased at 2°C min21 to 320°C, and held at 320°C for 14 min. The totalamount of cuticular wax was expressed per unit area of the leaf surface.Leaf area was measured using an LI-3000C Portable Area Meter (LI-CORBiosciences).

    Multiple Sequence Alignments and Phylogenetic Analysis

    Multiple sequence alignments were constructed using the ClustalX2software. A phylogenetic analysis was conducted by MEGA version 4.0using the neighbor-joining method with 1000 bootstrap replications. Seealignments in Supplemental Files 2 to 4.

    Yeast Two-Hybrid Assay

    ThecodingsequenceofDHSwascloned into theEcoRIandPstI sitesof thepGBKT7 vector to generate the BD-DHS construct. The coding sequenceofROC4was cloned into the EcoRI andXhoI sites of the pGADT7 vector togenerate the AD-ROC4 construct. The resulting constructs were trans-formed into yeast strain Y2H Gold. The presence of the transgenes wasconfirmed by growth on an SD/-Leu/-Trp plate. To assess protein inter-actions, the transformedyeastcellsweresuspended in liquidSD/-Leu/-Trpto OD600 = 1.0. The suspended cells were spread on plates containingSD/-His/-Leu/-Trpmedium. Interactionswereobservedafter4dof incubationat 30°C.

    Pull-Down Assay

    The full-length coding region of ROC4 in pENTR/D-TOPO was subclonedinto the expression vector pDEST15 to generate the glutathioneS-transferase (GST)-ROC4 fusion vector. The coding region of DHS wasligated into the pMAL-c2x vector (New England Biolabs) to generate theMBP-DHS construct. The resulting vectors were transformed intoEscherichia coli strain BL21 (DE3) to express the protein. The recombinantproteins MBP-DHS and GST-ROC4 were affinity purified using amyloseresin (NewEnglandBiolabs; E8021S) andglutathioneSepharose4Bbeads(GE Healthcare), respectively.

    For the in vitro pull-down assay, bacterial lysates containing ;2 mg ofMBP-DHS,;2 mg of GST-ROC4 fusion proteins, and amylose resin wereadded to pull-down buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mMEDTA, 1mMPMSF, and 1mMDTT) with continuous rocking at 4°C for 1 h.The beads were washed five times with wash buffer (20 mM Tris, pH 7.4,500 mM NaCl, 1 mM EDTA, and 1% Triton X-100), and the pull-downedprotein was separated by 10% SDS-PAGE and detected by immunoblotanalysiswith anti-GST (1:5000;Abmart;M20007) andanti-MBPantibodies(1:3000; CWBIO; CW0288), respectively.

    Protein Gel Blot Analysis

    For the secondary antibody in the protein gel blot assay, peroxidase-labeled goat anti-rabbit antibody (1:4000; Abcam; ab6789 XXX) or goatanti-mouse (1:4000; Abcam; ab6721) was utilized. Membranes were de-veloped with the SuperSignal West Pico Chemiluminescent Substrate Kit(PierceBiotechnology)and thesignalwasdetectedbychemiluminescenceimaging (Tanon 5200).

    Luciferase Complementation Imaging Assays

    For the luciferase (LUC) complementation imaging assays, the codingregions of DHS and ROC4 were ligated into pCAMBIA-nLUC andpCAMBIA-cLUC, respectively, and the nLUC-DHS and cLUC-ROC4constructs were generated. The nLUC-/cLUC-derivative constructs weretransformed intoAgrobacteriumstrainGV3101. After overnight culture, theagrobacteria were suspended in infiltration buffer (0.2% MgCl2, 100 mMacetosyringone, and 10 mM MES) at OD600 = 1.0. Equal volumes ofagrobacteria resuspension carrying the nLUC and cLUC derivative con-structs were mixed and coinfiltrated into Nicotiana benthamiana leaves.LUC activity in infiltrated leaves was analyzed at 48 h after infiltration usingchemiluminescence imaging (Tanon 5200).

