INVESTIGATION

Opaque7 Encodes an Acyl-Activating Enzyme-LikeProtein That Affects Storage Protein

Synthesis in Maize EndospermGang Wang,*,1 Xiaoliang Sun,*,1 Guifeng Wang,* Fei Wang,* Qiang Gao,* Xin Sun,* Yuanping Tang,*

Chong Chang,* Jinsheng Lai,† Lihuang Zhu,‡ Zhengkai Xu,* and Rentao Song*,2

*Shanghai Key Laboratory of Bioenergy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, China,†State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100101, China, and

‡The State Key Laboratory of Plant Genomics and National Center for Plant Gene Research,Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,

Beijing 100101, China

ABSTRACT In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins,the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeinsaccumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulationand high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functionalcomplementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulationof o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7mutant was produced by the reduction of protein body size and number caused by a decrease in the a-zeins concentrations. Analysisof amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting a-ketoglutaric acid andoxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced loweramounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cerealcrops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target forthe genetic manipulation of storage protein contents in cereal seeds.

THE texture and protein quality of maize (Zea mays L.)endosperm are important factors affecting grain ship-

ping, insect and fungal pathogen resistance, and nutritionalquality. Much evidence indicates that the reduction in theamount of zeins in the endosperm leads to a decrease in theendosperm hardness and an increase in lysine content(Mertz et al. 1964; Misra et al. 1972; Schmidt et al. 1987;Dombrink-Kurtzman and Bietz 1993; Holding and Larkins2006; Wu and Messing 2010; Wu et al. 2010). Maize have

a number of opaque or floury endosperm mutants that affectthe texture and protein quality of endosperm by alteringzeins accumulation. Our understanding of the underlyingmechanisms determining zeins accumulation comes fromthe study of seed mutants.

There are .18 mutants that can exhibit an opaque orfloury endosperm (Thompson and Larkins 1994; Hunteret al. 2002). Among them are the recessive opaque mutants(o1, o2, o5, o7, o9-o11, and o13-o17), the semidominantfloury mutants (fl1, fl2, and fl3), and the dominant mutantsMucronate (Mc) and Defective endosperm B30 (De-B30) (Mottoet al. 1996; Gibbon and Larkins 2005). The cloning and char-acterization of some of the opaque mutants has revealed im-portant regulatory mechanisms for zeins accumulation inmaize endosperm. The O2 gene, which encodes a defectivebasic-domain-leucine-zipper transcription factor, regulatesseveral endosperm-expressed genes, in particular the 22-kDaa-zeins (Schmidt et al. 1987, 1990; Damerval and De Vienne

Copyright © 2011 by the Genetics Society of Americadoi: 10.1534/genetics.111.133967Manuscript received August 18, 2011; accepted for publication September 21, 2011Supporting information is available online at http://www.genetics.org/content/suppl/2011/09/27/genetics.111.133967.DC1.Sequence data have been deposited to EMBL/GenBank under accession nos.JN578265, JN578266, and HQ234502.1These authors contributed equally to this study.2Corresponding author: Shanghai Key Laboratory of Bioenergy Crops, School of LifeSciences, Shanghai University, 333 Nanchen Rd., Shanghai 200444, China.E-mail: rentaosong@staff.shu.edu.cn

Genetics, Vol. 189, 1281–1295 December 2011 1281

1993; Habben et al. 1993). The o2 mutant reduces the pro-duction of zeins by repressing the expression of 22-kDa a-zeingenes (Schmidt et al. 1987, 1990). Other mutants, such as fl2,De-B30, and Mc, have structural defects in the zeins them-selves, which results in improper zeins deposition, protein bodydeformation, and the “unfolded protein response” (UPR)(Lending and Larkins 1992; Coleman et al. 1995; Gillikinet al. 1997; Hunter et al. 2002; Kim et al. 2004, 2006), caus-ing a general translational repression of zeins. However,mechanisms other than reduced zeins do exist. For example,the fl1 mutation does not affect the amount and compositionof zeins; it only changes the location of 22-kDa a-zeins withinthe protein body (Holding et al. 2010). The o5 mutant phe-notype is caused by a reduction in the galactolipid content ofthe maize endosperm, with no change in zeins (Myers et al.2011). Cloning and characterization of additional mutantscould reveal novel regulatory mechanisms for opacity forma-tion, zeins synthesis, and accumulation in maize endosperm.

The opaque7 (o7) is an important opaque mutant withlarge effects on zeins accumulation (Burr and Burr 1982). Itis considered one of the three most valuable high-lysineopaque mutants, along with the aforementioned o2 andfl2. The o7 mutant arose from a spontaneous mutation inW22 (Tsai and Dalby 1974) and has a starchy endosperm atmaturity. The o7 mutant induces a reduction in endospermweight and total protein content (Di Fonzo et al. 1979) andexhibits a general inhibition of the synthesis of all zein clas-ses (Misra et al. 1972; Lee et al. 1976; Burr and Burr, 1982;Hartings et al. 2011). Previous studies suggested that o7might have a very different mechanism for zeins regulationcompared to that of o2 and fl2. Studying this mechanismcould help us to understand the regulatory mechanisms ofzeins accumulation and endosperm opacity. However, the o7mutant is more difficult to study because its mutant pheno-type is only stable in a very narrow genetic background(McWhirter and Brink 1978).

In this study, we cloned the O7 gene by map-based clon-ing and confirmed its identity by transgenic functional com-plementation. The O7 gene encodes an acyl-activatingenzyme like (AAE3-like) protein, and the o7 mutant allelediffers from the wild-type (wt) allele by a 12-bp in-framedeletion. The deletion does not affect o7 transcript levels,but it greatly reduces the amount of o7 protein in the endo-sperm. Interestingly, transgenic RNAi targeting the O7 genewas effective in downregulating seed protein content andproducing opaque endosperm both in maize and rice, sug-gesting that the function of O7 is conserved among differentcereal crops. The O7 gene could serve as a target for thegenetic manipulation of seed proteins in cereal crops.

