Genome-wide identification of the basic leucine zipper transcription factor genes related to starch synthesis in wheat (Triticum aestivum L.)

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Jinjin Ding, Xiaolei Chen, Hassan Karim, Guzmán Carlos, Wendy Harwood, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3045123/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jan, 2024 Read the published version in Plant Growth Regulation → Version 1 posted 5 You are reading this latest preprint version Abstract The growth and development of plants are dependent upon the transcription factors. In plants, the basic leucine zipper (bZIP) family of transcription factor is known for its largest size and diversity. Using newly available wheat genome data, we compared our identification of 181 Triticum aestivum bZIP (TabZIP) genes to those reported in earlier studies. One gene was characterized, and some duplicate genes and incorrect annotations in previous studies are supplemented and corrected. The 181 bZIP genes identified were divided into 13 known groups and 5 unknown groups by phylogenetic analysis. All of the bZIP genes exhibit a minimum of one bZIP motif in their motif distribution and gene structure. Spatial and temporal expression patterns of bZIP family members during various stages of plant growth vary, as suggested by transcriptome data. As per the expression data obtained via quantitative reverse transcription PCR (qRT-PCR), over 10 TabZIP genes showed similarity with starch synthesis in wheat. In vitro binding activity of TabZIP68 to the promoter of TaWaxy was demonstrated by a dual-luciferase reporter (LUC) assay. Given its potential involvement in starch synthesis, the TabZIP68 gene presents itself as a strong candidate for further investigation. Wheat Basic leucine zipper Transcription factors Gene interaction Starch synthesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction In plants, transcription factors (TFs) are vital for regulating transcription, as they bind to cis-acting elements of their intended target genes (Gonzalez 2016 ; Jakoby et al. 2002 ). Of these, the basic region/leucine zipper (bZIP) TFs have conserved domains and one of the largest gene families(Izawa et al. 1993 ). These were classified and named based on their conserved DNA-binding domains, which utilize a leucine zipper dimerization motif to bind to DNA (Hurst 1995 ; Wei et al. 2012 ). The conserved basic region, which is responsible for sequence-specific DNA binding, comprises about 16 amino acid residues containing a conserved N-x7-R/K motif (Nijhawan et al. 2008 ). In plants, bZIPs TFs exist mainly in the form homo- and heterodimers (Hurst 1995 ) and bind preferentially to the A-box (TACGTA), C-box (GACGTC), and G-box (CACGTG), which have core ACGT sequences (Baloglu et al. 2014 ). With the development of whole genome sequencing technology, bZIP transcription factor family members have been comprehensively identified or predicted in many plants (Liao et al. 2008 ). In Arabidopsis, the bZIP gene family is divided into 10 groups based on the sequence similarity of their basic regions and the presence of additional conserved motifs (Jakoby et al. 2002 ). Many studies have identified members of the bZIP family in various species including 75 in Arabidopsis (Agarwal et al. 2019 ), 125 in maize (Jain et al. 2018 ), 187 in wheat (Li et al. 2015 ), 89 in rice (Liu et al. 2014 ), 92 in barley (Zhong et al. 2021 ), 55 in grapevine (Wang et al. 2011 ), and 64 in cucumber (Xiao et al. 2011 ), but only a small number of genes from this family have been functionally characterized. This transcription factor family is associated with numerous developmental processes in plants (CAO et al. 2012), including but not limited to flower development (Gupta et al. 2012 ), seed maturation (Li et al. 2018 ), and responses to both biotic (Lim et al. 2015 )and abiotic factors (Wigge et al. 2005 ). Bread wheat (Triticum aestivum L.) is an inbreeding crop with distinctive evolutionary limitations, which results in comparatively low genetic diversity. Despite this, it is a significant global source of both carbohydrates and proteins. In 2018, the International Wheat Genome Sequencing Consortium (IWGSC) publicly disclosed the genome sequencing map of Chinese Spring, a six-row malting wheat originating from the United States, marking the first time the genome had been fully sequenced and made available. In 2020, Chinese Spring chromatin accessibility data using MNase are available in open access. In, 2021, the IWGSC RefSeq v2.1 assembly and annotation became available to download, BLAST and display in a browser. In 2022, a new version of RepetDB containing 54 species became available. The major features include 36 new genomes annotated using REPET v3.0, manual annotation tags, and old annotations were updated using PASTEClassifier v2. In previous studies, They identified 187 and 191 TabZIP members using the wheat genome (2018 version) (Agarwal et al. 2019 ; Li et al. 2015 ). Starch is an important carbohydrate that is an essential source of energy for humans. Crop quality is dependent upon the type and characteristics of starch. Starch biosynthesis is a complex process conducted by a series of starch synthetases. There is a well-organized system for starch synthesis in cereal, which includes three gene families including starch synthase, granule bound starch synthase (GBSS), starch synthase (SS), and ADP-glucose pyrophosphorylase (AGPase). While another gene family is starch branching enzymes (SBE) including several isomers (Preiss 2018 ). First, sucrose is decomposed into fructose and UDP glucose in the endosperm cells by sucrose synthetase. Subsequently, G-1-P(1-phosphoglucose) is produced by UDp-GlC pyrophosphorylase. Thereafter, ADP-Glc pyrophosphorylase (AGPase) forms the direct substrate of starch synthesis, adenosine diphosphate glucose (ADPG). ADPG in the cytoplasm is transported to the powder by the adenylate transporter (BT1) on the powder membrane. Under the action of a series of starch synthetases, amylose and amylopectin are formed successively (Soliman et al. 2014 ). The synthesis of amylose is completed under the action of GBSS, located inside starch granules, causing the synthesized amylose to remain unbranched (Sonnewald and Kossmann 2013 ). The synthesis of amylopectin is relatively complex, involving the interaction of three enzymes SS, SBE, and DBE (Guzmán and & Alvarez 2016; Liu et al. 2016b ; Preiss 2018 ). Also the isoamylase 1 (ISA1) is involved in amylopectin production via trimming glucan cluster (Huang et al. 2021 ). Several studies have found OsbZIP58(Wang et al. 2013 ) and OsbZIP76 (Niu et al. 2020 ) to be involved in starch regulation in rice. Studies have also found the following genes to be onvolved in maize starch synthesis: ZmNAC36, ZmNAC128, ZmNAC130 ZmbZIP22, ZmbZIP91, Opaque 2, ZmEREB156 (Chen et al. 2016 ; Dong et al. 2019 ; Huang et al. 2016 ; Zhang et al. 2019 ). Moreover, TubZIP28 and TabZIP28 have been found to be transcriptional activators of starch synthesis in wheat (Liu et al. 2016a ; Liu et al. 2020 ; Song et al. 2020 ). One study showed that SUSIBA2 (sugar signaling in barley) from WRKY TF was identified only in barley whereas no evidence for it was found in rice and maize (Sun et al. 2003 ). The products of SUS-IBA2 bind to the promoter of ISA1 and influence the starch synthesis by regulating gene expression in barley. Studies have been published, which showed various bZIP family members involved in starch synthesis are already indentified in rice, maize and wheat. Analysis of the bZIP family is necessary to screen the genes related to starch synthesis. We used recently published assembly of the Chinese Spring wheat genome. Compared to previous studies, the study focused on the phylogenetic relationships, structural features, chromosomal location, and gene expression pattern among bZIP genes. We analyzed the gene expression pattern of the TabZIP family across various developmental stages and tissues, focusing on their co-expression with starch synthase genes. Hence, this study showed new insight into TabZIP genes, which may play essential roles in the production of starch synthesis in wheat. 2. Materials and methods 2.1 BZIP transcription factor genes identificate in Chinese Spring wheat To identify members of the bZIP gene family in the Chinese Spring genome, we conducted a search using URGI ( http://wheat-urgi.versailles.inra.fr/Tools ). To obtain genomic sequences, including DNA FASTA and GFF3 files, for Chinese Spring, we retrieved them from the URGI database ( https://www.wheatgenome.org/News/Latest-news/IWGSC-RefSeq-v2.1-Assembly-and-Annotation-now-freely-available-at-URGI-and-NCBI ). To process the biological data, we utilized TBtools, a toolkit for handling biological data (Chen et al. 2020), we conducted an initial search of the Chinese Spring wheat genomes from the PlantTFDB v5.0 database using BLAST (with an e-value cutoff of 1e − 10 ) ( http://planttfdb.gao-lab.org/index.php ), which includes protein sequence for bZIP TFs from Arabidopsis thaliana and Triticum aestivum L . We manually deleted redundant sequences. Based on the results from the first BLAST search using PlantTFDB v5.0 database, the protein sequences were extracted using TBtools and used as a query for the second BLAST search, which was performed using NCBI-BLASTP with an e-value threshold of 1e − 10 . The non-bZIP members were deleted manually after downloading the alignment results. The conserved bZIP domain were analyzed between the two BLAST via NCBI Conserved Domain Database server ( http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml ) (Marchler-Bauer et al. 2011). Protein search was performed to confirm that all the selected were bZIP TFs. 2.2 Comparison of TabZIP genes identified in different wheat genome versions Earlier research (Li et al. 2015 ; Pourabed et al. 2015), involved a search for and retrieval of sequences of TabZIP genes from the wheat genomic database ( http://www.gramene.org/ ). The bZIP gene identified in previous studies was used as a query sequence to perform a BLAST search of wheat V2 for comparison with previous results. Query the same sequence as having an Expect = 0 value gene. Next, we used the Chinese Spring V2 genome to identifiy the bZIP genes for a BLAST search (e-value ≤ 1e 10 ) of the URGA HC_genes_CDS_ Seq_2020, LC_genes_CDS_Seq_2020, and full-length cDNA ( http://wheat-urgi.versailles.inra.fr/morgoth/Data ) databases. Differences in TabZIP family members between the old and new versions were determind using BLAST. 