Transcriptional Regulation of Zein Gene Expression inMaize through the Additive and Synergistic Action of opaque2,Prolamine-Box Binding Factor, and O2Heterodimerizing Proteins

Zhiyong Zhang,1 Jun Yang,1 and Yongrui Wu2

National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for BiologicalSciences, Chinese Academy of Sciences, Shanghai 200032, China

Maize (Zea mays) zeins are some of the most abundant cereal seed storage proteins (SSPs). Their abundance influenceskernel hardness but compromises its nutritional quality. Transcription factors regulating the expression of zein and other SSPgenes in cereals are endosperm-specific and homologs of maize opaque2 (O2) and prolamine-box binding factor (PBF). Thisstudy demonstrates that the ubiquitously expressed transcription factors, O2 heterodimerizing proteins (OHPs), specificallyregulate 27-kD g-zein gene expression (through binding to an O2-like box in its promoter) and interact with PBF. The zeincontent of double mutants OhpRNAi;o2 and PbfRNAi;o2 and the triple mutant PbfRNAi;OhpRNAi;o2 is reduced by 83, 89, and90%, respectively, compared with the wild type. The triple mutant developed the smallest zein protein bodies, which weremerely one-tenth the wild type’s size. Total protein levels in these mutants were maintained in a relatively constant rangethrough proteome rebalancing. These data show that OHPs, O2, and PBF are master regulators of zein storage proteinsynthesis, acting in an additive and synergistic mode. The differential expression patterns of OHP and O2 genes may causethe slight differences in the timing of 27-kD g-zein and 22-kD a-zein accumulation during protein body formation.

INTRODUCTION

Seed storage proteins are synthesized in great abundanceduring seed development and serve as a nitrogen sink for thegerminating seedling; they are also a staple protein source forhumans and livestock. Maize (Zea mays) is one of the mostproductive crops in the world. Its seeds contain 10% protein, ofwhich >60% are alcohol-soluble proteins called zeins. Theabundance of zein proteins is largely attributed to their high levelof gene transcription. Zeins are classified into four subfamilies,a-zeins (19 and 22 kD), g-zeins (50, 27, and 16 kD), b-zeins (15kD), and d-zeins (10 and 18 kD), based on their molecular massand structures (Esen, 1987; Coleman and Larkins, 1999).a-Zeins and g-zeins are the two major proteins, accounting for60 to 70% and 20 to 25% of the total zein fraction, respectively,depending on the genetic background (Thompson and Larkins,1994). Consistent with their protein mass, zein RNA sequencesconstitute nearly 50% of total endosperm transcripts, of whicha- and g-zeins comprise 30 and 15%, respectively (Hunter et al.,2002). High-throughput sequencing revealed that three-quartersof zein genes (copies) are among the 100 most highly expressedgenes in the endosperm, with a- and g-zein subfamilies con-tributing the most abundant transcripts (Chen et al., 2014).

Although high-level expression of zein genes is critical for theformation of a hard endosperm, which confers strength to with-stand mechanical damage during harvesting, transportation, andstorage, zein abundance compromises the nutritional value of thegrain (Wu and Messing, 2012b), because these proteins are de-void of the essential amino acids lysine and tryptophan (Osborneet al., 1914; Wu et al., 2012).Opaque2 (O2) is an endosperm-specific transcription factor

(TF) belonging to the bZIP family. It has long been known toregulate the 22-kD a-zein and 15-kD b-zein genes by recog-nizing the O2 box (TCCACGT) in their promoters (Schmidt et al.,1992; Neto et al., 1995). In the o2 mutant, the levels of 22-kDa- and b-zein transcripts and proteins are dramatically reduced.Although the classic O2 box has not been found in the 19-kDa-zein gene promoters, their expression was always observed tobe markedly downregulated in the o2 mutant (Schmidt et al.,1992; Or et al., 1993; Wu et al., 2010). Prolamine-box bindingfactor (PBF), another endosperm-specific DOF (DNA bindingone zinc finger) TF, was speculated to play a central role inmediating the synchronized expression of all zein genes by 10 dafter pollination (DAP), since most zein genes contain a P box(TGTAAAG) cis-element (Vicente-Carbajosa et al., 1997). How-ever, it was reported recently that silencing Pbf with RNA in-terference (RNAi) only resulted in decreased expression of 22-kDa-zeins and 27-kD g-zeins (Wu and Messing, 2012a). SinceRNAi is not a knockout mutation, the normal expression of somezein genes with a P box could be explained by the possibilitiesthat either the residue PBF is sufficient for their activation orother DOF TFs play redundant roles with PBF. The combina-tion of o2 and PbfRNAi caused further reductions in 22-kD

1 These authors contributed equally to this work.2 Address correspondence to yrwu@sibs.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: Yongrui Wu (yrwu@sibs.ac.cn).www.plantcell.org/cgi/doi/10.1105/tpc.15.00035

The Plant Cell, Vol. 27: 1162–1172, April 2015, www.plantcell.org ã 2015 American Society of Plant Biologists. All rights reserved.

a-zein levels, confirming that this subfamily is cooperativelyregulated by the two TFs through protein-protein interaction(Supplemental Figure 1) (Wu and Messing, 2012a). Sequencecomparison identified a cis-element, TTTACGT, in the 27-kD g-zeinpromoter, which is similar to the O2 box (TCCACGT) in 22-kDa-zein promoters (Ueda et al., 1992); therefore, it was designatedas an O2-like box. O2 does not have a strong binding affinity to theO2-like box, consistent with its constitutive expression in the o2mutant (Ueda et al., 1992; Hunter et al., 2002). However, when theO2-like box was modified to create an exact O2 binding sequence,the transcription of a reporter gene driven by the 27-kD g-zeinpromoter was significantly enhanced when coexpressed with O2(Ueda et al., 1992). It seems that 22-kD a-zein and 27-kD g-zeinpromoters both contain a bifactorial motif that is composed of theclosely linked P box and the O2 or O2-like box (SupplementalFigure 1) (Wu and Messing, 2012a), reminiscent of a similar regu-latory apparatus in which an unknown bZIP TF might target the O2-like box and cotransactivate the 27-kD g-zein gene with PBF.

Two maize genes encoding O2 heterodimerizing proteins(OHP1 and OHP2) were identified by screening an endospermcDNA library with an O2 probe (Pysh et al., 1993). OHP1 andOHP2 were found to be constitutively expressed and could formheterodimers with O2; however, their biological functions areunclear (Pysh et al., 1993), mainly due to the lack of null mutants.Our work here shows that both OHP1 and OHP2 are able to bindthe O2-like box and cotransactivate the 27-kD g-zein promoterthrough protein-protein interaction with PBF. Using RNAi toknock down the expression of the two Ohp genes, both RNAtranscript and protein levels of 27-kD g-zein were dramaticallyreduced. We created different combinations of mutants with o2and PbfRNAi and found that the three TFs regulated the ex-pression of 90% of zein gene family members in an additive andsynergistic way. Time-course analysis revealed that Ohp1 andOhp2 are expressed at significantly higher levels than O2 be-tween 8 and 12 DAP, consistent with the earlier expression of27-kD g-zeins than 22-kD a-zeins. Perhaps this temporal dif-ference in expression explains the early appearance of g-zein inprotein bodies.

