| INVESTIGATION

Quality Protein Maize Based on Reducing Sulfur inLeaf Cells

Jose Planta and Joachim Messing1

Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854

ABSTRACT Low levels of the essential amino acids lysine (Lys) and methionine (Met) in a maize-based diet are a major cost to feed andfood. Lys deficiency is due to the abundance of Lys-poor proteins in maize kernels. Although a maize mutant, opaque-2 (o2), hassufficient levels of Lys, its soft kernel renders it unfit for storage and transportation. Breeders overcame this problem by selectingquantitative trait loci (QTL) restoring kernel hardness in the presence of o2, a variety called Quality Protein Maize (QPM). Although atleast one QTL acts by enhancing the expression of the g-zein proteins, we could surprisingly achieve rebalancing of the Lys content anda vitreous kernel phenotype by targeting suppression of g-zeins without the o2 mutant. Reduced levels of g-zeins were achieved withRNA interference (RNAi). Another transgenic event, PE5 expresses the Escherichia coli enzyme 39-phosphoadenosine-59-phosphosulfatereductase involved in sulfate assimilation, specifically in leaves. The stacked transgenic events produce a vitreous endosperm, which hashigher Lys level than the classical opaque W64Ao2 variant. Moreover, due to the increased sulfate reduction in the leaf, Met level iselevated in the seed. Such a combination of transgenes produces hybrid seeds superior to classical QPMs that would neither require acostly feed mix nor synthetic Met supplementation, potentially creating a novel and cost-effective means for improving maize nutritionalquality.

KEYWORDS High-lysine maize; high-methionine maize; quality protein maize

INmany developing countries, maize serves as an importantsource of nutrition in human and animal diets. Cereals like

maize are limiting in essential amino acids (EAAs) Lys, Met,and tryptophan (Trp), whereas legume crops like soybean aredeficient inMet. Therefore, corn is usually supplementedwithsoybean and synthetic free Met to provide a balanced aminoaciddiet for animal feed.Althoughhumans could live onadietof beans and corn, they cannot consume synthetic free Met,which, as a racemic substance, would not be allowed forhealth reasons. Met as a limiting nutrient had been demon-strated in the effects of methionine fortification of a soyisolate-based formula in infant feeding. Normal infants fedsoy isolate-based formula fortified with methionine had sig-nificant weight gain compared to infants fed not supple-mented soy isolate-based formula (Fomon et al. 1979).

Therefore, a lot of research efforts has been expended ongenetic improvements to the amino acid balance of maizekernels, either through conventional breeding or the use ofrecombinant DNA technology.

The bulk of proteins in mature maize kernels, prolamins,called zeins in maize, possess an amino acid imbalance to-wardproline, glutamine, alanine, and leucine residues. Zeinsare classified into four distinct classes ofa- (19- and22-kDa),b- (15-kDa), g- (16-, 27-, and 50-kDa), and d-zeins (10- and18-kDa) and coalesce into discrete spherical structures calledprotein bodies (PBs) (Kim et al. 2002). Mature PBs have ashell of g- and b-zeins surrounding the core of a-zeins. The27-kDa g-zein controls PB initiation, whereas the 50-kDag-zein plays a significant role in PB expansion, the bulk ofwhich is driven by the 19-kDa a-zein. The 22-kDa a-zeinprovides structural support in packaging the 19-kDa a-zein(Guo et al. 2013). On the other hand, the nonzein proteinfraction in the endosperm (glutelins, globulins, and albu-mins) is relatively balanced in their amino acid composition(Prasanna et al. 2001). This abundance of zeins effectivelydilutes the contribution of other endosperm proteins to thekernel Lys and Trp contents. Therefore, alterations in theaccumulation of zeins have led to the identification of

Copyright © 2017 by the Genetics Society of Americadoi: https://doi.org/10.1534/genetics.117.300288Manuscript received September 13, 2017; accepted for publication October 17, 2017;published Early Online October 20, 2017.Supplemental material is available online at www.genetics.org/lookup/suppl/doi:10.1534/genetics.117.300288/-/DC1.1Corresponding author: Waksman Institute of Microbiology, Rutgers, The StateUniversity of New Jersey, 190 Frelinghuysen Rd., Piscataway, NJ 08854. E-mail:messing@waksman.rutgers.edu

Genetics, Vol. 207, 1687–1697 December 2017 1687

mutants with altered nutritional quality. For instance, in-creased accumulation of the b-, g-, and d-zeins relative tothe more abundant a-zeins (Supplemental Material, TableS1 in File S1) has raised the sulfur (S) amino acid contentsof kernels and their nutritional value (Planta et al. 2017).Reduction of a-zeins either through transcriptional regula-tion (e.g., loss of O2 transcription factor), or a transgenethat reduces their transcript accumulation through RNA in-terference (RNAi), result in more nutritionally balanced en-dosperm proteins.

One of the zein-reduction mutants, the recessive o2 mu-tant, has about a 50% reduction in zeins (Tsai et al. 1978),and approximately double the amount of Lys compared tonormal genotypes (Mertz et al. 1964). It primarily affectsthe synthesis of a-zeins. The 22-kDa a-zeins are reduced toa very low level, whereas the 19-kDa a-zeins are also re-duced, but to a lesser extent (Jones et al. 1977). The o2mutant, aside from a reduced Lys-poor zein content, has acompensating increase in the levels of Lys- and Trp-rich non-zein proteins. However, its soft and starchy endospermmakesit susceptible to fungi and insect infestation, both in storageand in the field (NRC 1988). Identification of o2 modifiers(mo2s) function as suppressors that can restore the normalkernel phenotype in the presence of o2, resulting in a newtype of maize germplasm, known as quality protein maize(QPM) (Prasanna et al. 2001).

