The GDSL Lipase MHZ11 Modulates Ethylene Signaling inRice Roots[OPEN]

He Zhao,a,b,1 Biao Ma,c,1 Kai-Xuan Duan,d Xin-Kai Li,a,b Xiang Lu,a Cui-Cui Yin,a Jian-Jun Tao,a Wei Wei,a

Wan-Ke Zhang,a Pei-Yong Xin,e Sin Man Lam,f Jin-Fang Chu,e Guang-Hou Shui,f Shou-Yi Chen,a,2 andJin-Song Zhanga,b,2

a State Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Innovative Academy of Seed Design, ChineseAcademy of Sciences, Beijing 100101, ChinabCollege of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing 100049, Chinac Biology and Agriculture Research Center, School of Chemistry and Biological Engineering, University of Science and TechnologyBeijing, Beijing 100024, China.d Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, ChinaeNational Center of Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,Beijing 100101, Chinaf State Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academyof Sciences, Beijing 100101, China

ORCID IDs: 0000-0003-4222-4371 (H.Z.); 0000-0003-0791-3235 (B.M.); 0000-0003-4803-240X (K.-X.D.); 0000-0002-3809-9021(X.-K.L.); 0000-0002-1176-5389 (X.L.); 0000-0001-5691-6366 (C.-C.Y.); 0000-0002-2828-6835 (J.-J.T.); 0000-0002-9471-9694(W.W.); 0000-0003-3475-2852 (W.-K.Z.); 0000-0003-4429-244X (P-Y.X.); 0000-0001-9037-904X (S.M.L.); 0000-0003-1677-8776(J.-F.C.); 0000-0002-1621-9643 (G.-H.S.); 0000-0002-3557-4245 (S.-Y.C.); 0000-0003-2165-3468 (J.-S.Z.)

Ethylene plays important roles in plant growth and development, but the regulation of ethylene signaling is largely unclear,especially in crops such as rice (Oryza sativa). Here, by analysis of the ethylene-insensitive mutant mao huzi 11 (mhz11), weidentified the GDSL lipase MHZ11, which modulates ethylene signaling in rice roots. MHZ11 localized to the endoplasmicreticulum membrane and has acyl-hydrolyzing activity. This activity affects the homeostasis of sterols in rice roots and isrequired for root ethylene response. MHZ11 overexpression caused constitutive ethylene response in roots. Genetically,MHZ11 acts with the ethylene receptor ETHYLENE RESPONSE SENSOR2 (OsERS2) upstream of CONSTITUTIVE TRIPLERESPONSE2 (OsCTR2) and ETHYLENE INSENSITIVE2 (OsEIN2). The mhz11 mutant maintains more OsCTR2 in thephosphorylated form whereas MHZ11 overexpression promotes ethylene-mediated inhibition of OsCTR2 phosphorylation.MHZ11 colocalized with the ethylene receptor OsERS2, and its effect on OsCTR2 phosphorylation requires ethyleneperception and initiation of ethylene signaling. The mhz11 mutant overaccumulated sterols and blocking sterol biosynthesispartially rescued themhz11 ethylene response, likely by reducing receptor-OsCTR2 interaction and OsCTR2 phosphorylation.We propose that MHZ11 reduces sterol levels to impair receptor-OsCTR2 interactions and OsCTR2 phosphorylation fortriggering ethylene signaling. Our study reveals a mechanism by which MHZ11 participates in ethylene signaling for regulationof root growth in rice.

INTRODUCTION

Ethylene regulates many aspects of plant growth, development,and stress responses. By identification of the key components ofethylene signaling and uncovering the biochemical mechanisms,a linear ethylene signaling pathway has been established in themodel plant Arabidopsis (Arabidopsis thaliana; Ju and Chang,2015). In this pathway, the ethylene signal is perceived by a familyof endoplasmic reticulum (ER) membrane-localized ethylene

receptors (Chang et al., 1993; Hua et al., 1995; Chen et al., 2002)and transduced from the negative regulator CONSTITUTIVETRIPLE RESPONSE1 (CTR1; Kieber et al., 1993) to the positiveregulator ETHYLENE INSENSITIVE2 (EIN2; Alonso et al., 1999). Inthe absence of ethylene, the receptor proteins activate thedownstream Raf-like Ser/Thr protein kinase CTR1 by interactingwith the N-terminal region of CTR1, which subsequently phos-phorylates the C-terminal of EIN2 to suppress ethylene signaltransduction (Hua and Meyerowitz, 1998; Qiao et al., 2009; Juet al., 2012). In the presence of ethylene, the receptors are in-activated upon ethylene binding and fail to activate the kinaseactivity of CTR1, resulting in the dephosphorylation of EIN2. TheEIN2 C-terminal domain is further cleaved off from the ERmembrane and translocated into the nucleus (Ju et al., 2012; Qiaoet al., 2012; Wen et al., 2012) to activate the EIN3 (ETHYLENEINSENSITIVE3)/EIN3-LIKE1 (EIL1)-mediated transcriptional reg-ulation cascade (Chao et al., 1997; Solano et al., 1998) by sta-bilizing EIN3 and EIL1 proteins (Guo and Ecker, 2003; Potuschak

1 These authors contributed equally to this work.2 Address correspondence to jszhang@genetics.ac.cn and sychen@genetics.ac.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Jin-Song Zhang(jszhang@genetics.ac.cn).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00840

The Plant Cell, Vol. 32: 1626–1643, May 2020, www.plantcell.org ã 2020 ASPB.

et al., 2003; An et al., 2010) and regulating histone acetylation(Zhang et al., 2016, 2017b). Meanwhile, the EIN2 C-terminal endcan also be transferred into the cytoplasmic processing-body tobind to the 39-UTR of the EBF1/2 mRNA for translational re-pression of EIN3-BINDINGF-BOXPROTEIN1 (EBF1) and EBF2 toreleaseEIN3/EIL1 functions (Li etal., 2015;Merchanteetal., 2015).Although great progress has been made in understanding thebiochemical functionofEIN2, the regulatorymechanismupstreamof EIN2, especially how CTR1 is regulated in vivo, remains largelyunclear.

Semiaquatic rice (Oryza sativa) is an important monocoty-ledonous crop worldwide. Ethylene plays essential roles in theadaptive responses to submergence-induced hypoxia in rice andfunctions in the regulation of multiple agronomic traits. Using anefficient system,wehave screeneda set of rice ethylene responsemutants that were namedmao huzi (mhz, meaning “cat whiskers”based on the appearance of their roots), and identified conservedand novel components of ethylene signaling, i.e., MHZ7/OsEIN2(Ma et al., 2013); MHZ6/OsEIL1 (Yang et al., 2015); and the pre-viously unidentified component MHZ3, a stabilizer of OsEIN2 (Maet al., 2018).

The GDSL-family esterases/lipases are hydrolytic enzymeswidely found in bacteria and plants. This family of lipase is definedbyadistinctGDSLmotifGxSxxxxGwith theactivesiteSer locatednear the N terminus (Upton and Buckley, 1995). With a flexibleactive site, this family of lipases may have broad substratespecificity (Mølgaard et al., 2000). Numerous GDSL lipases havebeen characterized in plants and members are involved in hor-mone signaling (Oh et al., 2005; Kwon et al., 2009; Lee et al., 2009;Kim et al., 2013, 2014), cuticle formation (Park et al., 2010), xylandeacetylation (Zhang et al., 2017a), and secondary metabolism(Clauss et al., 2008; Kikuta et al., 2012). The secretedGDSL family

lipase AtGLIP1 is involved in ethylene signaling in regulatingArabidopsis resistance to Alternaria brassicicola (Oh et al., 2005;Kwon et al., 2009; Kim et al., 2013, 2014).In this article, we characterized the root ethylene-insensitive

mhz11mutant.MHZ11 encodes a previously uncharacterized ERmembrane GDSL-family lipase with acyl-hydrolyzing activity.MHZ11 facilitates ethylene signaling through modulating theethylene receptor-mediated OsCTR2 phosphorylation. Thisfunction is likely achieved partially by affecting the membranesterol homeostasis. Our findings reveal a potential mechanism bywhich theGDSL-type lipaseMHZ11modulates ethylene signalingthrough its acyl-hydrolyzing activity.

RESULTS

Phenotypic Analysis and Gene Identification of themhz11 Mutant

Rice mhz11 was identified in a genetic screen for rice ethylene-responsemutants from our ethyl methanesulfonate (EMS)mutantpopulations (Zhouet al., 2019). In air, dark-grownseedlingsofwildtypeandmhz11were similar in coleoptile and root growth. In 10mLL-1 of ethylene treatment, root growth of wild type was drasticallyrepressed comparedwith that in air, whilemhz11 root growthwasbarely inhibited, indicating a complete ethylene-insensitive phe-notype in primary roots of the mutant (Figure 1A). Coleoptilegrowth of mhz11 exhibits similar ethylene response with that ofwild type (Figure 1A). Rice ETHYLENE-RESPONSE-FACTOR002(OsERF002),RELATED-TO-AP28 (OsRAP2.8), andARABIDOPSIS-RESPONSE-REGULATOR5 (OsRRA5) were previously describedas ethylene-responsive genes (ERGs; Ma et al., 2013; Yin et al.,

MHZ11 Modulates Ethylene Signaling in Rice 1627

2015). Ethylene-induced expression of these genes inmhz11 rootswas largely abolished or hampered as compared with those of thewild type, further confirming the ethylene abnormality of mhz11roots (Figure 1B).