RESULTS

Temporal and Spatial Expression Patterns of Ohps, O2,and Pbf

OHPs were first thought to encode bZIP TFs that, along withPBF, regulate the expression of the 27-kD g-zein gene, sincethey are homologous to O2 (Xu and Messing, 2008). OHP1 andOHP2 are located on chromosomes 1L and 5S, respectively,and are expressed in all tissues tested (Pysh et al., 1993). Ourdata showed that OHP1 and OHP2 are more highly expressed incob, root, and leaf than in endosperm, tassel, and stalk (Figure1). Time-course expression patterns of Ohp1 and Ohp2 in en-dosperm are similar, but they are totally different from those ofPbf and O2. The expression of Ohp1 and Ohp2 was at maximumat 8 DAP and declined afterward, while that of O2 and Pbf in-creased steadily, reaching a peak around 25 DAP (Figure 1).Ohps are expressed at much higher levels than O2 and Pbf,

especially before 12 DAP; at a late stage of endosperm de-velopment (32 DAP), the abundance of their transcripts is lowerthan that of O2 and Pbf (Figure 1). Ohp1 expression wassomewhat higher than Ohp2 expression before 18 DAP butdeclined afterward (Figure 1).Endosperm is a highly differentiated organ and is composed

of four cell types, aleurone layer, starchy endosperm cells,transfer cell layers, and embryo surrounding cells, each speci-fied to fulfill certain functions during seed development andgermination (Olsen and Becraft, 2013). Our results revealed thatthe mRNAs of O2, Pbf, Ohp1, and Ohp2 have overlappingspatial distributions in starchy endosperm cells, being higher inthe outer than the inner area, coincident with the expressionpatterns of 22-kD a-zein and 27-kD g-zein genes (Figure 2). Theexpression of Ohp1 and Ohp2 was also detected in embryo(Figure 2), indicating that they function broadly in seeddevelopment.

OHP1 and OHP2 Bind the O2-Like Box in the 27-kDg-Zein Promoter

To test whether the O2-like box is specifically recognized byOHP1 and OHP2, a 50-bp oligonucleotide (2320 to 2271)containing this motif from the 27-kD g-zein promoter was ex-amined by electrophoretic mobility shift assay (EMSA) (Figure 3).Binding of GST-OHP1 and GST-OHP2 fusion proteins to thisDNA sequence could be visualized as retarded bands in the gel.The results revealed that both monomer (band 1) and dimer(band 2) of OHPs are able to bind to this fragment (Figure 3).When two bases of the O2-like box (TTTACGT) were mutated(TTTAAGG), the retarded bands were abolished (Figure 3). Thebinding specificity of OHPs was verified through the addition ofunlabeled intact probes (20 and 1003) in the reaction, whichresulted in a gradual loss of all retarded bands, whether rec-ognized by monomer or dimer OHPs. These data indicate thatOHP1 and OHP2 can specifically bind the O2-like box in the27-kD g-zein promoter.

Figure 1. The Expression Patterns of Ohps, O2, and Pbf.

Quantitative RT-PCR analysis of Ohp1, Ohp2, O2, and Pbf in developingendosperms from 8 to 32 DAP and other tissues. All expression levelsare normalized to Actin. Four replicates for each sample were made andare illustrated as 6SD.

Three Master Regulators of Zein Gene Expression 1163

OHP1 and OHP2 Interact with PBF and Cotransactivate the27-kD g-Zein Promoter

The P box and O2-like box are separated by 48 bp in the pro-moter, suggesting that PBF and OHPs could interact (Uedaet al., 1992; Wu and Messing, 2012a). To test this possibility,pull-down assays were performed. As shown in Figure 4A, GST-tagged OHP1 and OHP2, but not the GST protein, were able topull down maltose binding protein (MBP)-tagged PBF, indicatingthat both OHPs can recognize PBF in vitro. This protein-proteininteraction was not detected in a previous study (Vicente-Carbajosa et al., 1997), probably due to the sensitivities of theexperimental systems employed. To examine these interactionsin vivo, we performed a luciferase complementation image (LCI)assay. OHP1, OHP2, and PBF were fused to the C- andN-terminal domains of LUCIFERASE (CLUC and NLUC, re-spectively). The results showed that cotransfection of PBF-NLUC with either OHP1-CLUC or OHP2-CLUC could producestrong luciferase activity, while individual infiltration of the threevectors with the corresponding empty construct failed to bringabout a visible signal (Figure 4B). These results demonstratedthat both OHP1 and OHP2 can physically interact with PBF.

To investigate the ability of OHPs and PBF to activate thetranscription of the 27-kD g-zein gene, its promoter was fusedwith the luciferase coding sequence, yielding the reporter vec-tor, P27-LUC, while the coding regions of Ohp1, Ohp2, and Pbfwere driven by the cauliflower mosaic virus 35S promoter, giving

rise to the effector plasmids. Injection of P27-LUC into a wildtobacco (Nicotiana benthamiana) leaf produced only basal lu-ciferase activity (Figure 4C). Cotransfection of P27-LUC with35S-PBF increases luciferase activity to a level that was ;2-foldhigher than the control, consistent with a previous study (Marzábalet al., 2008). When the reporter vector was cotransfected with35S-OHP1 or 35S-OHP2, the luciferase activities were elevatedeven more than with 35S-PBF, 4- or 5-fold higher than thecontrol, respectively, indicating that OHPs are also trans-activators of 27-kD g-zein expression, comparable to the role ofO2 in regulating the 22-kD a-zein genes (Schmidt et al., 1992).Strikingly, transactivation of the 27-kD g-zein promoter by OHP1and OHP2 was significantly reinforced by the addition of PBF;both luciferase activities were 6-fold higher than the control,indicating that the interaction of PBF and OHPs had an additiveaction on the expression of the 27-kD g-zein gene (Figure 4D).

Silencing of Ohps Results in Dramatically ReducedExpression of the 27-kD g-Zein Gene

To genetically substantiate the regulatory function of OHPs, nullmutants are required to demonstrate their effect on 27-kD g-zeingene expression. Ohp2 was identified as an allele with a Muinsertion in position2120 relative to the start codon (SupplementalFigures 2A and 2B). However, the Mu insertion did not appear todisrupt Ohp2 transcription (Supplemental Figure 2C); there-fore, no discernible change in zein accumulation was observedin homozygous ohp2-Mu1 (Supplemental Figure 2D). SinceOHP1 and OHP2 are likely to be functionally redundant (Figure4), we resorted to RNAi to simultaneously knock down theirexpression based on the sequence similarity of the two genes(see Methods). Four transgenic events were recovered, and allexhibited silenced expression of Ohp1 and Ohp2 (Figure 5A).Among them, event 6 accumulated 90% less Ohp1 and Ohp2transcript than the control, while event 3 showed moderatesuppression of the two genes. The mRNA levels of 27-kDg-zein were significantly reduced in all the events, with event 6being the most affected (Figure 5B). The zein accumulation

Figure 2. RNA in Situ Hybridization of O2, Pbf, Ohp1, Ohp2, 22-kDa-Zein, and 27-kD g-Zein Genes in Developing Seeds.

