NAC-type transcription factors regulate accumulationof starch and protein in maize seedsZhiyong Zhanga,1, Jiaqiang Donga,1, Chen Jib,c, Yongrui Wub,c, and Joachim Messinga,2

aWaksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854; bNational Key Laboratory of Plant Molecular Genetics, Institute of PlantPhysiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032 Shanghai, China; and cChinese Academy of SciencesCenter for Excellence in Molecular Plant Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032 Shanghai, China

Contributed by Joachim Messing, April 19, 2019 (sent for review March 22, 2019; reviewed by L. Curtis Hannah and Alan Myers)

Grain starch and protein are synthesized during endosperm develop-ment, prompting the question of what regulatory mechanismunderlies the synchronization of the accumulation of secondary andprimary gene products. We found that two endosperm-specific NACtranscription factors, ZmNAC128 and ZmNAC130, have such a regu-latory function. Knockdown of expression of ZmNAC128 andZmNAC130 with RNA interference (RNAi) caused a shrunken kernelphenotype with significant reduction of starch and protein. We couldshow that ZmNAC128 and ZmNAC130 regulate the transcription ofBt2 and then reduce its protein level, a rate-limiting step in starchsynthesis of maize endosperm. Lack of ZmNAC128 and ZmNAC130also reduced accumulation of zeins and nonzeins by 18% and24% compared with nontransgenic siblings, respectively. AlthoughZmNAC128 and ZmNAC130 affected expression of zein genes in gen-eral, they specifically activated transcription of the 16-kDa γ-zeingene. The two transcription factors did not dimerize with each otherbut exemplified redundancy, whereas individual discovery of theirfunction was not amenable to conventional genetics but illustratedthe power of RNAi. Given that both the Bt2 and the 16-kDa γ-zeingenes were activated by ZmNAC128 or ZmNAC130, we could identifya core binding site ACGCAA contained within their target promoterregions by combining Dual-Luciferase Reporter and ElectrophoreticMobility Shift assays. Consistent with these properties, transcriptomicprofiling uncovered that lack of ZmNAC128 and ZmNAC130 had apleiotropic effect on the utilization of carbohydrates and amino acids.

maize endosperm | gene regulation | starch synthesis | protein

Endosperms, up to 90% of cereal grains, not only supportembryo and seedling development but also provide the primary

resource of carbohydrate and protein for humans and livestock.Carbohydrate is mainly stored as starch granules (SGs) in amyloplastsof endosperm cells. Prolamins, the most abundant storage proteins inmost grains, are deposited in protein bodies (PBs) or protein storagevacuoles (PSVs). Therefore, SGs and PBs/PSVs are the most abun-dant organelles in grain endosperm cells. Due to the importance ofhuman food, they have been comprehensively studied (1, 2).Maize (Zea mays) prolamins, also called zeins, account for

∼60 to 70% of endosperm proteins (1). Maize endosperm con-tains four types of prolamins, α-, β-, γ-, and δ-zeins, which areencoded by a large set of genes (3, 4). Because their expression isstrictly regulated in a temporal and spatial pattern, the tran-scriptional regulation has been widely investigated by identifyingconserved cis-elements in their promoters and correspondingtranscription factors (TFs). Several conserved cis-elements havebeen found in promoters of most zein genes, like the prolamin-box (P-box) (TGTAAAG), Opaque2-box (O2-box) (TCCACGT),or O2-like box (TTTACGT) (5). The corresponding TFs have alsobeen successively identified. O2, an endosperm-specific bZIP TF,binds to the O2-box to transactivate the expression of 22-kDaα-zein and 15-kDa β-zein genes (6–8). Recently, a genome-widestrategy of ChIP-Seq combined with differential expression anal-ysis also identified other O2 binding motifs, like TGACGTGG (9, 10).O2 can regulate the expression of most zeins except for 16-kDaγ-zein, illustrating a central role for O2-regulating zein expression.