2.3 Analysis of the TabZIP gene structure and phylogenetic TBtools was used to compare the coding sequence of each TabZIP gene with its genome sequence in order to construct an exon/intron map. The ClustalW program implemented in MEGAX was used to compare all amino acid sequences ( http://www.megasoftware.net/ ) to determine the evolutionary relationship between wheat and Athaliana bZIP proteins. The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replications, based on the JTT matrix-based model, and the amino acid sequences were aligned using the ClustalW program implemented in MEGAX. 2.4 Localization Conserved motif analysis and conserved motif analysis of bZIP genes on Chinese Spring chromosomes The motif analysis of the TabZIP protein sequences was conducted using the online software MEME 5.1.1 ( http://meme-suite.org/tools/meme ) and we selected these parameters: number of repetitions-any, maximum number of motifs = 20, and optimum motif width ≥ 6 and ≤ 200 (Ma et al. 2017). Chen et al was followed to analyze the chormosomal location using protein sequences against the genome annotation GFF3 file in TBtools (Chen et al. 2020). 2.5 Expression analysis We used the transcriptome database for the Wheat Reference Transcript (IWGSC Annotation v1.1) dataset ( http://202.194.139.32/expression/wheat.html ) to compare all CDS sequences for the TabZIP genes. The RNA-seq data of 13 tissues including root (10 cm seedlings), root 2 (4 week seedling), shoot (10 cm seedlings), rachis (5 weeks pa), senescing leaf(2months), tillers (3rd internode), inflorescence-1 (0.5 cm), inflorescence-2 (1–1.5 cm), embryo (germinating), palea (6 weeks pa), epidermis (4 weeks), grain (5 DPA), and grain (15 days postanthesis [DPA]) of Chinese Spring were retrieved from The James Hutton Institute ( http://www.wheat-expression.com/ ), and the log 2 transcripts per million value of each bZIP gene was visualized as a heatmap with a blue-yellow-red gradient. 2.6 Plant growth, RNA extraction, and quantitative reverse transcription PCR analysis The expression level of wheat starch synthase genes was measured. The gene expression of RNA-seq was verified by using Quantitative reverse transcription PCR (qRT-PCR) and also TabZIP genes were screened. The experiment was conducted using Chinese Spring wheat under the following growth conditions: 16 hours of light and 8 hours of dark with day and night temperatures of 24°C and 18°C, respectively. The grain samples were collected at different developmental stages of 5, 10, 15, 20, 25, and 30 DPA and these were transferred to -80°C from the liquid nitrogen. Total RNA was extracted using an RNA kit following the manufacturer's protocol (Biofit, Chengdu, China). For each developmental stage, three replications were tested. First-strand cDNA was synthesized using PrimeScript™ RT reagent kits with gDNA Eraser (TaKaRa, Dalian, China) and qRT-PCR was performed using SYBRVR Premix Ex Taq™ II (TaKaRa) on a CFX 96 Real-Time System (Bio-Rad, Hercules, USA). We analyzed and calculated the expression of the qRT-PCR data using CFX Manager Software (Bio-Rad, Hercules, USA). The expression level calculated using the 2 Ct method. Wheat-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes were used as internal reference genes to normalize the expression of the candidate genes. 2.7 Dual-luciferase reporter (DLR) assay A 1.5 kb portion of the promoter sequence from TaAGP-S , TaAGP-L , TaSS1 , TaSS2a , TaWaxy , TaSBE2a , and TaSBE1 was cloned into the pGreenII 0800-Luc vector to create promoter firefly luciferase reporter constructs. pGreenII 62-SK-TabZIP68 was created by cloning the TabZIP68 CDS into the pGreenII 62-SK vector and was used as the effector vector. Both vectors were co-expressed in tobacco. Subsequently, we cultured the co-transfected tobacco overnight in the dark. The DLR assay system (Promega, USA) was used to measure LUC and REN activities. All experiments were independently repeated six times, and each assay had three technical replicates. 2.8 Subcellular localization analysis For the analysis we ligated the TabZIP68 without termination codons into pJIT163 vector and then we transformed the A. tumefaciens strain GV3101 with pJIT163-GFP, pJIT163-TabZIP68-GFP, and plasmids. By following the Cao et al., maize protoplasts of the leaf sheaths of 15-day-old wheat seedlings were used in transforming the vectors to express fused GFP. A confocal laser-scanning microscope was used to monitor GFP flourescence (Zeiss LSM 800, Jena, Germany). 3. Results 3.1 Identification of bZIP genes in Chinese Spring The characterization of members of the TabZIP family can be more precise by utilizing the recently released Chinese Spring genome data from URGI in comparison to the previous version (Agarwal et al. 2019 ; Li et al. 2015 ). We identified 181 nonredundant TabZIP genes in Chinese Spring wheat based on a genome-wide search and the validation results. As there is no established standard nomenclature, each gene in the dataset was assigned a generic name based on the order of its original sequence ID ( TabZIP1-TabZIP181 ). Sequence analyses showed that all the TabZIP genes have the bZIP domain, and the amino acid (aa) length of these TabZIP genes ranged from 129 ( TabZIP58 ) to 652 ( TabZIP138 ), with an average size of 329 amino acids, protein mass between 14.11 ( TabZIP58 ) and 68.90 Kd ( TabZIP138 ), and pI ranging from 4.85 ( TabZIP134 ) to 11.63 ( TabZIP82 ). A subcellular localization analysis predicted that most genes are expressed in the nucleus, whereas TabZIP53 , TabZIP54 , TabZIP55 , TabZIP70 , TabZIP101 , TabZIP120 , TabZIP122 , and TabZIP123 are expressed in the nucleus and chloroplasts; however five genes ( TabZIP8 , TabZIP9 , TabZIP11 , TabZIP12 , and TabZIP13 ) did not have a predicted location (Table S1 ). 3.2 Identified the TabZIP genes in different wheat genome versions to comparison A comparative analysis of different versions of TabZIP genes in old studies (Agarwal et al. 2019 ; Li et al. 2015 ) was used to correct duplicates of one gene into separate genes, correcting family size and misnamed genes. In total, According to this resulets in the wheat genome were identified 187 and 192 TabZIP family genes. We further validated our results by comparing the TabZIP gene identified in the Chinese Spring V2 genome with previously identified TabZIP genes (Tables S1 and S2 ). 70 different TabZIP genes were identified in the Chinese Spring V2.1 genome, whereas 32 of the old bZIP genes did not match with the new version. In many cases, one gene in the old version corresponded to two genes in the new version. Traes_3AL_FC5523394.2 corresponded to the genes, TabZIP1 and TabZIP5 , Traes_5DL_67A38E9B7.1 corresponded to the TabZIP2 and TabZIP73 , the Traes_5BL_BBFA6D506.1 is corresponds to the TabZIP3 and TabZIP122 . The multiple sequence alignment results (Fig. S1 ) showed that the 2012 genome may be caused by incorrect splicing of this phenomenon. Compared with previous studies, the number of TabZIP genes identified from Chinese Spring V2.1 was more accurate and the sequence was more complete. 3.3 Phylogenetic analysis of TabZIP genes The bZIP family genes in Arabidopsis genome have been well classified according to the structural characteristics of their protein sequences (Jakoby et al. 2002 ). TabZIP was distributed in Groups A, B, C, D, E, F, G, H, I, and S, as well as four unknown groups (Groups U1, U2, U3, and UH). Groups A, D, I, and S had more member genes than the other groups (Fig. 1 ), containing 17, 15, 14, and 27 TabZIP genes, respectively. The other genes were divided into nine groups of varying numbers, with group B and U2 being the two smallest, each containing six genes. A phylogenetic tree analysis revealed that one member of Group H and U, and two members of Group A were not clustered together in Arabidopsis. Three subgroups were found in the Group S by members of Group C and G. One study also presented similar results by phylogenetic analysis of the wheat and barley bZIP family (Li et al. 2015 ; Zhong et al. 2021 ). We hypothesized that gene classification of Arabidopsis was based on protein sequence therefore, this study analyzed the basis of the amino acid alignments. 3.4 Gene and protein structural features of the wheat bZIP genes The gene structure characteristics of 181 bZIP family genes identified in this study were analyzed using gene annotation information. Of the 181 genes, only 25 did not contain introns, while the remaining 156 contained between 1 and 12 introns (Fig. S2 ). According to the results of evolutionary analysis results, most of the genes in the same group had a similar gene structure, length, and distribution of introns and exons. Ten conserved motifs were set to identify TabZIP family members, among which motif 1 was the core motif of bZIP and was shared by all family members (Figs. 2 A and 2 B). The number of motifs in different groups was different, and the maximum number for the S group was 9 (Fig. S3 ). There was only one motif in TabZIP80 , TabZIP81 , TabZIP90 , and TabZIP91 . All bZIP family genes were extracted from wheat genome with high confidence protein sequences. A chromosomal distribution analysis revealed that bZIP family factors were evenly distributed on each chromosome and the maximum number of genes on chromosome 5A was 15 (Fig. 3 ). 