Longitudinal sections of W64A kernels at 12 DAP were hybridized withantisense RNA probes of O2, Pbf, Ohp1, Ohp2, 22-kD a-zein, and 27-kDg-zein. The positive signals (blue violet) of the six genes were mainlyobserved in the peripheral area of starchy endosperm. The expression ofOhp1 and Ohp2 was also detected in the embryo, but that of the otherfour genes was not. No signal was seen in the two sections hybridizedwith mixed sense probes. e, embryo; se, starchy endosperm; Senseprobes-1, mixed sense probes of 22-kD a-zein and 27-kD g-zein; Senseprobes-2, mixed sense probes of O2, Pbf, Ohp1, and Ohp2. Bar = 1 mm.

Figure 3. EMSA of OHP1 and OHP2 with the O2-Like Box.

DNA fragments containing the intact O2-like box and one with two nu-cleotide substitutions were used as probes. The relative amounts of theunlabeled intact probe used for competition are indicated. 1, probebound by OHP1 or OHP2 monomer; 2, probe bound by OHP1 or OHP2dimer; Mp, mutated probe.

1164 The Plant Cell

patterns are exemplified by events 3 and 6, where 27-kD g-zeintranscripts were downregulated to a moderate and a low level,respectively. As shown by SDS-PAGE in Figure 5C, 27-kDg-zein was barely detected in event 6, while its levels weresignificantly reduced but still detectable in event 3, consistentwith their mRNA levels (Figure 5B). The accumulation of otherzeins was not affected in either event, indicating that OHPsspecifically regulate 27-kD g-zein gene expression. Sinceevent 6 had the best silencing effect, it was used in subsequentexperiments.

Effects of o2, PbfRNAi, and OhpRNAi on the Expression ofZein Genes

To compare the synthesis of the main zein components (i.e.,27-kD g-zein and 22- and 19-kD [19-kD z1A, z1B, and z1D]a-zeins) in the three mutants, which constitute 80 to 90% oftotal zeins (Thompson and Larkins, 1994), PbfRNAi and OhpR-NAi were introgressed into W64A and W64Ao2 for severalgenerations (see Methods). The gene expression and proteinlevels of 22-kD a-zeins and 27-kD g-zein were dramatically re-duced in o2 and OhpRNAi, respectively, and they were bothstrongly downregulated in PbfRNAi (Figures 6A and 6B) (Wu andMessing, 2012a). However, these mutations had variable effectson the expression of other zein genes. Although 19-kD a-zein

genes had not been found to contain the O2 box or other ele-ments recognized by O2 (Schmidt et al., 1992), their expressionwas markedly affected in o2 (Or et al., 1993; Wu et al., 2010).Consistent with the transcript levels, the accumulation of 19-kDa-zeins was perceptibly decreased, although not as much as the22-kD a-zeins, consistent with prior observations (Or et al.,1993; Wu et al., 2010). Neither the mRNA nor the protein levelsof 27-kD g-zein in o2 were significantly altered compared withthe wild-type levels; in OhpRNAi, the 22- and 19-kD a-zeingenes were observed to produce somewhat lower levels ofmRNAs (P < 0.05) but normal amounts of proteins (Figures 5Cand 6), indicating that this amount of decrease of their tran-scripts is still not sufficient to cause translational reduction.PbfRNAi had no significant effect on overall mRNA levels of 19-kD a-zein genes, except for a decrease in z1D transcript levels(P < 0.05). Again, the accumulation of 19-kD a-zein proteins wasnot affected (Figures 6A and 6B).To study the effects of the genetic interaction of O2, Pbf, and

Ohps on zein gene expression, three double mutants (PbfRNAi;o2, OhpRNAi;o2, and PbfRNAi;OhpRNAi) and one triple mutant(PbfRNAi;OhpRNAi;o2) were created. The most striking doublemutant combinations affecting zein gene expression werePbfRNAi;o2 and OhpRNAi;o2. As expected of the two doublemutants, 27-kD g-zein mRNA and protein accumulated at verylow levels but did not exhibit further reduction compared with

Figure 4. Interaction of OHPs and PBF and Their Activation of the 27-kD g-Zein Promoter.

(A) Pull-down experiment showing that OHPs can interact with PBF. Recombinant MBP-PBF was incubated with GST-OHP1 or GST-OHP2, and theboiled supernatants were analyzed with GST and PBF antibodies.(B) LCI showing that OHPs and PBF interact. Fluorescence signal intensities represent their interaction activities.(C) Transactivation of the 27-kD g-zein promoter by OHPs and PBF. A representative image of an N. benthamiana leaf 48 h after infiltration is shown.(D) Quantitative analysis of the luminescence intensities shown in (C). Three independent determinations were assessed by ImageJ. Error bars rep-resent 6SD.

Three Master Regulators of Zein Gene Expression 1165

PbfRNAi and OhpRNAi single mutants, indicating that the im-pacts of PbfRNAi and OhpRNAi on 27-kD g-zein expressionwere epistatic to the effect of o2. In contrast, the mRNA andprotein levels of 22- and 19-kD a-zeins were dramatically furtherreduced compared with those in the single mutants (Figures 6Aand 6B). Some 22-kD a-zeins could be detected in o2 by SDS-PAGE, but they were completely missing in the double mutantsPbfRNAi;o2 and OhpRNAi;o2 (Figure 6B); the 19-kD a-zeintranscripts and proteins were also reduced to barely detectablelevels, compared with the sizable amounts that remained in o2,indicating that either PbfRNAi or OhpRNAi has additive andsynergistic effects on a-zein gene expression in o2. The com-bination of PbfRNAi and OhpRNAi was seen to have discernibleadditive effects on the expression of the 22-kD and 19-kD z1Da-zeins but far less than in the other two double mutants (Figure6A); as a consequence, a-zeins detected by SDS-PAGE hardlyshowed further reduction compared with the single mutants(Figure 6B). In the triple mutant, transcript and protein levels of19-kD a-zeins were further reduced, although the extent wasmuch less (Figure 6B).