Materials and Methods

Plant materials

The o7 mutant stock was kindly provided by Frances Burr ofBrookhaven National Laboratory, Upton, NY. These segregat-

ing F2 seeds were derived from a cross between o7/o7 in theW22 background and an unknown flint line. Selfed ears ofplants from these F2 seeds were selected for solid opaquephenotype segregation and verification of the expected seg-regation ratio. At least two more rounds of selection werecarried out to obtain suitable parental lines to construct pop-ulations for genetic mapping. The backcross (BC) populationwas prepared from a cross between o7/o7 and o7/+ plants.Genomic DNA samples from o7/o7, o7/+ and the 13,933descendants of the BC population were extracted from leaftissue by the CTAB method (Murray and Thompson 1980) atthe seedling stage and used for marker analysis.

Seeds of the Hi-II parent A (pA) and Hi-II parent B (pB)lines for maize genetic transformation were initiallyobtained from the Maize Genetic Cooperation Stock Centerand amplified on the campus of Shanghai University. F2immature zygotic embryos (1.5–2.0 mm) of the maize Hi-II hybrid genotype (Armstrong et al. 1991) were asepticallydissected from ears harvested 10–12 days after pollination(DAP) and were used for genetic transformation.

Scanning electron microscopy and transmission electronmicroscopy analysis

For scanning electron microscopy (SEM) analysis, wt and o7mutant kernels were prepared as described previously (Lendingand Larkins 1992), with some modifications. Briefly, maizekernels were excised with a razor and immediately placed in2.5% glutaraldehyde. Mature samples were critical-point dried,and 21-DAP samples were cryofractured, sputter coated withgold in an E-100 ion sputter, and observed with a scanningelectron microscope (S3400N; Hitachi).

For transmission electron microscopy (TEM) analysis,previously published methods were used on immaturekernels from wt and o7 mutant with some modifications(Lending and Larkins 1992). Briefly, 21-DAP kernels of wtand mutant were fixed with paraformaldehyde and post-fixed in osmium tetraoxide. Fixed samples were dehydratedin an ethanol gradient up to 100% and then transferred toa propylene oxide solution and slowly embedded in acrylicresin (London Resin Company), where they were allowed topolymerize for at least 48 hr. Thin sections (70 nm) weretaken using a diamond knife microtome (Reichert UltracutE). Sections were placed on 100-mesh copper grids andstained for 30 min with uranyl acetate and for 15 min withlead citrate. Sections were visualized using a Hitachi H7600transmission electron microscope.

Measurement of starch, lipid, and protein contentsStarch analysis: Thirty-gram mature kernels from wt andmutant were collected from well-filled, mature ears. Thesamples were analyzed by the Cereal Quality Supervisionand Testing Center, Ministry of Agriculture, People’s Repub-lic of China. The analysis method was based on the GB7684-87 and GB5006-85 protocols developed by the Ministry ofAgriculture, People’s Republic of China. The starch analysiswas replicated three times for the wt and opaque kernels.

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Lipid analysis: Five mature kernels of the wt or mutant werecollected from well-filled, mature ears. One hundred milli-grams of dried endosperm flour were used for lipid extraction.The extraction procedure followed a standard protocol for lipidextraction from Arabidopsis seeds (http://www.k-state.edu/lipid/lipidomics/AT-seed-extraction.html). The extracted sam-ples were dried completely, stored in vials filled with nitrogengas, and shipped to the Kansas Lipidomics Research Center foranalysis. The lipids analysis was done according to lipid pro-filing protocols established by the facility (http://www.k-state.edu/lipid/lipidomics/profiling.htm). The lipid analysis wasreplicated three times for the wt and mutant kernels.

Protein analysis: Twenty mature kernels of the wt or mutantwere collected from well-filled, mature ears. Total protein,zein, and nonzein proteins were extracted from 40 mg ofdried endosperm flour, according to the method of Wallaceet al. (1990). Protein quantification analyses of the total ex-tract, zeins, and nonzein fractions were performed as de-scribed by Smith et al. (1985) using a BCA standard kit(Pierce). SDS–PAGE was performed in 12% polyacrylamidegels, and the gels were stained with Coomassie brilliant blueR250 (Bradford 1976).

Complementation test

For functional complementation tests, a 4066-bp genomicDNA fragment containing the entire coding region, an 1856-bpupstream sequence, and a 507-bp downstream sequencewere isolated from BAC clone ZMMBBb0342E21 (GenBankaccession no. HQ234502) by digesting with BamHI andKpnI. The genomic DNA was cloned into pTF102 (Frameet al. 2002), which carries a bar resistance marker anda CaMV 35S promoter. The resulting pTF102-O7 gene con-struct was introduced into the Agrobacterium tumefaciensstrain LBA4404. Agrobacterium-mediated maize transforma-tion using F2 immature zygotic embryos of the Hi-II hybrid(pApB) was carried out according to Frame et al. (2002). Forthe molecular identification of transgenic plants, a markerwas designed to identify the O7 gene with the primer pairTrans6837 F3 and Trans6837 R3, which bind the 35S pro-moter and O7 gene, respectively (see Supporting Informa-tion, Table S1 for primer sequences). The marker Indel6819,linked tightly with the O7 locus, was designed to identify thegenotype of the O7 gene on chromosome 10.

Maize RNAi transformation

To generate the RNAi construct, a PCR fragment containing303 bp from the cDNA of the O7 gene (nucleotides 1277–1579) was amplified using the first-strand cDNA derivedfrom W22 mixed endosperm (12 DAP, 15 DAP, and 18DAP) as the template and the following pairs of primersmaize P3 F and maize P3 R. The PCR fragment was sequen-tially cloned into the SpeI/KpnI and BamHI/SacI sites of thepTCK303 (Wang et al. 2004) vector in both the sense andantisense orientations. Then the fragment containing the

sense PCR fragment, rice intron, and antisense PCR fragmentwere cloned into the SacI site of the pHB vector (Mao et al.2005). The O7 gene promoter was then amplified by theprimer pair O7 promoter F/promoter R and used to replacethe 2·35S promoter of pHB by cloning into the EcoRI andBamHI sites. The resulting pHB–O7RNAi construct was intro-duced into the Agrobacterium strain LBA4404. Agrobacterium-mediated maize transformation using F2 immature zygoticembryos of the Hi-II hybrid (pApB) was carried out accord-ing to Frame et al. (2002). For the molecular identificationof T0 transgenic plants, a marker was designed to identifythe O7 gene with the primer pair rice intron F and maizeP3 R (which bind the rice intron and O7 gene, respectively).