By map-based cloning and sequencing of the candidate geneswithin a53.3-kb regiononchromosome5, a singleC insertionwasrevealed in the third exon of LOC_Os05g11950 (Figure 1C), re-sulting in a frame-shift mutation of the gene. A construct carrying

Figure 1. Ethylene Response Analysis and Gene Identification of mhz11.

(A) Ethylene response phenotype of mhz11 mutant. Dark-grown seedlings of the wild type (WT) and mhz11 were treated with varied concentrations ofethylene for 2.5 d. Representative seedlings grownwith or without 10 mL L-1 of ethylene are shown (left). Relative coleoptile (middle) and root (right) lengthsare means 6 SD (n > 25) calculated from at least 25 seedlings.(B)Ethylene-inducedgeneexpressionofOsERF002,OsRAP2.8, andOsRRA5 is abolishedorhampered inmhz11 roots.Two-d–oldetiolatedseedlingsweretreatedwithorwithout 10mLL-1of ethylene.Data aremeans6 SD,n53 (threebiological replicates, six seedlingsper replicate; *P<0.05, **P<0.01,Student’st test; compared with each corresponding wild-type [WT] control).(C) Fine mapping of the MHZ11 gene. The mutation sites are indicated in schematic diagrams. mhz11 (mhz11-1) was obtained by screening our EMS-mutagenized lines. mhz11-2 is a T-DNA insertion mutant requested from the POSTECH Biotech Center (http://www.postech.ac.kr/eng/).(D) Functional complementation ofmhz11withMHZ11 genomicDNA. Vector carrying thewild-typeMHZ11genomicDNAwas transformed into themhz11mutant, rescuing the ethylene responseofmhz11-etiolated seedlings in transgenic lines (top). Themhz11background in the transgenic lineswas confirmedby dCAPS analysis (lower representation). The fragment of mhz11 mutant was 18-bp shorter than that of wild type (WT).Scale bars 5 10 mm.

1628 The Plant Cell

the wild-type genomic sequence of the gene (3,016-bp sequenceupstreamof ATGcodon, 2,756-bp genomic coding sequence, and1,578-bp sequence downstream of stop codon) was transformedinto mhz11 and rescued the ethylene-insensitive phenotype ofmhz11, demonstrating that MHZ11 is LOC_Os05g11950. Themhz11 background of the transgenic lines was confirmed by de-rived cleaved amplified polymorphic sequence analysis (dCAPS;Figure 1D).

A rice mutant PFG_1A-21225.R from the POSTECH BiotechCenter (Yi and An, 2013) was further identified and namedmhz11-2, following the original mutant mhz11-1 (mhz11 for simplicity).This mhz11-2 had a T-DNA insertion 106-bp upstream of theATG codon of LOC_Os05g11950 and showed hardly detectableLOC_Os05g11950 transcripts compared with wild type.mhz11-2exhibited similar ethylene responses as mhz11 (SupplementalFigure 1A). This further confirmed that alteration of LOC_Os05g11950 is responsible for the mhz11 phenotype.

MHZ11 Encodes a Putative ER Membrane-IntegratedGDSL Lipase

Examination of sequence alignment suggested that MHZ11encodes a putative GDSL lipase (Supplemental Figure 1B).Conserved residues Ser, Gly, Asn, and His in four conservedblocks I, II, III, and V were found within MHZ11 (SupplementalFigure 1C), suggesting it belongs to the SGNH subgroup hy-drolases, which usually have broad substrate specificity dueto their flexible active sites (Mølgaard et al., 2000). MHZ11is predicted to be a type-II membrane protein by the server pro-gram TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/;Supplemental Figure 1D). To determine whether MHZ11 ismembrane-localized, MHZ11-FLAG/mhz11 transgenic lines weregeneratedby transforming theProMHZ11:MHZ11-FLAGconstructinto mhz11. These transgenic lines exhibited similar ethylene re-sponses as the wild type, indicating that the expressed MHZ11-FLAG proteins are functional (Figure 2A). Total proteins of rootswere subsequently extracted and separated into soluble and mi-crosomal fractions by ultracentrifugation. A small amount ofMHZ11-FLAGproteinwasdetected inthecytosol (thesupernatant),but MHZ11-FLAG protein was mainly detected in the microsomalfraction, indicating that MHZ11 is a membrane-associated protein(Figure 2B). To further determine the nature of the membrane as-sociation, microsomes extracted from MHZ11-FLAG/mhz11transgenic lines were further subjected to various extractions.Results showed that compared with the Triton X-100 or SDStreatments extracting the membrane protein MHZ11 (Figure 2C,lanes 5 to 8), neither high salt nor alkali treatments, which are ef-fective in extracting most peripherally associated proteins, wereable to remove MHZ11-FLAG from the membranes (Figure 2C,lanes1 to4), indicating thatMHZ11 is integrated into themembranerather than peripherally associated with it.

MHZ11-GFP protein was further transiently expressed in epi-dermal cells of Nicotiana benthamiana leaves and GFP fluores-cence was mainly detected in a reticular network-like structurelabeled by the ERmarker proteinmCherry-HDEL, suggesting thatMHZ11-GFP is mainly localized to the ER membrane (Figure 2D).The localization of different truncated versions of MHZ11 fusedwith GFP revealed that MHZ11 is bound to the ER membrane

through its N-terminal transmembrane domain (Figure 2E). Be-cause the N-terminal hydrophobic sequences in some GDSLfamily members may function as a signal peptide for secretion ofthe protein (Oh et al., 2005; Lee et al., 2009), the possibility thatMHZ11 may be secreted to the intercellular space was furthertested. N. benthamiana leaf cells expressing MHZ11-YFP weretreated with 30% Suc for plasmolysis, and the yellow fluores-cent protein (YFP) fluorescence was barely detected in the in-tercellular space, suggesting thatMHZ11 isan intracellular protein(Supplemental Figure 2A). All these results support the predictionthat MHZ11 is an integral ER membrane protein.

MHZ11 Expression Is Induced by Ethylene, and ItsOverexpression Confers Constitutive Ethylene Response

MHZ11 transcripts were induced by ethylene in both roots andshoots ofwild-type–etiolated seedlings, and the induction in rootswas stronger than in shoots (Figure 2F). Promoter-GUS analysisfurther confirmed the ethylene induction ofMHZ11 (SupplementalFigure 2B) in roots and coleoptiles of etiolated seedlings. Theinduction is abolished in the ethylene signaling mutants mhz7/Osein2 and mhz6/Oseil1 (Figure 2G), suggesting that ethyleneinduced MHZ11 expression through the canonical ethylene sig-naling pathway.MHZ11 fusedwith theFLAG tagdrivenby the35Spromoterwas

constructed in plant expression vector and transformed intomhz11 to study gene function. Three lines with high MHZ11 ex-pression (MHZ11-OE lines) were selected for phenotype analysis(Figure 2H, Supplemental Figure 2C). Compared with wild type,the OE lines exhibited significantly shorter roots when grown inthe air or upon ethylene treatment (Figure 2H). Treatment with theethylene perception inhibitor 1-methylcyclopropene (1-MCP)suppressed the short root phenotype of MHZ11-OE lines(Figure 2I), suggesting that the constitutive short root pheno-type was caused by enhanced ethylene response and MHZ11function requires ethylene signal perception. Because theethylene production in the OE lines was not enhanced com-pared with wild type (Supplemental Figure 2D), it is likely thatthe short root phenotype in theMHZ11-OE lines was due to anenhanced ethylene signaling. These results suggest thatMHZ11 facilitates ethylene signal transduction in rice roots.

MHZ11 Functions through Its Acyl-Hydrolyzing Activity toModulate Lipid Metabolism and Ethylene Response

As MHZ11 encodes a putative SGNH subfamily hydrolase thatmay have broad substrate specificity, a structure-based pre-diction of its specific substrates would be difficult. Therefore, weapplied a lipidomics approach to study the possible substrates forMHZ11 in vivo. By analyzing the lipidomics data of the wild type,mhz11, and theoverexpression lineOE-4, a variety of lipids relatedto MHZ11 were identified and divided into three groups: (1)Phospholipids including phosphatidylcholines (PCs), phospha-tidylethanolamines (PEs), phosphatidylserines (PSs), and phos-phatidylglycerols (PGs) etc.; (2) Triacylglycerols (TAGs) anddiacylglycerols (DAGs); and (3) Free phytosterols and phytosterylesters (Figures 3A to 3C; Supplemental Data Set 1). Under air

MHZ11 Modulates Ethylene Signaling in Rice 1629

Figure 2. MHZ11 Is Localized to the ER Membrane and Positively Regulates Ethylene Signaling.

