Qinlin Xio,Yyun Wng,Hui Li,Chunxi Zhng,Bin Wi,Yongbin Wng,Hunhun Hung,Yngping Li,Guowu Yu,Hnmi Liu,Junji Zhng,Yinghong Liu,Yufng Hu, Yubi Hung,*

aState Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University, Chengdu 611130,Sichuan,China

bCollege of Agronomy and Biotechnology, Southwest University,Chongqing 400716,China

cCollege of Agronomy,Sichuan Agricultural University,Chengdu 611130,Sichuan,China

dCollege of Life Science,Sichuan Agricultural University, Ya'an 625014,Sichuan,China

eMaize Research Institute,Sichuan Agricultural University,Chengdu 611130,Sichuan,China

fSichuan Seed Station,Chengdu 610041,Sichuan,China

Keywords:Maize Starch synthesis ZmNAC126 Co-expression Transcription regulation

ABSTRACT Maize(Zea mays L.)is one of the most important food crops in the world,and starch is the main component of its endosperm.Transcriptional regulation plays a vital role in starch biosynthesis.However, it is not well understood in maize. We report the identification of the transcription factor ZmNAC126 and its role in regulation of starch synthesis in maize. Transcriptional expression of ZmNAC126 was higher in maize endosperm and kernels than in roots or stems.ZmNAC126 shared a similar expression pattern with starch synthesis genes during seed development,and its expression pattern was also consistent with the accumulation of starch.ZmNAC126 is a typical transcription factor with a transactivation domain between positions 201 and 227 of the amino acid sequence,is located in the nucleus,and binds to CACG repeats in vitro.Yeast one-hybrid assay revealed that ZmNAC126 bound the promoters of ZmGBSSI, ZmSSIIa,ZmSSIV, ZmISA1, and ZmISA2. Transient overexpression of ZmNAC126 in maize endosperm increased the activities of promoters pZmSh2, pZmBt2, pZmGBSSI, pZmSSIIIa, and pZmBT1 but inhibited the activities of pZmISA1 and pZmISA2. ZmNAC126 thus acts in starch synthesis by transcriptionally regulating targeted starch synthesis-related genes in maize kernels.

1.Introduction

Starch is the end product of photosynthesis and the most abundant storage carbohydrate in crop grain. Its content is also closely associated with yield and quality of crops. Maize(Zea mays L.)starch is not only a human food,but also used in feed,fuel,and industry.Starch biosynthesis occurs in plastids in higher plants via the coordinated activity of four enzymes:ADP-glucose pyrophosphorylase (AGPase), starch synthase(SS), starch-branching enzyme (SBE), and starch debranching enzyme (DBE) [1-4]. AGPase catalyzes the synthesis of ADP-glucose (ADPG) and pyrophosphate (PPi) from Glc-1-P and ATP, in a key step of starch biosynthesis. Maize AGPase is a heterotetramer(α2β2)that consists of two large(LSU)and two small (SSU) subunits [5,6]. Brittle2 (Bt2) and Shrunken2 (Sh2)encoding SSU and LSU, respectively, are highly expressed in maize endosperm [6,7]. Loss of function of Bt2 or Sh2 significantly reduces the starch content in maize seed [8,9].Starch synthase (SS) is divided into two classes, granulebound starch synthase (GBSS) and soluble starch synthase(SSS). GBSS, also known as waxy protein, is responsible mainly for the synthesis of amylose [10]. In contrast, SSS enzymes act in amylopectin synthesis by mediating sugar chain elongation, and can be further classified into five subgroups, SSI, SSII, SSIII, SSIV, and SSV [11-13]. SBE catalyzes the formation of α-1,6-glucosidic linkages of amylopectin by breaking internal α-1,4 bonds and transferring the released reducing ends to C-6 hydroxyls [14]. DBE selectively removes branches formed at improper positions on a glucan chain. There are two types of DBEs, isoamylase and pullulanase, which show different substrate specificity.Isoamylase hydrolyzes β-dextrins, whereas pullulanase hydrolyzes pullulan [15-18]. Besides the four main starch biosynthesis enzymes, starch phosphorylase (SP) also acts in starch synthesis[19-21].