RT–PCR and real-time quantitative PCR analysis

Root, stem, leaf, husk, silk, tassel, ear, and kernels (3 DAP–36 DAP) from adult W22 plants were collected and flashfrozen in liquid nitrogen. Kernels (12 DAP, 18 DAP, and24 DAP) from adult wt and o7 mutant plants were alsocollected and flash frozen in liquid nitrogen. RNA extraction,purification, and quantification were performed accordingto Feng et al. (2009) and Wang et al. (2010, 2011). ForRT–PCR and real-time PCR, �2 mg of total RNA was reversetranscribed to cDNA with oligo d(T)18 primer using Rever-Tra Ace reverse transcriptase (Toyobo). The expression pat-tern was analyzed using real-time quantitative RT–PCR withSYBR Green Realtime PCR Master Mix (Toyobo). The reac-tion was performed on a Mastercycler ep realplex 2 (Eppen-dorf) with the primer 6837Q, and Ubiquitin (GenBankaccession no. BT018032) was used as a reference gene (Ta-ble S1). All of the real-time RT–PCR experiments were per-formed with two independent sets of RNA samples.Quantification of the relative changes in gene expressionwas performed using the 2-DDCt method as described pre-viously (Livak and Schmittgen 2001). The specificity of thePCR amplification procedures was verified after PCR by us-ing a heat dissociation protocol (from 65� to 95�) to ensurethat each amplicon was a single product.

Sequence homology analysis

Related sequences were identified in the NCBI nonredun-dant (nr) protein sequences database by performinga BLASTp search (Camacho et al. 2009) with O7 proteinsequences. Selected sequences were aligned using ClustalWin the MEGA5 package (Kumar et al. 2008). Conserved res-idues in the alignment were shaded using the SMS2program (http://www.bioinformatics.org/sms2/color_align_cons.html) (Kumar et al. 2008). The phylogenetic tree wasproduced with MEGA5 using the bootstrap analysis (1000replicates), although neighbor joining gave an identical to-pology (Tamura et al. 2007).

O7 antibody preparation

The full-length O7 ORF was amplified from cDNA derivedfrom W22 mixed endosperm (12 DAP, 15 DAP, and 18 DAP)

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using primers 6837FLcDNA F and 6837FLcDNA R. The se-quence correctness of the fragment was confirmed and wasdigested with BamHI and EcoRI and ligated into pGEX-4T-1(Amersham Biosciences) to create an in-frame fusion withGST. The selected clones were sequenced to exclude thepossibility of PCR-induced errors, and a single clone wassubsequently used for large-scale induction of the fusion pro-tein. The fusion protein was purified using a standard tech-nique (Amersham Biosciences). Antibodies were produced inwhite rabbits at Shanghai ImmunoGen Biological Technol-ogy. The polyclonal antibodies were affinity purified usingtheir respective bacterially expressed GST fusion proteinsaccording to standard procedures (Harlow and Lane 1988).

Total endosperm protein extraction andimmunoblot analysis

Developing endosperms (12 DAP, 15 DAP, 18 DAP, and 21DAP) were dissected as described above. Total protein fromwt and mutant endosperms was extracted according to themethod of Bernard et al. (1994). Protein samples wereloaded onto SDS gels (10% acrylamide) and run using theMini-protean system (Bio-Rad). Proteins were then blottedonto nitrocellulose using the Mini-transblot system (Bio-Rad)according to standard protocols. The immunoblot analyseswith the O7 antibody, a-tubulin antibody (Sigma-Aldrich),and BiP antibody (Santa Cruz Biotechnology) were per-formed according to Holding et al. (2007).

Soluble amino acids analysis

The soluble amino acids (SAAs) were analyzed according tothe method of Holding et al. (2010) with some modification.Briefly, 3-mg aliquots of each sample were refluxed for 24 hrin 6N HCl. Samples are typically hydrolyzed at 110� for24 hr in a vacuum or inert atmosphere to prevent oxidation.Hydrolysates were evaporated to dryness. The residue wasdissolved in 10 ml of pH 2.2 citrate buffer and the aminoacids were profiled on a Hitachi-L8900 amino acid analyzerat the Instrumental Analysis Center of Shanghai JiaotongUniversity. Amino acid analyses were replicated three timeson the wt and opaque kernels. The SAAs of 21-DAP endo-sperm samples were analyzed by the same methods.

a-Ketoglutaric acid and oxaloacetate analysis

Three wt or mutant kernels (21 DAP and 24 DAP) werecollected from each of three wt or mutant ears. One hundredmilligrams of dried endosperm flour were used for metab-olite extraction according to Chen et al. (2011) with some

modification. Briefly, dried extracts were dissolved in mixedsolution of water/isopropanol/methanol (1:3:6, v/v). Theanalysis of extracts by mass spectrometry was carried outaccording to Broeckling et al. (2005). The a-ketoglutaricacid (a-KG) and oxaloacetate (OAA) analyses were eachreplicated three times for the wt and opaque kernels.

Rice RNAi analysis

To generate the RNAi construct, a PCR fragment contain-ing 258 bp (nucleotides 967–1224) of the cDNA ofOs04g0683700 (OsAAE3) was amplified from the first-strand cDNA derived from Nipponbare young panicle byusing the following pair of primers: RiceP3 F and RiceP3R. The PCR fragment was sequentially cloned into theSpeI/KpnI and BamHI/SacI sites of the pTCK303 vector(Wang et al. 2004) in both the sense and antisense orienta-tions, yielding the pTCK303–OsAAE3 RNAi vector. ThepTCK303–OsAAE3 RNAi construct was introduced into theAgrobacterium strain LBA4404. Agrobacterium-mediated ricetransformation using the calli of Kitaake (a japonica ricevariety) was carried out according to Wang et al. (2004).The regenerated plants were further confirmed by PCR us-ing rice intron F and rice P3 R.