Alternatively, transforming maize with an RNAi transgenetargeting the a-zeins also enhances levels of Lys and Trp inmaize kernels (Segal et al. 2003; Huang et al. 2004, 2006).The observed increase in Lys and Trp in the a-zein-reducedkernels was also due to the replacement of the Lys-poor zeinswith the Lys-containing nonzein proteins (Huang et al.2006). Doubling the Lys levels without changing the proteincontent in corn could add up to $480 million in annual grossvalue to US corn in the global feed market (Johnson et al.2000).

Among the maize opaque kernel mutants that affect theaccumulation of the zeins, only a few have been reported thatalter the synthesis of the g-zeins. The maize Mucronate mu-tation is a deletion in the 16-kDa g-zein that produces anabnormal 16-kDa g-zein, whereas opaque-15 reduces the27-kDa g-zein synthesis and appears to be a mutation of ano2modifier gene (Dannenhoffer et al. 1995; Kim et al. 2006).Near-isogenic lines of several high-Lys opaque mutants in theW64A genetic background showed that o2 has the highestkernel Lys content among the opaque mutants (Hunter et al.2002). Efforts to improve the protein quality of maize seedshave focused on o2 seeds as other opaque mutants offer noadditional advantage over o2 in terms of Lys content andnutritional quality.

Given these results, we sought to generate a sole crop dietof corn that is enriched in both Lys and Met. We crossed thehigh-Metmaize line PE5 (Planta et al. 2017)with several zeinRNAi lines. The RNAi lines used target a-, b-, g-, a-/g-, org-/b-zeins. The opaque PE5;a-/g- kernels have higher Metand Lys contents than the vitreous PE5;g- kernels, and both

kernel genotypes have higher Lys and Met contents than theo2 mutant. Moreover, PE5 with RNAi targeting of g-zeinscould potentially be a preferred QPM variety because ofhigher Lys and Met levels and their dominance for introgres-sion into elite lines.

Materials and Methods

Genetic stocks

The a-, g-, and b2RNAi transgenic plants were from ourlaboratory stocks, and have been described elsewhere (Wuand Messing 2010, 2011; Wu et al. 2012). Both the gRNAiand bRNAi are homozygous for A654-Dzs10, a nonfunctionalallele of the 10-kDa d-zein gene from the inbred A654. ThebRNAi plant used for crosses is homozygous for the RNAitransgene as all kernel progenies tested positive for bRNAigenotyping, whereas the gRNAi is hemizygous, as about halfof the tested kernel progenies from crosses with gRNAi hadthe RNAi transgene. aRNAi, on the other hand, is in a Hi-IIA3B hybrid background, and is hemizygous for the RNAitransgene. The hybrid genotype Hi-II A3B was used formaize transformation.

The transgenic event PE5 was backcrossed twice to thehigh-Met inbred line B101 prior to being crossed with theRNAi lines. It expresses the Escherichia coli enzyme 39-phos-phoadenosine-59-phosphosulfate reductase, designated asEcPAPR (Martin et al. 2005), driven by the PepC promoter(Sattarzadeh et al. 2010). EcPAPR is involved in assimilatorysulfate reduction, and maize plants expressing this enzymeshows increased kernel Met content when used as the mater-nal parent (Tarczynski et al. 2003; Martin et al. 2005).

Crosses between the maternal PE5 and the paternal RNAilines were performed during the summer of 2014 and 2015.PE5; g- plants generated during the summer of 2014 werecrossed with aRNAi during the summer of 2015. The rest ofthe crosses of g/bRNAi, bRNAi, and aRNAi with PE5 weremade during the summer of 2014. These crosses gave fivedistinct ears that were used for analysis: (1) PE5;aRNAi, (2)PE5;b-, (3) PE5;g-, (4) PE5;g-/b-, and (5) PE5;g-/a-.

Genotyping

Genomic DNAwas isolated frommaize leaves at the three- tofour-leaf stage using a modified CTAB extraction method(Sawa et al. 1997). For extraction of genomic DNA frommature maize kernels, a portion of the kernel that is mostlyendosperm with no embryo tissues were ground to a fine pow-der and subjected to DNA extraction with the Nucleospin PlantII kit (Takara Bio). Transgenic plants were screened for thepresence of both RNAi and EcPAPR transgenes using the primerpairs 59-ACAACCACTACCTGAGCAC-39/59-ATTAAGCTTTGCAGGTCACTGGATTTTGG-39 (Wu and Messing 2010) and 59-CTCCCCATCCCTATTTGAACCC-39/59-GGTAGGTTTCCGGGAACAAGTA-39, respectively. PCR amplification for the RNAi trans-genes produced amplicons of the sizes 365, 913, and 1096 bpcorresponding toa-,b-, and gRNAi lines, respectively, whereasscreening for PE5 yields a 696-bp product. Kernels from an ear

1688 J. Planta and J. Messing

segregating and nonsegregating for the RNAi transgenes werepooled separately andused for DNAextraction and genotyping.

To determine possible allelic variation between differentmaize genotypes, amplified DNA fragments of the 27-kDag-zein gene were digested with PstI to display restrictionfragment length polymorphisms (Konieczny and Ausubel1993). Primers 59-CCACCTCCACGCATACAAG-39 and 59-ATGGACTGGAGGACCAAGC-39 were used to amplify a 487-bpfragment of the 27-kDa g-zein gene spanning positions 50–546 of the coding region (Das et al. 1991). Digestion with PstIwould produce three DNA fragments (487, 292, and 195 bp),when the gene exists as a tandem copy, or only two fragmentsfor a single-copy gene (292 and 195 bp).

Protein extraction, SDS-PAGE analysis, andwestern blotting

Total protein frompooled endosperm samples ofmaturemaizekernels were extracted with an alkaline sodium borate extrac-tion buffer (Wallace et al. 1990), whereas the alcohol-solublezeins from the endosperm of mature maize kernels were

fractionated and separated in SDS-PAGE as previously de-scribed (Wu and Messing 2010).