Another well-known endosperm-specific DNA-binding with onefinger (Dof) TF is the Prolamin-Box Binding Factor (PBF). PBFspecifically binds the P-box to regulate the expression of 22-kDaα-zein and 27-kDa γ-zein genes (5, 11). Besides O2 and PBF, anadditional four TFs were recently identified to regulate the ex-pression of zeins. Three of them are bZIP TFs: i.e., O2 hetero-dimerizing proteins (OHP1 and OHP2) and ZmbZIP22 (12, 13).OHP1 and OHP2 can recognize an O2-like box in the promotersof the 27-kDa γ-zein and 22-kDa α-zein whereas ZmbZIP22 bindsto the ACAGCTCA box in the 27-kDa γ-zein promoter, forming acomplex with O2, PBF, OHP1/OHP2 (13). ZmMADS47, aMADS box-containing TF, binds the CATGT motif in the pro-moters of α-zein and 50-kDa γ-zein, but its interaction with O2 isrequired for the transcriptional activation of these genes (14).However, there are still examples of missing regulatory factors: forinstance, Dzr1, regulating δ-zein accumulation (15) or the tran-scriptional activators of 16-kDa γ-zein.Moreover, how are synthesis and accumulation of both zeins

and starch coordinated at the filling stage from 10 to 35 d afterpollination (DAP) (16–18)? Unlike the synthesis of zeins, thestarch biosynthetic pathway is more complicated and involvescarbohydrate metabolism, such as the oxidative pentose phos-phate pathway (2). Indeed, there is less knowledge about thetranscriptional regulation of starch biosynthetic genes in maizeor other crops although O2 not only regulates the expression ofzeins but also affects the regulation of other storage compounds(9, 10). Direct evidence for O2 modulating starch synthesis is

Significance

World food supply depends on improving grain yield andquality, which are determined by accumulation of starch andproteins in maize endosperm, respectively. Because initiationof synthesis of the two compounds occurs 8 to 10 d after pol-lination in starchy endosperm cells, regulatory factors mustcontrol their coordinated accumulation. This work shows thattwo maize endosperm-specific transcription factors can co-ordinate the accumulation of starch and proteins by regulatingthe expression of key starch biosynthetic enzymes and themajor seed proteins.

Author contributions: Z.Z., J.D., Y.W., and J.M. designed research; Z.Z., J.D., and C.J.performed research; C.J. contributed new reagents/analytic tools; Z.Z., J.D., Y.W., andJ.M. analyzed data; and Z.Z., Y.W., and J.M. wrote the paper.

Reviewers: L.C.H., University of Florida; and A.M., Iowa State University.

The authors declare no conflict of interest.

Published under the PNAS license.

Data deposition: RNA-Seq data reported in this paper have been deposited in the Na-tional Center for Biotechnology Information Gene Expression Omnibus database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE127525).1Z.Z. and J.D. contributed equally to this work.2To whom correspondence may be addressed. Email: messing@waksman.rutgers.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1904995116/-/DCSupplemental.

Published online May 20, 2019.

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that O2 binds the O2-box in the promoter of SSIII to regulateits expression (19). PBF binds the core elements of P-box nearO2-box in the promoter of SSIII to coordinate its expression withO2. The synergetic regulatory role of PBF and O2 also applies tozein genes (19). The previous studies also tried to identify TFsregulating synthetic starch genes by coexpression analysis and bio-chemistry experiments, like the Electrophoretic Mobility Shift Assay(EMSA) and the Dual-Luciferase Reporter (DLR) assay. Therehave been candidate TFs, including one barley WRKY transcriptionfactor of SUSIBA2, and three maize transcription factors ofZmNAC36, ZmbZIP91, and ZmEREB156, although their regula-tory function on starch synthesis remains to be validated (20–23).In the search for TFs that regulate both starch biosynthetic

and zein genes, we took an approach different to the abovestudies by accounting for gene amplification. We investigatedRNA expression at early stages of endosperm development andfound that the two TFs, ZmNAC128 and ZmNAC130, are spe-cifically and strongly expressed at the filling stage. Because of thehigh identities of ZmNAC128 and ZmNAC130 in amino acidsequence, we used RNA interference (RNAi) to reduce theirexpression because it overcomes gene amplification and is domi-nant (24). Indeed, nacRNAi leads to a shrunken kernel phenotypewith significant reduction of starch and protein accumulationduring endosperm development, which would not have been de-tected by a single mutant screen. Molecular and biochemistryevidence uncovered that ZmNAC128 and ZmNAC130 coordinatethe accumulation of starch and protein through the transcriptionalregulation of at least the Bt2 and the 16-kDa γ-zein genes.