To determine global effects on protein synthesis, quantitativemeasurements of zein and nonzein proteins were performed forthese mutants. In the single mutants, o2 had the most effect onthe accumulation of zeins, followed by PbfRNAi and OhpRNAi.In W64A, seed flour contained 6.74% zeins based on seed dryweight (zeins/seed flour 3 100%), while o2 had 2.92%, 57%lower than the wild type. PbfRNAi and OhpRNAi accumulated4.90 and 5.70% zeins, 27 and 15% lower, respectively, than the

wild type (Table 1). In the double mutants, zein contents drop-ped drastically in PbfRNAi;o2 and OhpRNAi;o2, to 0.76 and1.15%, respectively, which is 89 and 83% less than that in thewild type; in PbfRNAi;OhpRNAi, the zein level was only slightlyreduced compared with PbfRNAi, consistent with the observa-tion from SDS-PAGE (Figure 6B, Table 1). Among all the mu-tants, the triple mutant accumulated the least amount of zeins,merely 0.70% of seed flour, which was 90% less than in the wildtype (Table 1). For all mutants, the levels of nonzein proteinswere proportionately increased to compensate for zein re-duction, maintaining total protein levels in a relatively fixedwindow apparently through proteome rebalancing (Figure 7,Table 1) (Holding and Larkins, 2009; Schmidt et al., 2011; Wuand Messing, 2012b, 2014; Wu et al., 2012).

Development of Protein Bodies and Effects onKernel Texture

Zeins are synthesized on the polyribosomes of the rough en-doplasmic reticulum and deposited into its lumen to form the

Figure 5. Silencing of Ohp1 and Ohp2.

(A) Four transgenic events showing silenced expression of Ohp1 andOhp2 at 18 DAP. Four biological replicates for each event were madeand are illustrated as 6SD. Asterisks indicate significant differences fromthe wild type (Student’s t test, P < 0.05).(B) Four transgenic events showing decreased accumulation of 27-kDg-zein transcripts at 18 DAP. Quantitative RT-PCR was performed asdescribed above.(C) SDS-PAGE analysis of zein proteins in events 3 and 6. Three kernelseach inheriting or not inheriting with the RNAi construct from self-crossed events 3 and 6 were analyzed. Total zein loaded in each lanewas equal to 200 mg of maize flour. The size of each band is indicated bynumbers.

Figure 6. Expression Analysis of Zeins in Different Combinations ofOhpRNAi, PbfRNAi, and o2.

(A) Quantitative RT-PCR measurement of the expression levels of a-zein(22-kD z1C, 19-kD z1A, 19-kD z1B, and 19-kD z1D) and g-zein genes inall mutant combinations at 18 DAP. The data shown are from two to fourbiological replicates per sample and are illustrated as 6SD. Asterisksindicate significant differences from W64A (Student’s t test, P < 0.05).(B) SDS-PAGE analysis of zein proteins in all mutant combinations. Totalzein loaded in each lane was from 200 mg of maize flour. Genotypescorresponding to each lane are indicated in (A). The size of each band isindicated by the number beside it. M, protein markers from top to bottomcorrespond to 37, 25, 20, 15, and 10 kD.

1166 The Plant Cell

protein bodies (PBs) (Larkins and Davies, 1975; Burr and Burr,1976; Larkins and Hurkman, 1978; Lending and Larkins, 1989). Ifzein protein accumulation is reduced, the most affected featureis the size of the PBs (Wolf et al., 1967; Wu and Messing, 2010a).Indeed, the average diameter of PBs in each mutant was posi-tively correlated with the amount of zeins (Figure 8; Table 1).Strikingly, the size of PBs in the triple mutant was only aboutone-tenth that in the wild type, consistent with the lowest levelof zeins (Figure 8; Table 1).

OhpRNAi had a vitreous phenotype, while all the other mu-tants were opaque, indicating that the sole loss of 27-kD g-zeinwas insufficient to cause opacity (Supplemental Figure 3). Thisresult is consistent with previous observations in which simul-taneous silencing of 27- and 16-kD g-zeins failed to create anopaque kernel phenotype (Wu and Messing, 2010a, 2010b).

DISCUSSION

Endosperm-Specific and Nonspecific TFs Regulate SeedStorage Protein Gene Expression

We previously speculated the existence of a novel bZIP TF,other than O2, that regulates 27-kD g-zein gene expressionthrough its interaction with PBF, based on the fact that 22-kDa-zein and 27-kD g-zein genes contain similar components ofcis-elements in their promoters (Ueda et al., 1992; Wu andMessing, 2012a). Since OHPs are related to O2 in the maizebZIP family, they were obviously candidates for investigation(Pysh et al., 1993; Xu and Messing, 2008). It turned out that theregulatory model hypothesized for 22-kD a-zein genes can beappropriated for 27-kD g-zein as well (Supplemental Figure 1).OHP1 and OHP2 can both bind the O2-like box and trans-activate the 27-kD g-zein gene through interaction with PBF(Figures 3 and 4). In the absence of null mutations in Ohp1 andOhp2, RNAi was an alternative approach to study multicopygene function (Segal et al., 2003; Wu and Messing, 2010a,2012b). Indeed, based on SDS-PAGE, the levels of 27-kD g-zeinwere dramatically reduced in the most highly RNAi-suppressedevent (Figure 5C).

All zein genes are specifically expressed in endosperm from10 to 12 DAP. It is believed that their temporal and spatialspecificities are conferred by endosperm-specific TFs (Vicente-Carbajosa et al., 1997). The discovery of genes homologous toO2 and PBF in other cereal species supports this empirical

hypothesis (Albani et al., 1997; Conlan et al., 1999; Oñate et al.,1999; Onodera et al., 2001; Mena et al., 2002; Yamamoto et al.,2006; Kawakatsu et al., 2009). However, in dicots, the seedspecificity of storage protein gene expression is not necessarilyregulated by seed-specific TFs. The main storage proteins inArabidopsis thaliana seed are 2S albumins and 12S cruciferins(Bäumlein et al., 1994). These proteins are specifically expressedin the seed and transcriptionally regulated by two classes ofTFs, including B3s such as ABSCISIC ACID INSENSITIVE3(ABI3), FUSCA3 (FUS3), and LEAFY COTYLEDON2 (LEC2)(Giraudat et al., 1992; Bäumlein et al., 1994; Parcy et al., 1994;Stone et al., 2001) and bZIPs (bZIP10 and bZIP25) (Lara et al.,2003). The B3 TFs ABI3, FUS3, and LEC2 are all seed-specific,but bZIP10 and bZIP25, which are O2-related TFs, are generallyexpressed in all tissues examined. In fact, their transcripts ac-cumulate in roots, shoots, and leaves, where their levels aremuch higher than in the developing seed (Lara et al., 2003). Inour work, Ohp1 and Ohp2 were found to be expressed about1 order of magnitude higher in cobs, roots, and leaves than in18-DAP endosperm (Figure 1). Since the protein-protein inter-actions of B3 and bZIP10/25 in Arabidopsis and PBF and OHPs

Table 1. Protein Contents and PB Diameters of Different Mutant Combinations

Material W64A PbfRNAi W64Ao2 OhpRNAi PbfRNAi;o2 PbfRNAi;OhpRNAi OhpRNAi;o2 PbfRNAi;OhpRNAi;o2

Zeina 6.74 6 0.03 4.90 6 0.00 2.92 6 0.11 5.70 6 0.06 0.76 6 0.01 4.61 6 0.03 1.15 6 0.04 0.70 6 0.08Nonzeina 4.26 6 0.10 4.74 6 0.09 6.21 6 0.42 4.61 6 0.11 8.04 6 0.52 4.43 6 0.12 8.70 6 0.49 6.93 6 0.31Total proteinb 11.00 9.64 9.13 10.31 8.80 9.04 9.85 7.63PB diameterc 1.10 6 0.21 0.57 6 0.19 0.37 6 0.05 0.99 6 0.17 0.28 6 0.07 0.46 6 0.11 0.26 6 0.04 0.13 6 0.03PBs counted 117 127 120 117 114 103 102 144aProtein contents in 100 mg of dry seed flour, averaged with three replicates and illustrated as 6SD.bSum of zein and nonzein contents.cPBs counted from the second to fourth starchy endosperm layers at 18 DAP (diameter = mm).