Results

Characterization of the o7 mutant endosperm

BC ears with progenies exhibiting 1:1 segregation of opaque(o7/o7) and wt (o7/+) (vitreous) kernel phenotype wereused for cytological analysis (Figure 1). SEM analysis

Figure 1 Phenotype of the BC ear, o7, and wt kernels.

Figure 2 Scanning electron microscopy of wt and o7 endosperm. (A) o7kernel at 21 DAP (·5000). (B) Wt kernel at 21 DAP (·5000). (C) Mature o7kernel (·3000). (D) Mature wt kernel (·3000). st, starch; pb, protein body.

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revealed that the protein bodies (PBs) and proteinaceouscytoskeletal matrix of o7 immature endosperm (21 DAP)were much smaller than those of the wt (Figure 2, A andB), resulting in loosely packed starch granules. SEM exam-ination of mature kernels showed that the mutant kernelhad smooth, spherical starch grains with very little matrixsurrounding them, whereas the wt kernel contained polyg-onal starch grains with a very dense proteinaceous matrixsurrounding them (Figure 2, C and D). TEM analysis re-vealed that in the mutant endosperm, there is a significantreduction in PB numbers (reduced 29%) and PB size (re-duced 13%) compared to the wt endosperm (Figure 3, A–F).

To determine the underlying biochemical reasons for theopaque phenotype of the mutant, we examined the majorcomponents in the endosperms of the mutant and wtkernels. First, we found that the 100-seed weight of opaquekernels was just 93% of the weight of wt kernels (obviously

significant decrease, P , 0.05, Student’s t-test). We thenanalyzed starch (amylose, amylopectin, and total starch)(Figure S1) and lipid content (Figure S2) per dry weightof opaque and wt kernels, and we found no obvious differ-ences between both opaque and wt kernels. However, theprotein content was found to be dramatically lower in opa-que kernels (Figure 4, A and B). Interestingly, there is a gen-eral reduction in zeins, nonzeins, and total proteins in opaquekernels (Figure 4, A and B). Quantitative analysis showed thatin the mutant kernels, zeins had the most severe decrease(33.4%), greater than that of nonzeins (15.1%) or total pro-teins (20.7%). This result is consistent with the cytologicalanalysis, which showed that the PBs numbers and size of o7were reduced about one-third. From the SDS–PAGE, wefound that there is a dramatic decrease of both 19-kDa and22-kDa a-zeins in o7 kernels (Figure 4A). The result showedthat o7 does not downregulate the 19-kDa a-zein specifically.

Figure 3 Transmission electron microscopy of wt and o7endosperm. (A) o7 kernel at 21 DAP (·2500). (B) Wt kernelat 21 DAP (·2500). (C) o7 kernel at 21 DAP (·6000). (D)Wt kernels at 21 DAP (·6000). cw, cell wall; pb, proteinbody; st, starch; rER, rough endoplasmic reticulum; m,mitochondrion. (E) Comparison of protein body numbersper 100 mm2 in o7 and wt 21-DAP endosperms (o7,protein body counts ¼ 36.87 6 6.30; wt, protein bodycount ¼ 26.20 6 4.21. *P , 0.05, Student’s t-test). (F)Comparison of protein body area in o7 and wt 21-DAPendosperms (o7, protein body area ¼ 0.547 6 0.345; n ¼203. wt, protein body area ¼ 0.624 6 0.246; n ¼ 270.**P , 0.01, Student’s t-test).

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Map-based cloning of the Opaque7 gene

The O7 gene was mapped between the markers umc2021and umc1038 on the long arm of chromosome 10 by ananalysis of 400 BC individuals (Figure 5A). Additional mo-lecular markers were then developed by comparing genomicsequence fragments near the O7 locus in both parental lines(Table S1). After characterizing a BC population with a totalof 13,933 individuals, the O7 gene was eventually mappedbetween the marker LM4 and the marker RM1, a regionencompassing a physical distance of �23 kb (Figure 5A).

Sequence analysis of this interval identified two predictedgenes (gene1 and gene2) (Figure 5A), but only gene2 hadsupport from expressed sequence tags (ESTs). We se-quenced the 23-kb regions from the mutant and wt haplo-types, and we found no sequence polymorphism withingene1. However, there was a 12-nucleotide in-frame dele-tion in the predicted second exon (codons 350–353) (Figure5B) of the o7 allele of gene2. Thus, gene2 appeared to bethe strongest candidate for the O7 gene.

Functionally complement the o7 mutant by transgeniccandidate gene

We used a functional complementation test involving genetictransformation to test the cloned candidate gene. In total, 13independent transgenic lines were obtained. These transgeniclines were then backcrossed to o7/o7 mutant plants for atleast two more generations to obtain individuals homozygousat the o7 locus. We characterized four independent o7/o7transgenic lines. In Figure 6, we present the analysis of oneof the transgenic lines. A randomly selected set of 20 vitreousand 10 opaque kernels (Figure 6A) from one transgenic linewere genotyped using molecular marker Indel6819 to confirmthat the kernels were homozygous at the o7 locus (Figure 6B).The primer set Trans6837 F3/Trans6837 R3 was used to de-tect kernels containing the O7 transgene. Without exception,all 20 vitreous kernels were found to be transgenic, whereasnone of the 10 opaque kernels were transgenic (Figure 6C).

Biochemical analysis of kernel proteins from transgenicfunctional complementation ears by SDS–PAGE showed that

Figure 4 Protein analysis of wt and o7 endosperm. (A) SDS–PAGE analysis of total proteins from wt and o7 endosperm. (B) Comparison of zein,nonzein, and total proteins from wt and o7 endosperm. Values are the mean values with standard errors (*P , 0.05; **P , 0.01, Student’s t-test).

Figure 5 Positional cloning of the O7 gene. (A) Fine map-ping of the O7 gene on chromosome 10. Names of themolecular markers, contig, BAC, and the recombinantsare indicated (n ¼ 13,933). ZMMBBb0166J07 andZMMBBb0342E21 (abbreviated as b0166J07 and b0342E21)are the names of BAC clones covering this locus. The O7locus was mapped to a �23-kb region containing gene1and gene2 between the molecular markers LM4 and RM1.(B) Schematic representation of the gene structure of O7.The mutant sequence has a 12-nucleotide in-frame dele-tion in the second exon. ATG and TGA represent the startand stop codons, respectively.

