Tianyi Wang,Yunfei Li,Yumin Huang,Xiaoming Zhao,Zhaobin Dong,Weiwei Jina,b,,d,*,Wei Huang,d,*

a State Key Laboratory of Plant Physiology and Biochemistry,National Maize Improvement Center of China,Beijing Key Laboratory of Crop Genetic Improvement,Key Laboratory of Crop Heterosis and Utilization,Ministry of Education,China Agricultural University,Beijing 100193,China

b College of Agronomy & Resources and Environment,Tianjin Agricultural University,Tianjin 300384,China

c Center for Crop Functional Genomics and Molecular Breeding,China Agricultural University,Beijing 100193,China

d Sanya Institute of China Agricultural University,Sanya 572025,Hainan,China

Keywords:Grain protein content Amino acid Maize Amino acid permease

ABSTRACT Grain protein content(GPC)is an indicator of cereal nutritional quality.Identification of genes involved in the regulation of GPC provides targets for molecular breeding for crop protein quality.We characterized a maize gene encoding the putative amino acid transporter ZmAAP6,a gene expressed mainly in immature seeds,especially in the basal endosperm transfer layer.Total protein and zein contents were decreased in ZmAAP6 null mutants and increased in ZmAAP6 overexpression(OE)lines,consistent with their changed in the size of protein bodies.Metabolic and transcriptomic analysis supported the regulatory role of ZmAAP6 in amino acid transportation.These results suggest that ZmAAP6 functions as a positive regulator of GPC in maize,shedding new light on the genetic basis of GPC regulation.

1.Introduction

Cereal grains provide protein and calories for human nutrition and animal feed[1].At present,nearly 30% of the world’s population suffers from protein malnutrition[2],and increasing grain protein content(GPC)is one of the most effective approaches to meeting nutrition requirements[3].Maize(Zea mays)is a staple cereal grown worldwide.More than 80%of maize kernels are composed of starch and protein[4,5].Identification of genes regulating GPC will improve the nutritional characteristics of maize seeds.

Amino acids are the constituent units of proteins,which mediate cell protein synthesis,metabolism,growth,and development[6].Transportation of amino acids into cells and cellular compartments depends on membrane transporter proteins[7],in particular amino acid transporters(AATs),which are found in bacteria,fungi,animals,and plants[8,9].In plants,the AAT family comprises two subfamilies,the amino acid polyamine choline transporter subfamily and the amino acid transporter subfamily,also known as the amino acid/auxin permease(AAAP)family[10,11].The plant AAAP family contains at least six subfamilies,including amino acid permeases(AAPs),lysine-histidine-like transporters,γ-aminobutyric acid transporters,proline transporters,ANT1-like aromatic and neutral amino acid transporters,and auxin transporters[12].These function in a variety of plant physiological processes including amino acid uptake[6,13],seed loading[14],xylem-phloem transfer[15]and phloem loading[16-17].

The variety of AAP expression patterns and functions indicates that AAP-family genes play diverse roles in the plant life cycle.In Arabidopsis,AtAAP5 was expressed throughout the plant and functioned in root absorption of amino acids[18,19].AtAAP6 is expressed in xylem parenchyma,may function in uptake of amino acids from xylem,and regulates sieve element composition[20,21].AtAAP3 was strongly expressed in root phloem and cotyledons[15].In contrast,AtAAP4 was expressed mainly in source leaves,stems,and flowers,suggesting that they act in longdistance amino acid transport,especially in phloem loading[22,23].AtAAP1,AtAAP2,and AtAAP8 are expressed in immature siliques and embryos,mediating the uptake of amino acids into growing embryos and endosperm and further affecting protein content of seeds[13-15,20,24,25].In rice,AAP6 functions as a regulator of GPC and nutritional quality[5].During seed development of Vicia faba,increases in VfAAP1 expression precedes amino acid concentration,indicating it has a primary role in supplying amino acids for storage protein synthesis[26].Moreover,ectopic expression of VfAAP1 in seeds of V.narbonensis and pea promotes amino acids uptaking,thereby increasing storage protein synthesis[27].Despite sustained interest in the role of AAPs,the regulatory mechanism that determine GPC remains elusive.In maize,ZmAAP4 and ZmAAP2 may function respectively in amino-N mobilization to the ear and in phosphorus-nitrogen crosstalk[28,29].But whether AAPs are involved in seed GPC regulation remains unknown.