3.5 Expression profiles of TabZIP genes during growth and development To determine test whether the identified bZIP family genes are normally expressed in wheat, we used transcriptomic data the wheat multiomics website (WheatOmics 1.0). The results showed that all the 181 genes had corresponding transcripts, indicating that the identified genes had normal function and were true genes. Using published RNA-seq database data, we further analyzed the spatial and temporal expression patterns of bZIP family genes and examined their expression profiles in different tissues (Table S3 ). TabZIP8 , TabZIP25 , TabZIP101 , TabZIP130 , and TabZIP132 were expressed throughout development, but at low levels(Fig. 4 ). During grain development, 52 genes were highly expressed, of which 32 were endosperm specific. We screened out some genes that were highly expressed during the grain development stages (5–25 DPA) and conducted real-time qPCR for further verification (Fig. 5 ). We also performed the same analysis on the genes encoding enzymes related to starch synthesis (Fig. 6 ), including TaAGP-L , TaAGP-S , TaWaxy , TaISA1 , TaSS1 , TaSS2a , TaSS3 , TaSBE2a , and TaSBE2b . Most of the selected genes expressed similar starch synthetase genes, but TabZIP13 , TabZIP41 , TabZIP69 , TabZIP148 , and TabZIP164 were different from the starch synthetase genes. 3.6 TabZIP68 bound specifically to the promoters of TaWx in DLR We used the DLR system to verify whether major starch synthetase genes interact with candidate TFs. According to the NCBI I-BLAST search results, TabZIP68 was a homologous gene with O2 , a transcription factor reported to regulate starch synthesis in maize (Zhang et al. 2016 ). In rice, TabZIP68 is homologous to the OsbZIP58 gene, which directly interacts with the promoter of six starch synthase genes and regulates their expression (Wang et al. 2013 ). However, the regulatory mechanism of TabZIP68 on grain starch synthesis in wheat has not been studied. The DLR results indicate that TabZIP68 binds to the cis-acting DNA element in the promoter region of starch synthetase Waxy gene. These results suggested that TabZIP68 might be involved in regulating the synthesis of wheat grain starch (Fig. 7 ). 3.7 TabZIP68 subcellular localization analysis We constructed the vector into TabZIP68-GFP and extracted wheat leaf protoplasts to locate the gene products. There was a strong GFP signal in the nuclei and plasma membranes (Fig. 8 ). On the other hand, the unfused GFP control exhibited GFP signals that were observed in various cellular compartments, including the plasma membrane, cytoplasm, and nuclei. 4. Discussion The accumulation of starch in wheat endosperm involves a complex mechanism of various starch synthesizing genes. Upto now many TFs are known for their specific roles in starch synthesis. Especially, rice, maize and wheat are those plant species in which bZIP family is identified. However, it is necessary to identify and analyze the TabZIP family genes to create valuable gene resources for starch synthesis. Among the studies on TabZIP, 192 TabZIP genes were identified in wheat (Agarwal et al. 2019 ). Subsequently, it was discovered that an earleir version of the wheat genome was used in these studies (Lim et al. 2015 ), and gaps were identified in this old version. Therefore, some important genes were considered missing in that version. The updated version of the Chinese Spring wheat genome data URGI (V2.1) includes the updated information regarding annotation and also have much more information than earlier V1. Therefore, V2.1 was used in this study, as it contains more precise information for characterizing of the TabZIP gene family. The present study identified 181 TabZIP genes in wheat. Although there were 11 fewer genes compared with the previous version, many misjudgments caused by incorrect splicing and assembly using the old version were found identified. Of these, 31 bZIP genes in the old version were not found to correspond with the gene in the V2.1 version, and there was one gene in the old version for every two genes in the new version. For example, Traes_3AL_FC5523394.2 is corresponded to the gene TabZIP1 and TabZIP5 genes, Traes_5DL_67A38E9B7.1 is corresponded to the TabZIP2 and TabZIP73 , the Traes_5BL_BBFA6D506.1 is corresponds to TabZIP3 and TabZIP122 . These findings demonstrate that the new version has more complete gene splicing and detailed annotation information compared with the old version, and our study can provide more accurate and detailed analysis for the bZIP genes in wheat. The diversity of bZIP genes was identified in the Arabidopsis genome, which represents flowering plants (Jakoby et al. 2002 ). The AtbZIP are classified into 10 groups on the basis of their structural and functional characterization. This classification system has been applied to various plants and has become the classification standard for members of the bZIP family. In previous studies, the conserved arginine in bZIP gene of Group U was replaced by a hydrophobic isoleucine in the DNA binding of the bZIP gene, which may affect DNA-binding characteristics. However, we did not find any gene that was highly expressed in all tissues through RNA-seq database. Our results indicate that TabZIP family genes are expressed in at least one tissue in wheat, showing tissue-specific differential expression. Thirty-two of these genes were specifically expressed in grain, suggesting that these genes may be involved in grain development and the accumulation of storage materials. Studies have reported the regulation of the gene expression of grain starch synthesis by bZIP family TFs. In rice, OsbZIP20 combines with starch synthase gene promoters Waxy and SBE1 to regulate grain starch content (Wang et al. 2013 ). The TubZIP28 and TabZIP28 are those genes which binds to promoter region of AGPase and enhances their transcriptional level in wheat. In maize, O2 regulates the expression of α and β-zein genes mainly by recognizing the O2 box in the α and β-zein promoter (Holdsworth et al. 1995 ), and O2 can also interact with other TFs or proteins to regulate starch synthesis in the grain (Zhang et al. 2016 ). It is stated in previous study Luo et al, that the key enzyme responsible for amylose synthesis is granule-bound starch synthase I (GBSSI) (Luo et al. 2019 ). Previous studies have shown that TabZIP68 and O2 are homologous genes and that O2 can bind multiple starch synthetase gene promoters to regulate grain starch synthesis. Using DLR, we demonstrated that TabZIP68 interacts with the starch synthetase gene promoter Waxy and theat the TabZIP68 gene may be a transcriptional regulatory factor for wheat starch synthesis. It is worth further study. Declarations Author Contributions JD was involved in retrieving the data, expression analysis and writing of manuscript, HK, XL and GC performed qRT-PCR, yeast one hybrid assay, and data analysis, respectively. WH helped in correction of expression data. HP and HX were involved in material planting. QJ designed and supervised the experiments. YZ, QX, YF, PQ, YJ, MD, and JM participated to improve the research design. JW, GY, YM and YLZ helped to revise manuscript. All of the authors are agreed to final version of manuscript. Acknowledgements This study received funding from the Sichuan Science and Technology Program of PR China (2022ZDZX0014 and 2022YFH0055), as well as the International Science & Technology Cooperation project of Chengdu, Sichuan Province, PR China (2019-GH02-00078-HZ). Conflict of Interest We declare that we do not have any conflicts of interest. Data availability All data generated or analyzed during this study are included in this article. 