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the total proteins and zeins levels in functionally comple-mented transgenic kernels were recovered to the proteinlevels of wt kernels and that the levels recorded in nontrans-genic kernels were similar to those of the mutant (Figure6, D and E). This result indicated that the o7 mutant phe-notype was fully rescued by transgenic candidate gene. Theanalysis of another three independent transgenic lines gavethe same results (data not shown). Therefore, the o7mutantallele was functionally complemented by the wt O7 allelethrough stable genetic transformation. This result demon-

strated that the candidate gene2, which was identified bymap-based cloning, was in fact the O7 gene.

RNAi against O7 reproduces the o7 phenotype

To further verify the function of the cloned O7 gene, weconstructed an RNAi transgenic vector to target the O7 genesequence, and we transformed the construct into maize.Seven independent transgenic events were obtained fromself-pollination. We characterized the seeds from two inde-pendent RNAi transgenic lines with opaque and vitreous

Figure 6 Phenotypic and molecular characterization of transgenic kernels from functional complementation experiment. (A) The phenotype of 20vitreous kernels and 10 opaque kernels, randomly selected from a transgenic line with homozygous o7 locus on chromosome 10. (B) Genotyping the 30kernels displayed in A for o7 locus on chromosome 10 using the molecular marker Indel6819. All 30 kernels are homozygous at the o7 locus onchromosome 10. (C) Characterization of transgenic status of the 30 kernels displayed in A using the molecular marker Trans6837F3R3. The vitreouskernels (lanes 1–20) are transgenic, and the opaque kernels (lanes 21–30) are nontransformed.2, H2O control;1, pTF102–O7Gene vector. (D) SDS–PAGEanalysis of total proteins from different kernels. T1, transgenic and o7/o7 homozygous kernel; T2, nontransformed and o7/o7 homozygous kernel. (E)Comparison of zein protein content in wt, o7, pApB, transgenic homozygote o7/o7 (T1), and nontransformed homozygote o7/o7 kernels (T2).

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kernel segregation (Figure 7A). In one of the transgenic lines,10 vitreous kernels and 10 opaque kernels selected randomlywere analyzed with the primer set rice intron F/maize P3 R todetermine whether the kernels contained the transgenic RNAiconstruct. The result indicated that all 10 opaque kernelswere RNAi transgenic, whereas none of the 10 vitreous ker-nels were RNAi transgenic (Figure 7B).

Analysis of the kernel proteins from RNAi transgenicevents indicated a decreased level of zeins compared to pApBcontrol seeds (R1 decreased 14.6%, R3 decreased 16.9%;Figure 7, C and D). These results showed that the o7 pheno-type could be caused by the quantitative reduction in zeins.Therefore, in both RNAi transgenic events, the seeds bearingthe RNAi construct produced the opaque phenotype (Figure

7, A–D). The results confirmed that the opaque phenotypewas indeed caused by the suppression of O7 gene function.

O7 encodes an AAE3-like protein

We obtained the full-length cDNAs of the O7 and o7 genes ofW22 by rapid amplification of cDNA ends (RACE) RT–PCR(GenBank accession nos. JN578265 and JN578266). We foundthat the W22 O7 cDNA is 1884 bp long, containing an openreading frame of 1584 bp, encoding a polypeptide of 527amino acids. Sequence comparison between the genomicDNA and cDNA indicated that the O7 gene contains two exons.

BLAST searches using the O7 protein sequence indicatedthat O7 belonged to the acyl-CoA synthetase (ACS) super-family and contained the AMP-binding and ACS conserved

Figure 7 Phenotypic and molecular characterization of transgenic kernels from maize RNAi transgenic experiment. (A) The vitreous and opaquephenotype of the 20 randomly selected kernels from an RNAi transgenic line. (B) Characterization of the transgenic status of the 20 kernels displayedin A using the molecular marker defined by the primer pair rice intron F/maize P3 R. The opaque kernels (lanes 1–10) are transgenic, and the vitreouskernels (lanes 11–20) are nontransformed. 2, H2O control; 1, pHB–O7RNAi vector. (C) SDS–PAGE analysis of total proteins from different kernels.R12CK and R32CK, nontransformed vitreous kernels from transgenic lines R1 and R3; R11 and R31, transgenic opaque kernels from transgenic linesR1 and R3. (D) Comparison of zein protein content in pApB, R12CK, R32CK, R11, and R31 kernels.

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domains (Black et al. 1992, 1997; Weimar et al. 2002;Shockey et al. 2003; Black and Dirusson 2006). We con-structed an ACS superfamily phylogenetic tree on the basisof the O7 protein sequence and the ACS proteins of Arabi-dopsis thaliana. The O7 protein was most similar to AAE3 inthis phylogenetic tree (Figure S3A). We then constructed anAAE3 family phylogenetic tree on the basis of homologsfrom fungi, bacteria, and plants (Figure S3B). In this phylo-genetic tree, we found that the AAE3 proteins of plants weremonophyletic. In this tree, there were AAE3 homologs fromother grass species, such as Sorghum bicolor (XP 002448800),Oryza sativa (NP 001054304 and Os04g0683700), and Hor-deum vulgare (BAK00674) (Figure 8). A detailed sequencealignment of these AAE3 homologs showed a highly con-served AMP-binding domain and ACS domains (Figure 9).The four-amino-acid deletion of o7 was also found to lie ina conserved sequence region (Figure 9). The deleted Val andGly residues in the o7 protein were highly conserved amongthe analyzed AAE3 homologs in plants (Figure 9).