In our previous transcriptomic study[30]of embryo development in maize,ZmAAP6 was highly expressed in the early embryonic stage of seeds.In the present study,ZmAAP6 was found by mRNA-in situ hybridization to be expressed predominantly in the basal endosperm transfer layer(BETL)and the placenta-chalazal(PC)region of developing seeds.To confirm its biological function during seeds development,we developed transgenic lines to knock out and overexpress ZmAAP6 in maize.ZmAAP6 knockout plants exhibited decreased GPC;in contrast,overexpressing ZmAAP6 increased protein accumulation in maize grain.Targeted metabolomic data showed that the accumulation of lysine,tryptophan,and various amino acids was altered in transgenic lines.These results suggest that ZmAAP6 functions in the GPC regulation in maize,and is a valuable candidate gene for improving the nutritional quality of maize.

2.Materials and methods

2.1.Plant materials and growth conditions

The maize inbred line LH244 was obtained from the Center for Crop Functional Genomics and Molecular Breeding of China Agricultural University and used as a transgenic recipient line.All constructs were introduced into LH244 by Agrobacterium-mediated transformation[31].For transformations using the overexpression construct,three independent positive transgenic lines(OE-1-3)were obtained by PCR amplification and measurement of expression level.For transformations using the CRISPR/Cas9 construct,three independent positive transgenic homozygous knockout lines(KO-1-3)with insertions or deletions in target sites were identified by PCR amplification and sequencing.All primers used for CRISPR/Cas9 vector construct are listed in Table S1.All plants were grown in the field at the experimental station of China Agricultural University in Beijing.

2.2.Scanning electron microscopy and transmission electron microscopy

Sample processing and electron microscopy observation of kernels followed previously described protocols[32].For scanning electron microscopy analysis,developing kernels(at 15 DAP)were dissected from wild-type,KO-1,and OE-1 plants,placed in 2.5%(v/v)glutaraldehyde overnight at 4 °C and dehydrated through a graded ethanol series of 30%,50%,70%,85%,95%,and 100%ethanol.The samples were dried at a critical point,coated with gold palladium,and observed under a Hitachi S-3400N scanning electron microscopy(SEM)at 10 kV.For transmission electron microscopy,immature wild-type,KO-1,and OE-1 seeds at 15 DAP were diced along the horizontal axis and fixed in paraformaldehyde.The samples were further processed and stained with osmic acid and observed under a Hitachi H7600 transmission electron microscope at Shanghai Normal University.

2.3.mRNA in situ hybridization

Developing kernels(at 6 and 9 DAP)were collected from wildtype plants and fixed in 3.7%[v/v]formalin-acetic acid-alcohol solution.The samples were dehydrated in a graded ethanol series(30% to 100%[v/v]),then embedded in paraffin and cut into 8-μm sections.A DIG-RNA Labeling kit(Roche)was used to synthesize and label antisense and sense probes(T7/SP6)for ZmAAP6,and mRNA in situ hybridization followed Zhang et al.[33].The primers used for probe preparation are listed in Table S1.

2.4.Measurement of total protein and starch

To measure the total GPC and starch content of mature seeds of the wild type and knockout and overexpression lines,20 seeds per line were used.Seed coats and embryos were removed from the kernels after soaking in water for 1 h and ground into fine powder in liquid nitrogen.At least three biological replicates were used for subsequent analysis.

For GPC measurements,total protein,zein,and nonzein proteins were extracted from 50 mg of dried endosperm flour as previously described[34].Protein quantification of the total extract,zeins,and nonzein fractions was performed with a Bradford Protein Assay Kit(catalog no.P0006C;Beyotime).Starch quantification followed the instructions of a total starch assay kit(catalog no.K-TSTA;Megazyme)for total starch measurements.All measurements were performed in three biological and technical replicates.