Mature maize kernels were imbibed in water for 2 days tofacilitate easier separation of the embryo from the endosperm.Proteins were isolated from embryos macerated in anSDS sample buffer [10% (v/v) glycerol, 2.3% (w/v) SDS,5% (v/v) b-mercaptoethanol, 62.5 mM Tris-Cl, pH 6.8] ata ratio of 50 mg tissue per milliliter of buffer and the ex-tracts processed as described previously (Belanger and Kriz1989; Puckett and Kriz 1991).

Total protein from three mature maize leaf discs wasextracted following the procedure of Conlon and Salter(2007). Ten microgram of total protein was separated in a12% Tris-glycine SDS-PAGE gel and immunoblotted with anantibody against EcPAPR kindly provided by Dr. Jens Schwen(Krone et al. 1991). For immunodetection, the secondaryantibody is a goat anti-rabbit peroxidase conjugate (Sigma-Aldrich) and was used at a 1:60,000 dilution while the pri-mary anti-EcPAPR antibody in a TBST buffer with 0.5% BSAwas used at a 1:4000 dilution.

Protein identification

Protein bands that have differential accumulation in kernelssegregating for thea-zein RNAi transgenewere excised out ofthe SDS-polyacrylamide gel and analyzed by trypsin-nano-LC-MS using Q Exactive HF hybrid quadrupole-Orbitrapmassspectrometer (Thermo Scientific). Proteins from the sampleswere identified at the Biological Mass Spectrometry Facilityat Rutgers University.

Amino acid composition analysis

Transgenic mature kernels were ground to fine powder and�10 mg were used for amino acid composition analysis con-ducted by the Proteomics and Mass Spectrometry Facility atthe Donald Danforth Plant Science Center (St. Louis, MO).Samples were pretreated with performic acid prior to acidhydrolysis, yielding the acid stable forms cysteic acid andMet sulfone that can be measured by separation on a UPLCcolumn. Trp was not detected following acid hydrolysis.

Data availability

The zein profiles of the RNAi lines and the different EcPAPRtransgenic events are presented in Figure S1 and Figure S2 inFile S1. Maize genetic stocks and reagents used in this studyare available upon request.

Results

Rebalancing of kernel sulfur using transgene stacking

PE5 is a high-Met transgenic maize line that specifi-cally expresses the PepC promoter-driven E. coli enzyme39-phosphoadenosine-59-phosphosulfate reductase in theleaf (EcPAPR; Figure 1A). This transgenic PE5 line exhibitsan increased kernel Met content when used as the mater-nal parent (Planta et al. 2017). PE5 plants were thereforecrossed with the zein reduction lines (Wu and Messing

Figure 1 Zein profiles of kernels from crosses of PE5 with RNAi linestargeting the a-, g-, b-, a-/g-, or g-/b-zeins. (A) Western blotting fordetection of the bacterial EcPAPR in leaf tissues of PE5 plants, which wereused as the maternal parents for crossing with the zein reduction lines.NT, nontransgenic null segregant of PE5. (B) SDS-PAGE zein profiles(upper panel) of segregating populations of kernels from PE53 zein RNAicrosses. Kernels nonsegregating (odd-numbered lanes) and segregating(even-numbered lanes) for the RNAi transgenes were pooled and used foranalysis. The RNAi transgenes in these segregating populations weredetected by electrophoresis in 1% (w/v) agarose gel (lower panel).

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2010) (Figure S1 in File S1) as the pollen donor to manifestthe increased seed Met phenotype in resulting ears. Five typesof distinct ears were obtained from crosses of PE5 with differ-ent zein reduction lines: (1) PE5;a-, (2) PE5;g-, (3) PE5;b-,(4) PE5;g-/a-, and (5) PE5;g-/b-. At least 14 kernels fromeach transgenic ear were analyzed individually by phenotyp-ing with SDS-PAGE or genotyping for the RNAi transgenes.Kernels were then pooled depending on whether theywere segregating or nonsegregating for the RNAi transgenes(Figure 1B).

Loss of b- and g-zeins promote a more pronounced in-crease in the accumulation of the Met-rich 10-kDa d-zeincompared to the reduction in the S-poor a-zeins (Figure1B). This increase in the accumulation of the 10-kDa d-zeinwas previously demonstrated in transgenic maize expressingthe b- and g-zein RNAi transgenes (Wu et al. 2012). Reduc-tion in both b- and g-zeins channels even more protein S tothe 10-kDa d-zein than the loss of either b- or g-zein. Al-though enhanced assimilation of S in the source leaf tissuesincreases accumulation of S-containing zeins in the seedsink tissues, loss of the S-containing zeins reallocate theprotein S to the remaining or available S-containing zeins(Planta et al. 2017).

PE5 influences kernel opacity depending on zeingene expression

As the loss of a- and g-zeins by RNAi induces a full or partialopaque seed phenotype, respectively (Segal et al. 2003; Wuand Messing 2010), hybrid kernels were inspected whether

the PE5 transgenic event affects endosperm modification.Opacity of the hybrid PE5;zein RNAi kernels were phenotypedwith a light box (Figure 2). Loss of b-zeins did not change thephenotype of the vitreous kernel, as indicated by the thickouter layer of the vitreous endosperm in sliced kernels (Figure2G). Reduction in g-zeins produced a partial opaque pheno-type. The regular pattern of endosperm modification extendsfrom the crown midway to the base of the kernel (Figure 2D),whereas reduction in both b- and g-zeins produced completeopaque kernels (Figure 2F). However, combining PE5with thereduction in both b- and g-zeins completely restored the vit-reous kernel phenotype (Figure 2I). This reversion to a morevitreous phenotypewas also observed in PE5;g- kernels (Figure2E). As bRNAi kernels are vitreous, combining it with PE5 didnot change its kernel phenotype (Figure 2H). Stacking of PE5with aRNAi (Figure 2B) or a/gRNAi (Figure 2C) did not alterthe opacity of the hybrid kernels. The PE5 transgenic event inPE5;g- or PE5;g-/b- can influence endosperm modificationdepending on which RNAi event is used.