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Theoretical and Applied Genetics 129:1203-1215 Liu Y, Hou J, Wang X, Li T, Majeed U, Hao C, Zhang XJJoeb (2020) The NAC transcription factor NAC019-A1 is a negative regulator of starch synthesis in wheat developing endosperm 71:5794-5807 Luo M, Ding J, Li Y, Tang H, Qi P, Ma J, Wang J, Chen G, Pu Z, Li W (2019) A single-base change at a splice site in Wx-A1 caused incorrect RNA splicing and gene inactivation in a wheat EMS mutant line. Theoretical and Applied Genetics 132:2097-2109 Nijhawan A, Jain M, Tyagi AK, Khurana JP (2008) Genomic survey and gene expression analysis of the basic leucine zipper transcription factor family in rice. Plant physiology 146:333 Niu B, Deng H, Li T, Sharma S, Yun Q, Li Q, E Z, Chen C (2020) OsbZIP76 interacts with OsNF‐YBs and regulates endosperm cellularization in rice (Oryza sativa). Journal of Integrative Plant Biology 62:1983-1996 Preiss J (2018) Plant starch synthesis. Starch in food:3-95 Soliman A, Ayele BT, Daayf F (2014) Biochemical and molecular characterization of barley plastidial ADP-glucose transporter (HvBT1). PloS one 9:e98524 Song Y, Luo G, Shen L, Yu K, Yang W, Li X, Sun J, Zhan K, Cui D, Liu DJNP (2020) TubZIP28, a novel bZIP family transcription factor from Triticum urartu, and TabZIP28, its homologue from Triticum aestivum, enhance starch synthesis in wheat 226:1384-1398 Sonnewald U, Kossmann J (2013) Starches—from current models to genetic engineering. Plant biotechnology journal 11:223-232 Sun C, Palmqvist S, Olsson H, Borén M, Ahlandsberg S, Jansson CJTPC (2003) A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter. The Plant Cell 15:2076-2092 Wang J-C, Xu H, Zhu Y, Liu Q-Q, Cai X-LJJoeb (2013) OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm 64:3453-3466 Wang J, Zhou J, Zhang B, Vanitha J, Ramachandran S, Jiang SY (2011) Genome‐wide Expansion and Expression Divergence of the Basic Leucine Zipper Transcription Factors in Higher Plants with an Emphasis on Sorghum F. Journal of integrative plant biology 53:212-231 Wei K, Chen J, Wang Y, Chen Y, Chen S, Lin Y, Pan S, Zhong X, Xie D (2012) Genome-wide analysis of bZIP-encoding genes in maize. DNA research 19:463-476 Wigge PA, Kim MC, Jaeger KE, Busch W, Schmid M, Lohmann JU, Weigel D (2005) Integration of spatial and temporal information during floral induction in Arabidopsis. Science 309:1056-1059 Xiao X, Wu Z-C, Chou K-C (2011) A multi-label classifier for predicting the subcellular localization of gram-negative bacterial proteins with both single and multiple sites. PloS one 6:e20592 Zhang Z, Dong J, Ji C, Wu Y, Messing JJPotNAoS (2019) NAC-type transcription factors regulate accumulation of starch and protein in maize seeds 116:11223-11228 Zhang Z, Zheng X, Yang J, Messing J, Wu Y (2016) Maize endosperm-specific transcription factors O2 and PBF network the regulation of protein and starch synthesis. Proceedings of the National Academy of Sciences 113:10842-10847 Zhong X, Feng X, Li Y, Guzmán C, Lin N, Xu Q, Zhang Y, Yang H, Qi P, Deng M, Ma J, Wang J, Chen G, Lan X, Wei Y, Zheng Y, & Jiang Q (2021) Genome-wide identification of bZIP transcription factor genes related to starch synthesis in barley (Hordeum vulgare L.). . Genome 64:1067–1080 Supplementary Files FigS1.jpg Supplementary Figure 1 Phylogenetic classification of wheat TabZIP and Arabidopsis AtbZIP proteins. FigS2.jpg Supplementary Figure 2 Gene structures of 181 wheat TabZIP genes with full-length coding sequences. Figs3.jpg Supplementary Figure 3 Conserved motifs of the 181 TabZIP genes. TableS1.xlsx Supplementary table 1 Basic information of 181 wheat bZIP family members. The coordinate position, gene length, Molecular weight and PI of each gene were analyzed TableS2.xlsx Supplementary table 2 Comparison of TabZIP family genes in different versions. TableS3.xlsx Supplementary table 3 TabZIP genes at different developmental stages in eight wheat tissues. Cite Share Download PDF Status: Published Journal Publication published 26 Jan, 2024 Read the published version in Plant Growth Regulation → Version 1 posted Reviewers agreed at journal 16 Jul, 2023 Reviewers invited by journal 24 Jun, 2023 Editor invited by journal 14 Jun, 2023 Editor assigned by journal 14 Jun, 2023 First submitted to journal 13 Jun, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3045123","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":212806283,"identity":"ff2e5a4e-8139-4002-8099-ceb61312687f","order_by":0,"name":"Jinjin Ding","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinjin","middleName":"","lastName":"Ding","suffix":""},{"id":212806284,"identity":"5ccc99c6-9f1b-43af-8512-47b34b09510e","order_by":1,"name":"Xiaolei Chen","email":"","orcid":"","institution":"Sichuan Agricultural 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jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIiWNgGAWjYDACZijFBiI/MEhAuDyEtByAamGcQZQWBogWiHa4Snxa+I4zP3v8ocaGnY/97OHXNn8s5PlnJDA+eNvGIG+OQ4vkYTZzgwPH0pjZePLSrHPbJAxn3EhgNpzbxmC4swG7FoPDDGYSB9gOA/2SY2ac2yCRwHAjgU2at40hweAALi3s3yQO/ANq4X9jZmzxRyJB/kYC+2/8WnjMJA62AbVI5Bg/ZmCTSDAA2sKMT4vkYZ4yibN9QL9IvDFj7AX6ZeOZh82Sc85JGG7AoYXv/PFtEhXfbJLl+3OMP/z4Uycvdzz54Ic3ZTbyuGyBxUkyELNB45GxAUhI4FCP0GIHxMwfcCsbBaNgFIyCkQwAM/tVcHRHAJoAAAAASUVORK5CYII=","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Qiantao","middleName":"","lastName":"jiang","suffix":""}],"badges":[],"createdAt":"2023-06-10 02:17:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3045123/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3045123/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10725-023-01111-z","type":"published","date":"2024-01-26T15:13:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":39304194,"identity":"bb59e40e-5758-42a7-b98e-4ade2ab51c4d","added_by":"auto","created_at":"2023-06-29 14:51:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1139397,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic classification of wheat TabZIP proteins. The 10 classes are represented by branches of different colors.\u003c/p\u003e","description":"","filename":"FIg1.png","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/55030685967103676f303825.png"},{"id":39303065,"identity":"453e6340-3ac4-41e2-b422-57d8b0708bc6","added_by":"auto","created_at":"2023-06-29 14:43:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1190962,"visible":true,"origin":"","legend":"\u003cp\u003eDomains and motifs of wheat TabZIP proteins.\u003c/p\u003e","description":"","filename":"FIg2.png","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/cd36bfe67a01e270a9894f68.png"},{"id":39304198,"identity":"57cfb3a1-61ec-4e3d-a175-da268a03618d","added_by":"auto","created_at":"2023-06-29 14:51:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2677497,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal distribution of the TabZIP genes in wheat.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/670542db531e5b5c2beda74d.png"},{"id":39304195,"identity":"d5a0236d-e869-4415-8cf7-6c090e05c576","added_by":"auto","created_at":"2023-06-29 14:51:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":761473,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmaps of the expression profiles of TabZIP genes at different developmental stages in eight wheat tissues. The color scale represents the expression value. * Indicates the 12 genes selected for qRT-PCR verification. Root (10 cm seedlings), Root 2 (4 weeks seedlings), Shoot (10 cm seedlings), Rachis (5 weeks pa), Senescing leaf (2 months), Tillers (third internode), Inflorescence-1 (0.5 cm), Inflorescence-2 (1–1.5 cm), Embryo (germinating), Palea (6 weeks pa), Epidermis (4 weeks), Grain (5 DPA) and Grain (15 DPA), DPA: days postanthesis, pa: postanthesis.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/97b7640ab0753294290cf367.png"},{"id":39303091,"identity":"134a48fc-69cd-4e7c-b31e-c19707ce9581","added_by":"auto","created_at":"2023-06-29 14:43:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":181964,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of 16 TabZIP genes.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/20688b3b9ea1f9567c92e21b.png"},{"id":39304200,"identity":"e62e83aa-5ad4-493c-b71b-232071527037","added_by":"auto","created_at":"2023-06-29 14:51:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":505820,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of key starch synthase genes. DPA: days post anthesis.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/c03e2dd36813c74c0b069c1d.png"},{"id":39304201,"identity":"30ca10f8-ccd4-4d5b-8f7d-014de4f6f069","added_by":"auto","created_at":"2023-06-29 14:51:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":316547,"visible":true,"origin":"","legend":"\u003cp\u003eTabZIP68 subcellular localization analysis.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/03bc0e66f3ff7d96d860da0a.png"},{"id":39305486,"identity":"f9b54851-dfd9-4bbe-bd2c-8af13962e96b","added_by":"auto","created_at":"2023-06-29 14:59:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":153986,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the interaction between the TabZIP68 protein and the promoters of starch synthase genes using a luciferase assay\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/d2d5c08bf2700fda972d9c04.png"},{"id":50314032,"identity":"7a56da6c-5183-4e8b-a1b8-194bf9fc539d","added_by":"auto","created_at":"2024-01-29 15:28:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4312524,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/eb9fddfd-3e1e-4f9f-acee-4119e139eefd.pdf"},{"id":39305487,"identity":"61b65ea1-e8a9-4c8e-a81b-e8107e9e3f5c","added_by":"auto","created_at":"2023-06-29 14:59:26","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":248197,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 1 Phylogenetic classification of wheat TabZIP and Arabidopsis AtbZIP proteins.\u003c/p\u003e","description":"","filename":"FigS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/d6480d28e1b273f0a8d66f3b.jpg"},{"id":39303096,"identity":"605cd18b-3abe-4db2-ac2e-640438e41c3f","added_by":"auto","created_at":"2023-06-29 14:43:26","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":112957,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 2 Gene structures of 181 wheat TabZIP genes with full-length coding sequences.\u003c/p\u003e","description":"","filename":"FigS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/1a76c5abd6b6e28eb7e6b253.jpg"},{"id":39305485,"identity":"39ad2533-1aee-40ac-a926-6db9c6205ad3","added_by":"auto","created_at":"2023-06-29 14:59:26","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":282899,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 3 Conserved motifs of the 181 TabZIP genes.