    In Vitro Ubiquitination Assay

    Purified MBP-DHS and MBP-DHSC95S were used for the ubiquitinationassay. TogenerateMBP-DHSC95S, theCys-95ofDHSwasmutated toSer-95 using a point mutation kit. The ubiquitination assay was performed asdescribed previously (Zhao et al., 2012). Briefly, purified wheat (Triticumaestivum) E1 (GI: 136632, ;40 ng), Arabidopsis thaliana Ubc10 (E2,;100ng), ArabidopsisUBQ14 (At4g02890) fusedwithHis tag (;1mg), andrecombinantMBP-DHS (;500ng)wereprepared for theE3ubiquitin ligaseactivity assay. The reactionwas stopped by adding 53SDS sample bufferand boiled before SDS-PAGE separation. Ubiquitinated proteins wereanalyzed using the anti-His antibody (1:4000; Santa Cruz Biotechnology;sc8036). The ubiquitination assay in the E. coli system was performedfollowing a recently published protocol (Han et al., 2017). DHS and ROC4were ligated into the appropriate Duet expression vectors. The autoubi-quitination of DHSwasanalyzed using anti-Myc (1:5000; Abmart;M20002)and anti-FLAG antibodies (1:5000; Abmart; M20008).

    Detection of ROC4 Ubiquitination in Vivo

    In vivo ubiquitination of ROC4 proteins was assayed as described pre-viously with somemodifications (Shen et al., 2008). Briefly,;1 gROC4OEcallus was treated with 20 mMMG132 for 4 h and ground into a powder inliquid nitrogen to extract protein using extraction buffer (100 mM sodiumphosphate, pH 7.8, 100 mM NaCl, 0.1% Nonidet P-40, 2 mM PMSF,complete protease inhibitor cocktail, and 50 mM MG132). Crude extractscontaining 500 mg proteins were coincubated with anti-ROC4 polyclonalantibodies and protein AMagBeads (GenScript) to immunoprecipitate theprotein complex. After 3 h incubation, the immunoprecipitated complexeswerewashed three timeswithwashbuffer (100mMsodiumphosphate, pH7.8, 100 mM NaCl, 0.5% Nonidet P-40, 2 mM PMSF, complete proteaseinhibitor cocktail, and 50 mMMG132), followed by the addition of 53 SDSbuffer and boiling for 5 min. The samples were separated on a 10% SDS-polyacrylamide gel and detected by immunoblot analysis with anti-ROC4(1:200; made by Abmart) and antiubiquitin (1:500; Santa Cruz Bio-technology; sc8017) antibodies.

    In Vitro Degradation Assay

    Total protein was isolated from ROC4 OE callus using degradation buffer(25 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mMMgCl2, 4 mM PMSF, 5 mM

    DHS Regulates Cuticular Wax and Drought Response 241

    http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1

  • DTT, and 10 mM ATP). At the indicated time, an aliquot of the extract wasremoved and 53 SDS buffer was added to stop the degradation, followedby boiling for 5 min. The samples were loaded onto a 10% SDS-PAGE gelfor immunoblotting with anti-ROC4 antibody (1:200; made by Abmart).

    To compare the degradation speeds of ROC4 in thewild type,DHSOE,and dhs, ROC4was extracted fromROC4OE callus and divided into equalparts. Each aliquot was incubated with an equal amount of crude proteinextract from wild-type, DHS OE, and dhs calli. The degradation of ROC4was stopped at the indicated time point and examined by immunoblottingwith anti-ROC4 (1:200; made by Abmart) and anti-HSP antibody (1:5000;BGI Tech; AbM51099).