Kernels from a cross of PE5 with aRNAi showed increasedlevel of the 27-kDa g-zein (Figure 1B). This increase in theg-zein was also observed in crosses of aRNAi with othertransgenic events overexpressing EcPAPR (Figure S2 in FileS1), and seems to be a response to the decreased levels ofa-zeins. However, reduced levels of 27-kDa g-zein in aRNAilines alone was attributed to segregation of the 27-kDa g-zeingene from the A and B lines used for transformation (Segalet al. 2003), and gene silencing of the 27-kDa g-zein gene dueto the use of the g-zein promoter in the transgenic expressioncassettes (Huang et al. 2004).

QPMs,whichhave reduced levels of the22-kDaa-zein, alsohave two- to threefold increases in the 27-kDa g-zein (Ortegaand Bates 1983; Wallace et al. 1990). This increase in the27-kDa protein correlates, and is necessary, if not sufficient,for endosperm modification and depends on the geneticbackground (Lopes and Larkins 1991; Wu et al. 2010). Forinstance, maize inbreds have one or two copies of the 27-kDag-zein gene that could influence its protein level (Das andMessing 1987; Das et al. 1991). Therefore, DNAwas isolatedfrom PE5 and aRNAi, their hybrid, and their parental lines todetermine the nature of g-zein alleles (Figure S3 in File S1).Two highly similar, tandemly duplicated genes of the 27-kDag-zein, “A” and “B,” in inbreds like W22 and A188, have dif-ferent PstI recognition sites, which were utilized for thecleaved amplified polymorphic sequence assay (CAPS; FigureS3A in File S1). The inbred line, A188, has a single rear-ranged B gene (Rb) originating from homologous recombi-nation at the highly conserved 59 regions of the two repeats(Das and Messing 1987; Das et al. 1991).

TheDNAgel profile in FigureS3B in File S1 shows thatHi-IIA3B, B101, PE5, aRNAi, and kernels from a PE53aRNAicross (Figure S3, B and C in File S1) have a single copy ofthe g-zein gene. There seems to be no copy number variationin the hybrids and inbreds related to PE5 and aRNAi, andthus, unlikely that modifiers of the opaque phenotype areassociated with copy number variations of the g-zein genes

Figure 2 Endosperm phenotypes of transgenic zein reduction kernels(upper panels) sliced in half to reveal the degree of vitreous endosperm(lower panels). (A, D, F, and G) Zein RNAi and (B, C, E, H, and I) PE5;zeinRNAi lines. gRNAi and bRNAi are in a hybrid Hi II A3 B and A654 back-grounds, aRNAi is in a Hi II A3 B background, and PE5 was backcrossedtwice to B101 prior to being crossed with the RNAi lines. PE5;a- is in a Hi IIA3 B and B101 backgrounds, whereas the other PE5;zein RNAi lines arein a hybrid background of Hi II A3 B, B101, and A654 (see Materials andMethods).

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in our crosses. Indeed, g-/b- is opaque (Figure 2F), butPE5;g-/b- is vitreous (Figure 2I), exemplifying restorationof the normal phenotype in the absence of the g-zeins, butin the presence of PE5. However, without PE5, duplication of the27-kDa g-zein gene in QPMs enhances its expression and pro-motes endosperm modification (Liu et al. 2016).

Protein accumulation patterns in PE5;zein RNAi kernels

To determine whether protein accumulation patterns aredifferent between transgenic zein reduction and nontrans-genic controls, kernels froman ear of a PE53RNAi crosswerepooled depending on the segregation of the RNAi transgenes.Total protein and the nonzein protein fractions were extract-ed from these kernels and separated in an SDS-PAGE gel(Figure 3). Aside from the reduction in zeins due to the RNAitransgenes, PE5;g- and PE5;b- kernels had similar proteinaccumulation profiles compared to their PE5;non-RNAi con-trols (Figure 3A). Reduction in a-zeins, however, in kernels ofPE5;a- and PE5;a-/g- had different seed protein accumula-tion patterns than their corresponding PE5;non-RNAi kernels(Figure 3, A and B). The loss of a-zeins conditioned an in-crease in the accumulation of nonzein proteins in the kernel.Proteins in the range of 60–70 and 45–55 kDa were differ-entially upregulated in the a-zein-mutant kernels.

Two protein bands with sizes of�60- and 65-kDa (arrowsin Figure 3A) were particularly increased in PE5;a- andPE5;a-/g- kernels, with the latter having more accumulationof these upregulated proteins than the former. These mix-tures of proteins were identified by mass spectrophotometricanalysis. Spectral quantitation showed that the upper bandhad 52.1% (316 out of 606), and the lower band had 29.8%(909 out of 3048) of its identified peptides to be fragments ofthe GLB1 (globulin-1) protein. The abundance of the iden-tified GLB-1 peptides makes it likely to be upregulated in

the a-zein-reduced kernels. GLB1 has no known enzymaticfunction, and, just like zeins, is thought to function as astorage protein (Kriz 1989). Pulse-chase labeling andin vitro translation studies showed that the primary trans-lation product of Glb1 undergoes at least three post- and/orcotranslational processing steps to produce the matureGLB1 protein (Kriz and Schwartz 1986). The lower bandof �60 kDa would therefore represent the mature GLB1,whereas the upper band of �65 kDa would be the process-ing intermediate GLB1’. GLB1 is mostly an embryo-specificprotein that is estimated to account for 10–20% of thetotal embryo protein along with GLB2 (Kriz 1989). Asthe deduced amino acid composition of GLB1 has 4.11%Lys (Belanger and Kriz 1989), an increased accumulationof this Lys-rich protein could contribute to an increase inthe content of protein-bound Lys in the kernel. Althoughthe 27-kDa g-zein was increased in PE5;a- kernels, the in-crease in kernel Lys could not be attributed to overaccumu-lation of this protein as g-zein is devoid of Lys (Table S1in File S1).