Expression analysis of the O7 gene

Real-time quantitative RT–PCR (real-time qRT–PCR) analy-sis revealed that O7 is expressed in a broad range of tissues,including root, stem, leaf, husk, silk, tassel, ear, and kernel(Figure 10A). The strongest expression was detected in im-mature seeds. However, root and tassel also had relativelyhigh expression levels (Figure 10A). Because the o7 mutantphenotype is related to maize endosperm, we further exam-ined the expression profile of O7 during seed development.The O7 transcript was first detectable at 3 DAP and sharplyincreased in abundance, peaking at �7 DAP (Figure 10B). Arelatively small but still significant amount of O7 expressionwas detected from 18 DAP to 36 DAP. We also compared the

expression levels of O7 and o7 at 12 DAP, 18 DAP, and 24DAP and found no significant difference in transcript levelsbetween wt and mutant alleles (Figure 10C).

Significant reduction of o7 protein in themutant endosperm

We further analyzed the O7 gene product of immature ker-nels (12 DAP, 15 DAP, 18 DAP, and 21 DAP) from the wt andmutant at the protein level. Immunoblot analysis employingantibody against a-tubulin was used to ensure equal loadingof total proteins for each sample. Western blot analysisemploying O7 protein antibody showed that the quantity ofo7 protein in the mutant kernel was significantly lower thanthe O7 protein in the wt kernel (Figure 11, A and B). There-fore, the 12-bp deletion in the o7 mutant allele caused sig-nificant reduction of o7 protein level in developing kernels.

At the same time, we analyzed the O7 protein fromthe 18-DAP kernels of transgenic homozygous o7/o7 plantsand nontransformed homozygous o7/o7 kernels from thefunctional complementation tests, and using the O7 anti-body, we found that the quantity of O7 protein in the trans-genic homozygous o7/o7 kernels (vitreous phenotype) wasrecovered but not in nontransformed homozygous o7/o7kernels (opaque phenotype; Figure 11, C and D). We alsoanalyzed the O7 protein in developing kernels (18 DAP)from the transgenic RNAi experiment. Indeed, the O7 pro-tein was significantly reduced in kernels bearing the trans-genic RNAi construct (Figure 11, E and F).

Normal UPR in the o7 endosperm

Because the UPR can cause a general translational repressionof zeins, we investigated whether zein reduction in the o7endosperm was caused by UPR. We used immunoblot analy-sis to compare the level of the ER lumen binding protein (BiP)(Kim et al. 2004) in 12-DAP, 15-DAP, and 18-DAP kernelsfrom wt and o7 plants by BiP antibody. The immunoblotshowed that BiP level was not obviously different betweenwt and o7 (Figure 12, A and B). The result indicated thatzeins reduction and phenotype of o7 was not caused by UPR.

Soluble amino acids and key metabolites analysis of theo7 endosperm

Amino acid supply was proposed as a possible cause fordownregulation of zeins (Motto et al. 1996). To observe thechanges in amino acid composition in o7 endosperm, weanalyzed the SAAs from wt and o7 mature seeds. SAAs anal-ysis showed that the mutant seeds have a higher content ofAsx (Asp + Asn) (+21.9%) and Lys (+38.2%), but a lowercontent of Glx (Glu + Gln) (214.5%) compared to wt seeds(Figure 13A). To understand the changes that contribute tothe final concentrations of SAAs in mature endosperm, wecharacterized the SAA levels and composition of Asx (Asp +Asn), Lys and Glx (Glu + Gln) of 21-DAP kernels. The resultsshowed that mutants have a higher content of Asx (+10.6%)and Lys (+12.8%) and have a lower content of Glx (210.2%)

Figure 8 Phylogenetic relationships of O7 (ZmAAE3) and its homologs.The O7 gene product (shown as Zm AAE3) was aligned with AAE3proteins from Capsicum annuum (AF354454.1), Vitis vinifera (XP002267459), Populus trichocarpa (XP 002322473), Ricinus communis(XP 002509782), Arabidopsis thaliana (NP 190468), A. lyrata subsp. lyrata(XP 002877640), Oryza sativa Japonica (NP 001054304), Hordeum vul-gare subsp. vulgare (BAK00674), Zea mays (NP 001152269.1), Sorghumbicolor (XP 002448800), A. thaliana AAE13 (At AAE13, NM 112487), andA. thaliana AAE14 (At AAE14, NM 102789).

The Cloning and Analysis of Maize Opaque7 1289

compared to wt (Figure 13B). The data were highly consistentbetween developing and mature endosperm.

Because amino acid biosynthesis is tightly linked to theconcentrations of certain metabolites, we analyzed some keymetabolites of the TCA cycle at 21 DAP and 24 DAP. Theanalysis showed that mutant kernels have a higher contentof OAA, the precursor substrate for Asx and Lys biosynthesis,but they have a lower content of a-KG acid, the precursorsubstrate for Glx biosynthesis (Figure 13, C and D).

O7 gene function is conserved between maize and rice

The synteny and phylogenetic tree analysis showed that theOsAAE3 (Os04g0683700) of rice is the orthologous gene ofmaize O7 (Figure 8). We generated RNAi transgenic plants

targeting OsAAE3 in the japonica rice variety Kitaake andobtained six independent transgenic lines. These transgenicplants showed segregation of the opaque and vitreous seedphenotypes (Figure 14, A and B). We randomly selected 10vitreous kernels and 10 opaque kernels from an RNAi trans-genic line and examined the transgenic RNAi construct bythe primer pair rice intron F/rice P3 R. On the basis of thisanalysis, all 10 opaque kernels were RNAi transgenic and all10 vitreous kernels were not RNAi transgenic (Figure 14C).Total seed proteins were analyzed by SDS–PAGE, and therewas an apparent decrease of total seed proteins in trans-genic kernels compared to nontransgenic kernels (Figure14D). SEM examination revealed that the opaque pheno-type kernel had loosely packed starch granules (Figure 14,

Figure 9 Protein sequence alignment of plantAAE3 proteins. ClustalW amino acid alignmentof Zm AAE3 protein and its homologs. Theblack shading with white lettering indicates res-idues conserved in all 10 members, whereasgray shading indicates conservation betweentwo or more of the family members. The 10sequences listed are from Zea mays (NP001152269.1), Oryza sativa (NP 001054304),Arabidopsis thaliana (NP 190468), Sorghum bi-color (XP 002448800), Hordeum vulgare subsp.vulgare (BAK00674), Ricinus communis (XP002509782), Capsicum annuum (AF354454.1),Populus trichocarpa (XP 002322473), Vitis vinif-era (XP 002267459) and A. lyrata subsp. lyrata(XP 002877640).