\u003c/p\u003e","description":"","filename":"Figs3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/f2d8735840eccfa0556c937e.jpg"},{"id":39303097,"identity":"d0eb805d-9bab-47f2-9a1a-63d71a5ad59f","added_by":"auto","created_at":"2023-06-29 14:43:26","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":26434,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary table 1 Basic information of 181 wheat bZIP family members. The coordinate position, gene length, Molecular weight and PI of each gene were analyzed\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/a4483542fb75ae0666f33e95.xlsx"},{"id":39303074,"identity":"cf52d7f6-5157-4760-9f01-513bb37152bb","added_by":"auto","created_at":"2023-06-29 14:43:26","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":19775,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary table 2 Comparison of TabZIP family genes in different versions.\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/06196581e81de899b861396a.xlsx"},{"id":39303098,"identity":"df5707cf-8bbd-41f1-b45a-c141de61c9ea","added_by":"auto","created_at":"2023-06-29 14:43:29","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":43485436,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary table 3 TabZIP genes at different developmental stages in eight wheat tissues.\u003c/p\u003e","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3045123/v1/3fd24b622dc1d62aa2b6a095.xlsx"}],"financialInterests":"","formattedTitle":"Genome-wide identification of the basic leucine zipper transcription factor genes related to starch synthesis in wheat (Triticum aestivum L.)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn plants, transcription factors (TFs) are vital for regulating transcription, as they bind to cis-acting elements of their intended target genes (Gonzalez \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jakoby et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Of these, the basic region/leucine zipper (bZIP) TFs have conserved domains and one of the largest gene families(Izawa et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). These were classified and named based on their conserved DNA-binding domains, which utilize a leucine zipper dimerization motif to bind to DNA (Hurst \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Wei et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The conserved basic region, which is responsible for sequence-specific DNA binding, comprises about 16 amino acid residues containing a conserved N-x7-R/K motif (Nijhawan et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In plants, bZIPs TFs exist mainly in the form homo- and heterodimers (Hurst \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) and bind preferentially to the A-box (TACGTA), C-box (GACGTC), and G-box (CACGTG), which have core ACGT sequences (Baloglu et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). With the development of whole genome sequencing technology, bZIP transcription factor family members have been comprehensively identified or predicted in many plants (Liao et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In Arabidopsis, the bZIP gene family is divided into 10 groups based on the sequence similarity of their basic regions and the presence of additional conserved motifs (Jakoby et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Many studies have identified members of the bZIP family in various species including 75 in Arabidopsis (Agarwal et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), 125 in maize (Jain et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), 187 in wheat (Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), 89 in rice (Liu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), 92 in barley (Zhong et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), 55 in grapevine (Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and 64 in cucumber (Xiao et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), but only a small number of genes from this family have been functionally characterized. This transcription factor family is associated with numerous developmental processes in plants (CAO et al. 2012), including but not limited to flower development (Gupta et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), seed maturation (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and responses to both biotic (Lim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)and abiotic factors (Wigge et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBread wheat (Triticum aestivum L.) is an inbreeding crop with distinctive evolutionary limitations, which results in comparatively low genetic diversity. Despite this, it is a significant global source of both carbohydrates and proteins. In 2018, the International Wheat Genome Sequencing Consortium (IWGSC) publicly disclosed the genome sequencing map of Chinese Spring, a six-row malting wheat originating from the United States, marking the first time the genome had been fully sequenced and made available. In 2020, Chinese Spring chromatin accessibility data using MNase are available in open access. In, 2021, the IWGSC RefSeq v2.1 assembly and annotation became available to download, BLAST and display in a browser. In 2022, a new version of RepetDB containing 54 species became available. The major features include 36 new genomes annotated using REPET v3.0, manual annotation tags, and old annotations were updated using PASTEClassifier v2. In previous studies, They identified 187 and 191 TabZIP members using the wheat genome (2018 version) (Agarwal et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStarch is an important carbohydrate that is an essential source of energy for humans. Crop quality is dependent upon the type and characteristics of starch. Starch biosynthesis is a complex process conducted by a series of starch synthetases. There is a well-organized system for starch synthesis in cereal, which includes three gene families including starch synthase, granule bound starch synthase (GBSS), starch synthase (SS), and ADP-glucose pyrophosphorylase (AGPase). While another gene family is starch branching enzymes (SBE) including several isomers (Preiss \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). First, sucrose is decomposed into fructose and UDP glucose in the endosperm cells by sucrose synthetase. Subsequently, G-1-P(1-phosphoglucose) is produced by UDp-GlC pyrophosphorylase. Thereafter, ADP-Glc pyrophosphorylase (AGPase) forms the direct substrate of starch synthesis, adenosine diphosphate glucose (ADPG). ADPG in the cytoplasm is transported to the powder by the adenylate transporter (BT1) on the powder membrane. Under the action of a series of starch synthetases, amylose and amylopectin are formed successively (Soliman et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The synthesis of amylose is completed under the action of GBSS, located inside starch granules, causing the synthesized amylose to remain unbranched (Sonnewald and Kossmann \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The synthesis of amylopectin is relatively complex, involving the interaction of three enzymes SS, SBE, and DBE (Guzm\u0026aacute;n and \u0026amp; Alvarez 2016; Liu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e; Preiss \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Also the isoamylase 1 (ISA1) is involved in amylopectin production via trimming glucan cluster (Huang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral studies have found OsbZIP58(Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and OsbZIP76 (Niu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) to be involved in starch regulation in rice. Studies have also found the following genes to be onvolved in maize starch synthesis: ZmNAC36, ZmNAC128, ZmNAC130 ZmbZIP22, ZmbZIP91, Opaque 2, ZmEREB156 (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Dong et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, TubZIP28 and TabZIP28 have been found to be transcriptional activators of starch synthesis in wheat (Liu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). One study showed that SUSIBA2 (sugar signaling in barley) from WRKY TF was identified only in barley whereas no evidence for it was found in rice and maize (Sun et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The products of SUS-IBA2 bind to the promoter of ISA1 and influence the starch synthesis by regulating gene expression in barley. Studies have been published, which showed various bZIP family members involved in starch synthesis are already indentified in rice, maize and wheat.\u003c/p\u003e \u003cp\u003eAnalysis of the bZIP family is necessary to screen the genes related to starch synthesis. We used recently published assembly of the Chinese Spring wheat genome. Compared to previous studies, the study focused on the phylogenetic relationships, structural features, chromosomal location, and gene expression pattern among bZIP genes. We analyzed the gene expression pattern of the TabZIP family across various developmental stages and tissues, focusing on their co-expression with starch synthase genes. Hence, this study showed new insight into TabZIP genes, which may play essential roles in the production of starch synthesis in wheat.