(A)MHZ11-FLAG protein rescued the ethylene-insensitive phenotype ofmhz11 roots. Two-kb promoter ofMHZ11was cloned to drive theMHZ11-FLAG.Vector carrying ProMHZ11:MHZ11-FLAG was transformed into mhz11 and ethylene responses of the homozygous lines, and checked. Dark-grownseedlings were treated with 10 mL L-1 of ethylene. Scale bar 5 10 mm.(B) Membrane association of MHZ11. Equal amounts of total protein (T), soluble protein (S), and microsomal membranes (M) were immunoblotted forMHZ11-FLAG, BiP (ER membrane marker), and UGPase (cytoplasm marker).(C)MHZ11 is an integral membrane protein. Microsomes were prepared from roots ofMHZ11-FLAG/mhz11 transgenic plants. The membrane pellet wasresuspended in 0.1MofNaCl, 0.1 ofMNa2CO3 at pH11, and1% (v/v) Triton X-100or 1% (w/v) SDS.Sampleswere separated into supernatant (S) andpellet(P) fractions by centrifugation at 125,000g for 1 h. Equal amounts of proteins were immunoblotted for MHZ11-FLAG.(D)ERmembrane localization ofMHZ11protein as revealed by transient expression ofMHZ11-GFP inN.benthamiana leaf epidermal cells.mCherry-HDELserved as ER marker. Scale bars 5 20 mm. Five randomly chosen regions of interest of infiltrated leaves were observed with similar results.(E)Localizationofdifferent truncatedversionsofMHZ11protein.TM, transmembranemotif ofMHZ11.Scalebars520mm.Five randomlychosen regionsofinterest of infiltrated leaves in each group were observed with similar results.

1630 The Plant Cell

treatment, thePS,DAG, and freephytosterol levels inmhz11weresignificantly higher than those in the wild type. PC, PE, and PGcontents in mhz11 were also slightly higher than the wild type(Figures 3A and 3C). Meanwhile, TAG and phytosteryl estercontents inmhz11were lower than those in wild type (Figures 3Band 3C). In the OE-4 line, PC, PE, PS, PG, and free sitosterolcontents were lower than those in the wild type (Figures 3A and3C),whileTAG, sitosteryl ester, andcampesteryl ester levelswerehigher than the wild type (Figures 3B and 3C). Under ethylenetreatment, levels of PC, PE, TAG, and steryl esters in all the wildtype, mhz11, and OE-4 lines were enhanced compared withthe corresponding values in air (Figures 3A to 3C). The patternchanges of lipid species in mhz11 and OE-4 compared with thewild typeafter ethylene treatment are similar to that in theseplantswithout treatment. In ethylene-treated mhz11, contents of PC,PE, PS, PG,DAG, and free sterols are higher than those in thewildtype,whilecontentsof steryl estersare lower (Figures3A to3C). Inethylene-treatedOE-4 line, PC, PE, PS and DAG levels are lowerthan those in the wild type, while TAG and campesteryl esterlevels are higher (Figures 3A to 3C). From these results, wepropose that MHZ11 may participate in the hydrolysis of phos-pholipids like PC, PE, PS, and PG, etc. to produce free fatty acids(FFAs), whichwere subsequently supplied for the biosynthesis ofTAG and phytosteryl esters using DAG and free phytosterols,respectively, as substrates, possibly with the help of other rel-evant enzymes.

FFAs and lyso-phospholipids are the hydrolysis products ofphospholipids (Ryu, 2004). In our lipid profile data, several FFAspecies (FFA18:3, FFA18:1, and FFA16:1) showed increases inMHZ11-OE plants but decreases in mhz11 in air and/or ethylene(Figure 3C). Regarding lyso-phospholipid species, while several(LPC16:0, LPE16:0, and LPS16:0) are roughly positively associ-ated with MHZ11, some other species exhibit opposite trends orare not closely associated with MHZ11 (Figure 3C).

TheabilityofMHZ11 tohydrolyzePCwas tested.MHZ11-FLAGprotein from rootsof thePro35S:MHZ11-FLAG/mhz11 transgenicplants were purified and tested for phospholipase A (PLA) activity.The MHZ11 protein exhibits strong PLA2 activity in the in vitroassay (Figure 4A). By contrast, the purified MHZ11(S39A)-FLAGprotein, with a mutation at the active site Ser, barely had anyenzyme activity (Figure 4A). These results indicate that MHZ11may function as a hydrolase with PLA2 activity and hydrolyzephospholipids to release FFAs for subsequent ester biosynthesis.Consideringthatavarietyof lipidspecieswerealteredbytheMHZ11mutationandoverexpression (Figures 3A to3C;SupplementalData

Set 1), it is also possible thatMHZ11may have an acyl-hydrolyzingactivity with broad substrate specificity.We further investigated the effect of the MHZ11(S39A) mutation

on ethylene response, and found that the MHZ11(S39A)-FLAGprotein could not rescue the ethylene-insensitive phenotype ofmhz11 inProMHZ11:MHZ11(S39A)-FLAG/mhz11 lines (Figure 4B).By contrast, normal MHZ11-FLAG protein can rescue the mhz11ethylene response phenotype in ProMHZ11:MHZ11-FLAG/mhz11lines. These results demonstrate that MHZ11 participates in eth-ylene responses through its acyl-hydrolyzing activity.

MHZ11 Functions Upstream of OsCTR2 in the EthyleneSignaling Pathway

To study the genetic relationship of MHZ11 with the knownethylene signaling components, genetic analyses were per-formed. In the wild type, overexpression of OsEIN2 and OsEIL1resulted in strong constitutive and enhanced ethylene responses(Figures 5A and 5B). By crossing the overexpression lines ofOsEIN2 and OsEIL1 with mhz11, lines overexpressing OsEIN2 orOsEIL1 in mhz11 background were generated. These lines ex-hibited a constitutive response phenotype in air and enhancedethylene responses in roots after ethylene treatment (Figures 5Aand 5B), suggesting that MHZ11 may function upstream ofOsEIN2 and OsEIL1. We further generated lines overexpressingMHZ11 in the Osein2 and Oseil1 backgrounds by crossing.The constitutive short roots and root ethylene response ofMHZ11OE-4 were completely abolished by Osein2 or Oseil1mutation, further confirming that MHZ11 acts upstream ofOsEIN2 and OsEIL1 in ethylene-regulated root growth (Figures5C and 5D). In rice, the ethylene-receptor loss-of-functionmutant Osers2 had mildly shorter roots compared with the wildtype either in air or under ethylene treatment (Figure 5E). Doublemutant analysis revealed that the Osers2 mhz11 exhibited longroots and ethylene insensitivity similar tomhz11, indicating thatMHZ11 may function at or downstream of ethylene receptors(Figure 5E).Rice OsCTR2 is the functional homolog of Arabidopsis CTR1

(Wang et al., 2013). To study the genetic relationship betweenMHZ11 and OsCTR2, Osctr2 single and Osctr2 mhz11 doublemutants were generated by mutating OsCTR2 in wild-type ormhz11 background using clustered regularly interspaced shortpalindromic repeats (CRISPR)/Cas9. Two Osctr2 lines and twoOsctr2mhz11 linesbearing the samemutation sitewithinOsCTR2(1039Tdeletion)wereused forphenotypeobservation. TheOsctr2

Figure 2. (continued).

(F) Gene expression ofMHZ11 is induced by ethylene as revealed by qPCR. Data are means6 SD, n5 3, six seedlings per replicate (*P < 0.05, **P < 0.01,Student’s t test; each compared with the corresponding 0-h control). Two-d–old dark-grown seedlings were treated with 10 mL L-1 of ethylene for varioustime lengths before being collected for RNA extraction.(G)Ethylene induction ofMHZ11 is abolished inOsein2 andOseil1mutants as revealed by qPCR. Two-d–old dark-grown seedlingswere treatedwith 10 mLL-1 of ethylene for 6 h. Data are means 6 SD, n 5 3, six seedlings per replicate (*P < 0.05, Student’s t test; compared with the corresponding air control).(H) Constitutive short root phenotype of MHZ11-OE lines. Vector carrying Pro35S:MHZ11-FLAG was transformed into the mhz11 mutant and threetransgenic lines overexpressing MHZ11 were used for phenotype analysis. Dark-grown seedlings were treated with 10 mL L-1 of ethylene for 2.5 d. Rootlengths are means 6 SD (n > 30) calculated from at least 30 seedlings (**P < 0.01, Student’s t test; compared with the wild type [WT]).(I)1-MCPsuppressed theshort rootphenotypeofMHZ11-OE.Dark-grownseedlingswere treatedwithorwithout10mLL-1of1-MCP for2d.Scalebars510mm.Root lengthsaremeans6SD (n>30)calculated fromat least30seedlings (**P<0.01,Student’s t test; eachcomparedwith thecorrespondingair control).

MHZ11 Modulates Ethylene Signaling in Rice 1631

Figure 3. MHZ11 Modulates Lipid Metabolism in Rice Roots.

(A) and (B)MHZ11-related lipids in roots of thewild type (WT),mhz11mutant, andMHZ11OE-4 transgenic line. Roots of 3-d–old etiolated seedlings treatedwith or without 10 mL L-1 of ethylene for 6 h were collected andweighed precisely. Polar lipids were analyzed using amodel no. 1260 HPLC system (Agilent

1632 The Plant Cell

mutant showed enhanced short root phenotype and Osctr2mutation suppressed the long root phenotype of mhz11 underethylene treatment, suggesting that MHZ11 may function up-stream of OsCTR2 (Figure 5F). All the genetic analyses suggestthat MHZ11 acts at or downstream of ethylene receptors, butupstream of OsCTR2, OsEIN2, and OsEIL1 to regulate ethyleneresponse of rice roots.