2.5.Semiquantitative PCR(sq-PCR)and quantitative real-time PCR(qPCR)

To examine the expression patterns of ZmAAP6,we collected various tissues from wild-type plants.Total RNA was extracted following the manufacturer’s instructions for the Quick RNA Isolation Kit(Huayueyang Biotechnology).For sq-PCR and qPCR,we used the Prime Script RT reagent kit with gDNA eraser(Takara,Dalian,China)to synthesize cDNA,and used TB Green PCR Master Mix(Takara)for qPCR.ZmActin1(Zm00001d010159)was used as an internal normalization control.Relative expression was calculated using the 2-ΔΔCTrelative quantification method[35].All primers are listed in Table S1.

2.6.RNA sequencing

Transcriptomes were generated by RNA sequencing(RNA-seq)in 13 DAP kernels of the wild type,KO-1,and OE-1.Twenty seeds were pooled for each biological replicate,and three biological replicates were collected for each genotype.Total RNA was extracted as described in Section 2.5.RNA-seq libraries were constructed according to the Illumina standard instructions at Novogene(Beijing,China),and sequenced on the Illumina NovaSeq 6000 platform based on the manufacturer’s instructions.TopHat(v2.1.0)[36]was used to map clean RNA-Seq data to the corn B73 reference genome(B73_v4)[37],and Cufflinks(v2.2.1)was used to calculate the comprehensive expression pattern of genes based on the previous method using default parameters[36].Cuffdiff in Cufflinks was used to compare gene expression profiles of samples at different stages.Differentially expressed genes(DEGs)were defined as those with q-value＜0.05.Gene Ontology(GO)enrichment analysis of the DEGs was performed using the AgriGO tool[38].RNA-seq data generated in this study have been deposited in the National Center for Biotechnology Information Sequence Read Archive(accession code PRJNA809102).

2.7.Amino acid targeted metabolomics

Samples(100 mg fresh weight)of 13 DAP kernels were homogenized in liquid nitrogen,added 10 times volume of ddH2O and vortexing,and then further diluted 50 times with ddH2O.Then 50μL of diluted sample was mixed by vortexing with 200μL of acetonitrile/methanol(1:1)containing internal standards.After incubation on ice for 30 min,the mixture was centrifuged at 12,000 rm in-1for 10 min and the supernatant was injected into an LC-MS/MS system for metabolite analysis according to a standard protocol at Tianjin Novogene Company.

3.Results

3.1.Expression of ZmAAP6

The maize genome contains 16 predicted AAP family protein members[39].We constructed a phylogenetic tree based on the AAP family protein sequences.Because in the tree,the protein product of Zm00001d012229 in maize showed the closest relationship to OsAAP6,we named this gene ZmAPP6(Fig.1A).It has been previously reported[5]that OsAAP6 affects grain protein content(GPC)by regulating the synthesis and accumulation of protein and starch.RNA-seq in our previous study[30,40]showed that ZmAAP6 was highly expressed in kernels,though its function was unclear.We wished to know whether ZmAAP6 performs a similar biological function to OsAAP6 for the regulation of GPC in kernels.

Fig.1.Expression analysis of ZmAAP6.(A)Phylogenetic analysis of AAP proteins from maize,rice,and Arabidopsis.The phylogenetic tree analysis was performed by MEGA6 using the maximum likelihood method based on the Poisson correction model.Bootstraps=1000.(B,D)Comparisons of ZmAAP6 expression levels in various tissues(B)and developing kernels(D)in the B73 inbred line using sq-PCR.The kernel samples in(B)were harvested at 15 DAP.Other tissues(root,stem,leaf,tassel,and ear)were collected from field-cultivated B73 plants.The kernel samples in(D)were collected at several developmental stages,as indicated by days after pollination.(C,E)qRT-PCR analysis of ZmAAP6 expression in various tissues(C)and developing kernels(E)in B73.The kernel samples in(C)were harvested at 15 DAP.Other tissues(root,stem,leaf,tassel and ear)were collected from field-cultivated B73 plants.The kernel samples in(E)were collected at several developmental stages,as indicated by days after pollination.ZmActin was used as the internal control.Three biological replicates were constructed with tissues from three plants.Error bars indicate±SD(n=3).(F)mRNA in situ hybridization of B73 kernels using a ZmAAP6-specific antisense probe and a negative control sense probe.Scale bars,100μm.Em,embryo;PC,placenta-chalazal;BETL,basal endosperm transfer layer.