    In Vivo Degradation Assay of ROC4

    ROC4 OE callus were treated with 50 mM CHX or 40 mM MG132 for 4 h.ROC4 protein was extracted in buffer (50 mM Tris-HCl, pH 7.5, 150 mMNaCl, 0.2% Nonidet P-40, 0.1% Triton X-100, 5 mM EDTA, completeprotease inhibitor cocktail, and 50mMMG132), followed by the addition of53 SDS buffer and boiling for 5 min. The samples were loaded on a 10%SDS-PAGE gel for immunoblotting with anti-ROC4 and anti-HSP anti-bodies. Simultaneously, to assess GFP fluorescence, 2-week-old ROC4OE seedlings were treated with 50 mM CHX or 50 mMMG132 for 4 h, andGFP fluorescence in ROC4 OE roots was observed and imaged undera Zeiss LSM 510 Meta UV confocal microscope.

    Transient Expression Assay in Rice Protoplasts

    The coding regions of ROC4, DHS, and DHSC95S were ligated into thepRT107 vector to generate the 35Spro:ROC4, 35Spro:DHS, and 35Spro:DHSC95S constructs. Rice protoplasts were isolated from stem and sheathtissues of young wild-type seedlings as described previously (Chen et al.,2006). Different combinations of plasmid DNA (;10 mg DNA of eachconstruct) were transiently expressed in protoplasts via polyethyleneglycol-mediated transfection. Following overnight incubation in the dark at28°C, total proteins were isolated from the protoplasts with extractionbuffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Nonidet P-40, 0.1%Triton X-100, 5mMEDTA, complete protease inhibitor cocktail, and 50mMMG132), followed by the addition of 53 SDS buffer and boiling for 5 min.The samples were loaded onto a 10% SDS-PAGE gel for immunoblottingwith anti-ROC4 and anti-HSP antibodies. For the transactivation assay inrice protoplasts, total RNA was extracted from protoplasts after overnightincubation and used for RT-qPCR analysis

    EMSA

    The full-length coding region of ROC4 in pENTR/D-TOPO was subclonedinto the expression vector pDEST17 to generate the histidine (His)-ROC4fusion vector. Purified His-ROC4 was used for the EMSA. Oligonucle-otide probes 48 bp long containing a wild-type L1-box (TAAATGYA) ormutated L1-box (TACGCGAA) motifs were synthesized and labeled withbiotin using an EMSA Probe Biotin Labeling Kit (Beyotime; catalog no.GS008). For competition with unlabeled probe, unlabeled probe wasadded to the reactions. EMSAwas performed using a chemiluminescentEMSA kit (Beyotime; catalog no. GS009). Probe sequences are shown inSupplemental Table 1.

    ChIP Assay

    ROC4-OEwas used for theChIP assay as previously described (Tian et al.,2017). Briefly, ;2 g of rice seedling tissue was cross-linked in 1% form-aldehyde under a vacuum. The cross-linking was stopped by the additionof 0.125 M glycine. The sample was ground to a powder in liquid nitrogenand used to isolate nuclei. Anti-ROC4 (1:200 dilution) was used to im-munoprecipitate the protein-DNA complex, and the precipitated DNAwas

    used for quantitative PCR. Chromatin precipitated without antibody wasused as a control. The data are presented as means 6 SE of three in-dependent experiments. Primers used for ChIP-qPCR are listed inSupplemental Table 1.

    Accession Numbers

    Sequence data from this article can be found in the GenBank Database orRice Genome Annotation Project under the following accession numbers:DHS, LOC_Os02g45780; Os-ROC4, LOC_Os04g48070; Os-BDG,LOC_Os06g04169; At-RHA2a, AEE29264.1; At-RHA2b, ABF58928.1;At-XERICO, AEC05812.1; Os-ROC5, BAC77158; At-HDG1, NP_191674;Zm-OCL1, CAG38614; and At-BDG, AAO63446.1.

    Supplemental Data

    Supplemental Figure 1. Phenotypic analysis of DHS OE plants.

    Supplemental Figure 2. Cuticular wax structure and compositionanalysis of DHS OE plants.