Although starch is synthesized coordinately by multiple enzymes, recent studies [22-24]indicate that the expression of coding genes of enzymes is also regulated by transcription factors. HvSUSIBA2, a transcription factor in barley (Hordeum vulgare L.),directly bound to the sugar-responsive elements of the iso1 promoter and served as a regulator in starch synthesis[23]. Heterologous expression of HvSUSIBA2 in rice (Oryza sativa L.) increased rice productivity [25]. OsbZIP58, a basic leucine zipper transcription factor in rice, regulated starch biosynthesis in endosperm by directly binding to the promoters of OsAGPL3, OsWx, OsSSIIa, OsSBE1, OsBEIIb, and OsISA2 [24]. OsRSR1 co-expressed with rice type I starch synthesis genes and served as a negative regulatory factor of those genes in rice seeds[22].In maize,ZmABI4 bound to the CACCG box and mediated the expression of the ZmSSI gene induced by abscisic acid [26]. ZmbZIP91 regulated starch synthesis by binding to the ACTCAT element in promoters of starch synthesis genes in maize [27]. ZmMYB14 not only regulated the promoter activity of the Brittle 1 (BT1) gene, but also modulated the promoters of key enzyme coding genes in starch synthesis [28]. O2 [29], ZmDof3 [30], ZmNAC128 and ZmNAC130 were highly expressed in maize endosperm, coexpressed with starch biosynthesis related genes,and participated in regulation of maize starch biosynthesis [31]. Coexpression is common in plants [32,33]and is also found in rice and maize starch biosynthesis[34,35].It has been used to identify transcription factors in specific metabolic pathways;for example, OsRSR1 was involved in transcriptional regulation of rice starch synthesis-related genes [22], and AtMyb28 and AtMyb29 were involved in the transcriptional regulation of glucosinolate biosynthesis in Arabidopsis[32].

NAC transcription factors are one of the largest families of plant-specific transcription factors. It consists of three subfamilies named no apical meristem(NAM),ATAF1-2,and cupshaped cotyledon (CUC) [36,37]. NAC transcription factors have a conserved DNA-binding domain and a variable C-terminal domain, and they are widely distributed in land plants and respond to biotic and abiotic stress[38,39].High or specific expression of maize NAC genes such as ZmaNAC36/ZmNAC130 and ZmNAC34/ZmNAC128 was found [31,40,41]to be involved in starch biosynthesis regulation.

In this study, we analyzed RNA microarray data from Sekhon and Lin [42]and RNA-sequence data in MaizeGDB(http://www.maizegdb.org).We focused on NAC transcription factors that are highly or specifically expressed in maize endosperm. Our objective was to identify the function of ZmNAC126 in maize starch biosynthesis.

2. Materials and methods

2.1.Expression and co-expression analysis of candidate genes and starch synthesis-related genes

The sequences of NAC transcription factors were retrieved from Plant Transcription Factor Database (http://planttfdb.gao-lab.org/) [43], and compared with the sequence information of previous research[44]. The data of expression and coexpression analysis were based mainly on the genome-wide transcriptional atlas during maize development, including 60 distinct tissues representing 11 major organ systems of inbred line B73, with gene expression omnibus (GEO) number GSE27004 [42]. Cluster 3.0 (http://bonsai.hgc.jp/～mdehoon/software/cluster/software.htm#ctv) and TreeView (http://jtreeview.sourceforge.net/) are programs that provide a computational and graphical environment for analyzing expression data,and they were used to analyze the expression data of maize NAC transcription factors in our study. Highly or specifically expressed genes in maize seed were identified from the analysis results.

Co-expression analysis was carried out following the descriptions by Fu and Xue [22]. Twenty-five genes involved in starch synthesis and showing high expression in maize seed were used as guide genes [2,4]. Pearson correlation coefficients(PCCs) between NAC transcription factors (Table S1) and starch synthesis-related genes were calculated by CORREL function in Microsoft Excel (https://www.microsoft.com/) based on the expression profile of them. Cytoscape_3.7.0 (https://github.com/cytoscape/cytoscape/releases/tag/3.7.0) was used to display the co-expression analysis results [45]. Phylogenetic analysis of maize NAC transcription factors were constructed by using MEGA 5.10, and neighbour-joining (NJ) method was used with the bootstrap replicates set to 1000[46].