To identify the function of ZmAAP6 during maize development,we investigated its expression in a broad range of tissues in wildtype plants in detail.Both sq-PCR and qRT-PCR results suggested that ZmAAP6 was predominantly expressed in kernels.However,it was barely detectable in other vegetative tissues including roots,stems,leaves,tassels,and ears(Fig.1B,C).We examined the expression profile of ZmAAP6 during seed development.ZmAAP6 was expressed throughout seed development and was most abundant in kernels at 3-6 DAP(Fig.1D,E).To further investigate the spatio-temporal expression pattern of ZmAAP6 in developing kernels,we performed mRNA in situ hybridization with 6 and 9 DAP kernels.ZmAAP6 transcripts were strongly detected in the BETL and the PC region,which are the two key maternal tissues that control the supply of nutrients to the central endosperm(Fig.1F)[41,42].The expression analysis suggested that ZmAAP6 might function in seed development.

3.2.ZmAAP6 mediated maize kernel protein content

To investigate the potential biological functions of ZmAAP6 in maize,we knocked ZmAAP6 out using the CRISPR/Cas9 system.A 19-bp sequence in the third exon of ZmAAP6 was selected as an editing target using the system as previously described[43].PCR amplification and sequencing identified three homozygous knockout lines with varied frameshift mutations,named KO-1,KO-2,and KO-3.KO-1 had a 25-bp nucleotide deletion in the juxtaposition of target site,and KO-2 and KO-3 had a 1-bp nucleotide insertion adjacent to the NGG motif(Fig.2A).To overexpress ZmAAP6,we amplified the full-length coding sequence(without the stop codon)of ZmAAP6 and cloned it into the AscI and KpnI sites of the binary vector pBCXUN-MYC to generate the pOE-Zm00001d012229-MYC construct driven by the Ubiquitin promoter.Three independent overexpression lines of ZmAAP6(OE-1,OE-2,and OE-3)were obtained.qPCR analysis indicated that the expression of ZmAAP6 was significantly greater in the seeds of ZmAAP6-OE lines than in wild-type seeds(Fig.2B).The morphology of ZmAAP6 transgenic lines showed no difference from that of the wild type(Fig.S3).Neither knockout nor overexpression of ZmAAP6 affected kernel size,weight,or texture(Fig.S4).

To quantify the effects of ZmAAP6 on the nutritional quality of grain,we measured the major protein components in the mature endosperm of wild-type,ZmAAP6-KO,and ZmAAP6-OE plants.Quantitative analysis of total(Fig.2C)and zein(Fig.2D)proteins showed that both were significantly reduced in ZmAAP6-KO lines but increased in ZmAAP6-OE lines compared with the wild type(Fig.2C,D).Nonzein was significantly decreased in ZmAAP6-KO lines but unchanged in ZmAAP6-OE lines(Fig.S1).Likewise,SDSPAGE revealed a similar alteration of protein content in ZmAAP6-KO and ZmAAP6-OE lines(Fig.S1).These results indicate that ZmAAP6 positively regulates protein accumulation in maize seed development.