    Supplemental Figure 3. Identification of dhs mutants generated byCRISPR/Cas9-mediated genome editing.

    Supplemental Figure 4. Wax content is increased in dhs versus thewild type.

    Supplemental Figure 5. Protein structure and bioinformatics analysisof DHS.

    Supplemental Figure 6. Ubiquitination assay in an Escherichia colisystem showing that DHS has E3 ligase activity.

    Supplemental Figure 7. Intact RING domain in DHS is essential for itsbiological function.

    Supplemental Figure 8. Interaction between DHS and varioustranscription factors in a yeast two-hybrid assay.

    Supplemental Figure 9. Protein structure and bioinformatics analysisof ROC4.

    Supplemental Figure 10. Identification of roc4 mutants generated byCRISPR/Cas9-mediated genome editing.

    Supplemental Figure 11. ROC4 positively regulates wax loads.

    Supplemental Figure 12. Characterization of the wax crystal structureand composition in the dhs roc4 double mutant.

    Supplemental Figure 13. Protein structure and bioinformatics anal-ysis of Os-BDG.

    Supplemental Table 1. Primers used in this study.

    Supplemental File 1. Uncut pictures of protein gel blot in this study.

    Supplemental File 2. Alignment used to produce the phylogenetictree shown in Supplemental Figure 5.

    Supplemental File 3. Alignment used to produce the phylogenetictree shown in Supplemental Figure 9.

    Supplemental File 4. Alignment used to produce the phylogenetictree shown in Supplemental Figure 13.

    ACKNOWLEDGMENTS

    We thank our laboratory members for their helpful comments and dis-cussions during the article preparation. We thank Jianmin Wan, XiaoquanQi, Lu Gan, and Lixin Duan for their assistance inmeasuring wax contents.We thank Yaoguang Liu for sharing the plasmid used for gene editing. We

    242 The Plant Cell

    http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1http://www.plantcell.org/cgi/content/full/tpc.17.00823/DC1

  • also thankDongpingLv for helpingwith theubiquitinationassay. This studywas supportedby theNationalNatural Science Foundation ofChina (Grants31701058 and 31671653), the Strategic Priority Research Program ofChinese Academy of Sciences (Grant XDA08040101), the Natural ScienceFoundation of Heilongjiang (Grants ZD2015005 and C2017071), and theHundred Talents Program of the Chinese Academy of Sciences to Q.B.

    AUTHOR CONTRIBUTIONS

    Q.B. conceived and supervised the entire project, analyzed the data, andwrote the article. Z.W. performed most of the experiments, analyzed thedata, and drafted the article. Q.Z. performed the autoubiquitination assay.Z.L., X.T., X.L., W.Z., Y.R., and J.T. performed some of the experiments andprovided technical assistance. J.F. andQ.X. helpedwith the discussion of thework. All authors discussed the results and contributed to the final article.

    Received October 23, 2017; revised December 11, 2017; acceptedDecember 11, 2017; published December 13, 2017.

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    244 The Plant Cell

  • DOI 10.1105/tpc.17.00823; originally published online December 13, 2017; 2018;30;228-244Plant Cell

    Qijiang Xu and Qingyun BuZhenyu Wang, Xiaojie Tian, Qingzhen Zhao, Zhiqi Liu, Xiufeng Li, Yuekun Ren, Jiaqi Tang, Jun Fang,

    by Promoting the Degradation of Transcription Factor ROC4 in RiceThe E3 Ligase DROUGHT HYPERSENSITIVE Negatively Regulates Cuticular Wax Biosynthesis

    This information is current as of April 1, 2021

    Supplemental Data /content/suppl/2019/01/30/tpc.17.00823.DC3.html /content/suppl/2017/12/13/tpc.17.00823.DC1.html

    References /content/30/1/228.full.html#ref-list-1

    This article cites 58 articles, 30 of which can be accessed free at:

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