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E and F). These results showed that RNAi-based repressionof OsAAE3 resulted in storage protein reduction and an opa-que endosperm phenotype in rice, as was observed in themaize o7 mutant and O7 RNAi transgenic endosperm. Theseresults suggested that the gene function is conserved be-tween maize and rice.

Discussion

In this study, we cloned the O7 gene by map-based cloningand confirmed it by functional complementation test. The o7allele is a recessive mutant, suggesting a loss-of-functionmutation. This is further supported by the result from theRNAi experiment with knocked-down O7, which reproducedthe o7 mutant phenotype. Sequence analysis indicated thatthe o7 mutant has a 12-nucleotide in-frame deletion in thesecond exon. The 12-bp deletion does not create a frame-

shift mutation or premature termination, but it removes fouramino acids from the O7 protein. Due to the unknown func-tion of the O7 protein, we were unable to determine theeffect of the deletion on its AAE activity. Transcript quanti-fication by real-time qRT–PCR analysis indicated that theexpression of O7 was not different from o7 at the transcriptlevel. However, at the protein level, we found a dramaticdecrease of o7 protein in the mutant endosperm. This in-dicated that the four-amino-acid deletion caused low accu-mulation of o7 protein in vivo. Thus, the molecular basis ofthe o7 phenotype is a dramatic decrease in o7 protein thatleads ultimately to a loss of O7 function.

The O7 gene was annotated as the AAE gene. In Arabi-dopsis, there are 44 genes belonging to the family, which canbe divided into seven groups on the basis of phylogeneticanalysis (Shockey and Browse 2011; Shockey et al. 2003).However, only half of them have been assigned definitive

Figure 10 Expression analysis of the O7 gene. (A) Real-time qRT–PCR analysis of O7 gene expression in root, stem, leaf, silk, tassel, ear, and kernel (7DAP). (B) Real-time qRT–PCR analysis of O7 gene expression from 3-DAP to 36-DAP kernels in the W22 background. (C) Real-time qRT–PCR analysis ofO7 gene expression in 12-DAP, 18-DAP, and 24-DAP samples from wt and o7 mutant kernels endosperm.

Figure 11 Immunoblot analysis of O7 pro-tein from endosperms of wt, o7, functionalcomplementation transgenic line, and RNAitransgenic line. (A) Immunoblot comparingthe accumulation of O7 protein in 12-DAP,15-DAP, 18-DAP, and 21-DAP kernels fromwt and o7 plants with an antibody againstO7 protein. (B) Immunoblot analysis usingan a-tubulin antibody. (C) Immunoblotcomparing the accumulation of O7 proteinin 18-DAP kernels with an antibody againstO7 protein. T1, transgenic homozygote o7/o7; T2, nontransformed homozygote o7/o7. (D) Immunoblot analysis using an a-tu-bulin antibody. T1, transgenic homozygoteo7/o7; T2, nontransformed homozygote

o7/o7. (E) Immunoblot comparing the accumulation of O7 protein in the 18-DAP kernels with an antibody against O7 protein. R12CK and R32CK,nontransformed kernels from transgenic lines R1 and R3; R1+ and R3+, transgenic kernels from transgenic lines R1 and R3. (F) Immunoblot analysisusing an a-tubulin antibody. R12CK and R32CK:, nontransformed kernels from transgenic lines R1 and R3; R1+ and R3+, transgenic kernels fromtransgenic lines R1 and R3.

The Cloning and Analysis of Maize Opaque7 1291

biochemical functions. On the basis of the phylogenetic anal-ysis, O7 is a maize ortholog of AAE3 and belongs in clade VII(Shockey et al. 2003). In Arabidopsis, clade VII containsthree genes, AAE3, AAE13, and AAE14 (Shockey et al.2003). The AAE14 and AAE13 genes encode the o-succinylbenzoyl-CoA ligase and malonyl-CoA synthetase respectively(Kim et al. 2008; Chen et al. 2011). Although AAE3 wasannotated as an ACS, its exact enzymatic activity is uncer-tain (Shockey et al. 2003). In Saccharomyces cerevisiae,Pcs60/Fat2, the ortholog of AAE3, showed no ACS activityon any of the saturated and unsaturated fatty acids tested(Blobel and Erdmann 1996). In Capsicum annuum, the AAE3homolog was found to be rapidly upregulated by treatmentwith salicylic acid or the pathogenic bacterium Xanthomonascampestris (Lee et al. 2001). In this study, although we could

not determine the exact biochemical function of maize AAE3(O7), the characterization of the o7 mutant phenotypeclearly indicated that the AAE3 gene function is closely re-lated to storage protein synthesis in the endosperm tissue inmaize.

During endosperm development, starch granules andprotein bodies are embedded in a proteinaceous cytoskeletalmatrix (Clore et al. 1996; Gibbon et al. 2003). The protein-aceous matrix is made up of protein bodies and clear, viscouscytoplasm. Therefore, any change in protein body size,quantity, or shape or the composition of the cytoplasm couldproduce loosely packed starch granules and an opaque phe-notype. Several maize mutants, such as o2, fl2, De-B30, andMc (Schmidt et al. 1987; Gillikin et al. 1997; Kim et al. 2004,2006), which have opaque kernels due to zeins reduction

Figure 12 Immunoblot analysis of BiP from wt ando7 endosperms. (A) Immunoblot comparing theaccumulation of BiP protein in 12-DAP, 15-DAP,and 18-DAP wt and o7/o7 kernels by using BiPantibody. (B) Immunoblot analysis using a-tubulinantibody.

Figure 13 The analysis of soluble Asx, Lys, Glx, a-ketoglutaric acid, and oxaloacetate from wt and o7 endosperms. Values are the means and standarderrors (n = 3; *P , 0.05; **P , 0.01, Student’s t-test). (A and B) Soluble Asp 1 Asn, Lys and Glu 1 Gln in mature and 21-DAP kernels of wt and o7.(C and D) a-Ketoglutaric acid and oxaloacetate analysis of 21-DAP and 24-DAP kernels of wt and o7.