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 BZIP transcription factor genes identificate in Chinese Spring wheat\u003c/h2\u003e \u003cp\u003eTo identify members of the bZIP gene family in the Chinese Spring genome, we conducted a search using URGI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://wheat-urgi.versailles.inra.fr/Tools\u003c/span\u003e\u003cspan address=\"http://wheat-urgi.versailles.inra.fr/Tools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To obtain genomic sequences, including DNA FASTA and GFF3 files, for Chinese Spring, we retrieved them from the URGI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.wheatgenome.org/News/Latest-news/IWGSC-RefSeq-v2.1-Assembly-and-Annotation-now-freely-available-at-URGI-and-NCBI\u003c/span\u003e\u003cspan address=\"https://www.wheatgenome.org/News/Latest-news/IWGSC-RefSeq-v2.1-Assembly-and-Annotation-now-freely-available-at-URGI-and-NCBI\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To process the biological data, we utilized TBtools, a toolkit for handling biological data (Chen et al. 2020), we conducted an initial search of the Chinese Spring wheat genomes from the PlantTFDB v5.0 database using BLAST (with an e-value cutoff of 1e\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://planttfdb.gao-lab.org/index.php\u003c/span\u003e\u003cspan address=\"http://planttfdb.gao-lab.org/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which includes protein sequence for bZIP TFs from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eTriticum aestivum L\u003c/em\u003e. We manually deleted redundant sequences. Based on the results from the first BLAST search using PlantTFDB v5.0 database, the protein sequences were extracted using TBtools and used as a query for the second BLAST search, which was performed using NCBI-BLASTP with an e-value threshold of 1e\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e. The non-bZIP members were deleted manually after downloading the alignment results. The conserved bZIP domain were analyzed between the two BLAST via NCBI Conserved Domain Database server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Marchler-Bauer et al. 2011). Protein search was performed to confirm that all the selected were bZIP TFs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Comparison of TabZIP genes identified in different wheat genome versions\u003c/h2\u003e \u003cp\u003eEarlier research (Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Pourabed et al. 2015), involved a search for and retrieval of sequences of TabZIP genes from the wheat genomic database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.gramene.org/\u003c/span\u003e\u003cspan address=\"http://www.gramene.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The bZIP gene identified in previous studies was used as a query sequence to perform a BLAST search of wheat V2 for comparison with previous results. Query the same sequence as having an Expect\u0026thinsp;=\u0026thinsp;0 value gene. Next, we used the Chinese Spring V2 genome to identifiy the bZIP genes for a BLAST search (e-value\u0026thinsp;\u0026le;\u0026thinsp;1e\u003csup\u003e10\u003c/sup\u003e) of the URGA HC_genes_CDS_ Seq_2020, LC_genes_CDS_Seq_2020, and full-length cDNA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://wheat-urgi.versailles.inra.fr/morgoth/Data\u003c/span\u003e\u003cspan address=\"http://wheat-urgi.versailles.inra.fr/morgoth/Data\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases. Differences in TabZIP family members between the old and new versions were determind using BLAST.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Analysis of the TabZIP gene structure and phylogenetic\u003c/h2\u003e \u003cp\u003eTBtools was used to compare the coding sequence of each TabZIP gene with its genome sequence in order to construct an exon/intron map. The ClustalW program implemented in MEGAX was used to compare all amino acid sequences (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.megasoftware.net/\u003c/span\u003e\u003cspan address=\"http://www.megasoftware.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to determine the evolutionary relationship between wheat and Athaliana bZIP proteins. The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replications, based on the JTT matrix-based model, and the amino acid sequences were aligned using the ClustalW program implemented in MEGAX.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Localization Conserved motif analysis and conserved motif analysis of bZIP genes on Chinese Spring chromosomes\u003c/h2\u003e \u003cp\u003eThe motif analysis of the TabZIP protein sequences was conducted using the online software MEME 5.1.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/tools/meme\u003c/span\u003e\u003cspan address=\"http://meme-suite.org/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and we selected these parameters: number of repetitions-any, maximum number of motifs\u0026thinsp;=\u0026thinsp;20, and optimum motif width\u0026thinsp;\u0026ge;\u0026thinsp;6 and \u0026le;\u0026thinsp;200 (Ma et al. 2017). Chen et al was followed to analyze the chormosomal location using protein sequences against the genome annotation GFF3 file in TBtools (Chen et al. 2020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Expression analysis\u003c/h2\u003e \u003cp\u003eWe used the transcriptome database for the Wheat Reference Transcript (IWGSC Annotation v1.1) dataset (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://202.194.139.32/expression/wheat.html\u003c/span\u003e\u003cspan address=\"http://202.194.139.32/expression/wheat.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to compare all CDS sequences for the TabZIP genes. The RNA-seq data of 13 tissues including root (10 cm seedlings), root 2 (4 week seedling), shoot (10 cm seedlings), rachis (5 weeks pa), senescing leaf(2months), tillers (3rd internode), inflorescence-1 (0.5 cm), inflorescence-2 (1\u0026ndash;1.5 cm), embryo (germinating), palea (6 weeks pa), epidermis (4 weeks), grain (5 DPA), and grain (15 days postanthesis [DPA]) of Chinese Spring were retrieved from The James Hutton Institute (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.wheat-expression.com/\u003c/span\u003e\u003cspan address=\"http://www.wheat-expression.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the log\u003csup\u003e2\u003c/sup\u003e transcripts per million value of each bZIP gene was visualized as a heatmap with a blue-yellow-red gradient.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Plant growth, RNA extraction, and quantitative reverse transcription PCR analysis\u003c/h2\u003e \u003cp\u003eThe expression level of wheat starch synthase genes was measured. The gene expression of RNA-seq was verified by using Quantitative reverse transcription PCR (qRT-PCR) and also TabZIP genes were screened. The experiment was conducted using Chinese Spring wheat under the following growth conditions: 16 hours of light and 8 hours of dark with day and night temperatures of 24\u0026deg;C and 18\u0026deg;C, respectively. The grain samples were collected at different developmental stages of 5, 10, 15, 20, 25, and 30 DPA and these were transferred to -80\u0026deg;C from the liquid nitrogen. Total RNA was extracted using an RNA kit following the manufacturer's protocol (Biofit, Chengdu, China). For each developmental stage, three replications were tested. First-strand cDNA was synthesized using PrimeScript\u0026trade; RT reagent kits with gDNA Eraser (TaKaRa, Dalian, China) and qRT-PCR was performed using SYBRVR Premix Ex Taq\u0026trade; II (TaKaRa) on a CFX 96 Real-Time System (Bio-Rad, Hercules, USA). We analyzed and calculated the expression of the qRT-PCR data using CFX Manager Software (Bio-Rad, Hercules, USA). The expression level calculated using the 2 Ct method. Wheat-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes were used as internal reference genes to normalize the expression of the candidate genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Dual-luciferase reporter (DLR) assay\u003c/h2\u003e \u003cp\u003eA 1.5 kb portion of the promoter sequence from \u003cem\u003eTaAGP-S\u003c/em\u003e, \u003cem\u003eTaAGP-L\u003c/em\u003e, \u003cem\u003eTaSS1\u003c/em\u003e, \u003cem\u003eTaSS2a\u003c/em\u003e, \u003cem\u003eTaWaxy\u003c/em\u003e, \u003cem\u003eTaSBE2a\u003c/em\u003e, and \u003cem\u003eTaSBE1\u003c/em\u003e was cloned into the pGreenII 0800-Luc vector to create promoter firefly luciferase reporter constructs. pGreenII 62-SK-TabZIP68 was created by cloning the TabZIP68 CDS into the pGreenII 62-SK vector and was used as the effector vector. Both vectors were co-expressed in tobacco. Subsequently, we cultured the co-transfected tobacco overnight in the dark. The DLR assay system (Promega, USA) was used to measure LUC and REN activities. All experiments were independently repeated six times, and each assay had three technical replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Subcellular localization analysis\u003c/h2\u003e \u003cp\u003eFor the analysis we ligated the TabZIP68 without termination codons into pJIT163 vector and then we transformed the A. tumefaciens strain GV3101 with pJIT163-GFP, pJIT163-TabZIP68-GFP, and plasmids. By following the Cao et al., maize protoplasts of the leaf sheaths of 15-day-old wheat seedlings were used in transforming the vectors to express fused GFP. A confocal laser-scanning microscope was used to monitor GFP flourescence (Zeiss LSM 800, Jena, Germany).