3.3.ZmAAP6 may regulate protein accumulation by enlarging protein bodies

As knockout and overexpression of ZmAAP6 greatly affected kernel protein content,we performed transmission electron microscopy(TEM)to observe whether the morphology of protein bodies(PBs)was altered in transgenic lines(Fig.3A).Although there was no apparent difference in PB number,the immature endosperm of ZmAAP6-KO-1 contained smaller PBs.ZmAAP6-OE-1 contained larger PBs than the wild type(Fig.3B,C),a finding consistent with the GPC levels.We focused on starch grains(SGs),the plant organelles where starch is produced and stored,in endosperm cells of 15 DAP kernels.Compared to the wild type,the mean number of SGs was changed negligibly(Fig.3D)in transgenic lines,with the mean SG size larger in ZmAAP6-KO-1 and smaller in ZmAAP6-OE-1(Fig.3E).Likewise,SEM indicated that only the size of SGs was altered in ZmAAP6 knockout and overexpression lines,whereas the overall morphology showed no apparent change,echoing the TEM observation.In agreement with the changes in SG size,the starch content was increased in ZmAAP6-KO-1 and decreased in ZmAAP6-OE-1 compared to that of the wild type(Fig.3G).Thus,the changes in GPCs possibly resulted from the change in PB size in ZmAAP6 null mutant and overexpression lines.

3.4.ZmAAP6 regulates the content and profiles of amino acids in developing seeds

AAPs recognize and allocate amino acids to supply cellular metabolism[11].To further determine whether ZmAAP6 affected the content and profiles of free amino acids in kernels,we used targeted metabolomics to determine whether amino acid contents were changed in ZmAAP6 transgenic lines(Fig.4A).Total amino acid content was significantly decreased in ZmAAP6-KO-1 kernels,but increased in ZmAAP6-OE-1 kernels(Fig.4B).Contents of Ser,Pro,Leu,Lys,Arg,Trp,His,Val,Orn,Ala,Ile,Asp,Thr,and Asn were significantly decreased in ZmAAP6-KO1.In contrast,the contents of Met,Pro,Lys,GABA,Val,Ala,and Gln were significantly increased in ZmAAP6-OE-1 kernels(Fig.4A).These results suggested that ZmAAP6 might be involved in transport of amino acids during seed development.

3.5.Transcriptome analysis of ZmAAP6 transgenic lines

To investigate the influence of ZmAAP6 on gene expression during maize endosperm development,we performed RNA-seq analysis at 13 DAP with immature endosperms from wild-type,ZmAAP6-KO-1,and ZmAAP6-OE-1 transgenic plants.Respectively 5780 and 1596 DEGs were identified in ZmAAP6-KO-1 and ZmAAP6-OE-1 relative to the wild-type(Fig.5A).GO enrichment analysis revealed that these DEGs were involved in multiple biological processes,including transport-related,stress-response,and energy metabolism processes(Fig.5B).Compared with the wild-type,multiple DEGs were enriched in amino acid transport-related GO terms in ZmAAP6-KO-1 or ZmAAP6-OE-1(Fig.5B),such as amino acid transmembrane transport,amide transport,and protein transport,suggesting that ZmAAP6 as an amino acid transporter gene might function in multiple regulatory pathways during seed development,especially the amino acid transport process.Several genes involved in endosperm development were differentially expressed in ZmAAP6 transgenic lines,including the zein accumulation and lysine content-associated genes opaque2(o2)and opaque7(o7)[34,44],and the soft starchy endosperm formation gene floury3(fl3)(Fig.5C)[45].o2 was upregulated in OE-1,while o7 was downregulated in KO-1.Their differential expression might contribute to the varied zein contents in transgenic seeds.fl3 was oppositely regulated in OE-1 and KO-1,a finding consistent with the starch content of transgenic kernels.These results suggested that the co-regulation of multiple pathways mediated by ZmAAP6 might determine seed nutritional quality during endosperm development.