ResultsShrunken Kernel Phenotype by Knockdown of ZmNAC128 andZmNAC130. A total of 112 putative proteins were annotated asthe NAC superfamily in the maize B73 V4 genome although thespatiotemporal transcriptome atlas showed that only two NACTFs, ZmNAC128 and ZmNAC130, were strongly and specificallyexpressed at the filling stage of endosperm cells (16, 25). The twogenes appear to have arisen from the allotetraploidization of maizeand are located on chromosome 3 (ZmNac128) and chromosome8 (ZmNAC130). The latter generated a tandemly duplicated copy,ZmNAC118 (SI Appendix, Fig. S1). However, only ZmNAC128and ZmNAC130, but not ZmNAC118, are strongly expressed inthe developing endosperm, suggesting that they possibly possessthe same molecular function (SI Appendix, Fig. S1). We also ver-ified their expression pattern by real-time qPCR (SI Appendix, Fig.S2). Phylogenetic analysis placed ZmNAC128 and ZmNAC130within a sister branch, and alignment of their protein sequencesshowed that there were high sequence similarities in the NACdomain and C-terminal regions (SI Appendix, Figs. S3 and S4). Ayeast transactivation assay showed that their C-terminal regionspossess transactivation activity (SI Appendix, Fig. S5). These resultsand their common ancestry suggested that the two NAC TFs mighthave redundant functions at the endosperm filling stage.To investigate such a redundancy, a five-amino acids deletion

within the conserved NAC domain of ZmNAC130 (nac130) wasgenerated with the CRISPR-Cas9 technology (SI Appendix, Figs.S4 and S6 A and B). No change of zein and nonzein proteinaccumulation or a kernel phenotype was apparent in this nac130mutant (SI Appendix, Fig. S6 D and E). Therefore, an RNAitransgenic construct was used to knock down the expression ofboth ZmNAC128 and ZmNAC130 in a dominant fashion (SIAppendix, Fig. S6). Two independent nacRNAi events (#2 and #4)were obtained and backcrossed to three inbred lines (B73, Mo17,and W64A) for two generations. Real-time qPCR results showedthat expression of ZmNAC128 and ZmNAC130 are significantlyreduced in the two nacRNAi lines, #2 and #4 (SI Appendix, Fig.S7). Mature nacRNAi transgenic cobs of B73 backcrosses exhibiteda shrunken phenotype of kernels compared with the plump kernelphenotype of nontransgenic seeds (NT) (Fig. 1A and SI Appendix,

Fig. S8). The same shrunken kernel phenotype was observed withnacRNAi transgenic cobs from backcrosses with Mo17 and W64A(SI Appendix, Fig. S8). Because the transgenic kernels exhibitedthe same shrunken phenotype for each transgenic event and indifferent inbred backcrosses, nacRNAi#4 in B73 background wasused as the genetic material for all subsequent experiments.

Reduction of Starch and Protein in nacRNAi. Kernel weight (KW)and test weight (TW) were considered as indicators of endospermfilling for grain yield and quality. Compared with NT with 0.27gram of KW and 1.12 g/mL TW, KW and TW of nacRNAi siblingswere reduced by ∼30% and 26%, respectively (Fig. 1B). Therefore,knockdown of ZmNAC128 and ZmNAC130 affected the filling ofendosperm, causing a shrunken kernel phenotype with a significantreduction of KW and TW. To investigate the mechanism un-derlying the shrunken kernel phenotype, starch and protein com-partmentalization in nacRNAi kernels was analyzed. Transmissionelectron microscopy (TEM) was employed to observe the devel-opment of SGs and PBs at the filling-stage endosperm cells. TheTEM observation showed that SGs were apparently smaller in the20-DAP nacRNAi endosperm cells than those in NT (SI Appendix,Fig. S9A). PBs in 20-DAP endosperm cells did not show a differ-ence in size and shape between NT and nacRNAi siblings, but theirnumber was reduced by 40% in nacRNAi (SI Appendix, Fig. S9 Band C). Furthermore, the content of starch and total seed proteins(including zeins and nonzeins) in mature dry seeds was measured.In 100 mg of mature seed flour of NT, there was 60.0 ± 2.4 mg ofstarch, 6.6 ± 0.4 mg of zein proteins, and 5.1 ± 0.5 mg of nonzeinproteins. Compared with NT, the content of starch, zeins, and