1292 G. Wang et al.

and small, misshapen protein bodies, support the relation-ship between vitreous endosperm and zeins content. In o7mutant endosperm, there is a significant reduction of zeinproteins. In tandem with the reduction in zeins, the mutantendosperm also shows significantly reduced PB size andnumber. The cytological and biochemical analysis indicatedthat the thinner proteinaceous cytoskeletal matrix in o7 mu-tant endosperm caused by PB size and number reductionwas the direct reason of the opaque phenotype.

Previous studies have suggested that most of the opaqueand floury genes that have been cloned regulate zeinsynthesis by affecting regulatory genes or structural genesfor storage proteins such as o2, fl2, De-B30, and Mc (Schmidtet al. 1987; Gillikin et al. 1997; Kim et al. 2004, 2006; Gibbonand Larkins, 2005). Motto et al. (1996) considered that otherregulatory mechanisms, such as amino acid supply, couldaffect zeins gene expression. Tonelli et al. (1986) predictedthat the starchy endosperm phenotype of proline1 (pro1) isassociated with a defect in amino acid biosynthesis. However,because pro1 has not been cloned yet, this prediction has notbeen validated. The Mto140 gene was found to encode arro-gate dehydrogenase, which provided more direct evidence

that a reduction in zein protein synthesis could be causedby limiting the supply of tyrosine (Holding et al. 2010).

The o7 mutant is different from the o2, fl2, De-B30, andMc mutants. The O7 gene encodes neither a regulatory genenor a structural gene for zeins and its endosperm has a nor-mal UPR. However, we did observe changes in amino acidbiosynthesis in the mutant endosperm, particularly for Glx,Asx, and Lys. The cue for such a change could lie upstreamof amino acid biosynthesis because the corresponding met-abolic precursors for these amino acids, such as a-KG andOAA, also showed correlated changes. The mutation mightact through a-KG and OAA, affecting the biosynthesis ofseveral amino acids. Changes in the levels of these aminoacids might lead to translational suppression of zeins, whichare highly biased for certain amino acids, such as Gln andLys. Thus, O7 may represent a new regulatory mechanismunlike those of known opaque mutants. Because the exactbiochemical function of O7 as an AAE3 protein remains un-known, the details of such a regulatory mechanism remainto be explored.

Phylogenetic analysis indicates that O7 orthologs in othermajor cereal crops are monophyletic. In other words, this

Figure 14 Phenotypic and molecular characterization of transgenic kernels from rice RNAi transgenic experiment. (A) The vitreous and opaquephenotype of the 20 randomly selected kernels from the RNAi transgenics. (B) Transverse sections of vitreous (top) and opaque (bottom) rice kernels.(C) Characterization of the transgenic status of the 20 kernels displayed in A using the molecular marker defined by the primer pair rice intron F/rice P3R. The opaque kernels (lanes 1–10) are transgenic, and the vitreous kernels (lanes 11–20) are nontransformed. 2, H2O control; 1, pTCK303–OsAAE3RNAi vector. (D) SDS–PAGE analysis of total proteins from different kernels. Lane 1, Kitaake kernel; lanes 2–4, nontransformed kernels; lanes 5–7,transgenic kernels. (E and F) SEM (·350) of a nontransformed vitreous rice kernel (E) and a transgenic opaque rice kernel (F).

The Cloning and Analysis of Maize Opaque7 1293

gene was present in the common ancestor of the major ce-real crops. The O7 gene might have a conserved functionamong cereal crops. Synteny analysis showed that theOsAAE3 of rice is orthologous to the maize O7 gene(ZmAAE3). To further test this hypothesis, we used RNAi toknockdown the rice OsAAE3 gene by genetic transformation.Indeed, transgenic rice seeds with the OsAAE3 RNAi con-struct not only exhibited the opaque endosperm phenotypebut also showed a general reduction in major seed storageproteins. These results are similar to those obtained for the o7mutant or the transgenic O7 RNAi lines in maize. The resultsnot only indicate that the O7 gene has a conserved functionin cereal endosperms, they also show that genetic manipula-tion of this gene is sufficient to change the seed protein con-tent in cereal crops. Therefore, the O7 gene is an importantgene for regulating seed storage proteins in cereal endo-sperm, and it is a target for the genetic manipulation of seedprotein content in cereal crops. The cloning of the O7 geneprovides new opportunities for improving the nutritionalvalue of cereal grains.

Acknowledgments

We thank Frances Burr at Brookhaven National Laboratoryfor his providing maize genetic stocks. We thank WeibinSong at the China Agricultural University and ChunhuaYang at the Institute of Genetics and Developmental Biology,Chinese Academy of Sciences, for their technical supportwith genetic transformation in maize and rice, respectively.We thank Jian Zhu at Tongji University for SEM technicalassistance. We thank Liangliang Zhou, Jianpin Xu, DianbinLin, Xiaowei Zhang, Zhihong Ren, and Lihua Wang atShanghai University for their technical assistance. We thankthe Kansas Lipidomics Research Center for lipid analysis.This work was supported by the Ministry of Science andTechnology of China (2006AA10A107, 2006AA10Z148, and2009CB118400) and the Natural Sciences Foundation ofChina (30700472, 30671303, 31000747, and 31171559).

Note added in proof: See Miclaus et al. 2011 (pp. 1271–1280)in this issue, for a related work.

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Communicating editor: J. A. Birchler

The Cloning and Analysis of Maize Opaque7 1295

GENETICSSupporting Information

http://www.genetics.org/content/suppl/2011/09/27/genetics.111.133967.DC1

Opaque7 Encodes an Acyl-Activating Enzyme-LikeProtein That Affects Storage Protein

Synthesis in Maize EndospermGang Wang, Xiaoliang Sun, Guifeng Wang, Fei Wang, Qiang Gao, Xin Sun, Yuanping Tang,

Chong Chang, Jinsheng Lai, Lihuang Zhu, Zhengkai Xu, and Rentao Song

Copyright © 2011 by the Genetics Society of AmericaDOI: 10.1534/genetics.111.133967