MHZ11 Is Essential for Ethylene-Induced Inhibition ofOsCTR2 Phosphorylation

Given that MHZ11 may act upstream of OsCTR2, whether andhow MHZ11 would affect OsCTR2 to regulate the ethylene re-sponse was further explored. A specific anti-OsCTR2 polyclonalantibody was generated by immunizing mice with polypeptidesof OsCTR2 N terminus-specific sequence DKGGDPADRPAG-SSGGGG. When testing the specificity of the antibody, an ad-ditional band above the original OsCTR2 band was detected,suggesting that the OsCTR2 protein is likely subject to post-translational modification (Figure 6A). The upper band vanishedafter 30 min of l-protein phosphatase treatment, suggesting thatthe upper band represents a phosphorylated version of OsCTR2(OsCTR2-P; Figure 6B).

Furthermore, we tested whether the phosphorylated OsCTR2resulted from its autophosphorylation. We generated a catalyti-cally inactive OsCTR2(D655E), which mimicked the ArabidopsisCTR1-1 mutant version CTR1(D694E; Huang et al., 2003). Inprotoplasts of wild-type rice, while the OsCTR2 suppressed ex-pression of the three ethylene responsive genes compared withcontrol, the OsCTR2(D655E) protein lost the ability to suppressthe expression of these genes (Figure 6C), indicating thatOsCTR2(D655E) is biologically inactive. Also, OsCTR2(D655E)failed to suppress OsEIN2 accumulation (Supplemental Figure 3),further indicating that OsCTR2(D655E) has lost the ability tosuppress ethylene signaling. Themutant protein OsCTR2(D655E)did not produce any phosphorylated form (Figure 6C), suggestingthat the phosphorylated OsCTR2 is a consequence of auto-phosphorylation and OsCTR2 phosphorylation represents itskinase activity.

We subsequently examined the effect of MHZ11 on OsCTR2phosphorylation status in roots in response to ethylene. In wild-type roots, phosphorylation levels of OsCTR2 decreased overtime upon ethylene treatment within 1 h (Figure 6D, top). Mean-while, the phosphorylation status of OsCTR2 stayed largelyunchanged at high level inmhz11 (Figure 6D, middle). InMHZ11-overexpressing line OE-4, an apparent decline of OsCTR2phosphorylation was observed in 15min after ethylene treatment,and the phosphorylated OsCTR2 completely disappeared in30 min after ethylene treatment (Figure 6D, bottom). After 1-MCPtreatment blocking ethylene perception, all plants showed similar

OsCTR2 phosphorylation levels, with a higher level of OsCTR2-Pcompared by OsCTR2. These results indicate that MHZ11 pro-motes ethylene inhibition of OsCTR2 phosphorylation/activity.Because mhz11- and 1-MCP–treated samples had more

OsCTR2-P than OsCTR2, we examined whether similar statuswas present in other ethylene-insensitivemutants identified in ourprevious studies. In air, the wild type and different mutants hadsimilar higher level of OsCTR2-P than OsCTR2, indicating thatmoreOsCTR2proteins are in active forms under normal condition(Figure 6E, bottom). After ethylene treatment (10 mL L-1, 1 h),phosphorylation levels of OsCTR2 decreased apparently in wildtype, indicating that ethylene inhibitedOsCTR2activity (Figure6E,top).While theOsein2andOseil1mutantshadsimilardecreasesofOsCTR2 phosphorylation as wild type in ethylene, the phos-phorylation status of OsCTR2 inmhz11 and Osers2d (a dominantOsERS2 gain-of-function mutant) remained largely unaffected byethylene (Figure 6E, top). These results indicate that MHZ11mutation and OsERS2 gain-of-function mutation may lockOsCTR2 in a phosphorylation state, whereas mutations indownstream components OsEIN2 and OsEIL1 do not affectethylene inhibition of OsCTR2 phosphorylation. This findingsuggests that bothMHZ11andethylene receptors are required forethylene inhibition of OsCTR2 phosphorylation, and MHZ11 mayact at ethylene receptor level for this function.Because MHZ11 affects OsCTR2 phosphorylation/activity, we

further examined whether the downstream OsEIN2 protein levelswere altered. In air, no or little OsEIN2 protein was detected in thewild type andmhz11, while OE-4 had OsEIN2 protein (Figure 6F).This is consistent with the observation that the majority ofOsCTR2 was in the active phosphorylation state in the wild typeandmhz11 in the air, while only half or less of theOsCTR2 inOE-4was phosphorylated (Figures 6D and 6E). The more phosphor-ylated OsCTR2 correlates with less OsEIN2 and vice versa. Inethylene, OsEIN2 levels were elevated in both the wild typeand OE-4 but not in mhz11 (Figure 6F). These results supportthat MHZ11 mediates ethylene-induced OsEIN2 accumulation,a likely downstream event after OsCTR2 dephosphorylation/inactivation by MHZ11 function.Taken together, our data indicate that MHZ11 is essential for

ethylene-induced OsCTR2 dephosphorylation/inactivation.

MHZ11 Functions at the Ethylene Receptor Level

Given that both MHZ11 and the ethylene receptor OsERS2 arerequired for ethylene-induced inhibition of OsCTR2 phosphory-lation, epistatic analysis was further performed to identify theirrelationship.MHZ11OE-4 linewascrossedwith theOsERS2gain-of-functionmutation (Osers2d) and theethylene responseofOE-4/Osers2d was examined. The constitutive short root and rootethylene response ofOE-4were completely abolished byOsers2d

Figure 3. (continued).

Technologies) coupledwitha triplequadrupole/ion trapmassspectrometer (QTRAP5500).Dataaremeans6 SD from fourbiological replicates (10seedlingsper replicate). Student’s t test indicates a significant difference compared with the corresponding WT control (*P < 0.05).(C)Heatmaps of MHZ11-related lipids in the wild-type (WT),mhz11mutant, andMHZ11 transgenic overexpression lineOE-4 according to the lipid profileanalysis.Bars indicate theZ-score.ET, ethylene;PI, phosphatidylinositols; LPS, lyso-PS;LPC, lyso-PC;LPE, lyso-PE.Student’s t test indicatesasignificantdifference compared with the corresponding WT control (*P < 0.05).

MHZ11 Modulates Ethylene Signaling in Rice 1633

(Figure 6G), indicating that Osers2d is epistatic to MHZ11 inethylene response. This was further confirmed by the presence ofmore phosphorylated OsCTR2 in OE-4/Osers2d after ethylenetreatment (Figure 6H). Thesefindingsareconsistentwith the resultthat 1-MCP treatment suppressed the short root phenotype ofOE-4 line (Figure 2I), suggesting that MHZ11’s positive role inmodulating ethylene response requires ethylene perception andsignal transduction initiation.While OsERS2 also requiresMHZ11for ethylene signaling (Figure 5E), and given the fact thatMHZ11 iscolocalized with OsERS2 on the ER membrane (Figure 6I), it maybe concluded thatMHZ11 functions at the ethylene receptor levelto modulate ethylene response.

Sterol Is Partially Responsible for the Ethylene-InsensitivePhenotype of mhz11

We demonstrated that the acyl-hydrolyzing activity of MHZ11 isrequired for ethylene responses in rice roots, and MHZ11 func-tions through affecting ethylene receptor-dependent OsCTR2phosphorylation tomodulate ethylene signaling.However, it is notclearwhat specific lipid species is actually involved in theprocess.Free sterols areprimarybilayer components regulatingmembranefluidity and flexibility and affecting the functions of membrane-associated components (Lindsey et al., 2003; Schaller, 2004).Sterol acylation plays crucial roles in maintaining the free sterolcontent of cell membranes (Lewis et al., 1987; Dyas and Goad,1993; Sturley, 1997; Schaller, 2004). Given that MHZ11 and otherethylene signaling components such as receptors andEIN2 are allER membrane-integrated, it is possible that accumulation of freesterols in mhz11 impaired the proper functions of ethylene sig-naling components and caused the ethylene-insensitive pheno-type of mhz11.