2.2. Plant materials and growth conditions

Line 18-599 is an elite maize inbred line in southwest China that was developed by the Maize Research Institute, Sichuan Agricultural University (Ya'an, Sichuan, China) in 1992 by selection from the American hybrid PN78599. 18-599 is not only high-yielding, high-quality, and disease-resistant, but also a good acceptor inbred line for transgenic maize in southwest China with good embryogenic callus rate, clonal ability and seedling rate in immature embryo culture. The line was grown during the summer under natural conditions on the experimental farm of Sichuan Agricultural University.Tassels and ears were bagged to ensure self-pollination at the silking stage. Roots, stems, and leaves were collected at the initial jointing stage of growth. Pollen and silk were collected during the flowering period prior to emergence of silk from husks. Pericarp, embryo, and endosperm were isolated from the seeds 15 days after pollination (DAP). All samples were immediately frozen in liquid nitrogen and stored at ?80 °C.

2.3. Gene cloning and sequence analysis

Total RNAs were isolated from different tissue samples using TRIzol reagent (Invitrogen, https://www.thermofisher.com/) according to the manufacturer's instructions. First-strand cDNA synthesis was performed using 1.5 μg of total RNA and the PrimeScript RT reagent Kit PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, http://www.takara-bio.com/). ZmNAC126 was cloned from the cDNA of 15-DAP seed. Genomic DNA of the inbred line 18-599 was used as the template to clone the promoters of maize starch synthesis genes. DNA fragments were cloned into pMD19-T (TaKaRa) using KOD enzymes (Toyobo, https://www.toyobo-global.com/). Sequences were verified by sequencing. The DNA binding domain of the ZmNAC126 protein sequence was identified with Smart (http://smart.embl-heidelberg.de/).

2.4. Expression analysis of ZmNAC126 and starch synthesisrelated genes in line 18-599

Freshly prepared RNA (1.5 μg) was used to synthesize firststrand cDNA. The primers ZmNAC126Q (Table S2), were used for semi-quantitative RT-PCR and quantitative real-time PCR(qRT-PCR) analysis. Maize β-actin was used as the internal control. Each PCR pattern was independently verified with at least three replicates under identical conditions. PCR products were separated in 1.5% agarose gel containing GoldView (http://www.solarbio.com/) and photographed under UV light.

The qRT-PCR analysis was performed using the iCycler instrument (model 5.0, Bio-Rad, https://www.bio-rad.com/) with SYBR Green PCR Master Mix (TaKaRa) in a total reaction volume of 10 μL. Relative transcription levels were calculated by the 2?ΔΔCqmethod based on the internal reference expression of maize βactin. Each independent experiment composed of four replicates,and three independent experiments were carried out. All primers used in the qRT-PCR analysis are listed in Table S2.

2.5. Analysis of ZmNAC126 functional properties

The pGBKT7 containing the binding domain (BD) in a GAL4 twohybrid yeast assay was used to analyze the self-activity of ZmNAC126. We made truncated ZmNAC126 by removing residues from either the N-terminus or the C-terminus and subcloned into the pGBKT7 vector with the primer ZmNAC126BKF and ZmNAC126BKR (Table S2). The restriction sites were NdeI and BamHI. The fusion carrier pGBKT7-ZmNAC126 was transformed into yeast strain AH109, to detect transcriptional activation.Transformants were screened on SD/-Trp plates and grown for three days in the dark at 28 °C. Monoclones were picked for propagation in 2 mL SD/-Trp liquid medium. Transcriptional activation activity was determined in monoclone cultured on SD/-Trp-His-Ura plates containing X-α-gal under the same conditions for three days.

To examine the subcellular localization of ZmNAC126, the coding sequence of ZmNAC126 without the stop codon was cloned between the KpnI and XbaI sites of pCAMBIA2300-35S-eGFP driven by the 35S promoter. Primers used are listed in Table S2. The construct pCAMBIA2300-35S-ZmNAC126-eGFP was introduced with a Helios Gene Gun System (Bio-Rad, https://www.bio-rad.com/) [26]into onion epidermal cells, which were then incubated in the dark for 16 h at 28 °C. A DNA fragment coding for ZmNAC126 and eGFP fusing protein was constructed into the pBI221-pUbi vector driven by the ubiquitin (Ubi) promoter by using ClonExpress II One Step Cloning Kit (Vazyme, http://www.vazymebiotech.com/). Primers used for ZmNAC126-eGFP cloning are listed in Table S2. The constructed vector was transformed into protoplasts prepared from maize leaves by the PEG-Ca2+method [47]. Transformants were cultured in the dark for 16 h at 25 °C for fluorescence detection. The subcellular localization of ZmNAC126-eGFP fusion protein was visualized with ECLIPSE 80i fluorescence microscope (Nikon, https://www.nikon.com/)under blue excitation at 488 nm.