Fig. 1. The phenotypes of nacRNAi. (A) Kernel phenotypes of nacRNAi#4 inthe B73 background. In the Upper, the white arrowheads point out thetransgenic nacRNAi kernels in the cob. The Lower Left and Right representthe nontransgenic (NT) and nacRNAi kernels from the same cob, respectively.(B) Kernel weight (Upper) and test weight (Lower) of NT and nacRNAi seeds.The data are measured from the three mature cobs of nacRNAi#4 in the B73background with ±SD. g, gram; g/mL, gram per milliliter. (C) The content ofstarch, zeins, and nonzeins in the mature kernels of NT and nacRNAi siblings.Percent represents mg per 100 mg mature dry kernels. In B and C, the as-terisk represents a significant difference from NT (Student’s t test, P < 0.05).

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nonzeins was reduced by ∼15%, 18%, and 24% in mature kernelsof nacRNAi siblings, respectively (Fig. 1C). Clearly, the two NACTFs reduced starch and protein accumulation.

ZmNAC128 and ZmNAC130 as TFs for the Bt2 Gene. Because nacRNAiaffected the size of SGs and starch content, we determined theexpression of genes contributing to starch biosynthesis in 20-DAPendosperms by real-time qPCR (SI Appendix, Fig. S10A). Tran-script levels of seven starch synthetic genes were significantlydown-regulated by an at least twofold threshold. These down-regulated genes included Bt2 (brittle2) encoding adenosine di-phosphate glucose pyrophosphorylase (AGPase) small subunit,SS1 encoding starch synthase 1, Zpu1 encoding pullulanase-typestarch-debranching enzyme 1, Sus1 encoding sucrose synthase 1,Sbe2b encoding starch branching enzyme 2b, Sbe1 encoding starchbranching enzyme 1, and GBSS1 (also called Waxy) encodinggranule-bound starch synthase 1. Among them, the transcript levelof Bt2 was the most reduced by about 200-fold in nacRNAicompared with NT (Fig. 2A). Consistent with the real-time qPCRresult, immunoblotting further showed that the protein accumu-lation of Bt2 dropped by 80 to 90% in the 20-DAP nacRNAiendosperm compared with NT (Fig. 2B). Because AGPase, whichis composed of Bt2 and Sh2 (AGPase large subunit), is the lim-iting enzyme for starch synthesis in maize endosperm, loss of Bt2could explain the shrunken phenotype of nacRNAi kernels. Todetermine whether this occurred through direct interaction of thetwo NAC TFs with the Bt2 promoter, we performed the DLRassay in Arabidopsis protoplast cells. The constructs of both re-porter and effector were transiently introduced into the sameprotoplast cells to measure the ratio of fluorescent signals of thetwo types of luciferases. The Renilla luciferase (REN) gene wasdriven by the 35S promoter as an internal control, and firefly lu-ciferase (LUC) was driven by the 1.5- kilo base pair (kbp) frag-ment upstream from the start codon of Bt2 (SI Appendix, Fig.S11A). Compared with the control, LUC activity was increased by38-fold in the presence of NAC128 and by 100-fold in presence ofNAC130. Although both together increased LUC activity by only72-fold (SI Appendix, Fig. S11B), these assays suggested that thetwo NAC TFs were functionally independent for the regulation ofBt2, albeit with different binding or transactivation activity.