Loss of a-zeins redistributes nitrogen, primarily stored inasparagine and glutamine in a-zeins (Table S1 in File S1), tothe nonzein protein fraction by compensatory increases ofproteins in this fraction. Puckett and Kriz (1991) andHunter et al. (2002) showed that GLB1 is upregulated in o2mutants, along with other Lys-rich proteins that contribute toan increase in kernel Lys content. Proteins that were upregu-lated in o2 kernels, such as GLB1 (Puckett and Kriz 1991;Hunter et al. 2002) and the Lys-rich (.8% Lys residues)glyceraldehyde-3-phosphate dehydrogenase (Damerval andLe Guilloux 1998), were also confirmed in the transgenica-zein reduction lines (Frizzi et al. 2010).

Amino acid composition analysis of PE5;zeinRNAi kernels

The o2 mutant has about twice the Lys content compared tonormal phenotype (Mertz et al. 1964), and mutant o2 allelesin different backgrounds display variations in Lys contentsand penetrance of the opaque phenotype (Balconi et al.1998; Hunter et al. 2002). Of the crosses of PE5with differentRNAi lines, only PE5;a-, PE5;g-, and PE5;a-/g- kernels havestatistically significant higher Lys content over nontransgenicA3B kernels (Figure 4A). The Lys contents in PE5;a- andPE5;a-/g- were 60 and 128.2% higher, respectively, comparedto their corresponding PE5;non-RNAi controls (Table 1). Thisgenotype-specific variation of the Lys contents in respect totheir controls suggests that hybrid genetic backgrounds havean impact on Lys accumulation. Compared to the Hi-II A3Bkernels, PE5;a- kernels had 151.9% and PE5;a/g- kernelshad 83.8% more Lys. PE5;a- kernels have higher Lys contentthan a- kernels, suggesting that the high-Met PE5 maternalbackground also influences Lys accumulation in the seeds(Figure 4A). Asmaize proteins contain�4 timesmore Lys thanTrp, either amino acid can be used as a single parameter forevaluation of protein quality (Hernandez and Bates 1969;Villegas et al. 1984).

Figure 3 Total seed proteins (A) and nonzein proteins (B) from transgenicprogeny kernels from crosses of PE5 with RNAi lines targeting the a-, b-,g-, b-/g-, or a-/g-zeins separated in a 12% SDS-polyacrylamide gel. Kernelsnonsegregating (odd-numbered lanes) and segregating (even-numberedlanes) for the RNAi transgenes were pooled and used for analysis. Arrowsindicate proteins in the �65–70 kDa range that have increased accumula-tion in kernels segregating for the a- and a-/g-zein RNAi transgenes. Theidentities of these protein bands (indicated by arrows) were determined bymass spectrophotometric analysis.

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PE5;g- kernels have significant increases in Met and Lyslevels, and are vitreous

W64Ao2 has reduced levels of kernel Met due to reducedamounts of the 10-kDa d- and 15-kDa b-zeins (Hunter et al.2002). PE5;a-/g- kernels have an increase of 26.4% moreMet than its PE5;non-RNAi control (Table 1 and Table 2),though levels are not as high as in the parental PE5 intro-gressed into the B101 background (Figure 4B). Therefore,positive regulators of expression of the 10-kDa d-zein in theB101 background might have been lost in the hybrid kernels.Still, PE5;a-/g- kernels have higher levels of bothMet and Lyscompared to the A3B and PE5;non-RNAi controls (Table 1).

BothMetandLyscanbe increasedby the synergistic effect ofa high-Met PE5 background and a combined a-/g-zein reduc-tion, with the hybrid genetic background of the kernel proge-nies affecting accumulation of these amino acids. Compared toother reports regarding increased Lys by transgenic zein re-duction in maize (Table S2 in File S1), our combination of PE5with reduction in the a-zeins seems to promote the highestincrease in Lys content. Although the higher Met and Lys con-tents of PE5;a-/g- wouldmake it a more balanced animal feedalternative than the W64Ao2 kernels (Figure 4), its opaquephenotype (Figure 2) may preclude its general use. Therefore,the higher Met and Lys contents of the vitreous PE5;g- wouldbe a better alternative than the W64Ao2 kernels in terms of itsnutritional quality and grain characteristics.

Knockdown of zeins by RNAi not only changes the accu-mulation profile of zeins (Figure 1) but also the accumu-lation of amino acids sequestered in the reduced zeins(Figure 5 and Table 2). PE5;a- has more changes in thekernel amino acid composition compared to PE5;g- andPE5;b- (Figure 5, A–C). The changes in the amino acid com-position of PE5;a- kernels were exacerbated by stacking itwith gRNAi (Figure 5D and Table 3). Surprisingly, the loss ofthe g- and b-zeins in PE5;g-/b- kernels (Figure 5E and Table3) did not produce a large change in amino acid compositioncompared to other PE5;zein RNAi kernels. The levels of seedamino acid composition in PE5;g-/b- were like its PE5;non-RNAi control (Figure 5 and Table 3) and this might havecontributed to the restoration of the vitreous phenotype inPE5;g-/b- kernels.

Amino acid changes were the lowest for PE5;g-/b- kernelscompared to other genotypes (Figure 5 and Table 3). Of the17 amino acids that were analyzed in PE5;a-/g- kernels, onlythree amino acids did not significantly differ from the PE5;non-RNAi control, whereas PE5;b-/g- kernels only had leu-cine as statistically different from normal kernels. In compar-ison to wheat grain, maize has a high leucine content, whichcontributes to its relatively poorer nutritional performancein human trials (Kies and Fox 1972). An excess of dietaryL-leucine acts as an antimetabolite of isoleucine as rats fedan excess of L-leucine in a low-protein diet, or diets deficientin isoleucine, exhibited growth retardations (Harper et al.1955). Only PE5;a- and PE5;a-/g- kernels had significantlyreduced leucine contents (Figure S3 in File S1 and Table 3).