Figure 7. Proteome Rebalancing in Different Mutant Combinations.

As zein levels decreased, the levels of nonzeins increased to compen-sate. Values shown are mg of protein per 100 mg of dry seed flour. Totalprotein content is the sum of zein and nonzein contents. Error barsrepresent 6SD with three replicates.

Three Master Regulators of Zein Gene Expression 1167

in maize are mandatory for the transactivation of their targetedgenes, it appears that only one class of TFs is seed-specific, andtheir cotransactivators need not be seed-specific to ensure theseed fidelity of storage protein synthesis. However, the 16-kDg-zein gene, a homeologous copy of the 27-kD g-zein genearising from maize allotetraploidization, has a deletion in thepromoter, which eliminates the P box and O2-like box, but it isstill specifically expressed in endosperm, indicating that otherregulators, possibly including epigenetic factors, can determinatethe endosperm specificity of zein gene expression (Wu andMessing, 2012a).

Differential Temporal Patterns of O2, Pbf, and OhpExpression in Maize Endosperm

The deposition of zeins in PBs is temporally and spatially or-ganized (Lending and Larkins, 1989, 1992). g-Zeins and b-zeinsappear first and are thought to initiate PB formation. Immuno-transmission electron microscopy revealed that g- and b-zeinsare localized in the peripheral region of PBs, while a- andd-zeins, synthesized subsequently, accumulate in the centralarea and enlarge the PBs (Lending and Larkins, 1989). Althoughthe spatial expression patterns of O2, Pbf, and Ohps in starchyendosperm cells are similar (Figure 2), their temporal patternsare different (Figure 1). In addition, since the o2 mutant hadmuch less effect on the transcription of the 27-kD g-zein and didnot cause a perceivable decrease in the protein level comparedwith OhpRNAi (Figures 5 and 6), O2 appears to be only a minorregulator of the 27-kD g-zein. From 8 to 12 DAP, Ohp1 andOhp2 are expressed at significantly higher levels than O2, andone could envision that, with the onset of Pbf expression, acti-vation of the 27-kD g-zein should be more rapid than that of the22-kD a-zeins. To test this, we performed RT-PCR to measuretheir expression. As shown in Supplemental Figure 4A, expression

of the 27-kD g-zein gene occurred by 10 DAP, while that of 22-kDa-zeins did not occur until 12 DAP. In the zein accumulation patternobserved by SDS-PAGE at 12 DAP, the predominant band is the27-kD g-zein, and 22-kD a-zeins do not yet appear (SupplementalFigure 4B). This difference in 27-kD g-zein and 22-kD a-zein ac-cumulation is consistent with the differential temporal expression

Figure 8. Transmission Electron Microscopy of PBs in Different Mutant Combinations.

The fourth starchy endosperm cell layer at 18 DAP was analyzed for each genotype. Each genotype is indicated above the corresponding panel. CW,cell wall; RER, rough endoplasmic reticulum; SG, starch granule. Bars in full images = 1 mm; bars in enlarged inset images = 200 nm.

Figure 9. Hypothetical Model Depicting the Transcriptional Regulationof the 27-kD g- and a-Zein Genes Mediated by O2, PBF, and OHPs.

Due to the significantly higher expression of OHPs than O2 before 12DAP, the 27-kD g-zein gene is expressed earlier than the a-zein genes;therefore, the 27-kD g-zein protein is more predominant than a-zeinproteins in the early-forming PBs. Afterward, the amount of a-zein pro-teins increases rapidly, enlarging the PBs, when the level of O2 is in-creased. For the regulation of 22-kD (and 19-kD) a-zein genes, O2 is themain transactivator, while PBF and OHP have additive and synergisticactions on their expression.

1168 The Plant Cell

patterns of O2 and Ohps and perhaps is related to the mechanismof PB formation (Figure 9).

Regulation of Seed Storage Protein Gene Expressionin Maize

One of the striking features of the zein gene family is its ex-pression level, with g- and a-zeins being the most highly ex-pressed subfamilies (Hunter et al., 2002; Chen et al., 2014). Inour work here, we found that the synthesis of most zeins can bemassively affected at the transcriptional level by the combina-tion of o2 with either PbfRNAi or OhpRNAi. The triple mutantsynthesized 90% less zeins than the wild type (Figure 6, Table1). However, due to proteome rebalancing, the total seed proteincontent approached a level that did not deviate much from thewild type by compensatory synthesis of nonzein proteins (Figure7, Table 1) (Holding and Larkins, 2009; Schmidt et al., 2011; Wuand Messing, 2012b, 2014; Wu et al., 2012). Despite this, thetriple mutant accumulated the lowest level of total protein, in-dicating that the combination of the three mutations can havepleiotropic effects on protein synthesis and proteome rebalanc-ing. As PBs are specialized organelles for the storage of zeinproteins, it is not surprising that their sizes correlate well with theremaining amounts of zeins.

As shown in Figure 6A, all the single mutants had variablenegative effects on transcript levels of 19-kD a-zeins, but onlyo2 accumulated discernibly reduced amounts of these proteins.However, the combination of o2 with either PbfRNAi or OhpRNAicaused a sharp decline in the levels of all a-zein transcripts andproteins (Figures 6A and 6B), indicating that O2 has the greatestimpact on the expression of all a-zein genes and PBF and OHPshave additive and synergistic effects on their expression (Figure9). However, a regulatory cis-motif in 19-kD a-zein promotersthat is specifically targeted by O2 has not been identified. Thus,the regulatory machinery involved in DNA-protein recognitionbetween O2, PBF, OHPs, and perhaps other unknown factorsand cis-elements remains to be identified for the 19-kD a-zeins(Figure 9).

METHODS

Genetic Materials

Coding sequences of Ohp1 (L00623) and Ohp2 (L06478) from maize (Zeamays) were used to search against the UniformMu Transposon Resource(http://www.maizegdb.org/documentation/uniformmu/index.php), andMu04042 and Mu06960 were found to contain a Mu insertion in Ohp2.Through PCR amplification and sequencing, the two insertions werefound to be in the same position, at2120 bp relative to the start codon ofOhp2. Therefore, the two mutants were designated as Ohp2-Mu1.