To test the hypothesis, free sterol contents were further mea-sured and compared amongwild-type,mhz11, and theOE-4 lines

using the sterol detection kit (cat. no. K603-100; BioVision).Consistent with the lipidomics data, the level of free sterols washigher inmhz11 but lower in OE-4 compared with that of the wildtype (Figure 7A). To testwhether theaccumulationof free sterols isresponsible for the ethylene-insensitive phenotype ofmhz11, weused the sterol biosynthesis inhibitor fenpropimorph (FEN; Heet al., 2003) to treat themhz11-etiolatedseedlings. In thepresenceof FEN, ethylene significantly suppressed the root growth ofmhz11, while the root growth of mhz11 under DMSO treatmentremained insensitive to ethylene (Figure 7B). The results indicatethat FEN treatment could partially rescue the ethylene-insensitivephenotype of mhz11, implying that reduction of sterol level mayfacilitate ethylene responses.As mhz11 accumulates excess sterol (Figures 3A, 3C, and 7A)

and the sterol biosynthesis inhibitor FEN partially restored theethylene response of mhz11 (Figure 7B), we tested the effect ofFENonOsCTR2phosphorylation. Under FEN treatment, ethyleneapparently inhibited the phosphorylation of OsCTR2 in mhz11roots (Figure 7C), indicating that sterol accumulation in mhz11may at least partially impair the ethylene-induced OsCTR2 de-phosphorylation. Because MHZ11 functions at the ethylene re-ceptor level tomodulate OsCTR2 phosphorylation, the possibilitythat FEN impairs the interaction between ethylene receptors andOsCTR2 was tested using the bimolecular fluorescence com-plementation (BiFC) assay. The results showed that FEN treat-ment obviously suppressed the interaction betweenOsERS2 andOsCTR2 in N. benthamiana epidermal cells using a BiFC system(Figure 7D), suggesting that low sterol levels could affect OsERS2and OsCTR2 interaction. The split luciferase complementationassay in N. benthamiana epidermal cells also demonstrated thatFEN treatment suppressed the interaction between OsERS2 andOsCTR2withoutaltering theproteinabundance (Figure7E). Takentogether, our results imply that MHZ11-derived low level ofmembrane sterol could disrupt interaction of ethylene receptors

Figure 4. MHZ11 has PLA2 Activity and the Enzyme Activity Is Essential for Its Function.

(A) PLA2 activity of MHZ11. (Left) MHZ11-FLAG and MHZ11(S39A)-FLAG proteins were purified from transgenic plants expressing MHZ11-FLAG andMHZ11(S39A)-FLAG, respectively. (Middle) PLA2 activity of MHZ11-FLAGwas tested. Proteins were incubated with a synthetic thiophospholipid at 37°C,producing a lysothiophospholipid that reactswith a fluorogenic probe to produce afluorescent product detectable in the visible range (Excitation5388nm;Emission5513nm).Beevenom (provided in thePLA2activity assaykit,BioVision) andwater servedaspositive andnegative controls, respectively. Dataaremeans6 SD calculated from three replicates. (Right) The specific activity of MHZ11-FLAG andMHZ11(S39A)-FLAGwas calculated. One unit of PLA2 is theamount of enzyme that generates 1 mmole of lysothiophospholipid metabolite per min at pH7.5 at 37°C. RFU, relative fluorescence units.(B) Ethylene responses ofMHZ11(S39A)-FLAG/mhz11 transgenic lines. Seedlings were grown in dark with 10 mL L-1 of ethylene for 2.5 d. Expression ofMHZ11-FLAGandMHZ11(S39A)-FLAGprotein in the transgenic lineswere testedusinganti-FLAGantibody.Actin servedas internal reference.Scalebar510 mm. WT, wild type.

1634 The Plant Cell

Figure 5. Genetic Relationship between MHZ11 and Ethylene Signaling Genes.

(A) and (B) Ethylene responses of OsEIN2-OE/mhz11 (A) and OsEIL1-OE/mhz11 (B) lines. The OsEIN2-OE/mhz11 and OsEIL1OE-1/mhz11 lines weregenerated by crossingmhz11withOsEIN2OE-44 (anOsEIN2 overexpression transgenic line) andOsEIL1OE-1 (anOsEIL1 overexpression transgenic line),respectively. Seedlings of each linewere grown in darknesswith 10 mL L-1 of ethylene for 2.5 d. Scale bars5 10mm.Data aremeans aremeans6 SD (n>30)calculated from at least 30 seedlings (**P < 0.01, Student’s t test).(C) and (D) Ethylene responses ofMHZ11OE-4/Osein2 (C) andMHZ11OE-4/Oseil1 (D) lines. TheMHZ11OE-4/Osein2 andMHZ11OE-4/Oseil1 lines weregenerated by crossingMHZ11OE-4 transgenic line withOsein2 andOseil1mutants, respectively. Seedlings were grown under darkness with 10 mL L-1 ofethylene treatment for 2.5 d. Scale bars 5 10 mm. Data are means 6 SD (n > 30) calculated from at least 30 seedlings (**P < 0.01, Student’s t test).(E)Ethylene responseofOsers2mhz11doublemutant (left) and root lengthquantification (right).Osers2mutanthasaDongjin (DJ)background.Seedlingsofwild type (Nipponbare [NIP] and DJ) andmutants were grown in darkness with or without 1 mL L-1 of ethylene for 2 d. Scale bars5 10mm. Root lengths aremeans 6 SD (n > 20) calculated from at least 20 seedlings (**P < 0.01, Student’s t test; compared with corresponding air control).(F)Osctr2 suppressed theethylene-insensitive roots ofmhz11.Osctr2 single andOsctr2mhz11doublemutantsweregeneratedbymutatingOsCTR2 in thewild type (WT) or mhz11 background using CRISPR/Cas9. Two Osctr2 lines and two Osctr2 mhz11 lines bearing the same mutation site within OsCTR2(1039Tdeletion)wereused forphenotypeobservation.Seedlingsofeach lineweregrown indarknesswith10mLL-1ofethylene for2.5d.Scalebars510mm.Data aremeans6 SD (n > 25) calculated from at least 25 seedlings. One-way ANOVAwas performed followed by a Tukey’smultiple comparisons test. **P <0.01, Student’s t test.

MHZ11 Modulates Ethylene Signaling in Rice 1635

with OsCTR2 and inhibit OsCTR2 phosphorylation for down-stream signaling on ER membrane (Figure 7F).

DISCUSSION

In this study, we identified an ER membrane-integrated GDSLlipase MHZ11 with acyl-hydrolyzing activity that facilitates eth-ylene signaling in rice roots. MHZ11 hydrolyzes phospholipids torelease fatty acids, which is likely further transferred to sterol to

produce sterol esters, affecting sterol homeostasis. During thisprocess, MHZ11 mediates the ethylene-inhibited OsCTR2 phos-phorylation and the subsequent OsEIN2-related signal trans-duction. This process requires ethylene signal perception andsignaling initiation. Blocking sterol biosynthesis can reduceOsERS2-OsCTR2 interaction, and suppress OsCTR2 phosphor-ylation in mhz11 in response to ethylene for restoration of eth-ylene response. Our study reveals a potential mechanism bywhich MHZ11 helps to maintain a low sterol level on ER

Figure 6. MHZ11 Modulates the Phosphorylation Status of OsCTR2.

(A) Specificity test of OsCTR2 antibody. The wild type (WT) indicates protoplast of wild-type rice. OsCTR2-GFP/WT indicates WT protoplast expressingOsCTR2-GFP protein.(B)Detection of OsCTR2 in roots of thewild type. Protein extractswere treatedwith l-PPase for different time lengths before being boiledwith SDS loadingbuffer.(C) The phosphorylation state of OsCTR2 is dependent on its kinase activity. OsCTR2(D655E) mimicked the Arabidopsis CTR1-1 mutant versionCTR1(D694E), which is catalytically inactive. OsCTR2-myc and OsCTR2(D655E)-myc were transiently expressed in protoplasts of wild-type rice andbiological activity of OsCTR2-myc andOsCTR2(D655E)-myc were compared by examining the inhibition effect they have on the expression of ERGs (left).The expressed proteins were detected with anti-myc antibody (right). Data are means 6 SD, n 5 3 (*P < 0.05, **P < 0.01, Student’s t test; compared withcontrol).(D)Phosphorylation time-courseofOsCTR2 in roots of thewild type (WT),mhz11, andMHZ11OE-4 (OE-4) in response to ethylene.Dark-grown seedlingsofwild-type,mhz11, andOE-4were treated with ethylene (ET) and 1-MCP for different time lengths and total proteins of roots were extracted for immunoblotanalysis.(E)OsCTR2 phosphorylation status in roots of different ethylene signaling mutants. Roots of 2-d–old dark-grown seedlings were treated with 10 mL L-1 ofethylene for 1 h before collected for protein extraction. ET, ethylene; WT, wild type.(F)Abundance of OsEIN2 protein in roots of thewild type (WT),mhz11, andMHZ11OE-4 (OE-4) etiolated seedlings. Two-d–old dark-grown seedlings weretreated with or without 10 mL L-1 of ethylene (ET) for 6 h. The unspecific band served as an internal reference.(G)Osers2d suppressed the short root phenotype ofMHZ11-OE. Dark-grown seedlings were treated with 10 mL L-1 of ethylene (ET) for 2 d. Scale bar5 10mm. Data are means 6 SD (n > 30) calculated from at least 30 seedlings (**P < 0.01, Student’s t test). WT, wild type.(H)Osers2dblocked the phosphorylation status change ofOsCTR2 inOE-4 in response to ethylene. Roots of 2-d–old dark-grown seedlings treatedwith 10mL L-1 of ethylene (ET) for 30 min were collected for protein extraction. WT, wild type.(I) MHZ11 colocalized with OsERS2 on the ER membrane. Scale bars 5 50 mm.

1636 The Plant Cell

Figure 7. Overaccumulated Sterol in mhz11 Is At Least Partially Responsible for the Ethylene-Insensitive Phenotype of mhz11 Roots.