The aRNAi lines, as well as o2, also had reduced leucinecontents (Segal et al. 2003; Huang et al. 2004, 2006). Zeinsin general, particularly a-zeins, are exceptionally rich in leu-cine (Table S1 in File S1) and, therefore, reduction in a-zeinswould also decrease seed leucine accumulation.

Discussion

The phenotypic plasticity of seed storage proteins in maizewas exploited to generate maize kernels that have enhancedaccumulation of the EAAs Lys andMet. To reduce supplemen-tation requirements of a corn-based diet, transgenic zeinreduction lines were crossed with the high-Met line PE5. Asseed storage proteinsmainly serve as the reservoir of nitrogenin the germinating maize seedling and not somuch of specificamino acids, it has been proposed that seeds are functionallyable to accommodate awide range of variations in amino acidcomposition (Shotwell and Larkins 1991). These propertiesof the seed storage proteins would make it a good target forimproving the nutritional quality of maize either through adirect manipulation of zein synthesis (e.g., by RNAi) or byincreasing the amino acid supply to the kernels due to in-creased source strength or through a combination of bothmethods. Out of the five genotypes that were tested for en-hanced accumulation of Met and Lys, only PE5;a-, PE5;g-,and PE5;a-/g- kernels had higher Lys and Met contents thanthe high-Lys W64Ao2. The latter, however, has Met levelsthat are lower than that of PE5 (Figure 4). PE5;a- andPE5;a-/g- have opaque kernels, whereas PE5;g- has vitreouskernels (Figure 2).

Amino acid composition of maize kernels varies widelyacross different genetic backgrounds. Balconi et al. (1998)and Hunter et al. (2002) reported that the opaque mutanto1 exhibits different Lys levels in different maize geneticbackgrounds. The o1 mutant has a Lys content that approx-imates that of the normal level when the allele is in a W64Abackground (Hunter et al. 2002). However, it increased the

Figure 4 Total seed Lys (A) and Met (B) contents. Values are means fromthree independent measurements of pooled samples and error barsindicate SD. The hybrid genotype Hi-II A3 B was used for maize trans-formation while PE5 had been previously characterized (Planta et al.2017). Statistical analysis was performed by using the one-way ANOVAwith post hoc Tukey HSD test; significant differences between samplesare indicated by different letters.

1692 J. Planta and J. Messing

Lys content of the inbred line A69Y as much as W64Ao2(Balconi et al. 1998), indicating a differential response ofthese backgrounds to the same mutant allele. Met accumu-lation in PE5 also varies when this transgenic event is indifferent backgrounds. Introgression of PE5 to the high-Metinbred line B101 (in at least four generations of backcrosses)has higher Met content than when it is in an A3B hybridbackground at the F3 generation (Planta et al. 2017). Resultsreported here refer to hybrid genetic backgrounds resultingfrom a combination of crosses of PE5 with the transgenic zeinreduction lines (see Materials and Methods).

In QPMs, the RNAi-induced loss of the 27-kDa g-zein ab-rogates the ability of the mo2s to restore kernel hardness,suggesting that it is necessary for endosperm modification(Wu et al. 2012). Although the 27-kDa gene does not requireO2 for its expression (Schmidt et al. 1992), the 27-kDa g-zeinlocus is linked to a QTL of mo2 in QPM (Holding et al. 2008).Gene duplication is implicated in the enhanced expression ofthe 27-kDa g-zein for endosperm modification (Liu et al.2016), whereas absence of the 27- and 50-kDa g-zein genesin a QPM deletion mutant abolished vitreous endosperm for-mation (Yuan et al. 2014). This QPM deletion mutant alsoshowed a happloinsufficient role for g-zein in o2 endosperm

modification. Alternatively, enhanced mRNA transcriptionor stability, rather than gene amplification, was hypothe-sized to be the reason for enhanced expression of theg-zein as modified and nonmodified o2 genetic back-grounds that were studied possess one or two copies ofthe gene (Geetha et al. 1991). However, cleaved amplifiedpolymorphisms exhibited no variation in 27-kDa g-zeingene copy number in the genetic background that was usedin this study (Figure S3 in File S1). Therefore, increasedlevels of g-zeins in PE5;a- (Figure 1B) and aRNAi (FigureS4 in File S1) are probably due to post-transcriptional reg-ulation of gene expression as previously described (Geethaet al. 1991).

Although the increase in 27-kDa g-zein expression in thepresence of aRNAi occurred independently of the o2 muta-tion, it can result in kernel modification in certain geneticbackgrounds (Figure S4 in File S1). It appears that kernelmodification in reduced levels of a-zeins requires at leasttwo factors: increased accumulation of the 27-kDa g-zeinand genetic modifiers of the opaque phenotype. The modi-fiers in Mo17 are probably dominant as it was used as thepaternal parent in a cross with the maternal aRNAi line.Different genotypes studied for inheritance of modified

Table 1 Percent changes in the Lys and Met contents of the hybrid PE5;RNAi+ kernels relative to the PE5;RNAi- and nontransgenic A3 Bkernels

AminoAcid

% Variation From

PE5;RNAi- Kernels (SD) Nontransgenic Hi-II A3 B Kernels (SD)

PE5;a- PE5;g- PE5;b- PE5; g/a- PE5; g/b- PE5;a- PE5;g- PE5;b- PE5; g/a- PE5; g/b-

Lys 60 (0.043) 32.83 (0.129) 220.54 (0.030) 128.16 (0.163) 217.26 (0.061) 151.85 (0.236) 63.89 (0.151) 21.97 (0.030) 83.80 (0.112) 2.08 (0.076)Met 23.97 (0.018) 24.12 (0.017) 230.64 (0.010) 26.38 (0.009) 4.64 (0.020) 0.79 (0.013) 11.61 (0.014) 219.35 (0.008) 48.61 (0.006) 22.12 (0.015)

Data shown are means (SD) of three pooled replicates.