4.Discussion

The value of grain is determined mainly by the endosperm,the primary storage tissue of seed[46,47].The BETL is a unique cell layer at the junction of maternal tissue and endosperm,whose function is to transfer apoplastic solutes from the outside of the maternal pedicel to the endosperm,and provide compounds needed for embryo development and storage reserve accumulation for seeds[48].ZmAAP6 was expressed mainly in BETL and PC cells(Fig.1F).In a previous study[40],ZmAAP6 was expressed mainly in the maternally-derived nucellus,the main component of the seed at the early development stage[49,50].Subsequently,as the nucellus was continuously degraded and reabsorbed,ZmAAP6 was expressed mainly in endosperm cells undergoing cell division and expansion(Figs.1E,S2)[51,52].The spatio-temporal expression pattern of ZmAAP6 expressoin suggests that it functions as a transporter allocating free amino acids from the maternal tissue to the seed to facilitate cellular differentiation in early seed development,and to contribute to metabolic processes required for cell specialization and endosperm development,such as signal transduction,protein synthesis,and energy metabolism.

Fig.2.Biochemical analysis of wild-type(WT),ZmAAP6-KO,and ZmAAP6-OE endosperm.(A)Gene structure of ZmAAP6 and sequences of three homozygous gene-edited lines with deletions or insertions in the target sites.(B)Expression of ZmAAP6 in 12 DAP WT and ZmAAP6-OE endosperms.Values are means with standard errors,n=3 individual plants(**,P＜0.01,Student’s t-test).(C)SDS-PAGE of total proteins from the wild-type(WT),ZmAAP6-KO and ZmAAP6-OE mature endosperm.(D)SDS-PAGE of zein proteins from WT,ZmAAP6-KO and ZmAAP6-OE mature endosperm.(E)Comparison of total proteins from WT,ZmAAP6-KO and ZmAAP6-OE mature endosperm.Means are of three biological replates,and 20 dried endosperms were used for each replicate(*,P＜0.05;**,P＜0.01,Student’s t-test).(F)Comparison of zein protein content in mature endosperm of WT and ZmAAP6 transgenic lines.Values are means with standard errors,n=3 biologically independent samples(*,P＜0.05;**,P＜0.01,Student’s t-test).

In addition to their role as substrates for the synthesis of proteins and enzymes,amino acids are precursors of plant hormones[53],which regulate broad aspects of plant development,metabolism and defense response to stress.In plants,proline accumulation positively correlates with tolerance to various environmental stresses,including salt,drought,temperature,heavy metal ions,and oxidative stress[53-58].The accumulation of proline accompanies the transition from vegetative to reproductive growth and seed development initiation,and can maintain cell division under long-term abiotic stress[59].Proline can facilitate seed germination under salt-stress conditions by maintaining osmotic balance and scavenging toxic free radicals[60].Proline also functions as a signaling molecule that induces activation of defensive mechanisms to alleviate the toxicity of cadmium to maize growth[61].Previous report[62]showed that the AAP family proteins function in proline uptake.In our study,proline concentrations were decreased in ZmAAP6-KO-1 but increased in ZmAAP6-OE-1 kernels(Fig.4A).The fold change of proline content among various amino acids was highest in ZmAAP6-OE-1.GO enrichment analysis revealed that the DEGs were involved in many stress-associated pathways,including temperature stimulus,salt stress,oxidative stress,metal ion,and water deprivation response(Fig.5B).Thus,ZmAAP6 may be involved in proline-mediated stress responses except for regulation of seed development through amino acid transport.

Fig.3.Cytological and biochemical characteristics of wild-type(WT),ZmAAP6-KO-1 and ZmAAP6-OE-1 endosperm.(A)Transmission electron microscopy of the fourth cell layer from the aleurone layer in the WT,ZmAAP6-KO-1 and ZmAAP6-OE-1 endosperm at 15 DAP.Scale bars,5μm.SG,starch grain;PB,protein body.(B)Comparison of protein body numbers per cell in WT,ZmAAP6-KO-1 and ZmAAP6-OE-1 endosperm at 15 DAP;ns,not significant.(C)Comparison of protein body area per cell in WT(n=101),ZmAAP6-KO-1(n=103)and ZmAAP6-OE-1(n=108)endosperm at 15 DAP(**,P＜0.01,Student’s t-test).(D)Comparison of starch body numbers per cell in WT,ZmAAP6-KO-1 and ZmAAP6-OE-1 endosperm at 15 DAP;ns,not significant.(E)Comparison of starch body area per cell in WT(n=69),ZmAAP6-KO-1(n=70)and ZmAAP6-OE-1(n=76)endosperm at 15 DAP.(**,P＜0.01,Student’s t-test).(F)Scanning electron microscopy of the central regions of WT,ZmAAP6-KO-1 and ZmAAP6-OE-1 endosperm at 15 DAP.Scale bars,10μm.SG,starch grain;PB,protein body.(G)Total starch contents of mature WT and ZmAAP6 transgenic lines endosperm.Values are means with standard errors,n=3 biological replicates(*,P＜0.05;**,P＜0.01,Student’s t-test).