Transcriptional Activation of the 16-kDa γ-Zein Gene by ZmNAC128and ZmNAC130. The Coomassie Brilliant Blue (CBB)-stainingprotein gel showed that zein proteins were apparently reduced innacRNAi whereas 16-kDa γ-zein was almost not visible in theCBB-staining protein gel of nacRNAi kernel (Fig. 3A). Consistent

with the reduction of zeins, real-time qPCR detection showedthat the transcript levels of most zeins, except for z1B, weresignificantly down-regulated by more than twofold in 20-DAPnacRNAi endosperm compared with NT (SI Appendix, Fig.S10B). The transcript level of the 16-kDa γ-zein gene was re-duced by even 40-fold (Fig. 3B), indicating that ZmNAC128 andZmNAC130 could directly regulate the 16-kDa γ-zein gene.Therefore, we also conducted the DLR assay with the 16-kDaγ-zein promoter. Compared with the control, LUC activity drivenby 16-kDa γ-zein promoter is increased by 735-fold withZmNAC128 and by 1,568-fold with ZmNAC130. Similar to theBt2 promoter, the LUC activity driven by the 16-kDa γ-zeinpromoter is not stronger in the presence of both NAC128 andNAC130 (SI Appendix, Fig. S11C).

ACGCAA as a Primary Cis-Element Binding Site of ZmNAC128 andZmNAC130. Given their specific binding to different promoters,the question arose whether there was a common cis-elementbinding site for ZmNAC128 and ZmNAC130, which would beconsistent with the high identity of amino acid sequences of thetwo TFs (SI Appendix, Fig. S4). Therefore, a DLR assay withEMSA was performed to narrow down the cis-element bindingsite (Fig. 4 and SI Appendix, Fig. S12).For the Bt2 promoter, four fragments upstream from the start

codon different in length were used. The result showed that,when the Bt2 promoter was shortened to a 500-bp promoterfragment, neither ZmNAC128 or ZmNAC130 could trans-activate LUC activity, compared with strong transactivation ac-tivities of 1,468-bp-, 1,005-bp-, and 500-bp-length promoterfragments, placing the cis-element in the Bt2 promoter in the500-bp region from −500 to −1 upstream from the start codon(Fig. 4A). The DLR assay was also performed to shorten thetarget region of the 16-kDa γ-zein promoter. The cis-element inthe 16-kDa γ-zein promoter was placed within the 383-bp regionfrom −480 to −98 upstream from the start codon (Fig. 4A).Then, the 500-bp promoter of Bt2 was divided into seven

80-bp fragments (Fig. 4B). After labeling the 3′ end of the sevenfragments using biotin, we performed EMSA with purifiedrecombinant His-ZmNAC128 and His-ZmNAC130. The shiftedbands in the lane of the sixth probe (P6) indicated thatZmNAC128 and ZmNAC130 bound P6 to form retarded bandsin the gel (Fig. 4B). The 383-bp promoter fragment of the 16-kDaγ-zein gene was divided into five 80-bp probes. ZmNAC128 andZmNAC130 retarded the fourth probe (P4) as shown by band shifts(Fig. 4C), locating the binding sites within the 80-bp fragments ofthe Bt2 and 16-kDa γ-zein promoter, respectively.

Fig. 2. Transcript and protein levels of Bt2 in 20-DAP endosperms of NT andnacRNAi. (A) Real-time qPCR analysis of the Bt2 expression. The data fromthree replicates are illustrated with ±SD. The asterisk represents a significantdifference from NT (Student’s t test, P < 0.05). (B) Immunoblotting analysisof the Bt2 protein. The Upper displays the immunoblotting result. Theprotein loading amount in NT is diluted by 0.8-, 0.6-, 0.4-, 0.2-, and 0.1-fold.The Lower is a piece of CBB-staining replicated gel as the loading control.

Fig. 3. The expression of 16-kDa γ-zein in nacRNAi. (A) The accumulation ofzein proteins in NT and nacRNAi. Samples 1 and 4, 2 and 5, and 3 and 6 are,respectively, from the three cobs of B73 background. 50γ, 50-kDa γ-zein; 27γ,27-kDa γ-zein; 22α, 22-kDa α-zein; 19α, 19-kDa α-zein; 16γ, 16-kDa γ-zein;15β, 15-kDa β-zein; 10δ, 10-kDa δ-zein. (B) Transcript levels of 16-kDaγ-zein in 20-DAP endosperms of NT and nacRNAi. The data from threereplicates are illustrated with ±SD. The asterisk represents a significant dif-ference from NT (Student’s t test, P < 0.05).