Table 2 Amino acid composition of transgenic maize kernels from a cross of the maternal PE5 plant with the zein RNAi lines (a-, g-, b-,g-/a-, or g-/b-zein RNAi)

Amino Acida

Mol % (SD)

W64Ao2 PE5;a- PE5;g- PE5;b- PE5;g-/a- PE5;g/b-

Ala 10.06 (0.110) 24.38 (1.000)b 29.24 (0.855) 29.54 (1.915)b 23.99 (0.398)b 26.83 (1.692)Arg 3.70 (0.133) 6.91 (0.745)b 5.01 (0.395) 3.22 (0.090) 5.05 (0.351)b 3.31 (0.751)Asp 9.83 (0.613) 3.28 (0.439) 2.88 (0.332) 2.58 (0.098) 7.63 (0.110)b 4.22 (0.949)Cya 3.21 (0.218) 5.36 (0.438) 3.57 (0.796) 2.49 (0.219) 3.58 (0.131) 2.51 (0.330)Glu 15.09 (0.266) 9.79 (0.870)b 10.20 (1.339)b 9.16 (0.160)b 7.40 (0.625)b 7.89 (0.792)Gly 9.28 (0.140) 3.30 (0.384) 2.38 (0.104) 1.98 (0.076) 5.22 (0.408)b 2.87 (0.704)His 2.93 (0.172) 2.59 (0.163) 1.35 (0.329) 1.64 (0.042) 2.24 (0.178) 1.35 (0.468)Ile 3.07 (0.032) 3.32 (0.240) 4.36 (0.523) 4.48 (0.454) 3.68 (0.067) 4.57 (0.578)Leu 7.94 (0.410) 5.03 (0.331)b 8.30 (1.855) 11.00 (1.695) 7.09 (0.269)b 12.86 (1.591)b

Lys 4.21 (0.519) 7.25 (0.711)b 4.72 (0.799) 2.82 (0.165) 5.29 (0.389)b 2.94 (0.624)MetS 2.57 (0.087) 3.39 (0.371) 3.72 (0.371) 2.69 (0.301) 4.95 (0.275)b 4.06 (0.389)Phe 2.54 (0.111) 0.71 (0.118) 1.05 (0.361) 1.18 (0.272) 1.42 (0.060)b 2.11 (0.846)Pro 9.48 (0.514) 14.36 (0.944) 13.95 (0.767)b 17.31 (0.551) 12.31 (0.374)b 14.97 (0.442)Ser 5.38 (0.085) 0.21 (0.049) 0.13 (0.007) 0.11 (0.006) 0.69 (0.070)b 0.69 (0.070)Thr 3.96 (0.050) 0.50 (0.017) 0.44 (0.038) 0.40 (0.015) 1.32 (0.030)b 0.88 (0.439)Tyr 0.23 (0.025) 0.42 (0.085) 0.35 (0.015) 0.34 (0.035) 0.30 (0.036) 0.34 (0.032)Val 6.46 (0.000) 9.27 (0.555)b 8.38 (0.150) 9.03 (0.139) 7.79 (0.119)b 7.71 (1.343)

Ground corn meal of bulked samples were from kernels segregating for the zein RNAi. Data shown are means (SD) of three pooled replicates. The high-Lys opaque-2 mutantin W64A background, W64Ao2, is added for comparison.a Acid hydrolysis of the sample yields the acid stable forms Cya (cysteic acid) and MetS (Met sulfone) from cysteine and Met, respectively.b Values that are statistically different from their corresponding controls (P , 0.01) of kernels that do not segregate for the RNAi transgene(s).

Amino Acids for Animal Food/Feed 1693

endosperm in o2 backgrounds exhibited a complex system ofgenetic control involving gene dosage effects in the triploidendosperm, cytoplasm effects, and unstable and incomplete

penetrance of the modifier genes. These modifier genes haveeither dominant, semidominant, synergistic, or recessivemodes of action (Belousov 1987; Lopes and Larkins 1991).

Figure 5 Amino acid composition analysis of segregat-ing kernels from a cross of the maternal PE5 with thezein RNAi lines. Kernels from an ear resulting from thecross and segregating or nonsegregating for the RNAitransgene(s) were pooled and used for analysis. Shownare changes in the amino acid composition of (A) PE5;a-,(B) PE5;g-, (C) PE5;b-, (D) PE5;g-/a-, and (E) PE5;g-/b- rel-ative to the corresponding controls of PE5;RNAi- kernels.Cya and MetS refer to cysteic acid andMet sulfone, respec-tively, the acid stable forms of cysteine and Met producedafter performic acid treatment of the sample. Values aremeans from three independent measurements of pooledsamples and error bars indicate SD. Boxes in gray denotethat values for the PE5;RNAi+ kernels are statistically differ-ent from the corresponding control at P , 0.01.

Table 3 Percent changes in the amino acid contents of the hybrid PE5;RNAi+ kernels relative to the PE5;RNAi- segregating kernels