Fig.4.Effects of ZmAAP6 on amino acid content in wild-type(WT),ZmAAP6-KO-1,and ZmAAP6-OE-1 lines.(A)Contents of free amino acids in kernels of WT,ZmAAP6-KO-1,and ZmAAP6-OE-1 lines.Values are means with standard errors,n=3 individual plants(*,P＜0.05;**,P＜0.01,Student’s t-test).(B)Insert indicates the total content of free amino acids.Values are means with standard errors,n=3 individual plants(**,P＜0.01,Student’s t-test).

Fig.5.Transcriptome analysis of ZmAAP6.(A)Number of DEGs(up-and down-regulated)between the wild-type(WT)and ZmAAP6-KO-1/OE-1 lines of 13 DAP kernels.DEGs were identified based on q-values＜0.05.(B)GO term enrichment of DEGs(up-and down-regulated)distinguishing the WT and ZmAAP6-KO-1/OE-1 lines.(C)Expression change of 5 DEGs involved in endosperm development,q-values＜0.05(*,P＜0.05;**,P＜0.01;***,P＜0.001,Student’s t-test).

Zein accounts for almost 60% of total endosperm protein,strongly influencing maize nutritional properties.The lack of lysine and tryptophan in zein results in low nutritional value[4].In view of the low lysine content of maize seeds,a major effort began in the middle of the 20th century to characterize high-lysine maize cultivars by genetic methods[4].These efforts led to the discovery of the opaque2 mutant with higher lysine content than normal maize[63].However,field analyses showed that opaque mutants had insurmountable adverse agronomic traits,including reduced yield and protein content,kernel damage,soft endosperm with increased susceptibility to diseases and insects,and poor food processing quality[64].To avoid these drawbacks,maize breeders have focused on introducing quantitative-trait loci known as o2 modifiers to restore seed hardness in the o2 mutant while maintaining the high-lysine trait,thereby creating quality protein maize(QPM)[65].However,fully restoring the hardness of o2 endosperm requires extensive efforts by breeders to integrate multiple o2 modifiers.In the present study,we identified an amino acid transporter gene,ZmAAP6,as a positive regulator of amino acid and protein contents in maize seeds.Overexpression of ZmAAP6 showed no severe drawbacks for plant growth and seed development(Figs.S3,S4),whereas total protein and free amino acids,especially lysine content,were increased(Fig.4A).ZmAAP6 could thus be used as a new gene resource for QPM improvement,in crop nutritional quality breeding.

CRediT authorship contribution statement

Tianyi Wang:Data curation,Formal analysis,Investigation,Methodology,Validation,Visualization,Writing-original draft,Writing-review & editing.Yunfei Li:Data curation,Formal analysis,Investigation,Methodology,Validation,Visualization.Yumin Huang:Data curation,Visualization,Methodology.Xiaoming Zhao:Methodology,Resources.Zhaobin Dong:Writing-review&editing.Weiwei Jin:Conceptualization,Funding acquisition,Project administration,Resources,Writing-review & editing.Wei Huang:Conceptualization,Funding acquisition,Project administration,Supervision,Writing-original draft,Writing-review &editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was supported by the 2022 Research Program of Sanya Yazhou Bay Science and Technology City(SYND-2022-10 to Wei Huang and SYND-2022-03 to Weiwei Jin).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.03.007.