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Fig. 4. Identification of the cis-element binding site of ZmNAC128 and ZmNAC130. (A) Transactivation of different truncated promoters of Bt2 and 16-kDa γ-zein(16γ) by ZmNAC128 and ZmNAC130. The length of all promoters is calculated from the first nucleotide of the start codon (ATG). In the transactivation assay of Bt2promoter, Control represents a 496-bp sequence downstream from the start codon. The relative LUC activity is the LUC-to-REM ratio, which is measured by usingthe same assay described in SI Appendix, Fig. S11. The promoter sequences of Bt2 and 16-kDa γ-zein are in SI Appendix, Fig. S12, and all of the probe sequences arein Dataset S2. (B) EMSA of five probes in the 500-bp promoter region of Bt2 from −500 to −1 with ZmNAC128 and ZmNAC130. (C) EMSA of five probes in the 383-bp promoter region of 16γ from −480 to −98 with ZmNAC128 and ZmNAC130. (D) EMSA of three probes truncated from P6 in the Bt2 promoter with ZmNAC128and ZmNAC130. (E) EMSA of three probes truncated from P4 in the 16γ promoter with ZmNAC128 and ZmNAC130. (F) EMSA of ZmNAC128 and ZmNAC130with a series of 30-bp probes of the Bt2 promoter from −102 to −73. (G) EMSA of ZmNAC128 and ZmNAC130 with a series of 30-bp probes of the 16γ promoterfrom −248 to −219. In F and G, The 6-bp box of ACGCAA center in the 30-bp probe (WT) and a series of mutant probes are produced by 1-bp mutations in the 6-bpbox. The 6-bp consensus sequence (ACGCAA) in the promoters of Bt2 and 16-kDa γ-zein is highlighted in the red and bold format.

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Furthermore, the 80-bp fragments of Bt2 and 16-kDa γ-zeinpromoter were further divided into the three probes, respectively.The EMSA results of the three probes in the P6 of Bt2 promotershowed that only P6c was retarded by ZmNAC128 and ZmNAC130(Fig. 4D). The cis-element of ZmNAC128 and ZmNAC130 wasfinally located within a 15-bp sequence in the Bt2 promoter (Fig.4D). The P4b of 16-kDa γ-zein promoter was retarded byZmNAC128 and ZmNAC130, narrowing down the cis-elementinto a 25-bp sequence in the 16-kDa γ-zein promoter (Fig. 4E).Common to both is a 6-bp consensus sequence of ACGCAA.To validate the consensus sequence ACGCAA in the two

promoters as the cis-element of ZmNAC128 and ZmNAC130,we subjected ACGCAA to point mutation analysis (Fig. 4 F andG). Every point mutation of ACGCAA abolished the bindinginteraction with ZmNAC128 and ZmNAC130, confirming the6-bp sequence of ACGCAA as the cis-element binding site. Havingthe same cis-element binding site also explained the redundancy ofZmNAC128 and ZmNAC130.

Metabolism of Carbohydrates and Amino Acids in the Presence ofnacRNAi. To investigate which other genes might be affected inthe absence of the two NAC TFs, we performed transcriptomicanalysis of 20-DAP nacRNAi endosperm as described in Materialsand Methods (26). The principal component analysis showed thatthe three biological replicates of nacRNAi and NT were clus-tered into one group (SI Appendix, Fig. S13A). Furthermore,2,138 up-regulated and 2,218 down-regulated genes were iden-tified by a threshold of P value < 0.05 and twofold change (SIAppendix, Fig. S13B and Dataset S1). Furthermore, the 2,218down-regulated genes were subjected to the enrichment analysisof Kyoto Encyclopedia of Genes and Genomes (KEGG) path-ways. The result showed that the enriched pathways center on themetabolisms of carbohydrates and amino acids (SI Appendix, Fig.S13C). Three or four carbohydrate metabolic pathways were alsosignificantly down-regulated in the presence of nacRNAi, in-cluding starch and sucrose metabolism, glycolysis, gluconeogen-esis, fructose, and mannose metabolism, which correlated withthe reduction of SG size and starch content. Biosynthesis ofalanine, tryptophan, arginine, proline, histidine, phenylalanine,tyrosine, valine, leucine, isoleucine, lysine, and asparagine wassignificantly down-regulated in developing nacRNAi endosperm,which interferes with protein synthesis in developing endosperm.Such a broad selection of amino acids explains also the reductionof both zein and nonzein proteins in mature nacRNAi kernelscompared with NT.