Amino Acida

% Variation (SD) from PE5;RNAi- Segregating Kernels

PE5;a- PE5;g- PE5;b- PE5;g-/a- PE5;g-/b-

Ala 214.81 (0.001) 6.78 (0.002) 7.86 (0.006) 110.10 (0.005) 22.02 (0.004)Arg 82.72 (0.051) 44.06 (0.083) 27.47 (0.030) 126.61 (0.038) 24.79 (0.077)Asp 224.25 (0.016) 232.87 (0.007) 239.86 (0.001) 22.92 (0.008) 21.71 (0.051)Cya 16.09 (0.011) 28.39 (0.077) 236.13 (0.020) 23.33 (0.002) 235.62 (0.024)Glu 32.06 (0.019) 45.92 (0.051) 31.09 (0.012) 259.72 (0.001) 12.92 (0.021)Gly 21.49 (0.019) 230.41 (0.005) 242.01 (0.003) 223.81 (0.004) 216.08 (0.048)His 35.30 (0.013) 229.37 (0.037) 213.81 (0.011) 221.61 (0.006) 229.2 (0.067)Ile 26.12 (0.005) 14.24 (0.020) 17.29 (0.015) 22.15 (0.001) 19.65 (0.024)Leu 237.57 (0.002) 216.28 (0.055) 11.03 (0.065) 233.72 (0.001) 29.8 (0.074)Lys 60 (0.043) 32.83 (0.129) 220.54 (0.030) 128.16 (0.163) 217.26 (0.061)Phe 240.45 (0.010) 228.47 (0.080) 219.13 (0.058) 251.15 (0.001) 44.19 (0.411)MetS 23.97 (0.018) 24.12 (0.017) 230.64 (0.010) 26.38 (0.009) 4.64 (0.020)Pro 27.33 (0.004) 215.20 (0.003) 5.25 (0.002) 9.39 (0.005) 29.00 (0.002)Ser 256.69 (0.016) 272.22 (0.003) 276.3 (0.002) 287.03 (0.000) 53.33 (0.109)Thr 247.74 (0.002) 258.28 (0.003) 261.46 (0.002) 261.06 (0.000) 216.24 (0.184)Tyr 217.65 (0.037) 210.92 (0.018) 214.29 (0.023) 91.49 (0.088) 215.13 (0.021)Val 20.7 (0.006) 9.79 (0.002) 18.35 (0.002) 43.88 (0.001) 1.05 (0.032)

Data shown are means (SD) of three pooled replicates.a Acid hydrolysis of the sample yields the acid stable forms Cya (cysteic acid) and MetS (Met sulfone) from cysteine and Met, respectively.

1694 J. Planta and J. Messing

One explanation for the increased expression of the native27-kDa g-zein protein could be the selection pressure on the27-kDa g-zein promoter used for expression of the aRNAicassette, in a phenomenon proposed as proxy selection(Bodnar et al. 2016). In proxy selection, the increased activityof a transgene under the control of a native promoter canenhance the protein levels of the native gene with the samepromoter. One complication in the use of aRNAi lines is theuse of sequences of the 27-kDa gene that drive the RNAiexpression cassette, resulting in variations of g-zein levelsin kernels of the progenies of these RNAi lines. In transgenica-zein reduction lines, gene silencing could occur or accumu-lation of variable levels of expression of the g-zein due tosegregation of its alleles (Segal et al. 2003; Huang et al.2004).

The increased accumulation of nonzein proteins in boththe endosperm and embryo of PE5;a- and PE5;a-/g- kernels(Figure 3) is a response to a-zein reduction, as also evident ino2 (Hunter et al. 2002). Because some of these nonzein proteinscontain some amount of Lys residues, the effective kernel Lysis increased (Kriz 2009). Because a-zeins make up .30% ofthe total seed proteins, their loss would also entail a majorreduction in the levels of N-transport amino acids like gluta-mine. However, o2-converted lines showed only a minor de-crease in the protein content compared with the analogousnormal inbred lines (Gupta et al. 1974), implying a protein Nredistribution from zein to nonzein proteins. We have foundthat GLB1 is likely to be upregulated in our transgenic a-zeinreduction lines, similar to what was observed in o2 (Puckettand Kriz 1991).

Reduction in g-zeins can also induce the opaque seed phe-notype, albeit at a less severe degree than the loss of a-zeins.Opacity of the b/gRNAi kernels is caused by incomplete em-bedding of the starch granules in the outer, vitreous endo-sperm rather than a reduction in the vitreous area observedin aRNAi kernels (Wu and Messing 2010; Guo et al. 2013).Kernels with reduced levels of g-zein in PE5;g- and PE5;g-/b-,but not in PE5;a-/g- kernels, have the vitreous phenotype(Figure 2). This endosperm modification is probably an indi-rect effect of the PE5 transgenic event, where it can overcomethe opaque phenotype mediated by gRNAi but not by aRNAior a/gRNAi. Zeins confer the distinct shape to PBs and canform intra- and intercellular disulfide bonds with other pro-teins. a-zeins are postulated to serve as the “brick” and theg-zeins the “mortar” in the seed during maturation and desic-cation. During desiccation, the rough ER associated with thePBs breaks down, mixing the zeins with other components ofthe cytosol and associating directly with the starch granules.The peripheral 27-kDa g-zein then serves as the “mortar” thatbonds the starch granules in a proteinaceous matrix in theouter vitreous zone of the kernel, imparting the hard endo-sperm phenotype to the kernel (Chandrashekar and Mazhar1999).

In tobacco leaves, the 10-kDa d-zein can form novel PBs,which is unlikely the case in PE5;g-/b- as the d-zein has astrong interaction with the a-zeins (Bagga et al. 1997; Kim

et al. 2002). It is more likely that the 15-kDa b-zein has aredundant functionwith the 27-kDa g-zein in terms of embed-ding the starch granules in a proteinaceous matrix because ofits cysteine content. We have previously shown that PE5 in-creases expression of cysteine-rich nonzein proteins (Plantaet al. 2017). It is possible that one of these proteins is anaccessory protein that associates with PBs and promotes itsstructural integrity.

Although QPMs have been proven to be effective, thecomplexity of introducing multiple, unlinked loci of mo2sinto a defined o2 background has slowed the creation andwidespread use of QPMs (Gibbon and Larkins 2005). Herewe report an alternative strategy to generate QPM with anadditional high-Met content without a reduction of a-zeins.PE5;g- kernels could be generated in one generation of cross-ing, whereas QPMs entail generations of backcrossing the o2mutant allele into a desirable germplasm and subsequentbackcrosses of the o2-converted germplasm with the mo2s.It would take �17 generations to convert a desirable germ-plasm into a QPM (Wu and Messing 2011). Even if both thegRNAi and PE5 lines are introgressed into a desirable germ-plasm, the eight generations it would take to make an intro-gression linewith both the PE5 and gRNAi transgenes are stillabout half the time it takes to generate a classical QPM.

Acknowledgments

This research was supported by the Selman A. WaksmanChair in Molecular Genetics of Rutgers University to J. M.

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Communicating editor: J. Birchler

Amino Acids for Animal Food/Feed 1697