Because coding sequences of Ohp1 and Ohp2 share 89% identity,a 664-bp Ohp1 cDNA fragment was amplified for OhpRNAi construction.The method for making PbfRNAi was described elsewhere (Wu andMessing, 2012a). The OhpRNAi cassette was coupled with the visiblegreen fluorescent protein (GFP) marker under the control of the 10-kDd-zein promoter to facilitate scoring transgenic-positive progeny seeds(Supplemental Figure 5) (Wu et al., 2013). Four positive events were re-covered following the standard transformation protocol (Frame et al.,2002). All primers used for plasmid construction are listed in Supplemental

Table 1. PbfRNAi was introgressed into W64A and W64Ao2 for threegenerations and self-crossed for two generations, yielding the homo-zygous single and double mutants W64APbfRNAi and W64APbfRNAi;o2.The newly generated OhpRNAi in the Hi-II genetic background wasbackcrossed into W64A and W64Ao2 for two generations, yielding thesingle and double mutants W64AOhpRNAi/+ and W64AOhpRNAi/+;o2.The positive OhpRNAi progeny were screened by viewing GFP fluo-rescence in kernels with Dark Reader visualization glasses under anSL9S Spot lamp (Clare Chemical Research). The selected progeny(W64AOhpRNAi/+ and W64AOhpRNAi/+;o2) were crossed withW64APbfRNAi and W64APbfRNAi;o2, yielding the double and triplemutantsW64APbfRNAi/+;OhpRNAi/+ andW64APbfRNAi/+;OhpRNAi/+;o2.Since RNAi is dominant, the eight genetic materials used in this study aredesignated asW64A,W64Ao2,PbfRNAi,OhpRNAi,PbfRNAi;o2,OhpRNAi;o2,PbfRNAi;OhpRNAi, and PbfRNAi;OhpRNAi;o2, regardless of the homo-zygosity or heterozygosity of RNAi.

Quantitative and Standard RT-PCR

Total RNA was extracted from developing endosperms and other tissuesusing TRIzol reagent (Invitrogen) and purified with the RNeasy Mini Kitafter DNase 1 digestion (Qiagen). The concentration and purity of the RNAwere determined using a NanoDrop 2000 spectrophotometer. TheSuperScript III First Strand Kit (Invitrogen) was used for reverse tran-scription. Standard RT-PCR was performed with REDTaq Reaction Mix(Sigma-Aldrich) and quantitative RT-PCR with SYBR Green I. The com-parative CT method (DDCT method) was employed for the relative quan-tification of gene expression, in which themaizeActin genewas used as thereference. Statistically significant differences of target gene expression fromdifferentmutants were calculated by Student’s t test. All primers are listed inSupplemental Table 1.

Expression and Purification of Recombinant Proteins

The expression constructs for GST-OHP1 and GST-OHP2 were gener-ated by cloning the corresponding open reading frames (ORFs) into theBamHI and EcoRI sites of pGEX-4T-1 (Amersham). The ORF of Pbf wasfused to the MBP tag of the expression vector pMAL-C2X (New EnglandBiolabs). All recombinant proteins were affinity-purified following themanufacturer’s manual.

EMSA

The two oligonucleotide probes of the 27-kD g-zein promoter described inFigure 3, containing the intact andmutated O2-like box, were synthesizedand labeled with biotin at the 39 end (Thermo). The probes weremixed withthe purified protein at 25°C for 20 min in a reaction buffer containing 13binding buffer, 2.5% glycerol, 5 mM MgCl2, 50 ng/mL poly(dI∙dC), and0.05% Nonidet P-40. The mixture was separated by 6% native PAGE in0.53 Tris/borate/EDTA buffer. Subsequent procedures were performedaccording to the instructions of the LightShift Chemiluminescent EMSAKit (Thermo). The luminescence was visualized on the Tanon-5200Chemiluminescent Imaging System (Tanon Science and Technology).

GST Pull-Down Assay

GST or GST-OHP fused proteins were mixed with MBP-tagged PBF in 1mL of GST binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,and 1.8 mM KH2PO4, pH 7.3), and the mixture was kept at 4°C for 4 h withrotating. After washing with GST binding buffer, Glutathione Sepharose4B (Amersham) was added to each mixture and then kept rotating foranother 2 h at 4°C. After washing three times with GST binding buffer, theGST resin was boiled in SDS loading buffer, and the supernatants were

Three Master Regulators of Zein Gene Expression 1169

analyzed by immunoblotting. For protein gel blot analysis, the elutedproteins were separated by 15 or 12% SDS-PAGE and transferred ontoa nitrocellulose membrane (Whatman). The anti-GST or anti-PBF proteinand the secondary antibody, anti-rabbit-IgG conjugated to horseradishperoxidase, was used at a 1:5000 dilution. Protein bands were visualizedusing the Tanon-5200 system.

LCI Assay and Transactivation of 27-kD g-Zein Promoter

ORFs of Pbf, Ohp1, and Ohp2 were cloned into JW771 (NLUC) andJW772 (CLUC), respectively, yielding PBF-NLUC and OHP1/OHP2-CLUC constructs for the LCI assay, following the protocol describedelsewhere (Gou et al., 2011).

The reporter P27-LUC was generated by insertion of the 27-kD g-zeinpromoter (1052 bp) into the HindIII and BamHI sites of the pLL00R vector.Effectors 35S-OHP1, 35S-OHP2, and 35S-PBF were created by cloningtheir corresponding coding sequences into pRI101 vector (Takara). Theempty vector was used as a negative control.

Agrobacterium tumefaciens GV3101 harboring the above constructswas infiltrated into 5-week-old Nicotiana benthamiana leaves using a nee-dleless syringe for LCI and transactivation analyses. After growing for 48 hunder the condition of 16 h of light and 8 h of dark, leaves were injected with0.94 mM luciferin, and the resulting luciferase signals were captured usingthe Tanon-5200 image system. These experiments were repeated at leastthree times with similar results. Quantitative analysis was performed usingImageJ software (http://rsb.info.nih.gov/ij/).

RNA in Situ Hybridization

W64A kernels at 12 DAP were used for RNA in situ hybridization. cDNAfragments of target genes were inserted into the pBluescript SK+ vectorfor RNA probe synthesis. The primers for amplifying these fragments arelisted in Supplemental Table 1. The antisense and sense RNA probesweresynthesized by in vitro transcription using T7 and T3 RNA polymerase,with DIG RNA Labeling Mixture (Roche). Tissue processing and in situhybridization experiments using 10-mm sections were performed ac-cording to the methods described previously (Cox and Goldberg, 1988;Langdale, 1994).

The sections were observed and imaged with a light stereomicroscope(Leica M165 FC).