DiscussionCoordinated Regulation of Zein and Starch Biosynthetic Genes.Maizeendosperm accumulates starch and proteins in a spatiotemporalpattern, requiring coordinated regulation of gene expression.Previously, it has been shown that mutations in the starch bio-synthetic pathway could also affect transcription levels of zeingenes (18). A mutation of bt2 increased transcripts of α-zein andstarch biosynthetic genes (Sh1, Sh2, etc.) in the developing en-dosperm. Here, we could show a direct regulatory effect throughtranscriptional activation by two specific TFs (Fig. 5). Moreover,reduction of Bt2 transcription in the nacRNAi lines did not in-crease transcripts of zein genes (SI Appendix, Fig. S10). On thecontrary, the expression of most zein genes and multiple starchbiosynthetic genes was significantly down-regulated in nacRNAicompared with NT. Failure of expressing the two endosperm-specific NAC TFs had no compensatory effect, as shown withreduction of zeins by RNAi in Illinois-High-Protein maize (27).Given that ZmNAC128 and ZmNAC130 transactivate the Bt2and 16-kDa γ-zein genes directly via a common DNA binding site(ACGCAA), we now could also ask whether such a binding siteexisted in promoters of other target genes, whose transcript levelswere reduced in nacRNAi. Indeed, of 15 starch biosynthetic genes

besides Bt2 (SI Appendix, Fig. S10), six other genes contained sucha conserved binding site within their 1-kbp-length promoter regions.The six potential target genes include Zpu1, GBSS1, Sh2, SS5encoding starch synthesis 5, ISA2 encoding isoamylase-type starch-debranching enzyme 2, and SS2a encoding starch synthase 2a. In-terestingly, the cis-element ACGCAA also exists within 500 bp of thepromoters of the 50-kDa γ-zein and 15-kDa β-zein genes. Therefore,the lower reduction of transcript levels of other genes than the Bt2and 16-kDa γ-zein genes in nacRNAi could be due to the mosaicstructure of promoters containing multiple cis-acting enhancer se-quences, requiring other TFs for full transcriptional activation.Syntenic alignments between cereal genomes has shown that

TFs like O2 have been conserved throughout cereal evolution(28). Indeed, rice OsbZIP58 like maize O2 controls rice storageaccumulation by regulating the expression of starch biosyntheticand storage-protein genes (29). Analysis of protein sequences ofZmNAC128 and ZmNAC130 could also identify two relatedNAC TFs in rice, Os01g01470 and Os01g29840, which have 85%similarity. Syntenic analysis of chromosomal regions of rice andthe homeologous regions in maize showed that the four NACTFs are collinear (SI Appendix, Fig. S1). Moreover, the ricespatiotemporal transcriptome atlas showed that Os01g01470 andOs01g29840 are specifically and strongly expressed in rice de-veloping seeds (30). Due to the conservation of regulatorymechanisms through the evolution of the grasses, each speciescan serve as a reference for the others (31).