(A)Freesterol contents in rootsof thewild-type (WT),mhz11mutant, andoverexpression lineOE-4. Sterolswereextracted fromrootsof 2-d–olddark-grownseedlingswithCHCl3/IPA/NP-40 (7:11:0.1)andmeasuredusingacolorimetricmethod.Dataaremeans6SD (n53),20seedlingsperbiological replicate (*P<0.05, **P < 0.01, Student’s t test).(B)Sterol biosynthesis inhibitor FEN could partially restore the ethylene-insensitive phenotype ofmhz11 roots. Representative seedlings grown in 10 mL L-1

of ethylene (ET) with DMSOor 10mMof FEN are shown (left). Lengths (middle) and relative lengths (right) of roots aremeans6 SD (n > 20) calculated from atleast 20 seedlings (**P < 0.01, ***P < 0.0001, Student’s t test; compared with the corresponding control).(C) FEN treatment partially restored the ethylene inhibition effect on OsCTR2 phosphorylation in mhz11. Roots of 2-d–old etiolated seedlings grown inDMSO or 5 mM of FEN were treated with air or 10 mL L-1 of ethylene for 1 h before being collected for protein extraction. WT, wild type.

MHZ11 Modulates Ethylene Signaling in Rice 1637

membrane, and then facilitates theethylene-triggeredand receptor-off-state–related OsCTR2 inactivation for downstream signaling(Figure 7F).

Our study discovered that MHZ11 affects the phosphorylationof rice OsCTR2, which is homologous to CTR1 in Arabidopsis(Wang et al., 2013). In air, OsCTR2 has two forms, i.e., thephosphorylated andnonphosphorylated, and thephosphorylatedform appeared to be the majority (Figure 6). Upon ethylenetreatment, the level of phosphorylated form (OsCTR2-P) is re-duced and the nonphosphorylated form seems to be enhanced,implying that the two states may be easily changeable at least atvery early stages of the treatment. This process is regulated byMHZ11. MHZ11 overexpression caused faster decrease ofOsCTR2-P level whereas MHZ11 mutation locked OsCTR2-P ata constantly higher level than nonphosphorylated OsCTR2. TheOsCTR2-P increases with increasing OsCTR2 kinase activity(Figure6C) andcanbe regardedasadefault state for ano-ethylenesituation or an ethylene receptor-on situation. By contrast, loss ofOsCTR2-P indicates the initiation of ethylene signaling, and thelower ratio of OsCTR2-P/OsCTR2 may reflect the progress ofsignaling or intensity/strength of the later ethylene response.Consistently, the OsCTR2-P level is negatively correlated with theOsEIN2 level (Figure 6), which is positively correlated with theethylene response (Qiao et al., 2009). This fact provides in vivoevidence for theconnectionbetweenOsCTR2dephosphorylation/inactivation and ethylene signaling activation, consistent with theidea that Arabidopsis CTR1 functions through its kinase activity tophosphorylate EIN2 and thus control ethylene signaling (Ju et al.,2012). Also, the findings thatOsERS2gain-of-functionmutation or1-MCPtreatmentkeepsmoreOsCTR2 in thephosphorylatedstateprovides the in vivo biochemical evidence for the relationshipbetween ethylene receptors and OsCTR2 activity, which is onlyspeculated in the canonical Arabidopsis model (Hua and Meyer-owitz, 1998).

MHZ11has demonstrated acyl-hydrolyzing activity and causeschanges of lipid profiles in rice roots. Among the MHZ11-relatedlipid species, we show that sterol plays a role in OsCTR2 regu-lation. Inhibition of sterol biosynthesis by the inhibitor FEN led topartial rescue of the mhz11 ethylene response, and this functionappears to be due to blocking of theOsERS2-OsCTR2 interaction

and suppression of OsCTR2 phosphorylation (Figure 7). Sterolsare primary components of bilayers where they regulate mem-brane fluidity, properties, and functions. Previous studies sug-gested that changes in sterol contents affect the functions ofmembrane-associated receptors or components of signal trans-ductionpathways (Lindseyet al., 2003;Schaller, 2004).Asakindofsterol modification, sterol acylation is essential in maintaining thefreesterolcontentofcellmembranesat theirphysiological levelsassteryl esters cannot participate in the bilayer of the membranes(Lewis et al., 1987; Dyas and Goad, 1993; Sturley, 1997; Schaller,2004). Phospholipids could serve as acyl donors for sterol ester-ification (Zimowski and Wojciechowski, 1981; Dyas and Goad,1993).We propose thatMHZ11 helpsmaintain a proper free sterollevel on the ERmembrane through affecting sterol esterification. Itis most likely that the low sterol levels caused by MHZ11 functionmay increasemembranefluidity andflexibility of themicrodomainshosting the ethylene receptors and CTRs, facilitating conforma-tional change and thus dissociation of the protein complex in re-sponse to ethylene for downstreamsignaling.Consistentwith this,Arabidopsis sterol biosynthesis mutants hydra1 and hydra2 ex-hibited abnormal root hair patterning that could be rescued byETHYLENE RESPONSE1 (ETR1) mutant etr1-1 or silver iontreatment (Souter et al., 2002; Lindseyet al., 2003), indicating thatsterolsmayplay roles in ethylene signal transduction. It shouldbementioned that the differences amongwild type,mhz11, andOE-4 are visually small but statistically significant, leading to thecorresponding change of ethylene response (Figures 3 and 7).Phospholipids and free sterols are the structural lipid compo-nents of cellular membranes and mild alterations of their abun-dancemay result in strong phenotype changes. Previous studiesshowed that mutations of the genes for patatin-related phos-pholipase OspPLAIIIa in rice and pPLAIIIb in Arabidopsis, bothwith PLA activities, caused similar slight changes of phospholi-pids but have a series of growth defects (Li et al., 2011; Liu et al.,2015).In addition to the structural role, campesterol also served as the

precursor for the biosynthesis of brassinosteroids (Yokota, 1997).While the brassinosteroids were detected at higher levels inmhz11comparedwith those inwild type (SupplementalFigure4A),brassinazole (Brz, a brassinosteroid biosynthesis inhibitor)

Figure 7. (continued).

(D) FEN treatment suppressed the interaction betweenOsERS2 andOsCTR2 in anN.benthamiana epidermal cell BiFC system. Different concentrations ofFEN were injected into N. benthamiana epidermal cells expressing OsERS2nYFP-FLAG and OsCTR2cYFP-myc. Confocal microscopy imaging wasperformed12hafterFENtreatment (left).OsCTR2cYFP-myc1nYFP-FLAGandOsERS2nYFP-FLAG1cYFP-mycservedasnegativecontrol.Scalebars550 mm. Immunoblot showed similar expression of OsCTR2cYFP-myc and OsERS2nYFP-FLAG proteins in each experimental group (right). Five randomlychosen regions of interest of infiltrated leaves in each group were observed with similar results.(E)FEN treatment suppressed the interactionbetweenOsERS2andOsCTR2 inN.benthamianaepidermal cell split LUCcomplementationsystem.Differentconcentrations of FEN were injected into N. benthamiana epidermal cells expressing OsERS2-nLUC-myc and OsCTR2-cLUC. (Left) Color bar indicatesrelative fold changes. Relative luminescences are means 6 SD from four leaves (**P < 0.01, Student’s t test; compared with DMSO; middle). Immunoblotshowed similar expression of OsERS2-nLUC-myc and OsCTR2-cLUC proteins in each experimental group (right).(F) A proposed working model for MHZ11. On the ER membrane, MHZ11 hydrolyzes phospholipids to release FFAs, which are used for sterol acylation,affecting sterol homeostasis. By maintaining a low sterol level on ER membrane, MHZ11 facilitates ethylene-triggered– and receptor-off-state–relatedOsCTR2 inactivation for downstream signaling. MHZ11 is transcriptionally activated by ethylene signaling, which may serve as a feedback regulation. Inmhz11 mutant, overaccumulated free sterols likely hampered the ethylene-induced conformation change of ethylene (ET) receptors, leaving OsCTR2constitutively active. OsEIN2 protein is thus constitutively phosphorylated and sent to degradation, and ethylene signal transduction is blocked. WT,wild type.

1638 The Plant Cell

treatment did not restore the ethylene response of mhz11(Supplemental Figure 4B), suggesting that mhz11 phenotype isnot due to the overaccumulation of brassinosteroids.

It should be noted that while we find a potential role forMHZ11-derived low sterol level in OsCTR2 inactivation in rice roots, thepossibility that other lipid species participate in this processcannot be excluded. The effect of PA on mhz11 was tested asa previous study reported that PA binds to CTR1 and inhibits itskinase activity in vitro in Arabidopsis (Testerink et al., 2007). Di-octanoylphosphatidic acid (a water-soluble PA) treatment couldnot rescue the ethylene response of mhz11 roots, suggestingthat PA may not be responsible for the mhz11 phenotype(Supplemental Figure 5A). This discrepancy may be becausedifferent plant species may adopt different mechanisms for reg-ulation. The effects of FFAs and lyso-phospholipids on mhz11phenotype were also tested as they are the direct hydrolysisproducts of phospholipids byMHZ11. LPC16:0 and FFA18:1, thepotential hydrolyzing products of MHZ11 whose contents arehigher in MHZ11 OE-4 plants compared with the wild type(Figure 3C), were selected for the rescue. Neither rescued themhz11 phenotype (Supplemental Figures 5B and 5C), suggestingthat these lipids are unlikely to be directly responsible for themhz11 phenotype.