2.6. Overexpression of recombinant ZmNAC126 in Escherichia coli

The prokaryotic expression vector, pET32a (YouBio, http://www.youbio.cn/), was used for the prokaryotic expression of ZmNAC126-His fusion protein. The primers used to insert ZmNAC126 between the BamHI and SacI sites of pET32a are listed in Table S2. E. coli Transetta (DE3) (Transgen Biotech,https://www.transgen.com.cn/) was used as host cells for prokaryotic expression. When the OD600value of the bacterial cultures reached 0.6, isopropyl β-D-1-thiogalactopyranoside(IPTG), at a final concentration of 0.5 mmol L?1, was added to induce the expression of the ZmNAC126-His fusion protein.The cells were then incubated overnight at 16 °C in a shaker at 120 r min?1. Cells were broken ultrasonically under 300 W for 10 min. The Ni-Agarose His label Kit (CWBIO, https://www.cwbiotech.com/) was used to purify the recombinant proteins according to the manufacturer's instructions.

2.7. Electrophoretic mobility shift assay (EMSA) of the ZmNAC126 binding to cascaded CACG motif

Stress-inducible NAC transcription factors in Arabidopsis regulated gene expression by binding to the harboring [C/T]ACG sequence of target genes [48-51]. A double-strand DNA fragment (5′-CACACGCACGCACGCACGCACGTG-3′) containing five tandem CACG repeats was used to test whether ZmNAC126 bound to the motif. The DNA fragment was labeled with biotin at the 3′-end with a Biotin 3′ End DNA Labeling Kit (ThermoFisher, https://corporate.thermofisher.com/en/home.html). The conjugation reaction and detection were performed with a LightShift Chemiluminescent EMSA Kit (ThermoFisher).

2.8. Particle bombardment and transient expression assay

ZmNAC126 and the promoters were subcloned into pBI221 for transient expression assay according to the method described previously [26]. Luciferase (Luc) and β-glucuronidase (Gus) were used as the reporter genes. The Gus reporter gene driven by Ubiquitin promoter (pUbi) was used as the internal control in the particle bombardment experiments. The Luc gene driven by the promoters of starch synthesis-related genes (Pro) was used as the control group. An Adh1 intron (Adh1), which had the function of enhancing promoter activity [52], was inserted between the promoter and the reporter genes to form the Pro-Adh1-Luc vector. The vector ratios in the co-transformation experiments were pUbi-Gus:Pro-Adh1-Luc (1:2) and pUbi-Gus:Pro-Adh1-Luc:pUbi:ZmNAC126 (1:2:2). LUC activity was detected with a Luciferase Assay System (Promega, https://www.promega.com). GUS activity was determined by using 4-methylumbelliferyl-β-D-galactopyranoside (MUG) as the substrate in a water bath at 37 °C for 0 h and 4 h. Each independent experiment consisted of four replicates, and four independent experiments were performed. The activity of promoters in the experimental and the control groups was determined using the formula of LUC activity / [GUS activity(4 h)-GUS activity (0 h)]. The difference with ZmNAC126 on the promoter activity of starch synthesis-related genes was tested by one-tailed t-test.

2.9. Yeast one-hybrid assay of interaction between ZmNAC126 and the promoters of starch synthesis-related genes

The promoters of the starch synthesis-related genes were amplified by PCR and subcloned into the yeast expression vector pHIS2 with different restriction sites. The ZmNAC126 coding region was amplified with the primer pair ZmNAC126R(Table S2), and subcloned into the pGADT7-Rec2 vector to obtain the pGADT7-Rec2-ZmNAC126 construct. The yeast strain Y187 was used as the host. The combination of pHIS2 vectors and pGADT7-Rec2-ZmNAC126 was used as a negative control, while the combination of pGADT7-Rec2-ZmNAC126 and pHIS2-promoter was included as the experimental group. The interactions between DNA and protein were detected by the growth of yeast cells cultured on SD/-His/-Leu/-Trp selective medium containing 50, 100,and 150 mmol L?13-amino-1,2,4-triazole (3-AT) for three days.