The 16-kDa γ-Zein as a Regulator of the PB Initiation. In developingendosperm cells, the four types of α-, β-, γ-, and δ-zeins aredeposited within the endoplasmic reticulum lumen to form theorderly spherical accretions called PBs. RNAi against differentzeins indicate that 27-kDa γ-zein RNAi has 60% reduction of PBnumber and is not involved in protein body filling (32). Combi-nations of different zein RNAi lines (without 27-kDa γ-zeinRNAi) have a weak effect on PB initiation except that thecombination of 27-kDa γ-zein RNAi with α-zeinRNAi;50-kDaγ-zeinRNAi;15-kDa δ-zeinRNAi has a 30% reduction in PB number(32). Therefore, 27-kDa γ-zein plays an important role in PB ini-tiation. There are the three (50-, 27-, 16-kDa) γ-zein genes in maize.Deletion of 27- and 50-kDa γ-zein genes still allowed 10% of PBs tobe formed compared with wild type (33), suggesting that additionalproteins could be involved in the initiation of PBs. Here, we foundthat nacRNAi dramatically reduced the accumulation of 16-kDaγ-zein and PB number in the developing endosperm cells ofnacRNAi, but the PB size and shape apparently remainedunchanged. Because 16-kDa γ-zein gene was likely the resultfrom unequal crossing-over with loss of the 50- and 27-kDa γ-zeingenes after allotetraploidization (3), its function was probably sim-ilar in PB formation. For instance, maizeMucronatemutation formsa misfolded 16-kDa γ-zein that leads to irregular PB formation, butit could be rescued to restore normal PB by silencing the expressionof the mutant 16-kDa γ-zein gene (34, 35). Although 16-kDa γ-zeinlost a large part of its N-terminal domain compared with 27-kDa

Fig. 5. A proposed model for ZmNAC128 and ZmNAC130 regulating thesynthesis of zeins and starch.

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γ-zein that was able to promote PB formation (36, 37), a recentstudy showed that 16-kDa γ-zein acquired a new function in PBassembly (38). Furthermore, the 16-kDa γ-zein promoter lost allknown conserved cis-elements present in other zein promoters (5)but acquired the cis-element ACGCAA, recognized by the corre-sponding NAC TFs, thereby restoring its endosperm-specific ex-pression and synchronization with starch storage.

Functional Redundancy of ZmNAC128 and ZmNAC130. The in vivoDLR assay has been used to determine whether two TFs have acooperative effect, requiring physical interaction between them(13, 19, 39). For example, interaction between O2 and PBF onthe expression of zein, PPDK, and SSIII genes can be illustratedwith DLR assays (19, 39). However, ZmNAC128 or ZmNAC130alone can already transactivate the Bt2 and 16-kDa γ-zein pro-moters to full strength (SI Appendix, Fig. S11). We further per-formed yeast-two-hybrid (Y2H) and bimolecular fluorescencecomplementation (BiFC) assays to determine whether the twoNAC TFs can interact with each other. Because their C-terminalshave transactivation activity, the Y2H assay used their NAC do-mains linked in the vector of BD to test the interactions with theirfull-length, N- or C-terminal regions linked in the vector of AD(SI Appendix, Fig. S14). The Y2H results showed that there was nointeraction. Similar to the Y2H results, the BiFC assay only de-tected weak interaction between NLUC-NAC128 and CLUC-

NAC128 (SI Appendix, Fig. S15). Therefore, ZmNAC128 andZmNAC130 are functionally redundant. This finding is also con-sistent with the gene balance hypothesis. In contrast to a pre-viously described cochaperone, there was no selection against theduplication after allotetraploidization because of the absence ofprotein–protein interaction (40). Furthermore, the two homeol-ogous regions in the maize genome exhibited a large expansion onchromosome 3 or contraction on chromosome 8, including asegmental inversion between the NAC118/NAC130 and itsflanking genes, a pattern previously described for the diploidiza-tion of the maize genome (41). This process might have alsoprevented the loss of the duplicated copy. Interestingly, in rice, asegmental duplication achieved the same purpose of having tworice NAC TFs (SI Appendix, Fig. S1), indicating some selectivepressure on redundancy.

Materials and MethodsGenetic materials and molecular procedures are described in SI Appendix, SIMaterials and Methods. The correct splicing information for Bt2 is based oncDNA analysis (42).

ACKNOWLEDGMENTS. We thank Janine R. Shaw (University of Florida) fordrawing our attention to the cDNA data of the Bt2 gene. This research wassupported by the Selman A. Waksman Chair in Molecular Genetics (to J.M.).

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