It should be mentioned that the secreted GDSL lipase AtGLIP1(GDSL LIPASE-LIKE 1), which is different from our present ER-membrane localized MHZ11, has been found to be involved inresistance to A. brassicicola and ethylene responses in Arabi-dopsis. This gene may contribute to the production of a systemicsignal(s) that function at thedownstreamof thecanonical ethylenesignaling pathway (Oh et al., 2005; Kwon et al., 2009; Kim et al.,2013, 2014).

While our mhz11 mutant is insensitive to ethylene in rootgrowth inhibition, it seems that the aerial parts are not affected.We examined the OsCTR2 phosphorylation status in shootsof the wild type, mhz11, and OE-4. In response to ethylene, theOE-4 line hadsimilar decrease trendofOsCTR2phosphorylationcompared with that of the wild type, while the OsCTR2 phos-phorylationwas largelymaintained inmhz11shoots (SupplementalFigure 6A). The fact that the mhz11 shoots maintained OsCTR2phosphorylation but showed normal ethylene response may bedue to the shoot/root differential responses to the change of en-dogenous OsCTR2 phosphorylation. The OsCTR2 phosphoryla-tion level in mhz11 shoots may not be sufficient to suppress theethylene response of coleoptile/shoot. In our RNA sequencing(RNA-seq) data, the ethylene responsiveness of 34% of the totalERGs in shoots is impaired in mhz11 mutant (SupplementalFigure 6B; Supplemental Data Set 2), suggesting that ethylenesignaling may be partially impaired in mhz11 shoots. q-PCRanalysis also reveals that several ERGs are differentially affectedin mhz11 shoots (Supplemental Figures 6C and 6D).

Collectively, we identified a novel ER-localized GDSL lipaseMHZ11 with acyl-hydrolyzing activity, which participates in eth-ylene signaling by modulating OsCTR2 phosphorylation. Thisfunction is likely achieved by affecting sterol homeostasis. Ourstudy discovers a previously unidentified mechanism and pro-vides insights into the understanding of howOsCTR2 is regulatedduring ethylene signal transduction.

METHODS

Plant Materials and Growth Conditions

mhz11 and Osers2d mutants were identified in our genetic screen for rice(Oryza sativa) ethylene-response mutants from our EMS-induced mutantpools of Nipponbare rice (Zhou et al., 2019). Osers2d harbors a dominantgain-of-function version of OsERS2(A32V) which is equivalent to Arabi-dopsis (Arabidopsis thaliana) etr1-3 (Hall et al., 1999). mhz7/Osein2 andmhz6/Oseil1arepreviously identifiedethylene-responsemutants in our lab(Maetal., 2013;Yangetal., 2015).mhz11-2andOsers2areT-DNA insertionmutants obtained from the POSTECH Biotech Center (Yi and An, 2013).The OsEIN2-OE/mhz11,OsEIL1OE-1/mhz11, andOsers2 mhz11 lines aregenerated by crossing mhz11 with OsEIN2OE-44 (an OsEIN2 over-expression transgenic line), OsEIL1OE-1 (an OsEIL1 overexpressiontransgenic line), and Osers2, respectively. The MHZ11OE-4/Osein2,MHZ11OE-4/Oseil1, and MHZ11OE-4/Osers2d lines were generated bycrossing MHZ11OE-4 transgenic line with Osein2, Oseil1, and Osers2d,respectively. Osctr2 single and Osctr2 mhz11 double mutants weregenerated by mutating OsCTR2 in wild-type or mhz11 background usingCRISPR/Cas9. TwoOsctr2 lines and twoOsctr2mhz11 lines fromdifferenttransformation events bearing the same mutation site within OsCTR2(1039T deletion) were used for phenotype observation.

All rice plants used in this studywere grown in the experimental fields ofthe Institute of Genetics andDevelopmental Biology in Beijing fromMay toOctober and in Hainan from November to next April each year between2014 and 2019. Ethylene treatment of rice seedlings was performed aspreviously described byMa et al. (2013, 2018). For FEN andBrz treatment,stocks solution of FEN (Cayman, 20,875) and Brz (Cayman, 16,804) wereprepared in DMSO and diluted into solutions of different concentrationswith water. Equivalent volumes of DMSO were added to the control.

Map-Based Cloning and Complementation Assay

F2 mapping populations used for map-based cloning was generated bycrossingmhz11with an indica rice, MH63. TheMHZ11 locus wasmappedto the short armof chromosome5,within a 53.3-kb genomic region. Genesand the relevant sequences in this region were amplified for sequencing.For genetic complementaryassay,wild-typegenomicsequenceofMHZ11(3,016-bp sequence upstream of ATG codon, 2,756-bp genomic codingsequence, and 1,578-bp sequence downstream of stop codon) wascloned into the pCAMBIA2300 vector and transformed into mhz11. Themhz11 background in the transgenic lines was confirmed by dCAPSanalysis. The fragment ofmhz11mutantwas18-bpshorter than that of thewild type. The primers are listed in Supplemental Table.

Gene Expression Analysis by Real-Time PCR

For gene expression analysis under ethylene treatment, 2-d–old etiolatedseedlings of wild type andmhz11were treated with or without 10 mL L-1 ofethylene. Roots and shoots of the seedlings were harvested for total RNAextraction using TRIZOL reagent (Invitrogen). RNA was treated withTURBO DNase (Invitrogen) for genomic DNA digestion. cDNAs weresynthesized using a Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific) and real-time PCR was conducted according to the in-structions of TransStart Green qPCR SuperMix (TransGen Biotech).OsActin2 was used as internal control. The primers are listed in theSupplemental Table. Each data point has three biological replicates.

GUS Staining

Etiolated seedlings treated with air or ethylene were fixed in 90% (v/v)acetone on ice for 15 min and washed with staining buffer (100 mM of Na3

MHZ11 Modulates Ethylene Signaling in Rice 1639

PO4buffer atpH7.0,10mMofEDTA,5mMofpotassiumferricyanide,5mMof potassium ferrocyanide, and 0.1% [v/v] Triton X-100) for two times.Samples were then soaked in staining solution, which is a staining buffercontaining 0.5mg/mL of X-Gluc (cat. no. B8049; Sigma-Aldrich) for 10minin a vacuum system before being incubated at 37°C in the dark for 1 h. Thesamples were observed using a stereo microscope (model no. M165 FC;Leica; Ma et al., 2014).

Phylogenetic Analysis

Alignment of MHZ11 and the GDSL-family members in Arabidopsis andrice were analyzed according to the sequences from Chepyshko et al.(2012) and Lai et al. (2017). The phylogenetic tree of plant GDSL lipases/esterases was built using maximum likelihood (ML) with the programMEGA6 (Tamura et al., 2013). One-thousand bootstrap replicates wereeach used in nearest-neighbor interchange searches for the best ML tree.The ML tree was visualized online with the program iTOL (http://itol.embl.de/; Letunic and Bork, 2019). Sequence alignments and the tree file areprovided as Supplemental Files 1 and 2, respectively.

Subcellular Localization

Microsomal membrane proteins were isolated according to Ma et al.(2018). In brief, 3 g of seedling roots were ground in liquid nitrogen to a finepowderanddissolved in3mLofextractionbuffer (100mMofTris-HCl atpH8.0, 150 mM of KCl, 5 mM of EDTA, 10% Glycerol [v/v], 3.3 of mMdithiothreitol, 0.6% [w/v] polyvinylpyrrolidone, and 2% [v/v] protease in-hibitor cocktail; Sigma-Aldrich). Homogenates were centrifuged at16,000g for 10 min at 4°C for three times to remove debris. Supernatantswere further centrifuged at 100,000g for 60min at 4°C and themicrosomalmembrane were washed three times with extraction buffer before dis-solved in 100 mL of extraction buffer containing 1% (v/v) Triton X-100 and0.1% (w/v) SDS. Sampleswere heated at 65°C for 10minwith SDS loadingbuffer before loaded for SDS-PAGE and immunoblot analysis.

For analyses of MHZ11 subcellular localization, MHZ11 and differenttruncated versions were fused to GFP or YFP and transiently expressed inNicotiana benthamiana leaf cells before fluorescence was observed in theepidermal cells. HDEL-mCherry was used as an ER marker. For plas-molysis analysis, N. benthamiana leaf cells expressing MHZ11-YFP weretreated with 30% (w/v) Suc for 5 min.

Ethylene Production Measurement

Ethylene production measurement was performed according to Yin et al.(2015). In brief, seedlings of wild-type,mhz11, andOE lines were grown in40-mL uncapped vials for 7 d in dark at 28°C. Vials were then sealed withrubber syringe caps for 17 h before 1 mL of headspace of each vial wasmeasured using a gas chromatograph (model no. GC-2014; Shimadzu).

Lipidomics Analysis

The lipidomics analysis of rice seedlings was performed as described byXiong et al. (2017). In brief, roots of 3-d–old etiolated rice seedlings werecollected for lipidextraction,whichwasperformedusingamodifiedversionof the Bligh and Dyer’s protocol as described by Lam et al. (2014b). Polarlipids were analyzed using a model no. 1260 HPLC system (AgilentTechnologies) coupled with a triple quadrupole/ion trap mass spectrom-eter (QTRAP 5500; SCIEX) as described by Lam et al. (2014a). Four bi-ological repeatswereperformed. Individual lipidspecieswerequantifiedbyreferencing to spiked internal standards.