3. Results

3.1. ZmNAC126 was highly expressed in maize endosperm and co-expressed with maize starch synthesis genes

From the TF database [43]and following Shiriga et al. [44], we collected the information of 132 NAC genes of maize with complete open reading frames,including expression patterns of 128 genes (Fig. S1A). The expression patterns showed that 12 genes were highly or specifically expressed in maize endosperm(Fig. S1A and Table S1).

Phylogenetic analysis revealed that maize NAC family transcription factors could be classified into four subgroups(Fig.S1B).ZmNAC126,ZmNAC128,and ZmNAC130 shared high sequence similarity and were assigned to the same branch of the phylogenetic tree. ZmNAC120, ZmNAC77, ZmNAC12,ZmNAC78,ZmNAC117,and ZmNAC66 were placed in the same evolutionary branch,and ZmNAC60 and ZmNAC6 were closely related in the tree. ZmNAC126 was expressed mainly in the maize endosperm during the seed filling period. The starch synthesis-related genes including Shrunken2(Sh2),Brittle2(Bt2),GBSSI, Brittle 1 (BT1), SSI, SSIIa, SSIIIa, SBEI, SBEIIb, ISA1, and PHOL were highly expressed in maize seed (Fig. 1A). Coexpression analysis based on the expression data from the previous study [42]revealed that ZmNAC126 exhibited a similar expression pattern to most of those starch synthesisrelated genes(Fig.1).Of them,Sh2,BT1,Bt2,SSIIa,GBSSI,ISA1,and SSV had a PCC greater than 0.5 with ZmNAC126.ZmNAC126 and BT1 had the greatest PCC(0.75).The genes SSI,SSIIIa,and SBEI showed either a negative correlation with ZmNAC126 or a PCC<0.5(Fig.1B).

We investigated the ZmNAC126 expression pattern in line 18-599 via semi-quantitative RT-PCR and qRT-PCR (Fig. 2).ZmNAC126 was highly expressed in seed and endosperm. Its transcripts were also detected in stem, pericarp, and embryo(Fig. 2A). The expression of ZmNAC126 in seeds at different DAP showed a parabola-like pattern and reached a peak in 15-DAP seed(Fig.2B).The starch synthesis-related genes,ZmBT1,ZmSh2, ZmBt2, and ZmSSI showed the highest level of transcripts in maize seeds at 15 DAP, and showed a parabola-like gene expression pattern similar to that of ZmNAC126 during maize seed development (Fig. 2C and Fig.S5A). Co-expression of ZmNAC126 and starch synthesisrelated genes is shown in Fig. 2D. The PCC values of ZmNAC126 with ZmSBEIIb, ZmBt2, ZmSBEI, ZmSSIIIa, ZmPHOL,ZmISA1, ZmGBSSI, ZmAGPLS4, ZmBT1, ZmSSI, ZmSSIIa and ZmSh2 were all greater than 0.8.

3.2. Cloning and sequence analysis of ZmNAC126

The coding sequence (CDS) of ZmNAC126 comprised 969 bp and encoded a 35.307 kD protein with 322 amino acid residues(Fig. S2A). The protein sequence contained a NAM conserved domain at the amino terminus, meaning that ZmNAC126 encoded a protein belonging to the NAC transcription factor family (Fig. S2B). The similarity between ZmNAC126 and ZmaNAC36/ZmNAC130 was 56% in CDS and 41% in protein sequence(Fig.S3).

3.3. ZmNAC126 possessed transcriptional active activity and was localized to the nucleus

Fig.1- Expression patterns and co-expression analysis of starch synthesis-related genes.(A)Expression pattern of ZmNAC126 and starch synthesis-related genes.(B)Co-expression of starch synthesis-related genes and ZmNAC126 based on the expression data of Sekhon et al.[42].The vertical distance from a gene to the Y axis indicates the size of the PCC.PCC,Pearson correlation coefficient.Genes marked in green showed positive correlation and those marked in yellow showed negative correlation.

The plasmid pGBKT7-ZmNAC126,positive control pGBKT7-GAL4 and three negative controls were transformed into the yeast strain AH109 cells and screened on SD/-Trp plates.Positive clones were first confirmed by PCR and then cultivated on SD/-Trp-His-Ura plates containing X-a-gal. Yeast cells containing pGBKT7-ZmNAC126 and pGBKT7-GAL4 degraded the substrate and turned blue after incubation at 28°C for three days in darkness,showing that ZmNAC126 had transcriptional activity (Fig. 3A).Truncation of ZmNAC126 showed that the core sequence of the ZmNAC126 activation site was located between amino acid positions 201 and 227(Fig.3B).