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Identification of bZIP genes in Chinese Spring\u003c/h2\u003e \u003cp\u003eThe characterization of members of the TabZIP family can be more precise by utilizing the recently released Chinese Spring genome data from URGI in comparison to the previous version (Agarwal et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We identified 181 nonredundant \u003cem\u003eTabZIP\u003c/em\u003e genes in Chinese Spring wheat based on a genome-wide search and the validation results. As there is no established standard nomenclature, each gene in the dataset was assigned a generic name based on the order of its original sequence ID (\u003cem\u003eTabZIP1-TabZIP181\u003c/em\u003e). Sequence analyses showed that all the \u003cem\u003eTabZIP\u003c/em\u003e genes have the bZIP domain, and the amino acid (aa) length of these TabZIP genes ranged from 129 (\u003cem\u003eTabZIP58\u003c/em\u003e) to 652 (\u003cem\u003eTabZIP138\u003c/em\u003e), with an average size of 329 amino acids, protein mass between 14.11 (\u003cem\u003eTabZIP58\u003c/em\u003e) and 68.90 Kd (\u003cem\u003eTabZIP138\u003c/em\u003e), and pI ranging from 4.85 (\u003cem\u003eTabZIP134\u003c/em\u003e) to 11.63 (\u003cem\u003eTabZIP82\u003c/em\u003e). A subcellular localization analysis predicted that most genes are expressed in the nucleus, whereas \u003cem\u003eTabZIP53\u003c/em\u003e, \u003cem\u003eTabZIP54\u003c/em\u003e, \u003cem\u003eTabZIP55\u003c/em\u003e, \u003cem\u003eTabZIP70\u003c/em\u003e, \u003cem\u003eTabZIP101\u003c/em\u003e, \u003cem\u003eTabZIP120\u003c/em\u003e, \u003cem\u003eTabZIP122\u003c/em\u003e, and \u003cem\u003eTabZIP123\u003c/em\u003e are expressed in the nucleus and chloroplasts; however five genes (\u003cem\u003eTabZIP8\u003c/em\u003e, \u003cem\u003eTabZIP9\u003c/em\u003e, \u003cem\u003eTabZIP11\u003c/em\u003e, \u003cem\u003eTabZIP12\u003c/em\u003e, and \u003cem\u003eTabZIP13\u003c/em\u003e) did not have a predicted location (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.2 Identified the\u003c/b\u003e \u003cb\u003eTabZIP\u003c/b\u003e \u003cb\u003egenes in different wheat genome versions to comparison\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eA comparative analysis of different versions of \u003cem\u003eTabZIP\u003c/em\u003e genes in old studies (Agarwal et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) was used to correct duplicates of one gene into separate genes, correcting family size and misnamed genes. In total, According to this resulets in the wheat genome were identified 187 and 192 \u003cem\u003eTabZIP\u003c/em\u003e family genes. We further validated our results by comparing the TabZIP gene identified in the Chinese Spring V2 genome with previously identified TabZIP genes (Tables S1 and S2 ). 70 different TabZIP genes were identified in the Chinese Spring V2.1 genome, whereas 32 of the old bZIP genes did not match with the new version. In many cases, one gene in the old version corresponded to two genes in the new version. \u003cem\u003eTraes_3AL_FC5523394.2\u003c/em\u003e corresponded to the genes, \u003cem\u003eTabZIP1\u003c/em\u003e and \u003cem\u003eTabZIP5\u003c/em\u003e, \u003cem\u003eTraes_5DL_67A38E9B7.1\u003c/em\u003e corresponded to the \u003cem\u003eTabZIP2\u003c/em\u003e and \u003cem\u003eTabZIP73\u003c/em\u003e, the \u003cem\u003eTraes_5BL_BBFA6D506.1\u003c/em\u003e is corresponds to the \u003cem\u003eTabZIP3\u003c/em\u003e and \u003cem\u003eTabZIP122\u003c/em\u003e. The multiple sequence alignment results (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) showed that the 2012 genome may be caused by incorrect splicing of this phenomenon. Compared with previous studies, the number of \u003cem\u003eTabZIP\u003c/em\u003e genes identified from Chinese Spring V2.1 was more accurate and the sequence was more complete.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Phylogenetic analysis of TabZIP genes\u003c/h2\u003e \u003cp\u003eThe bZIP family genes in Arabidopsis genome have been well classified according to the structural characteristics of their protein sequences (Jakoby et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). TabZIP was distributed in Groups A, B, C, D, E, F, G, H, I, and S, as well as four unknown groups (Groups U1, U2, U3, and UH). Groups A, D, I, and S had more member genes than the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), containing 17, 15, 14, and 27 \u003cem\u003eTabZIP\u003c/em\u003e genes, respectively. The other genes were divided into nine groups of varying numbers, with group B and U2 being the two smallest, each containing six genes. A phylogenetic tree analysis revealed that one member of Group H and U, and two members of Group A were not clustered together in Arabidopsis. Three subgroups were found in the Group S by members of Group C and G. One study also presented similar results by phylogenetic analysis of the wheat and barley bZIP family (Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhong et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We hypothesized that gene classification of Arabidopsis was based on protein sequence therefore, this study analyzed the basis of the amino acid alignments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Gene and protein structural features of the wheat bZIP genes\u003c/h2\u003e \u003cp\u003eThe gene structure characteristics of 181 bZIP family genes identified in this study were analyzed using gene annotation information. Of the 181 genes, only 25 did not contain introns, while the remaining 156 contained between 1 and 12 introns (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). According to the results of evolutionary analysis results, most of the genes in the same group had a similar gene structure, length, and distribution of introns and exons. Ten conserved motifs were set to identify TabZIP family members, among which motif 1 was the core motif of bZIP and was shared by all family members (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The number of motifs in different groups was different, and the maximum number for the S group was 9 (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). There was only one motif in \u003cem\u003eTabZIP80\u003c/em\u003e, \u003cem\u003eTabZIP81\u003c/em\u003e, \u003cem\u003eTabZIP90\u003c/em\u003e, and \u003cem\u003eTabZIP91\u003c/em\u003e. All bZIP family genes were extracted from wheat genome with high confidence protein sequences. A chromosomal distribution analysis revealed that bZIP family factors were evenly distributed on each chromosome and the maximum number of genes on chromosome 5A was 15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Expression profiles of \u003cem\u003eTabZIP\u003c/em\u003e genes during growth and development\u003c/h2\u003e \u003cp\u003eTo determine test whether the identified bZIP family genes are normally expressed in wheat, we used transcriptomic data the wheat multiomics website (WheatOmics 1.0). The results showed that all the 181 genes had corresponding transcripts, indicating that the identified genes had normal function and were true genes. Using published RNA-seq database data, we further analyzed the spatial and temporal expression patterns of bZIP family genes and examined their expression profiles in different tissues (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). \u003cem\u003eTabZIP8\u003c/em\u003e, \u003cem\u003eTabZIP25\u003c/em\u003e, \u003cem\u003eTabZIP101\u003c/em\u003e, \u003cem\u003eTabZIP130\u003c/em\u003e, and \u003cem\u003eTabZIP132\u003c/em\u003e were expressed throughout development, but at low levels(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). During grain development, 52 genes were highly expressed, of which 32 were endosperm specific. We screened out some genes that were highly expressed during the grain development stages (5\u0026ndash;25 DPA) and conducted real-time qPCR for further verification (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We also performed the same analysis on the genes encoding enzymes related to starch synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), including \u003cem\u003eTaAGP-L\u003c/em\u003e, \u003cem\u003eTaAGP-S\u003c/em\u003e, \u003cem\u003eTaWaxy\u003c/em\u003e, \u003cem\u003eTaISA1\u003c/em\u003e, \u003cem\u003eTaSS1\u003c/em\u003e, \u003cem\u003eTaSS2a\u003c/em\u003e, \u003cem\u003eTaSS3\u003c/em\u003e, \u003cem\u003eTaSBE2a\u003c/em\u003e, and \u003cem\u003eTaSBE2b\u003c/em\u003e. Most of the selected genes expressed similar starch synthetase genes, but \u003cem\u003eTabZIP13\u003c/em\u003e, \u003cem\u003eTabZIP41\u003c/em\u003e, \u003cem\u003eTabZIP69\u003c/em\u003e, \u003cem\u003eTabZIP148\u003c/em\u003e, and \u003cem\u003eTabZIP164\u003c/em\u003e were different from the starch synthetase genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 \u003cem\u003eTabZIP68\u003c/em\u003e bound specifically to the promoters of \u003cem\u003eTaWx\u003c/em\u003e in DLR\u003c/h2\u003e \u003cp\u003eWe used the DLR system to verify whether major starch synthetase genes interact with candidate TFs. According to the NCBI I-BLAST search results, \u003cem\u003eTabZIP68\u003c/em\u003e was a homologous gene with \u003cem\u003eO2\u003c/em\u003e, a transcription factor reported to regulate starch synthesis in maize (Zhang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In rice, \u003cem\u003eTabZIP68\u003c/em\u003e is homologous to the \u003cem\u003eOsbZIP58\u003c/em\u003e gene, which directly interacts with the promoter of six starch synthase genes and regulates their expression (Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, the regulatory mechanism of \u003cem\u003eTabZIP68\u003c/em\u003e on grain starch synthesis in wheat has not been studied. The DLR results indicate that \u003cem\u003eTabZIP68\u003c/em\u003e binds to the cis-acting DNA element in the promoter region of starch synthetase \u003cem\u003eWaxy\u003c/em\u003e gene. These results suggested that \u003cem\u003eTabZIP68\u003c/em\u003e might be involved in regulating the synthesis of wheat grain starch (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 \u003cem\u003eTabZIP68\u003c/em\u003e subcellular localization analysis\u003c/h2\u003e \u003cp\u003eWe constructed the vector into TabZIP68-GFP and extracted wheat leaf protoplasts to locate the gene products. There was a strong GFP signal in the nuclei and plasma membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). On the other hand, the unfused GFP control exhibited GFP signals that were observed in various cellular compartments, including the plasma membrane, cytoplasm, and nuclei.