Protein Quantification

At least 10 mature kernels for each sample were ground into fine powderwith a coffee grinder, and 100 mg of flour was weighed for the extractionof zein and nonzein proteins. The patterns of zein accumulation wereanalyzed by SDS-PAGE. Protein quantification was performed with theCompat-Able Protein Assay Preparation Reagent Kit and the BCA ProteinAssay Kit (Pierce). All measurements were replicated at least three times.

Transmission Electron Microscopy

Kernels at 18 DAP of W64A and different mutants were sliced, fixed,dehydrated, and embedded as described previously (Wu and Messing,2010a). Ultrathin sections of the samples were cut with a diamond knife ona Leica EMUC6-FC6 ultramicrotome and imaged at 80 kV with a HitachiH-7650 transmission electron microscope.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBLdata libraries under accession numbers NM_001111951 for O2,NM_001111930 for Pbf, L00623 for Ohp1, and L06478 for Ohp2.

Supplemental Data

Supplemental Figure 1. A working model for transcriptional regulationof the 22-kD a- and 27-kD g-zein genes.

Supplemental Figure 2. Mu insertion in Ohp2.

Supplemental Figure 3. Kernel phenotypes of different mutantcombinations.

Supplemental Figure 4. Expression patterns of 27-kD g-zein and22-kD a-zeins in endosperm.

Supplemental Figure 5. OhpRNAi construct.

Supplemental Table 1. Primer list.

ACKNOWLEDGMENTS

We thank the reviewers for their critical review of this article and theirinvaluable suggestions on the editing of the language. The PbfRNAi usedin the work was originally generated and obtained from JoachimMessing’s laboratory at the Waksman Institute of Microbiology, RutgersUniversity. This research was supported by the National Natural ScienceFoundation of China (Grants 31371630, 91335109, and 31422040 toY.W.) and a Chinese Thousand Talents Program grant (to Y.W.).

AUTHOR CONTRIBUTIONS

Z.Z., J.Y., and Y.W. designed and performed the research. Z.Z., J.Y., andY.W. analyzed the data. Z.Z., J.Y., and Y.W. wrote the article.

Received January 14, 2015; revised April 6, 2015; accepted April 9, 2015;published April 21, 2015.

REFERENCES

Albani, D., Hammond-Kosack, M.C., Smith, C., Conlan, S., Colot,V., Holdsworth, M., and Bevan, M.W. (1997). The wheat tran-scriptional activator SPA: A seed-specific bZIP protein that recog-nizes the GCN4-like motif in the bifactorial endosperm box ofprolamin genes. Plant Cell 9: 171–184.

Bäumlein, H., Miséra, S., Luerßen, H., Kölle, K., Horstmann, C.,Wobus, U., and Müller, A.J. (1994). The FUS3 gene of Arabidopsisthaliana is a regulator of gene expression during late embryogene-sis. Plant J. 6: 379–387.

Burr, B., and Burr, F.A. (1976). Zein synthesis in maize endosperm bypolyribosomes attached to protein bodies. Proc. Natl. Acad. Sci.USA 73: 515–519.

Chen, J., Zeng, B., Zhang, M., Xie, S., Wang, G., Hauck, A., and Lai,J. (2014). Dynamic transcriptome landscape of maize embryo andendosperm development. Plant Physiol. 166: 252–264.

Coleman, C., and Larkins, B. (1999). The prolamins of maize. In SeedProteins, P. Shewry and R. Casey, eds (Dordrecht, The Netherlands:Springer), pp. 109–139.

Conlan, R.S., Hammond-Kosack, M., and Bevan, M. (1999). Tran-scription activation mediated by the bZIP factor SPA on the endo-sperm box is modulated by ESBF-1 in vitro. Plant J. 19: 173–181.

Cox, K.H., and Goldberg, R.B. (1988). Analysis of plant gene ex-pression. In Plant Molecular Biology: A Practical Approach, C.H.Shaw, ed (Oxford, UK: Oxford IRL Press), pp. 1–35.

Esen, A. (1987). A proposed nomenclature for the alcohol-solubleproteins (zeins) of maize (Zea mays L.). J. Cereal Sci. 5: 117–128.

1170 The Plant Cell

Frame, B.R., Shou, H., Chikwamba, R.K., Zhang, Z., Xiang, C.,Fonger, T.M., Pegg, S.E., Li, B., Nettleton, D.S., Pei, D., andWang, K. (2002). Agrobacterium tumefaciens-mediated trans-formation of maize embryos using a standard binary vector system.Plant Physiol. 129: 13–22.

Giraudat, J., Hauge, B.M., Valon, C., Smalle, J., Parcy, F., andGoodman, H.M. (1992). Isolation of the Arabidopsis ABI3 gene bypositional cloning. Plant Cell 4: 1251–1261.

Gou, J.Y., Felippes, F.F., Liu, C.J., Weigel, D., and Wang, J.W.(2011). Negative regulation of anthocyanin biosynthesis in Arabi-dopsis by a miR156-targeted SPL transcription factor. Plant Cell 23:1512–1522.

Holding, D., and Larkins, B. (2009). Zein storage proteins. In Mo-lecular Genetic Approaches to Maize Improvement, A. Kriz and B.Larkins, eds (Berlin: Springer), pp. 269–286.

Hunter, B.G., Beatty, M.K., Singletary, G.W., Hamaker, B.R.,Dilkes, B.P., Larkins, B.A., and Jung, R. (2002). Maize opaqueendosperm mutations create extensive changes in patterns of geneexpression. Plant Cell 14: 2591–2612.

Kawakatsu, T., Yamamoto, M.P., Touno, S.M., Yasuda, H., andTakaiwa, F. (2009). Compensation and interaction between RISBZ1and RPBF during grain filling in rice. Plant J. 59: 908–920.

Langdale, J. (1994). In situ hybridization. In The Maize Handbook,M. Freeling and V. Walbot, eds (New York: Springer), pp. 165–180.

Lara, P., Oñate-Sánchez, L., Abraham, Z., Ferrándiz, C., Díaz, I.,Carbonero, P., and Vicente-Carbajosa, J. (2003). Synergisticactivation of seed storage protein gene expression in Arabidopsisby ABI3 and two bZIPs related to OPAQUE2. J. Biol. Chem. 278:21003–21011.

Larkins, B.A., and Davies, E. (1975). Polyribosomes from peas. V. Anattempt to characterize the total free and membrane-bound poly-somal population. Plant Physiol. 55: 749–756.

Larkins, B.A., and Hurkman, W.J. (1978). Synthesis and depositionof zein in protein bodies of maize endosperm. Plant Physiol. 62:256–263.

Lending, C.R., and Larkins, B.A. (1989). Changes in the zein com-position of protein bodies during maize endosperm development.Plant Cell 1: 1011–1023.

Lending, C.R., and Larkins, B.A. (1992). Effect of the floury-2 locuson protein body formation during maize endosperm development.Protoplasma 171: 123–133.

Marzábal, P., Gas, E., Fontanet, P., Vicente-Carbajosa, J., Torrent,M., and Ludevid, M.D. (2008). The maize Dof protein PBF activatestranscription of gamma-zein during maize seed development. PlantMol. Biol. 67: 441–454.