The subcellular localization of ZmNAC126 was investigated in onion cells and maize leaf protoplast (Fig. 4). In the two assays,the eGFP signals of ZmNAC126 were found only in nuclei compared to the control, indicating that ZmNAC126 functions in the nucleus.

3.4. ZmNAC126 binds to CACG repeats in vitro

Fig.2-Expression and co-expression of ZmNAC126 and starch synthesis-related genes in maize inbred line 18-599.(A)Semiquantitative RT-PCR analysis of ZmNAC126 in different tissues of maize.R, root;St,stem;L,leaf;Se,seeds(10 DAP;S, silk;A,anthers;P,pericarp 15 DAP;Em,embryo15 DAP;En,endosperm 15 DAP.(B)qRT-PCR analysis of the expression pattern of developing maize seeds.(C)qRT-PCR analysis of starch synthesis-related genes in developing maize seeds.DAP represents days after pollination.(D)Co-expression of ZmNAC126 starch synthesis-related genes using expression during seed development.The vertical distance from a gene to the Y axis indicates the size of the PCC.PCC,Pearson correlation coefficient.Genes in green show positive correlation,and those in yellow indicate negative correlation.

ZmNAC126 recombinant protein was purified from E.coli(Fig.5A)and was verified by Western blotting (Fig.5B). To identify the DNA binding motif of ZmNAC126, a DNA fragment containing five tandem CACG repeats was used in the EMSA assay (Fig. 5C). Adding the labeled probe formed a clear free probe fragment, and the labeled probe could be retarded by ZmNAC126. Addition of unlabeled probe increased the intensity of the free probe fragment and reduced that of the blocking fragment, and the changes in intensity corresponded to the amount of unlabeled free probe.These results indicated that ZmNAC126 bound to the CACG repeat sequences in vitro.

3.5. Interaction between ZmNAC126 and the promoters of starch synthesis genes

The distribution of CATGT and CACG motifs in promoters of maize starch synthesis-related genes was determined from the cloned promoter sequence.There were several CACTG and CACG motifs in the promoters (Fig. S4). The yeast one-hybrid assay showed that the combination of ZmNAC126 with pZmGBSSI, pZmSSIIa, pZmSBE1, pZmISA1, and pZmISA2 grew normally on SD/-Trp-Leu and SD/-Trp-Leu-His plates in the presence of different concentrations of 3-AT (Fig. 5D). However,yeast cells co-transformed with ZmNAC126 with pZmSh2,pZmBt2,pZmSSI,and pZmSSIIIa could not grow on SD/-Trp-Leu-His plates containing 3-AT. These results suggested that ZmNAC126 bound to pZmGBSSI, pZmSSIIa, pZmSBE1, pZmISA1,and pZmISA2 in yeast.

3.6. Particle bombardment and transient expression assay

The plasmids pUbi-Gus and pUbi-ZmNAC126 were separately co-transformed with Pro-Adh1-Luc into the maize endosperm and the activities of GUS and LUC were then detected. The ratio of LUC activity/[GUS activity (4 h) ?GUS activity (0 h)]served as the standard to detect whether the ZmNAC126 activated or inhibited promoter activity.As shown in Fig.S5B,the relative activities of pZmSh2, pZmGBSSI, pZmSSI, and pZmBT1 were high, while those of pZmBt2, pZmSSIIIa and pZmSBEI were low. Co-transformation of ZmNAC126 with Pro-Adh1-Luc changed the relative activities of these promoters(Fig.6).These results suggested that ZmNAC126 increased the activities of pZmSh2, pZmBt2, pZmGBSSI, and pZmSSIIIa and pZmBT1. In contrast ZmNAC126 inhibited the activity of pZmISA2 and pZmISA1. However, ZmNAC126 had no apparent effect on pZmSSI and pZmSBE1.Owing to the lack of promoter activity, the effects of ZmNAC126 on pZmSSIV and pZmSSIIa were not detected following the particle bombardment and transient expression.

4. Discussion

Fig.3-Determination of transcriptional activity and activity sites of ZmNAC126.(A)Transcription activity assays of ZmNAC126 in yeast strain AH109.The schematic diagram indicates that the AH109 yeast contained the constructs. The pGBKT7-GAL4 served as positive control and pGBDKT7-Lam,pGBDKT7-53 and pGBDKT7 as negative controls.(B) Identification of transcriptional activation sites of ZmNAC126.Vertical bars represent the amino acid sequence of ZmNAC126 and the gray box represents the NAM domain.

Co-expression analysis has been successfully exploited to identify important regulators in Arabidopsis [53,54], which provides a guide for the research of gene transcriptional regulation. In this study, ZmNAC126 is also identified as a candidate gene involved in the regulation of starch biosynthesis through co-expression analysis.Transcription factors,as a type of important regulators, can be divided into many families, which participate in gene transcriptional regulation[43],and play important roles in plant development and stress response [55-57]. NAC transcription factors are also a very important and specific family in plant [50,51].

Fig.4- Subcellular localization of ZmNAC126.(A)The ZmNAC126-eGFP fusion protein was driven by the 35S promoter and transiently expressed in onion epidermal cells.The eGFP driven by 35S promoter was bombarded into the onion epidermal cells as a control.(B)The ZmNAC126-eGFP fusion protein was driven by the ubiquitin(Ubi)promoter and transiently expressed in the protoplasts of maize leaves.The eGFP driven by the Ubi promoter transformed into the protoplasts of maize was used as a control.The labels of ZmNAC126-eGFP correspond to the distribution of ZmNAC126-eGFP in the onion epidermal cells and protoplasts under the fluorescent field,bright field, and merge.

Transcriptional regulation is an important way to regulate starch biosynthesis in crops. Rice Starch Regulator1 (RSR1),encoding an AP2 transcription factor,is a negative regulator of starch synthesis in rice and is co-expressed with many starch synthesis-related genes [22]. OsbZIP58, a basic leucine zipper transcription factor, participates in regulation of starch synthesis in rice endosperm, and also shows similar expression patterns with starch synthesis genes [24]. The expression pattern of starch synthesis-related genes and transcription factors was similar to the starch accumulation pattern in maize kernels(Figs.2,S1 and S6),suggesting that transcription factors play an important role in the regulation of maize starch synthesis. Transcriptional regulators known to participate in the transcriptional regulation of starch synthesis in maize include ZmbZIP91[27],ZmMYB14[28], O2[29], and ZmDof3 [30]. These transcriptional regulators were specifically or highly expressed in maize kernels and endosperm[58].

In a recent study [41], a NAC family transcription factor,ZmNAC130, was highly expressed in maize endosperm, was co-expressed with ZmBt2, ZmSh2 and ZmSSI, and increased their transcription levels in transient transformation.ZmNAC128 and ZmNAC130 bound to cis-acting elements in the promoter of ZmBt2 and participated in the regulation of promoter activity and maize starch biosynthesis [31].ZmNAC126 belongs to the same subgroup as ZmNAC128 and ZmNAC130 (Fig. S2). It shares high similarity in CDS and protein sequences with ZmNAC130(Fig.S4),and has similar gene expression patterns with ZmNAC128 and ZmNAC130(Fig.S1).ZmNAC126 also shared similar expression patterns with the starch synthetic genes in maize endosperm(Figs.1 and 2). These results suggest that ZmNAC126 is involved in the transcriptional regulation of starch synthesis-related genes.

The CACATG motif and the motif containing the core sequence [T/C]ACG have been identified as the core binding site of stress-inducible NAC in Arabidopsis[48-51].In the EMSA analysis,ZmNAC126 bound to the CACG motif directly in vitro(Fig.5).CACATG and CACG are distributed in the promoters of maize starch synthesis-related genes. Some CACATG and CACG form tandem repeats (Fig. S5), suggesting that ZmNAC126 binds directly to these promoters for function. In the yeast one-hybrid assay, ZmNAC126 bound directly to the promoters of ZmGBSSI, ZmSSIIa, ZmSBEI, ZmISA1, ZmISA2, and ZmSSIV(Fig.5),but differed from ZmNAC128 and ZmNAC130,which bound to the promoter of Bt2 [31]. Some transcription factors transactivate target gene promoters and are involved in starch biosynthesis; ZmbZIP91, OsbZIP58, OsRSR1, O2 and PBF bound to promoters to participate in the transcription regulation of starch biosynthesis[22,24,27,29].Thus,the direct binding of ZmNAC126 to promoters provides an opportunity for participation in transcriptional regulation during maize starch biosynthesis.

Cloning and activity analysis of a gene promoter allows study of its transcriptional regulation [26,27]. In this study,ZmNAC126 promoted the activity of the promoters of ZmBt2, ZmSh2, ZmGBSSI, ZmSSIIIa, and ZmBT1 and inhibited the activity of promoter of ZmISA1 and ZmISA2 (Fig. 6). The active effect of ZmNAC126 on the activity of pZmBt2 is similar to that of ZmNAC128 and ZmNAC130 [31]. However, the binding results of the yeast one-hybrid assay were not completely consistent with that of the particle bombardment of endosperm. These results suggest that the direct binding of ZmNAC126 to a specific promoter may not affect activity and that the impact on activity may not depend on direct binding. They also suggest that a transcriptional regulation in maize starch biosynthesis is complex, in agreement with previous reports [59,60]. For example, RPBF interacted with RISBZ1 to regulate the expression of starch synthesis-related genes [60,61]. MYC interacted with EREBP protein and co-regulated the transcription of the rice Wx gene [59]. The transcription factors O2interacted with PBF and co-regulated the biosynthesis of maize starch [29]. ZmNAC128 and ZmNAC130 also interacted with each other to regulate maize starch and protein biosynthesis [31].

Fig.5-Binding of ZmNAC126 to promoters of starch synthesis-related genes.(A)Recombinant proteins were induced from E.coli.The numbers of 1 to 5 represent no induction and induction at 2,4,6,and 8 h,respectively.(B)Detection of purification of recombinant proteins by Western blotting.The numbers 1 to 6 represent no induction,total protein,the liquid after binding,the liquid of washing buffer,and the liquid of elution buffer with 200 mmol L?1 and 300 mmol L?1 iminazole, respectively.(C)The binding results of ZmNAC126 recombinant protein with the CACG repeats in vitro.“+and number” represents the added composition and amount, “?” means the corresponding component not added.(D)Yeast one-hybrid analysis of interaction between ZmNAC126 and the promoters of starch synthesis-related genes.The purple triangle indicates that the yeast solution with four gradients was obtained by isometric dilution in multiples of 10.

5.Conclusions

We have identified the transcription factor ZmNAC126 as a regulator of maize starch biosynthesis based on co-expression analysis.Our study suggests that ZmNAC126 is a typical NAC transcription factor,is highly expressed in maize endosperm,is localized to the nucleus, possesses a transactivation domain, and binds to CACG repeats for its action. Transient expression assays and yeast one-hybrid assay demonstrated that ZmNAC126 positively or negatively regulated the activity of starch synthesis gene promoters by direct or indirect binding. Thus, ZmNAC126 is a key factor in transcriptional regulation of maize starch synthesis-related genes.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Fig. 6 - Transient assay for the interaction between ZmNAC126 and the promoter of starch synthesis-related genes in maize endosperm. (A) Schematic diagram of carrier construction. The promoters of starch synthesis-related genes were inserted into the reporting vector and ZmNAC126 was subcloned into the effect vector. (B) ZmNAC126 affected the activities of starch synthesis-related gene promoters in transient expression assays. The effect of ZmNAC126 on the promoter activity of starch synthesis-related genes was tested by one-tailed t-test at ( P <0.05; **, P <0.01).

Author contributions

Yubi Huang, Qianlin Xiao, Yayun Wang and Hui Li designed the research. Qianlin Xiao, Yayun Wang, Hui Li and Chunxia Zhang performed the experiments. Bin Wei, Yongbin Wang,Huanhuan Huang, and Yangping Li analyzed the data. Yubi Huang, Qianlin Xiao, and Yayun Wang prepared the manuscript. Guowu Yu, Hanmei Liu, Junjie Zhang, Yinghong Liu,Yufeng Hu, and Yubi Huang revised the manuscript. All the authors read and approved the manuscript.

Acknowledgments

We gratefully acknowledge using the expression profile of Dr.Rajandeep S. Sekhon (Department of Energy Great Lakes Bioenergy Research Center,University of Wisconsin Madison,USA) to perform the co-expression analysis. This work was supported by the National Natural Science Foundation of China (31571757) and the National Key Basic Research Program of China(2014CB138202).

Appendix A. Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.04.014.