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe accumulation of starch in wheat endosperm involves a complex mechanism of various starch synthesizing genes. Upto now many TFs are known for their specific roles in starch synthesis. Especially, rice, maize and wheat are those plant species in which bZIP family is identified. However, it is necessary to identify and analyze the TabZIP family genes to create valuable gene resources for starch synthesis. Among the studies on TabZIP, 192 TabZIP genes were identified in wheat (Agarwal et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Subsequently, it was discovered that an earleir version of the wheat genome was used in these studies (Lim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and gaps were identified in this old version. Therefore, some important genes were considered missing in that version. The updated version of the Chinese Spring wheat genome data URGI (V2.1) includes the updated information regarding annotation and also have much more information than earlier V1. Therefore, V2.1 was used in this study, as it contains more precise information for characterizing of the TabZIP gene family. The present study identified 181 TabZIP genes in wheat. Although there were 11 fewer genes compared with the previous version, many misjudgments caused by incorrect splicing and assembly using the old version were found identified. Of these, 31 bZIP genes in the old version were not found to correspond with the gene in the V2.1 version, and there was one gene in the old version for every two genes in the new version. For example, \u003cem\u003eTraes_3AL_FC5523394.2\u003c/em\u003e is corresponded to the gene \u003cem\u003eTabZIP1\u003c/em\u003e and \u003cem\u003eTabZIP5\u003c/em\u003e genes, \u003cem\u003eTraes_5DL_67A38E9B7.1\u003c/em\u003e is corresponded to the \u003cem\u003eTabZIP2\u003c/em\u003e and \u003cem\u003eTabZIP73\u003c/em\u003e, the \u003cem\u003eTraes_5BL_BBFA6D506.1\u003c/em\u003e is corresponds to \u003cem\u003eTabZIP3\u003c/em\u003e and \u003cem\u003eTabZIP122\u003c/em\u003e. These findings demonstrate that the new version has more complete gene splicing and detailed annotation information compared with the old version, and our study can provide more accurate and detailed analysis for the bZIP genes in wheat.\u003c/p\u003e \u003cp\u003eThe diversity of bZIP genes was identified in the Arabidopsis genome, which represents flowering plants (Jakoby et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The AtbZIP are classified into 10 groups on the basis of their structural and functional characterization. This classification system has been applied to various plants and has become the classification standard for members of the bZIP family. In previous studies, the conserved arginine in bZIP gene of Group U was replaced by a hydrophobic isoleucine in the DNA binding of the bZIP gene, which may affect DNA-binding characteristics. However, we did not find any gene that was highly expressed in all tissues through RNA-seq database. Our results indicate that TabZIP family genes are expressed in at least one tissue in wheat, showing tissue-specific differential expression. Thirty-two of these genes were specifically expressed in grain, suggesting that these genes may be involved in grain development and the accumulation of storage materials.\u003c/p\u003e \u003cp\u003eStudies have reported the regulation of the gene expression of grain starch synthesis by bZIP family TFs. In rice, \u003cem\u003eOsbZIP20\u003c/em\u003e combines with starch synthase gene promoters Waxy and SBE1 to regulate grain starch content (Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The \u003cem\u003eTubZIP28\u003c/em\u003e and \u003cem\u003eTabZIP28\u003c/em\u003e are those genes which binds to promoter region of AGPase and enhances their transcriptional level in wheat. In maize, O2 regulates the expression of α and β-zein genes mainly by recognizing the O2 box in the α and β-zein promoter (Holdsworth et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), and O2 can also interact with other TFs or proteins to regulate starch synthesis in the grain (Zhang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It is stated in previous study Luo et al, that the key enzyme responsible for amylose synthesis is granule-bound starch synthase I (GBSSI) (Luo et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Previous studies have shown that TabZIP68 and O2 are homologous genes and that O2 can bind multiple starch synthetase gene promoters to regulate grain starch synthesis. Using DLR, we demonstrated that TabZIP68 interacts with the starch synthetase gene promoter Waxy and theat the TabZIP68 gene may be a transcriptional regulatory factor for wheat starch synthesis. It is worth further study.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJD was involved in retrieving the data, expression analysis and writing of manuscript, HK, XL and GC performed qRT-PCR, yeast one hybrid assay, and data analysis, respectively. WH helped in correction of expression data. HP and HX were involved in material planting. QJ designed and supervised the experiments. YZ, QX, YF, PQ, YJ, MD, and JM participated to improve the research design. JW, GY, YM and YLZ helped to revise manuscript. All of the authors are agreed to final version of manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received funding from the Sichuan Science and Technology Program of PR China (2022ZDZX0014 and 2022YFH0055), as well as the International Science \u0026amp; Technology Cooperation project of Chengdu, Sichuan Province, PR China (2019-GH02-00078-HZ).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that we do not have any conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLinking the International Wheat Genome Sequencing Consortium bread wheat reference genome sequence to wheat genetic and phenomic data\u003c/li\u003e\n\u003cli\u003eAgarwal P, Baranwal VK, Khurana P (2019) Genome-wide analysis of bZIP transcription factors in wheat and functional characterization of a TabZIP under abiotic stress. 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Proceedings of the National Academy of Sciences 113:10842-10847\u003c/li\u003e\n\u003cli\u003eZhong X, Feng X, Li Y, Guzm\u0026aacute;n C, Lin N, Xu Q, Zhang Y, Yang H, Qi P, Deng M, Ma J, Wang J, Chen G, Lan X, Wei Y, Zheng Y, \u0026amp; Jiang Q (2021) Genome-wide identification of bZIP transcription factor genes related to starch synthesis in barley (Hordeum vulgare L.). . Genome 64:1067\u0026ndash;1080\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Wheat, Basic leucine zipper, Transcription factors, Gene interaction, Starch synthesis","lastPublishedDoi":"10.21203/rs.3.rs-3045123/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3045123/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe growth and development of plants are dependent upon the transcription factors. In plants, the basic leucine zipper (bZIP) family of transcription factor is known for its largest size and diversity. Using newly available wheat genome data, we compared our identification of 181 \u003cem\u003eTriticum aestivum\u003c/em\u003e bZIP (TabZIP) genes to those reported in earlier studies. One gene was characterized, and some duplicate genes and incorrect annotations in previous studies are supplemented and corrected. The 181 bZIP genes identified were divided into 13 known groups and 5 unknown groups by phylogenetic analysis. All of the bZIP genes exhibit a minimum of one bZIP motif in their motif distribution and gene structure. Spatial and temporal expression patterns of bZIP family members during various stages of plant growth vary, as suggested by transcriptome data.\u003c/p\u003e\n\u003cp\u003eAs per the expression data obtained via quantitative reverse transcription PCR (qRT-PCR), over 10 TabZIP genes showed similarity with starch synthesis in wheat. In vitro binding activity of TabZIP68 to the promoter of TaWaxy was demonstrated by a dual-luciferase reporter (LUC) assay. Given its potential involvement in starch synthesis, the \u003cem\u003eTabZIP68\u003c/em\u003egene presents itself as a strong candidate for further investigation.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification of the basic leucine zipper transcription factor genes related to starch synthesis in wheat (Triticum aestivum L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-29 14:43:21","doi":"10.21203/rs.3.rs-3045123/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-07-16T11:15:27+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-06-24T11:36:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Growth Regulation","date":"2023-06-15T03:46:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-06-14T17:40:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Growth Regulation","date":"2023-06-13T09:53:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f2da7906-d13a-4635-91a4-23d00b818cd6","owner":[],"postedDate":"June 29th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-01-29T15:25:00+00:00","versionOfRecord":{"articleIdentity":"rs-3045123","link":"https://doi.org/10.1007/s10725-023-01111-z","journal":{"identity":"plant-growth-regulation","isVorOnly":false,"title":"Plant Growth Regulation"},"publishedOn":"2024-01-26 15:13:21","publishedOnDateReadable":"January 26th, 2024"},"versionCreatedAt":"2023-06-29 14:43:21","video":"","vorDoi":"10.1007/s10725-023-01111-z","vorDoiUrl":"https://doi.org/10.1007/s10725-023-01111-z","workflowStages":[]},"version":"v1","identity":"rs-3045123","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3045123","identity":"rs-3045123","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}