Enzyme Activity Assay

The PLA2 activity of MHZ11 was tested using a PLA2 Activity Assay Kit(cat. no. K400-100; BioVision). MHZ11-FLAG and MHZ11(S39A)-FLAGproteins were purified from transgenic plants using the anti-FLAG M2Magnetic Beads (Sigma-Aldrich) and concentrated. For PLA2 activitymeasurement, proteins were incubated with a synthetic thiophospholipidat 37°C, producing a lysothiophospholipid that reacts with a fluorogenicprobe to produce a fluorescent product detectable in the visible range(Excitation 5 388 nm/Emission 5 513 nm), and measured in relativefluorescence units. Bee venom provided in the PLA2 activity kit and waterserved as positive and negative controls, respectively.

Measurement of Brassinosteroids

Measurements of brassinosteroids in rice seedlings were performed aspreviously described by Xin et al. (2016). Roots of 3-d–old etiolated riceseedlings were collected and ground in liquid nitrogen to a fine powderbefore brassinosteroid extraction. Brassinosteroid contents wereanalyzed based on ultra-performance liquid chromatography-massspectrometry.

OsCTR2 Antibody Generation and Immunoblot Analysis

The specific anti-OsCTR2 and anti-OsEIN2 polyclonal antibodies weregenerated by immunizingmicewith synthetic KLH-conjugated peptides ofOsCTR2 N terminus-specific sequence DKGGDPADRPAGSSGGGG andOsEIN2-specific sequence PNILESDNKPLGGNNPS, respectively. Forimmunoblot analysis, proteins were heated at 65°C for 5 min with SDS-PAGE loading buffer and separated using SDS-PAGE. Primary antibodydilutions were in PBS containing 3% (w/v) milk. The anti-OsCTR2 and anti-OsEIN2 antibodies were diluted in Immunoreaction Enhancer Solution I(Toyobo). The primary antibodies used were: anti-FLAG (FLA-1, 1:3,000,cat. no. M185-3; MBL), anti-BiP (1:5,000, ER marker, cat. no. AS09 481;Agrisera), anti-UGPase (1:5,000, cytoplasm marker, cat. no. AS05 086;Agrisera), anti-GFP (7G9, 1:5,000, cat. no. M20004H; Abmart), anti-c-Myc(19C2, 1:2,000, HRP-conjugated, cat. no. M20019L; Abmart), anti-OsCTR2 (1:10,000), and anti-OsEIN2 (1:10,000). Secondary goat anti-mouse-IgG-horseradish peroxidase (cat. no. M210021; Abmart) antibodywas used at 1:10,000 dilutions in PBS containing 3% (w/v) milk to detectFLAG-tagged proteins, while also being used at 1:10,000 to detectOsCTR2 and OsEIN2. Secondary goat anti-rabbit-IgG-horseradish per-oxidase (cat. no. M210011; Abmart) antibody was used at 1:10,000 dilu-tions in PBS containing 3% (w/v) milk. The signals were detected bychemiluminescence method using WesternBright ECL Detection Kit (cat.no. K-12045-D50; Advansta) while the WesternBright Sirius Detection Kit(cat. no. K-12043-D10; Advansta) was used to detect OsCTR2 andOsEIN2.

BiFC Assays

For interaction of OsERS2 and OsCTR2, OsERS2 and OsCTR2 codingsequences were fused to the N-terminal half of YFP (nYFP) and C-terminalhalf (cYFP), respectively. The nYFP and cYFP were tagged with flag andmyc, respectively. Plasmids were transformed into N. benthamiana leafcells throughAgrobacterium tumefaciens strainGV3101. The combinationof OsCTR2cYFP-myc and nYFP-FLAG, OsERS2nYFP-FLAG, and cYFP-mycservedasnegative controls. TransformedN.benthamianaplantsweregrown at 23°C for 48 h before being infiltratedwith different concentrationsof FEN or DMSO. YFP fluorescence was detected 12 h later usinga confocal microscope.

1640 The Plant Cell

Split Luciferase Complementation Assay

OsERS2 andOsCTR2 coding sequences were fused to the N-terminal halfof Luciferase (LUC) andC-terminal half, respectively. TheN-terminal half ofLUC was tagged with myc tag. Plasmids were transformed into N. ben-thamiana leaf cells through A. tumefaciens strain GV3101. TransformedN.benthamianaplantsweregrownat23°C for48hbeforebeing infiltratedwithdifferent concentrations of FEN or DMSO. LUC activity was observed witha low-light cooled charge-coupled device imaging apparatus (iXon; AndorTechnology).

Statistical Analysis

The relative root or coleoptile length of each mutant is analyzed relative tothe length in untreated conditions. All of the data were analyzed usingANOVA or Student’s t test. Detailed descriptions of statistical analyses arepresented in Supplemental Data Set 3.

RNA-Seq Analysis

For RNA-seq analysis, 2-d–old etiolated seedlings of wild type andmhz11were treatedwith air or 10mLL-1 of ethylene for 8 h. Roots andshoots of theseedlings were collected separately and subjected to RNA-seq analysiswith three biological replicates. Genes with at least 2-fold changes ofexpression in ethylene treatment compared with those in the air (falsediscovery rate#0.05)were identifiedasERGs. ERGsno longer inducedbyethylene in mhz11 were identified as MHZ11-dependent ERGs.

Data Availability

The rawsequencingdata reported in this article havebeendeposited in theSequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under a Na-tional Center for Biotechnology Information BioProject accession number(PRJNA600591) and National Center for Biotechnology Information Bio-Sample accession numbers (SAMN13826478 to SAMN13826513).

Accession Numbers

Sequence data from this article can be found in the GenBank library underthe following accession numbers: Os05g11950 (MHZ11), Os06g08340(OsERF002), Os11g05740 (OsRAP2.8), Os07g26720 (OsRRA5),Os05g06320 (OsERS2), Os07g06130 (OsEIN2), Os03g20790 (OsEIL1),and Os02g32610 (OsCTR2).

Supplemental Data

Supplemental Figure 1. MHZ11 is a GDSL lipase that positivelymodulates ethylene response supports Figures 1 and 2).

Supplemental Figure 2. MHZ11 is localized to the ER membrane andis induced by ethylene (supports Figure 2).

Supplemental Figure 3. OsCTR2 kinase activity is required for itsfunction in ethylene signaling (supports Figure 6).

Supplemental Figure 4. MHZ11 affects the brassinosteroids levels inrice roots (supports Figure 7).

Supplemental Figure 5. PA, LPC, and FFA and treatment failed torescue the ethylene insensitivity of mhz11 roots (supports Figure 7).

Supplemental Figure 6. OsCTR2 phosphorylation status in shootsand expression of ERGs in roots/shoots of rice (supports Figures 6and 7).

Supplemental Table. Primers used in this study.

Supplemental Data Set 1. Lipid profile analysis of wild-type, mhz11,and OE-4 roots in air and ethylene.

Supplemental Data Set 2. RNA-seq analysis of ERGs in wild type andmhz11 mutant.

Supplemental Data Set 3. Summary of statistical tests.

Supplemental File 1. Alignment of GDSL family lipases from rice andArabidopsis.

Supplemental File 2. Phylogenetic relationships among GDSL familylipases from rice and Arabidopsis.

ACKNOWLEDGMENTS

Thiswork is supportedby theNationalNatural ScienceFoundationofChina(31530004, 31670274, and 31600980), 973 Projects (2015CB755702, andthe State Key Lab of Plant Genomics.

AUTHOR CONTRIBUTIONS

J.-S.Z., H.Z., B.M., and S.-Y.C. designed the research; H.Z. performedmost of the research; B.M. and K.-X.D. isolated the mutants; X.-K.L.performed the rice protoplast isolation and transformation; X.L. performedRNA-seq analysis; C.-C.Y., J.-J.T., W.W. and W.-K.Z. helped performedthe genetic analyses; G.-H.S. andS.M.L. performed the lipidmeasurementand analysis; J.-F.C. and P.-Y.X. performed the brassinosteroid measure-ments; all authors contributed to material preparation and data analysis;H.Z. and J.-S.Z. wrote the article; all authors read and approved the article.

Received October 28, 2019; revised March 2, 2020; accepted March 15,2020; published March 17, 2020.

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MHZ11 Modulates Ethylene Signaling in Rice 1643

DOI 10.1105/tpc.19.00840; originally published online March 17, 2020; 2020;32;1626-1643Plant CellJin-Song Zhang

Wan-Ke Zhang, Pei-Yong Xin, Sin Man Lam, Jin-Fang Chu, Guang-Hou Shui, Shou-Yi Chen and He Zhao, Biao Ma, Kai-Xuan Duan, Xin-Kai Li, Xiang Lu, Cui-Cui Yin, Jian-Jun Tao, Wei Wei,

The GDSL Lipase MHZ11 Modulates Ethylene Signaling in Rice Roots

 This information is current as of July 24, 2020

 

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