Mena, M., Cejudo, F.J., Isabel-Lamoneda, I., and Carbonero, P.(2002). A role for the DOF transcription factor BPBF in the regulationof gibberellin-responsive genes in barley aleurone. Plant Physiol.130: 111–119.

Neto, G.C., Yunes, J.A., da Silva, M.J., Vettore, A.L., Arruda, P.,and Leite, A. (1995). The involvement of Opaque 2 on b-prolamingene regulation in maize and Coix suggests a more general role forthis transcriptional activator. Plant Mol. Biol. 27: 1015–1029.

Olsen, O.-A., and Becraft, P.W. (2013). Endosperm development. InSeed Genomics, P.W. Becraft, ed (Hoboken, NJ: Wiley-Blackwell),pp. 43–62.

Oñate, L., Vicente-Carbajosa, J., Lara, P., Díaz, I., and Carbonero,P. (1999). Barley BLZ2, a seed-specific bZIP protein that interactswith BLZ1 in vivo and activates transcription from the GCN4-likemotif of B-hordein promoters in barley endosperm. J. Biol. Chem.274: 9175–9182.

Onodera, Y., Suzuki, A., Wu, C.Y., Washida, H., and Takaiwa, F.(2001). A rice functional transcriptional activator, RISBZ1, re-sponsible for endosperm-specific expression of storage proteingenes through GCN4 motif. J. Biol. Chem. 276: 14139–14152.

Or, E., Boyer, S.K., and Larkins, B.A. (1993). opaque2 modifiers actpost-transcriptionally and in a polar manner on gamma-zein geneexpression in maize endosperm. Plant Cell 5: 1599–1609.

Osborne, T.B., Mendel, L.B., Ferry, E.L., and Wakeman, J.A. (1914).Nutritive properties of proteins of the maize kernel. J. Biol. Chem.18: 1–16.

Parcy, F., Valon, C., Raynal, M., Gaubier-Comella, P., Delseny, M.,and Giraudat, J. (1994). Regulation of gene expression programsduring Arabidopsis seed development: Roles of the ABI3 locus andof endogenous abscisic acid. Plant Cell 6: 1567–1582.

Pysh, L.D., Aukerman, M.J., and Schmidt, R.J. (1993). OHP1: Amaize basic domain/leucine zipper protein that interacts with opa-que2. Plant Cell 5: 227–236.

Schmidt, M.A., Barbazuk, W.B., Sandford, M., May, G., Song, Z.,Zhou, W., Nikolau, B.J., and Herman, E.M. (2011). Silencing ofsoybean seed storage proteins results in a rebalanced proteincomposition preserving seed protein content without major collat-eral changes in the metabolome and transcriptome. Plant Physiol.156: 330–345.

Schmidt, R.J., Ketudat, M., Aukerman, M.J., and Hoschek, G.(1992). Opaque-2 is a transcriptional activator that recognizesa specific target site in 22-kD zein genes. Plant Cell 4: 689–700.

Segal, G., Song, R., and Messing, J. (2003). A new opaque variant ofmaize by a single dominant RNA-interference-inducing transgene.Genetics 165: 387–397.

Stone, S.L., Kwong, L.W., Yee, K.M., Pelletier, J., Lepiniec, L.,Fischer, R.L., Goldberg, R.B., and Harada, J.J. (2001). LEAFYCOTYLEDON2 encodes a B3 domain transcription factor that in-duces embryo development. Proc. Natl. Acad. Sci. USA 98: 11806–11811.

Thompson, G., and Larkins, B. (1994). Characterization of zein genesand their regulation in maize endosperm. In The Maize Handbook,M. Freeling and V. Walbot, eds (New York: Springer), pp. 639–647.

Ueda, T., Waverczak, W., Ward, K., Sher, N., Ketudat, M., Schmidt,R.J., and Messing, J. (1992). Mutations of the 22- and 27-kD zeinpromoters affect transactivation by the Opaque-2 protein. Plant Cell4: 701–709.

Vicente-Carbajosa, J., Moose, S.P., Parsons, R.L., and Schmidt,R.J. (1997). A maize zinc-finger protein binds the prolamin box inzein gene promoters and interacts with the basic leucine zippertranscriptional activator Opaque2. Proc. Natl. Acad. Sci. USA 94:7685–7690.

Wolf, M.J., Khoo, U., and Seckinger, H.L. (1967). Subcellular structure ofendosperm protein in high-lysine and normal corn. Science 157: 556–557.

Wu, Y., and Messing, J. (2010a). RNA interference-mediated changein protein body morphology and seed opacity through loss of dif-ferent zein proteins. Plant Physiol. 153: 337–347.

Wu, Y., and Messing, J. (2010b). Rescue of a dominant mutant withRNA interference. Genetics 186: 1493–1496.

Wu, Y., and Messing, J. (2012a). Rapid divergence of prolamin genepromoters of maize after gene amplification and dispersal. Genetics192: 507–519.

Wu, Y., and Messing, J. (2012b). RNA interference can rebalance thenitrogen sink of maize seeds without losing hard endosperm. PLoSONE 7: e32850.

Wu, Y., and Messing, J. (2014). Proteome balancing of the maizeseed for higher nutritional value. Front. Plant Sci. 5: 240.

Three Master Regulators of Zein Gene Expression 1171

Wu, Y., Holding, D.R., and Messing, J. (2010). Gamma-zeins areessential for endosperm modification in quality protein maize. Proc.Natl. Acad. Sci. USA 107: 12810–12815.

Wu, Y., Wang, W., and Messing, J. (2012). Balancing of sulfur storagein maize seed. BMC Plant Biol. 12: 77.

Wu, Y., Yuan, L., Guo, X., Holding, D.R., and Messing, J. (2013).Mutation in the seed storage protein kafirin creates a high-valuefood trait in sorghum. Nat. Commun. 4: 2217.

Xu, J.H., and Messing, J. (2008). Diverged copies of the seedregulatory Opaque-2 gene by a segmental duplication in the pro-genitor genome of rice, sorghum, and maize. Mol. Plant 1: 760–769.

Yamamoto, M.P., Onodera, Y., Touno, S.M., and Takaiwa, F.(2006). Synergism between RPBF Dof and RISBZ1 bZIP activatorsin the regulation of rice seed expression genes. Plant Physiol. 141:1694–1707.

1172 The Plant Cell

DOI 10.1105/tpc.15.00035; originally published online April 21, 2015; 2015;27;1162-1172Plant Cell

Zhiyong Zhang, Jun Yang and Yongrui WuSynergistic Action of opaque2, Prolamine-Box Binding Factor, and O2 Heterodimerizing Proteins

Transcriptional Regulation of Zein Gene Expression in Maize through the Additive and

 This information is current as of May 24, 2020

 

Supplemental Data /content/suppl/2015/04/29/tpc.15.00035.DC1.html

References /content/27/4/1162.full.html#ref-list-1

This article cites 42 articles, 30 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists