Genome-wide identification and analyses of ZmAPY  genes reveal their roles involved in maize development and abiotic stress responses

Genome-wide identification and analyses of ZmAPY genes reveal their roles involved in maize development and abiotic stress responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide identification and analyses of ZmAPY genes reveal their roles involved in maize development and abiotic stress responses Zhenghua He, jie Zhang, Haitao Jia, Shilong Zhang, Xiaopeng Sun, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4222821/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Apr, 2024 Read the published version in Molecular Breeding → Version 1 posted 4 You are reading this latest preprint version Abstract Apyrase is a class of enzyme that catalyzes the hydrolysis of nucleoside triphosphates/diphosphates (NTP/NDP), which widely involved in regulation of plant growth and stress responses. However, apyrase family genes in maize have not been identified, and their characteristics and functions are largely unknown. In this study, we identified 16 apyrases (named as ZmAPY-ZmAPY16 ) in maize genome, and analyzed their phylogenetic relationships, gene structures, chromosomal distribution, upstream regulatory transcription factors and expression patterns. Analysis of the transcriptome database unveiled tissue-specific and abiotic stress-responsive expression of ZmAPY genes in maize. qPCR analysis further confirmed their responsiveness to drought, heat, and cold stresses. Association analyses indicated that variations of ZmAPY genes may regulate maize agronomic traits and drought responses. Our findings shed light on the molecular characteristics and evolutionary history of maize apyrase genes, highlighting their roles in various biological processes and stress responses. This study forms a basis for further exploration of apyrase functions in maize. maize apyrase abiotic stress response association analysis metabolic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Apyrase (APY) enzymes, classified as nucleoside triphosphate (NTP) diphosphohydrolases, belong to the superfamily of guanosine diphosphatase 1 (GDA1)-cluster of differentiation 39 (CD39) nucleoside phosphatase. These enzymes have the capability to remove the terminal phosphate from nucleoside triphosphates (NTPs) and nucleoside diphosphates (NDPs) but not from nucleoside monophosphates (NMPs). Based on their subcellular localization, APYs can be broadly categorized into ecto-apyrases and endo-apyrases (Hideaki et al. 1984 ; Tong et al. 1993 ; Thomas et al. 1999 ). ecto-apyrase are located on the cell surface, whereas endo-apyrase are usually located in the endoplasmic reticulum, Golgi and intracellular vesicles (Leal et al. 2005 ). Some ecto-apyrase possess transmembrane domains at their N- and C-terminals, which are crucial for correct protein folding, membrane targeting, cellular allocation and enzyme activity (Wu et al. 2005 ; Knowles 2011 ). The cellular ATP level serves a dual role in providing energy and regulating various cellular processes associated with responses to abiotic stress (Sun et al. 2012 ). Therefore, the maintenance of cellular ATP homeostasis, regulated in part by apyrases, is essential for preserving normal cell function. Apyrases are evolutionarily highly conserved (Clark et al. 2014 ). Their presence in plants was initially identified in potatoes several decades ago (Hideaki et al. 1984 ). Subsequently, members of the APY family were discovered in the genomes of various plants, including potato (Handa and Guidotti, 1996 ; Riewe et al. 2008 ), wheat (Liu et al. 2019 ), soybean (Day et al. 2000 ), Arabidopsis (Yang et al. 2013 ), cotton (Clark et al. 2010 ), rice (Chowdhury et al. 2023 ), and peanut (Sharif et al. 2023 ). Transcriptome analysis has revealed specific spatio-temporal expression patterns of APY genes and regulation of these genes by biological and abiotic stresses (Liu et al. 2019 ; Chowdhury et al. 2023 ; Clark et al. 2024 ), indicating potential roles in plant growth, development, and stress responses. Further studies have elucidated the functions of APY genes in plant growth and development. Arabidopsis APY genes, APY1 and APY2 , are predominantly expressed in rapidly growing tissues or those with high auxin levels. Suppression of their expression affects root and shoot growth (Wu et al. 2007 ). Additionally, APY1 / APY2 also participate in the phytochrome-mediated signaling pathway that induces differential growth changes in etiolated seedling tissues (Weeraratne et al. 2022 ). Another member of the APY gene family, APY7 , acts as a negative regulator of cell growth. APY7 modulates the growth-inhibiting effects of RALF1 (Rapid ALkalinization Factor), influencing cell wall architecture, composition, and alters the pH of the extracellular matrix (Gupta et al. 2024 ). Constitutive expression of pea ectoapyrase, psNTP9, in Arabidopsis and soybeans leads to a more extensive root system architecture (RSA) (Veerappa et al. 2019 ). Furthermore, the catalytic activity of GS52 ecto-apyrase is crucial for the early infection process of B. japonicum , nodule primordium development initiation, and subsequent nodule organogenesis in soybean (Govindarajulu et al. 2009 ). In addition to participating in the regulation of plant growth and development, the APY gene also plays a crucial role in stress response (Clark et al. 2021 ). Overexpression of the pea APY gene psNTP9 significantly enhances drought resistance and field yield in soybean under both normal watering and drought conditions (Veerappa et al. 2019 ). Populus euphratica APY genes, PeAPY1 and PeAPY2 , enhance drought tolerance by modulating stomatal aperture in Arabidopsis (Zhang et al. 2021b ). Furthermore, overexpression of PeAPY2 improves cold resistance by modulating vesicular trafficking and extracellular ATP in Arabidopsis (Deng et al. 2015 ). Proteome analysis of soybean roots under waterlogging has demonstrated that APY responded to waterlogging stress in soybean (Alam et al. 2010 ). These findings underscore the multifaceted roles of the APY genes in plant stress responses, highlighting its potential as a key player in enhancing plant resilience to various environmental challenges. Maize, a primary global food crop, faces significant threats from abiotic stresses. Identifying additional abiotic stress resistance genes and further understanding response system in maize remain crucial. While APY genes has been reported to be involved in various abiotic stresses in Arabidopsis , their presence and biological functions in maize have not been reported. This study aims to characterize APY genes and elucidate their roles. Here, 16 APY family members were identified in maize genome by phylogenetic analysis (Fig. 1 ; Table S1). APY family expansion patterns and genetic characteristics were analyzed comprehensively (Fig. 1 ; Fig. 2 ; Table S2). In addition, potential upstream regulators of ZmAPYs were predicted (Fig. 4 ). Transcriptome analysis revealed tissue-specific and abiotic stress-responsive expression of ZmAPY genes in maize (Fig. 3 ; Fig. 5 ). Association analysis suggested that variations in ZmAPY genes could impact maize agronomic traits and drought responses (Fig. 6 ; Fig. 7 ). These findings establish a basis for further investigations into the functions of maize APY genes. Materials and methods Plant materials and growth condition The maize inbred line B73 was used in this study. Maize seeds are germinated on moist filter paper in a plant growth chamber at 28°C for 2–3 days. Uniformly germinated seeds are then transplanted into soil-filled pots (10 cm×10 cm×9 cm) and grown in the chamber until the three-leaf stage for subsequent stress treatments. For drought stress, water is withheld from three-leaf stage maize seedlings for 10 days, while the control group is watered normally. Leaf samples are collected for RNA extraction to analyze ZmAPY genes expression levels. For heat and cold treatments, three-leaf stage maize seedlings were placed in growth chambers at 50°C and 4°C for 4 hours and 24 hours, respectively, while the control group continues growing at 28°C. Leaf samples are then collected for RNA extraction to evaluate ZmAPY genes expression level. Identification and Phylogenetic analysis of the ZmAPY genes in maize Gene ID of all APY genes in Arabidopsis thaliana and Oryza sativa was collected previous study (Chowdhury et al. 2023 ), protein sequences of these APYs were downloaded from Ensemble Plants ( http://plants.ensembl.org/ ) and aligned for constructing HMM model. Protein sequences for maize ( Zea mays ) were also collected from Ensemble Plants, and ZmAPY genes were identified using HMMsearch with default parameters. Proteins identified by HMMsearch that lacked conserved domains were removed after submission to SMART ( https://smart.embl.de/ ). Subsequently, MEGA X was used for multiple sequence alignment and phylogenetic tree construction of the identified 18 protein sequences with those from rice and Arabidopsis thaliana's APY proteins. The tree was built using Neighbor-Joining (NJ) method, pair-wise deletion, and bootstrap value set to 1000. As a result, 16 genes were identified as ZmAPY genes based on the evolutionary tree and named as ZmAPY1 - ZmAPY16 , respectively. The Synteny analysis of ZmAPY genes was employed by MCScanX by using default parameter. Prediction of physicochemical properties of ZmAPY proteins To obtain data such as the number of amino acids, molecular weight, isoelectric point, and average hydrophobicity index of the ZmAPY proteins, a physicochemical property analysis of ZmAPY protein sequences was employed by using an online tool ProtParam on ExPASy ( https://web.expasy.org/protparam/ ). Gene structure analysis was performed by GSDS2.0 ( https://gsds.gao-lab.org/ ). The subcellular localization of ZmAPY proteins were predicted by WoLF PSORT ( https://wolfpsort.hgc.jp/ ) and CELLO ( http://cello.life.nctu.edu.tw/ ). Expression analysis of ZmAPY genes in different tissues The expression data for the ZmAPY genes were obtained from various tissues, including seedling, roots, leaves, seeds, shoot apical meristems, internodes, tassel, cob, coleoptite, pericarp and anthers. These data were sourced from the qTeller ( https://qteller.maizegdb.org/ ). The FPKM values of the ZmAPY genes were used to draw a heat map by using HEMI (Deng et al. 2014 ). Upstream transcription factor prediction of ZmAPY genes The 2kb upstream sequences of each ZmAPY gene were obtained from the maize genome for analysis. The PlantTFdb ( https://planttfdb.gao-lab.org/ ) and jbrowse on maizeGDB ( https://jbrowse.maizegdb.org/ ) were used to analyze the retrieved sequences and identify. All predicted upstream transcription factors were visualized by using Gephi ( https://gephi.org/ ). Then expression correlation coefficients between potential upstream transcription factors and ZmAPY genes were calculated by using the expression profiles collected from qTeller. Analysis of ZmAPY genes expression in maize under abiotic stresses. Expression profiles data of ZmAPY genes under well-water and drought treatment were collected from qTeller and previous study (Zhang et al. 2019 ). The FPKM / TPM values of the ZmAPY genes were used to draw a heat map by using HEMI. For qPCR validation, the FastPure Universal Plant Total RNA Isolation kit (Vazyme) was employed for RNA extraction. HiScript III qRT SuperMix for qPCR (Vazyme) was used for reverse transcription to synthesize cDNA. Specifc primers were designed for the 16 ZmAPY genes and utilized for qRT-PCR analysis. The qRT-PCR reactions were performed using the SYBR Green master mix (ChamQ SYBR qPCR Master Mix, Vazyme). Three independent replicates were performed for each treatment. The primers used are described in Table S4. Association analysis of ZmAPY genes Association analysis for ZmAPYs was performed by using a maize association mapping population containing 540 inbred lines in a previous study (Liu et al. 2017). Among 1,227,480 high-quality SNP data with minor allele frequency (MAF) ≥ 0.05, 502 SNPs were found in the gene region of all 16 ZmAPYs . maize agronomic traits and drought tolerance trait were also collected from previous studies (Yang et al. 2014 ). The MLM model were chosen to detect the SNPs significantly associated with maize agronomic traits and drought tolerance by using the TASSEL5.0 program (Bradbury et al. 2007). Results Identification of maize APY genes Totally, we identified 16 ZmAPY family members from the Ensembl Plant database ( https://plants.ensembl.org/index.html ), named ZmAPY1 - ZmAPY16 (Table S1). Their physicochemical properties, including gene ID, protein size, molecular weight (MW), isoelectric point (pI), the grand averages of hydropathicity (GRAVY), instability index and and localization prediction, were characterized and shown in Table S1. The ZmAPY proteins varied in length from 81 to 701 amino acids, with molecular weights ranging from 8.74822 kDa to 76.9779 kDa. Isoelectric points ranged from 4.70 to 11.62 acidic. Most ZmAPY proteins were hydrophilic (GRAVY 0). The estimated instability index ranged from 30.91 to 72.05. Subcellular localization predictions indicated that nine ZmAPY proteins were likely localized in the chloroplast, four in the plasma membrane, three in the mitochondria, one in the extracellular space, and one in the nucleus (Table S1), indicating diverse functional roles for these ZmAPY genes. To explore the phylogenetic relationships of ZmAPYs with other species, a phylogenetic tree was constructed, incorporating 16 ZmAPYs, 7 AtAPYs, and 9 OsAPYs (Fig. 1 A; Table S2). The phylogenetic tree topology classified these APY proteins into three Groups: Group I, II, III. In addition, there are two APY-like proteins (ZmAPYL1, ZmAPYL2) in maize that do not belong to Group I-III. Therefore, we classify them as an outgroup (Fig. 1 A, Table S2). The expansion of APYs in maize, compared to AtAPYs and OsAPYs, suggests the potential importance of this gene family to regulate biological processes. Distribution and collinearity analysis of maize APY genes To investigate features of the ZmAPYs gene family, we analyzed the chromosome distribution of each ZmAPY gene. Our investigation showed that the ZmAPY genes in the maize genome were unevenly distributed across all 9 chromosomes, with the exception of chromosome 8 (Fig. 1 B). The number of APY genes varied on each chromosome. Specifically, there is a single APY gene located on chromosomes Chr5, Chr6, Chr7, and Chr9. Chromosomes Chr2, Chr3, and Chr10 each contain two APY genes, while chromosomes Chr1 and Chr4 each have three APY genes. Gene duplications play a crucial role in the expansion of gene families (Konrad et al. 2011 ). Segmental duplications lead to the presence of large repetitive chromosomal blocks in the genome and are often associated with chromosomal rearrangements and polyploid events (Lallemand et al., 2020 ). Colinearity analysis indicated the occurrence of three segmental duplication events involving five ZmAPY genes across the maize genome, while no tandem duplications were observed (Fig. 1 B). APY gene structures and predicted protein motifs Structural differences in exon-intron arrangement serve as sources of gene family variation and species diversity, leading to alterations in gene expression and function. To investigate the conservation and diversity of gene structure within the maize APY gene family, the exons and introns of 16 APY genes were analyzed based on their coding sequences and genomic data. The number of exons varied among the APY genes, ranging from 1 to 10 (Fig. 2 A). Most members contained 6–10 exons, with two members in Group I ( ZmAPY5 and ZmAPY6 ) and one member in Group III ( ZmAPY13 ) having only 1 and 2 exons, respectively (Fig. 2 A). The number of introns ranged from 0 to 9. Two transposon insertions of 9.56 kb and 33.57 kb were identified in the intron regions of ZmAPY5 and ZmAPY10 , respectively. ZmAPY genes with collinearity exhibited similar gene structures. Additionally, members within the same subgroup typically displayed similar motifs and lengths, suggesting functional similarities (Fig. 2 A). Analysis of the protein sequences of all ZmAPY gene family members revealed a conserved GDA1_CD39 domain in all proteins, with varying numbers of transmembrane regions. Specifically, members of Group I had one transmembrane region, most members of Group II had two transmembrane regions, and family members of Group III had no transmembrane regions (Fig. 2 B). This structural variation may contribute to the functional distinctions observed among different subgroups. Expression patterns of APY genes in maize The investigation into tissue-specific gene expression patterns provides valuable insights into the potential biological roles of the ZmAPY genes. Analysis of the expression patterns within the ZmAPY gene family revealed distinct expression profiles among different members (Fig. 3 ), highlighting their diverse functions. Specifically, ZmAPY8 , ZmAPY9 , and ZmAPY12 exhibited specific expression in roots, indicating a potential role for these genes in root-related processes. On the other hand, ZmAPY15 and ZmAPY16 showed significantly higher expression levels in anthers compared to other tissues, suggesting their involvement in anther-related functions. Moreover, there was a notable trend of high expression of ZmAPY11 in seeds and endosperm, implying a potential role in seed development and maturation. Conversely, minimal to no detectable expression was observed for ZmAPY3 , ZmAPY4 , ZmAPY6 , and ZmAPY7 across all tissues and organ. Interestingly, similar tissue-specific expression patterns were observed between collinear ZmAPY genes, such as ZmAPY1 / ZmAPY2 / ZmAPY5 and ZmAPY15 / ZmAPY16 , suggesting potential functional conservation or shared regulatory mechanisms among these gene clusters. The differential tissue-specific expression patterns observed among the ZmAPY genes indicate their diverse biological roles and potential contributions to various developmental processes and physiological functions in maize. Prediction of upstream regulators of APY gene The analysis of variations in expression patterns among different ZmAPY genes has led to the identification of potential upstream regulators that may control APY gene transcription in maize. By utilizing planttfdb software and existing ChIP-seq data of 104 transcription factors (Tu et al. 2020 ), a total of 251 upstream regulators were predicted (Fig. 4 A, Table. S3). Subsequently, the correlation between the expression levels of these predicted regulators and ZmAPY gene expression was examined. Significantly, NACTF78, ZIM36, bZIP79, and CCHH26 displayed strong correlations with the transcription levels of ZmAPY2 (R 2 = 0.51), ZmAPY5 (R 2 = 0.56), ZmAPY9 (R 2 = 0.67), and ZmAPY14 (R 2 = 0.60), respectively (Fig. 4 B). Notably, NACTF78 has been previously reported to regulate Fe concentrations in maize kernels, potentially enabling the cultivation of maize varieties with both high yield and high Fe concentrations in their kernels using a molecular marker in the NACTF78 promoter (Yan et al. 2023 ). Additionally, Vélez-Bermúdez et al. reported that ZML2 (ZIM36) regulates wound-induced lignin genes in maize (Vélez-Bermúdez et al. 2015 ), while ZmTGA9-1 (bZIP79) has been shown to regulate male sterility in maize (Jiang et al. 2021 ). The observed correlations between the expression levels of ZmAPY genes and these transcription factors suggest that ZmAPY genes may also play a role in regulating these biological processes, indicating a potential link between APY gene expression and the modulation of Fe concentrations, lignin gene regulation, and male sterility in maize. These findings provide valuable insights into the regulatory network involving ZmAPY genes and their upstream regulators in maize, shedding light on the diverse biological processes influenced by these genes. Expression analysis of ZmAPYs under drought, cold, heat stresses To investigate the potential role of APY genes in regulating maize abiotic stress responses, we analyzed the transcription levels of APY genes under drought, cold, and heat stress using RNA-seq data from the maize inbred line B73. Our findings revealed the transcription of the ZmAPY gene is responsive to drought, cold, and heat stress, displaying distinct response profiles (Fig. 5 A; 5 B). Subsequently, we validated the transcription of ZmAPY genes under drought, cold, and heat stress through qRT-PCR. The results revealed significant upregulation of 6 ZmAPY genes, ZmAPY1 , ZmAPY2 , ZmAPY8 , ZmAPY13, ZmAPY14 and ZmAPY15 under severe drought (DT4), while the transcription of ZmAPY15 was suppressed by drought (Fig. 5 C). Cold stress induced the transcription of ZmAPY1 but inhibited the transcription of ZmAPY5 , ZmAPY8 , ZmAPY11 , ZmAPY13 and ZmAPY15 (Fig. 5 C). Moreover, heat stress significantly inhibited the transcription of 7 ZmAPY genes, ZmAPY1 , ZmAPY2 , ZmAPY5 , ZmAPY8 , ZmAPY11 , ZmAPY14 , and ZmAPY16 , while the transcription of ZmAPY15 was induced by heat (Fig. 5 C). It is noteworthy that some of the expression analysis results for the ZmAPY genes are absent from Fig. 5 C due to their expression levels falling below the detection threshold of qRT-PCR. The stress-responsive expression patterns of ZmAPY genes suggest their potential regulatory roles in drought, cold, heat, and salt stress responses. ZmAPYs was associated with agronomic traits and drought resistance of maize To further investigate the impact of APY on maize agronomic traits and drought resistance, we examined the relationship between SNPs in the ZmAPY gene region and 17 agronomic traits as well as drought phenotypes using the MLM model. Our analysis revealed a significant association between the SNP (chr9_124751424, CC/TT) in ZmAPY16 and maize plant height (Fig. 6 A). Subsequent analysis indicated notable differences not only in plant height but also in ear height, drought resistance, and ZmAPY16 expression between the "CC" and "TT" genotypes. Plants with the "TT" allele, showing high ZmAPY6 expression, exhibited greater plant height, ear height, and drought survival rates compared to those with the "CC" allele (Fig. 6 B-E), suggesting a positive regulatory role of ZmAPY16 in maize plant height, ear height, and drought resistance. Additionally, we observed a significant association between the SNP (chr1_54070081, CC/TT) in ZmAPY5 and the spinemaking period in maize (Fig. 6 F). Plants with the "TT" allele and high ZmAPY5 expression displayed a delayed spinemaking period and enhanced drought survival rates compared to plants with the "CC" allele and low ZmAPY5 expression (Fig. 6 G-I), indicating a negative regulation of maize spinemaking and a positive regulation of maize drought resistance by ZmAPY5 . Genetic variation within ZmAPYs regulate the content of drought-induced metabolites Metabolites, as small molecules that serve as the end products of metabolic processes and physiological pathways, are known to play crucial roles in plant drought resistance (Kim et al. 2017 ; Todaka et al. 2017 ). These compounds can act as osmoprotectants, antioxidants, signaling molecules, and regulators of various stress-responsive pathways in plants (Nakabayashi et al. 2014 ; Obata et al. 2013 ; Fàbregas et al. 2018 ). Analyzing the genome-wide metabolite profiles of 385 maize natural inbred lines grown under well-watered and drought-stressed conditions (Zhang et al. 2021a ), we identified metabolite quantitative trait loci (mQTL) for 18 metabolites that co-located with the ZmAPY genes, indicating a potential relationship between ZmAPYs and the levels of these metabolites (Fig. 7 A). Further investigation into these metabolites revealed that four drought-induced metabolites were influenced by genetic variations within the ZmAPYs gene region. Specifically, genetic variations chr1.S_65348503 and chr1.S_65352471 within the ZmAPY15 gene region were found to regulate the contents of metabolites PN_group_05106 and PN_group_17082, while another genetic variation chr1.S_65352471 within the ZmAPY11 gene region was associated with the regulation of the metabolite PN_group_00505. Additionally, a genetic variation chr10.S_71730551 within the ZmAPY7 gene region was linked to the regulation of the metabolite PN_group_11670 (Fig. 7 B). These findings suggest that ZmAPY genes may impact maize drought resistance by modulating the contents of these drought-induced metabolites. This insight highlights the potential role of ZmAPY genes in mediating maize response to drought stress through the regulation of key metabolites involved in stress adaptation and tolerance. Discussion Apyrase (APY) is widely existed in eukaryotes and is highly conserved throughout the evolution of eukaryotes. In plants, apyrase genes have been reported to regulate a variety of biological processes, including root hair development, stomatal movement, and defense responses (Clark et al. 2014 ; Lim et al. 2014 ; Wu et al. 2007 ). However, no apyrase genes have been identified in maize, and their functions remain unclear. In this study, through sequence alignments and phylogenetic analysis, we identified 16 apyrase genes in maize genome and further divided these genes into three subgroups (Fig. 1 A). The number of group I ZmAPY genes far exceeds the number of group I apyrase genes in Arabidopsis and rice that identified in a previous study (Clark et al. 2014 ; Chowdhury et al. 2023 ). Syntenic analysis revealed that most ZmAPY genes are of the dispersed repeat type, but segmental duplication also played a significant role in the expansion process of group I ZmAPY genes (Fig. 1 B). Meanwhile, genes with synteny show higher similarity in expression patterns (Fig. 3 ). These results suggest that APY genes in maize may have produced functional redundancy through expansion, and since apyrase plays an important regulatory role in plant growth and development and stress responses, the expansion and functional redundancy of gene subgroups generated by segmental and dispersed duplication may enhance the robustness of the maize regulatory network. The members of the ZmAPY genes are predicted to encode proteins ranging from 81 to 701 amino acid in length, and the PI ranging from 4.70 to 11.62 (Table S1). These results suggest that the differences among ZmAPY genes may be more pronounced than those in rice (in which OsAPY proteins ranging from 451 to 702 amino acid in length, and the PI ranging from 5.44 to 9.34) (Chowdhury et al. 2023 ). The predicted gene structures of the ZmAPY genes contain one exon to ten exons (Fig. 2 A), similar to the gene structures in rice, with two to twelve exons (Chowdhury et al. 2023 ). Similar to OsAPY proteins, the ZmAPY proteins were predicted to localize to various cellular compartments, including the chloroplast, mitochondrial, plasma membrane, cytoplasm, nuclear and extracellular (Table S1). In summary, ZmAPY genes share similar characteristics with apyrase in rice, but exhibits greater diversity among family members, suggesting that although ZmAPY genes maintain functional conservation, they may also have undergone functional divergence. Transcription factors are core elements that regulate transcriptional levels during various stages of plant life processes. Analysis of the promoter regions of all ZmAPY genes revealed the presence of diverse upstream transcription factors which may regulate ZmAPY genes expression (Fig. 4 ). Among the 251 potential upstream transcription factors, several genes have been reported to regulate maize growth and stress response progresses (Table S3). For example, WRKY48 / ZmWRKY40 was predicted to binding the promoter sequences of seven ZmAPY genes, overexpression ZmWRKY40 could enhance drought tolerance in transgenic Arabidopsis by regulating stress-related genes, and the reactive oxygen species (ROS) content in transgenic lines was reduced compared with wild-type plants under drought stress (Wang et al. 2018 ). ZmBES1/BZR1-5 was predicted to bind the promoter sequences of six ZmAPY genes, ZmBES1/BZR1-5 decreases ABA sensitivity and confers tolerance to osmotic stress in transgenic Arabidopsis. Meanwhile, ZmBES1/BZR1-5 can also positively regulates kernel size (Sun et al. 2020 ; Sun et al. 2021 ). A bZIP transcription factor id1 was predicted to binding the promoter sequences of ten ZmAPY genes, previous study showed that id1 can regulate maize flowering time and floral inductive signals (Colasanti et al. 1998 ; Muszynski et al. 2006 ). These results further suggested potential roles of ZmAPY genes in maize growth and stress response progresses. Maize is a world major food crop, and its yield is seriously threatened by abiotic stresses (Lesk et al. 2016 ). Although a large number of abiotic stress response genes and loci have been identified in recent years through genetic and molecular biology techniques, due to limited understanding of the plant abiotic stress response system, only a few genes have been successfully applied in commercialization (Zhang et al. 2020 ). Therefore, it remains essential to further elucidate the abiotic stress response system of maize. Previous studies have shown that the extracellular ATP (eATP) level rises when plants are subjected to stress, leading to the production of reactive oxygen species (ROS) and cell death. Apyrase can regulate the production of ROS by hydrolyzing eATP, and participate in various stress response processes in plants (Deng et al. 2015 ). By analyzing the transcriptomic data from previous studies, we investigated the expression patterns of ZmAPY genes under different abiotic stresses (Makarevitch et al. 2015 ; Zhang et al. 2019 ). The results showed that seven ZmAPY genes responded to drought stress, six to cold stress, and eight to heat stress, and the stress responsive ZmAPY genes were further confirmed by qPCR (Fig. 5 C). These results indicated that, like other plants, ZmAPY genes could also be involved in regulating maize's abiotic stress response. Of course, more researches are required to clearly elucidate the function of ZmAPY genes in maize abiotic stress responses. Natural variations significantly affect maize agronomic traits and stress resistance, and numerous elite alleles have been identified through genetic approaches which significantly accelerated the breeding process (Xiao et al. 2017 ). To explore the potential application value of ZmAPY genes and their genetic variations in maize breeding, we collected drought tolerance and agronomic traits data of maize association panels published in previous study (Zhang et al. 2019 ; Yang et al. 2014 ), and utilized high-density SNP markers to perform association analyses on the ZmAPY gene and its flanking regions. The result indicated that natural variations in ZmAPY16 gene and flanking region were significantly associated with maize plant height (Fig. 6 A), further analysis revealed that these variations were also correlated with the ZmAPY16 expression and maize drought tolerance (Fig. 6 B-E). Additionally, we found that ZmAPY5 was significantly associated with maize silking time (Fig. 6 F), and associated variations were also correlated with the ZmAPY5 expression and maize drought tolerance (Fig. 6 G-I). These findings suggest that variations of ZmAPY gene expression may influence maize agronomic traits and drought tolerance. Furthermore, since many metabolic pathways involve energy conversion, while apyrase can affect the levels of ATP/ADP. To investigate the effects of apyrase on maize metabolites, we performed co-localization analysis between ZmAPY variations and previously reported metabolite regulation sites (Zhang et al. 2021a ), which revealed that seven ZmAPY genes were co-localized with 18 metabolite regulation sites (Fig. 7 A). Some of these metabolites showed significant content change under drought stress (Fig. 7 B). Although the functions of these metabolites are not well understood at present, these results suggest that ZmAPY genes and their genetic variations may participate in maize drought response by affecting the levels of drought-responsive metabolites. In summary, we revealed the variations of ZmAPY gene expression among maize populations, which were associated with multiple important maize traits. These results indicate that ZmAPYs and their genetic variations may have potential applications in maize breeding. Declarations Funding This work was supported by the Key Technologies Research and Development Program, China (2022YFE0100500), The National Natural Science Foundation of China (31971954, 32061143031). Author information Zhenhua He and Jie Zhang contributed equally to this work. Authors and Affiliations Hubei Key Laboratory of Food Crop Germplasm and Genetic Improvement & Key Laboratory of Crop Molecular Breeding, Ministry of Agriculture and Rural Affairs, Food Crops Institute, Hubei Academy of Agricultural Sciences, Wuhan 430064, China Zhenhua He, Haitao Jia, Shilong Zhang & Xiaopeng Sun National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China Jie Zhang, Hui Zhang & Mingqiu Dai Hubei Hongshan Laboratory, Wuhan 430070, China. Jie Zhang, Xiaopeng Sun, Hui Zhang & Mingqiu Dai Author contribution MD, HZ, ZH and XS conceived and designed the research. ZH and JZ performed the experiments and collected the data. HZ and XS supervised the experiments. HZ and XS wrote the manuscript. MD modified manuscript. HJ and SZ polished the manuscript and images. All authors have read and agreed to the published version of the manuscript. Corresponding authors Correspondence to Mingqiu Dai or Hui Zhang Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval We declare that these experiments comply with the ethical standards in China. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Cite Share Download PDF Status: Published Journal Publication published 30 Apr, 2024 Read the published version in Molecular Breeding → Version 1 posted Reviewers agreed at journal 10 Apr, 2024 Reviewers invited by journal 10 Apr, 2024 Editor assigned by journal 10 Apr, 2024 First submitted to journal 09 Apr, 2024 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-4222821","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289927905,"identity":"e9978dce-15d2-4d93-913a-886b7095160f","order_by":0,"name":"Zhenghua He","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhenghua","middleName":"","lastName":"He","suffix":""},{"id":289927906,"identity":"74642bc0-cd76-497e-8965-d69c9dac8b46","order_by":1,"name":"jie Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"jie","middleName":"","lastName":"Zhang","suffix":""},{"id":289927907,"identity":"463e59bb-acd1-4559-ad9c-b20c96098b39","order_by":2,"name":"Haitao Jia","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Haitao","middleName":"","lastName":"Jia","suffix":""},{"id":289927908,"identity":"e2f9c94e-afc7-4356-8064-802f41597ed7","order_by":3,"name":"Shilong Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shilong","middleName":"","lastName":"Zhang","suffix":""},{"id":289927909,"identity":"04def877-53fc-4973-9dc7-f7cf97bc068f","order_by":4,"name":"Xiaopeng Sun","email":"","orcid":"","institution":"Hubei Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaopeng","middleName":"","lastName":"Sun","suffix":""},{"id":289927910,"identity":"4651a325-0bb4-4b6a-a4f5-b8f4a417f0c3","order_by":5,"name":"Hui Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIiWNgGAWjYBACAwbGBhBiYGAHEgkFDAkQcTZitPAcAGoxIEoLEIC1SCSAuYS1mEskNzD+3GGXJx/5OvHDAwOGPPn2MwYMH8oOM/DPbsCqxXJGYgOD5JnkYsPbuZslgA4rZuzJMWCcce4wg8SdA9gddgOoxbCNOXHj7NwNIC2JzQw5Bsy8bYcZDCQScGtJbKtP3Djz7OYfIC1t/G8MmP8S0nKw7XDifAnebWBbeiSAtjDi03LmYQNjY9vxxA08udssEgwkEmdIPCs42HMunUfiBg4tx9MfMP5sq06c3352880fFTaJ8/uTNz74UWYtxz8DuxYgYP8B1gsJHgkwCWLz4FIPB/INBJWMglEwCkbBSAUAjJdh1XmTCsMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0008-1903-5162","institution":"HZAU: Huazhong Agriculture University","correspondingAuthor":true,"prefix":"","firstName":"Hui","middleName":"","lastName":"Zhang","suffix":""},{"id":289927911,"identity":"d6e9a0b0-6995-4e9f-9fab-67a993d6bd1e","order_by":6,"name":"Mingqiu Dai","email":"","orcid":"","institution":"Huazhong Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"Mingqiu","middleName":"","lastName":"Dai","suffix":""}],"badges":[],"createdAt":"2024-04-05 12:00:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4222821/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4222821/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11032-024-01474-9","type":"published","date":"2024-05-01T00:40:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54699117,"identity":"0dc627eb-b856-4864-ac6e-5cfa482b9928","added_by":"auto","created_at":"2024-04-15 11:59:06","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":104109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis, chromosomal distribution and collinearity analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmAPY\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes. A \u003c/strong\u003ePhylogenetic analysis of APYs from \u003cem\u003eZ. mays\u003c/em\u003e,\u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eO. sativa\u003c/em\u003e. A total number of 16 ZmAPYs from maize, 7 AtAPYs from Arabidopsis and 9 OsAPYs from rice were used to construct the phylogenetic tree. All APY transporter members were classified into four groups. Group Ⅰ-III and outgroup are distinguished by different colors. \u003cstrong\u003eB \u003c/strong\u003eChromosomal distribution and collinearity analysis of \u003cem\u003eZmAPY\u003c/em\u003egenes. The chromosomal location of each \u003cem\u003eZmAPY\u003c/em\u003e gene was mapped according to the maize genome. The chromosome number is indicated at the top of each chromosome. The syntenic \u003cem\u003eZmAPY\u003c/em\u003e gene pairs are connected by gray dotted lines.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4222821/v1/7f7729d465082c58a0fb4382.jpeg"},{"id":54699112,"identity":"9c0eb32b-af6f-4f82-bce7-8ce53fa6d85b","added_by":"auto","created_at":"2024-04-15 11:59:06","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115226,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExon-intron structure and conserved motifs of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmAPY\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene family. A\u003c/strong\u003e Exon–intron structures of \u003cem\u003eZmAPY\u003c/em\u003e genes. Red boxes represent exons (CDS), black lines represent introns, and blue boxes represent 5’ and 3’UTR regions. \u003cstrong\u003eB\u003c/strong\u003e Predicted conserved protein domains of ZmAPY proteins. The black lines represent the length of each protein sequence, and the conserved protein kinase domains are depicted with colored boxes. blue boxes represent transmembrane region and yellow boxes represent GDA1_CD39 domain.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4222821/v1/1c42271b24d226de49c5eac8.jpeg"},{"id":54699116,"identity":"ca663105-1722-4dd3-a984-01622aba392c","added_by":"auto","created_at":"2024-04-15 11:59:06","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmAPY \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenes in different tissues. \u003c/strong\u003eThe genes were labeled on the left and the tissues were displayed at the top of each column. The gene expression values are reported as square root transformations of the fragments per kilo bases per million mapped reads (FPKM). Different colors in map represent FPKM values, as shown in the bar to the right.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4222821/v1/d0e75c18f168ec81a4cc51cc.jpeg"},{"id":54699115,"identity":"f8651064-e5a0-4ae7-85ca-6166e4cc4dc9","added_by":"auto","created_at":"2024-04-15 11:59:06","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":139849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrediction of upstream regulators of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAPY\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes. A\u003c/strong\u003e Upstream regulators of \u003cem\u003eZmAPYs\u003c/em\u003e were predicted by ChIP-seq data of 104 transcription factors and planttfdb software. Orange dots represent \u003cem\u003eZmAPYs \u003c/em\u003eand blue dots represent potential upstream regulators of\u003cem\u003e ZmAPY \u003c/em\u003egenes. The red line connects the transcription factors and \u003cem\u003eZmAPY \u003c/em\u003egenes that may have regulatory relationship analyzed by ChIP-seq data, while the blue line connects the transcription factors and\u003cem\u003eZmAPY \u003c/em\u003egenes that may have regulatory relationship predicted by planttfdb software. \u003cstrong\u003eB \u003c/strong\u003eCorrelation analysis between the expression levels of potential upstream regulators \u003cem\u003enacttf78\u003c/em\u003e, \u003cem\u003ezim36\u003c/em\u003e, \u003cem\u003ebzip79\u003c/em\u003e, \u003cem\u003ecchh26\u003c/em\u003e and \u003cem\u003eZmAPYs.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4222821/v1/26ea902957b454d9a5434e6d.jpeg"},{"id":54699121,"identity":"61c074d8-c2e7-4bca-8394-3141df9d46e8","added_by":"auto","created_at":"2024-04-15 11:59:07","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":178385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmAPYs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eunder drought, cold, heat stresses.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Expression analysis of \u003cem\u003eZmAPY\u003c/em\u003e genes under three different drought degree stresses\u003cstrong\u003e. B \u003c/strong\u003eExpression analysis of \u003cem\u003eZmAPY\u003c/em\u003e genes under cold and heat stresses\u003cstrong\u003e. \u003c/strong\u003eThe genes were labeled on the left and the abiotic stresses were displayed at the top of each column. The FPKM / TPM values of the \u003cem\u003eZmAPY \u003c/em\u003egenes were used to draw a heat map by using HEMI. \u003cstrong\u003eC\u003c/strong\u003e qRT-PCR in analyzing the expression of \u003cem\u003eZmAPYs \u003c/em\u003eunder drought, cold and heat stress. Statistical significance was determined by Student’s \u003cem\u003et\u003c/em\u003e test: “*”\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and “**”\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4222821/v1/f8017e492c48017ff80772a4.jpeg"},{"id":54699120,"identity":"3ce8564b-9780-48b9-8277-a71926089c5f","added_by":"auto","created_at":"2024-04-15 11:59:06","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":138352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eZmAPYs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ewas associated with agronomic traits and drought resistance of maize.\u003c/strong\u003e \u003cstrong\u003eA. \u003c/strong\u003eAssociation of SNPs from \u003cem\u003eZmAPY16\u003c/em\u003e with plant height. chr9_124751424 is most significantly associated with plant height compared with other SNPs. SNPs significant associated with plant height (p\u0026lt;0.01) were marked in red. \u003cstrong\u003eB-E \u003c/strong\u003eGenetic variation of \u003cem\u003eZmAPY16 \u003c/em\u003e(chr9_124751424) regulates plant length (B), ear-height (C), drought resistance (D) and \u003cem\u003eZmAPY5\u003c/em\u003eexpression (E) in maize. \u003cstrong\u003eF \u003c/strong\u003eAssociation of SNPs from \u003cem\u003eZmAPY5\u003c/em\u003e with day to silking (DTS). Chr1_54070081 is most significantly associated with DTS compared with other SNPs. SNPs significant associated with DTS (p\u0026lt;0.01) were marked in red. \u003cstrong\u003eG-I \u003c/strong\u003eGenetic variation of \u003cem\u003eZmAPY5 \u003c/em\u003e(chr1_54070081) regulates silking period (G), drought resistance (H) and \u003cem\u003eZmAPY5\u003c/em\u003eexpression (I) in maize.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4222821/v1/1364a71264048d5d77bc9d1d.jpeg"},{"id":54699119,"identity":"dada6c74-99a9-48c9-9661-ba61adc02f53","added_by":"auto","created_at":"2024-04-15 11:59:06","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":105137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic variation in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmAPYs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eregulate the content of drought-induced metabolites. A \u003c/strong\u003eCo-localization analysis of mQTLs with \u003cem\u003eZmAPY\u003c/em\u003e genes. The red lines represent the collinearity relationship of mQTLs under drought stress with\u003cem\u003e ZmAPY\u003c/em\u003egenes, while the blue lines represent the collinearity relationship of mQTLs under control conditions with \u003cem\u003eZmAPY\u003c/em\u003e genes. \u003cstrong\u003eB\u003c/strong\u003e Genetic variations in the \u003cem\u003eZmAPY \u003c/em\u003egene region regulate the contents of metabolites induced by drought.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4222821/v1/bc6ed3612241154485cff0ca.jpeg"},{"id":56416536,"identity":"5009e2be-e8cb-40aa-a818-d494bfda5533","added_by":"auto","created_at":"2024-05-14 00:40:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1714010,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4222821/v1/6c4d01b1-c6a7-48a9-a0d4-c3251a5acdbe.pdf"},{"id":54699113,"identity":"893a26e6-528d-485b-b141-9bc3f3630832","added_by":"auto","created_at":"2024-04-15 11:59:06","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":44416,"visible":true,"origin":"","legend":"\u003cp\u003eTable. S1 Physicochemical properties of \u003cem\u003eZmAPY\u003c/em\u003egenes in maize.\u003c/p\u003e\n\u003cp\u003eTable. S2 \u003cem\u003eAPY\u003c/em\u003egenes in Arabidopsis, rice and maize.\u003c/p\u003e\n\u003cp\u003eTable. S3 Potential upstream transcription facotors (TFs) in regulation of \u003cem\u003eZmAPYs.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTable. S4 Premers used in the study.\u003c/p\u003e","description":"","filename":"SupplementalTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4222821/v1/3208c74d1bd891ace9d8bf2c.xlsx"}],"financialInterests":"","formattedTitle":"Genome-wide identification and analyses of ZmAPY genes reveal their roles involved in maize development and abiotic stress responses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eApyrase (APY) enzymes, classified as nucleoside triphosphate (NTP) diphosphohydrolases, belong to the superfamily of guanosine diphosphatase 1 (GDA1)-cluster of differentiation 39 (CD39) nucleoside phosphatase. These enzymes have the capability to remove the terminal phosphate from nucleoside triphosphates (NTPs) and nucleoside diphosphates (NDPs) but not from nucleoside monophosphates (NMPs). Based on their subcellular localization, APYs can be broadly categorized into ecto-apyrases and endo-apyrases (Hideaki et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Tong et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Thomas et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). ecto-apyrase are located on the cell surface, whereas endo-apyrase are usually located in the endoplasmic reticulum, Golgi and intracellular vesicles (Leal et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Some ecto-apyrase possess transmembrane domains at their N- and C-terminals, which are crucial for correct protein folding, membrane targeting, cellular allocation and enzyme activity (Wu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Knowles \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The cellular ATP level serves a dual role in providing energy and regulating various cellular processes associated with responses to abiotic stress (Sun et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Therefore, the maintenance of cellular ATP homeostasis, regulated in part by apyrases, is essential for preserving normal cell function.\u003c/p\u003e \u003cp\u003eApyrases are evolutionarily highly conserved (Clark et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Their presence in plants was initially identified in potatoes several decades ago (Hideaki et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Subsequently, members of the APY family were discovered in the genomes of various plants, including potato (Handa and Guidotti, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Riewe et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), wheat (Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), soybean (Day et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), Arabidopsis (Yang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), cotton (Clark et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), rice (Chowdhury et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and peanut (Sharif et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Transcriptome analysis has revealed specific spatio-temporal expression patterns of \u003cem\u003eAPY\u003c/em\u003e genes and regulation of these genes by biological and abiotic stresses (Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chowdhury et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Clark et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), indicating potential roles in plant growth, development, and stress responses.\u003c/p\u003e \u003cp\u003eFurther studies have elucidated the functions of \u003cem\u003eAPY\u003c/em\u003e genes in plant growth and development. \u003cem\u003eArabidopsis APY\u003c/em\u003e genes, \u003cem\u003eAPY1\u003c/em\u003e and \u003cem\u003eAPY2\u003c/em\u003e, are predominantly expressed in rapidly growing tissues or those with high auxin levels. Suppression of their expression affects root and shoot growth (Wu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Additionally, \u003cem\u003eAPY1\u003c/em\u003e/\u003cem\u003eAPY2\u003c/em\u003e also participate in the phytochrome-mediated signaling pathway that induces differential growth changes in etiolated seedling tissues (Weeraratne et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Another member of the \u003cem\u003eAPY\u003c/em\u003e gene family, \u003cem\u003eAPY7\u003c/em\u003e, acts as a negative regulator of cell growth. \u003cem\u003eAPY7\u003c/em\u003e modulates the growth-inhibiting effects of RALF1 (Rapid ALkalinization Factor), influencing cell wall architecture, composition, and alters the pH of the extracellular matrix (Gupta et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Constitutive expression of pea ectoapyrase, psNTP9, in \u003cem\u003eArabidopsis\u003c/em\u003e and soybeans leads to a more extensive root system architecture (RSA) (Veerappa et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, the catalytic activity of GS52 ecto-apyrase is crucial for the early infection process of \u003cem\u003eB. japonicum\u003c/em\u003e, nodule primordium development initiation, and subsequent nodule organogenesis in soybean (Govindarajulu et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to participating in the regulation of plant growth and development, the \u003cem\u003eAPY\u003c/em\u003e gene also plays a crucial role in stress response (Clark et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Overexpression of the pea \u003cem\u003eAPY\u003c/em\u003e gene \u003cem\u003epsNTP9\u003c/em\u003e significantly enhances drought resistance and field yield in soybean under both normal watering and drought conditions (Veerappa et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003ePopulus euphratica APY\u003c/em\u003e genes, \u003cem\u003ePeAPY1\u003c/em\u003e and \u003cem\u003ePeAPY2\u003c/em\u003e, enhance drought tolerance by modulating stomatal aperture in \u003cem\u003eArabidopsis\u003c/em\u003e (Zhang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Furthermore, overexpression of \u003cem\u003ePeAPY2\u003c/em\u003e improves cold resistance by modulating vesicular trafficking and extracellular ATP in \u003cem\u003eArabidopsis\u003c/em\u003e (Deng et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Proteome analysis of soybean roots under waterlogging has demonstrated that \u003cem\u003eAPY\u003c/em\u003e responded to waterlogging stress in soybean (Alam et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These findings underscore the multifaceted roles of the \u003cem\u003eAPY\u003c/em\u003e genes in plant stress responses, highlighting its potential as a key player in enhancing plant resilience to various environmental challenges.\u003c/p\u003e \u003cp\u003eMaize, a primary global food crop, faces significant threats from abiotic stresses. Identifying additional abiotic stress resistance genes and further understanding response system in maize remain crucial. While \u003cem\u003eAPY\u003c/em\u003e genes has been reported to be involved in various abiotic stresses in \u003cem\u003eArabidopsis\u003c/em\u003e, their presence and biological functions in maize have not been reported. This study aims to characterize \u003cem\u003eAPY\u003c/em\u003e genes and elucidate their roles. Here, 16 \u003cem\u003eAPY\u003c/em\u003e family members were identified in maize genome by phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table S1). \u003cem\u003eAPY\u003c/em\u003e family expansion patterns and genetic characteristics were analyzed comprehensively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table S2). In addition, potential upstream regulators of \u003cem\u003eZmAPYs\u003c/em\u003e were predicted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Transcriptome analysis revealed tissue-specific and abiotic stress-responsive expression of \u003cem\u003eZmAPY\u003c/em\u003e genes in maize (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Association analysis suggested that variations in \u003cem\u003eZmAPY\u003c/em\u003e genes could impact maize agronomic traits and drought responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings establish a basis for further investigations into the functions of maize \u003cem\u003eAPY\u003c/em\u003e genes.\u003c/p\u003e "},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth condition\u003c/h2\u003e \u003cp\u003eThe maize inbred line B73 was used in this study. Maize seeds are germinated on moist filter paper in a plant growth chamber at 28\u0026deg;C for 2\u0026ndash;3 days. Uniformly germinated seeds are then transplanted into soil-filled pots (10 cm\u0026times;10 cm\u0026times;9 cm) and grown in the chamber until the three-leaf stage for subsequent stress treatments. For drought stress, water is withheld from three-leaf stage maize seedlings for 10 days, while the control group is watered normally. Leaf samples are collected for RNA extraction to analyze \u003cem\u003eZmAPY\u003c/em\u003e genes expression levels. For heat and cold treatments, three-leaf stage maize seedlings were placed in growth chambers at 50\u0026deg;C and 4\u0026deg;C for 4 hours and 24 hours, respectively, while the control group continues growing at 28\u0026deg;C. Leaf samples are then collected for RNA extraction to evaluate \u003cem\u003eZmAPY\u003c/em\u003e genes expression level.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification and Phylogenetic analysis of the\u003c/b\u003e \u003cb\u003eZmAPY\u003c/b\u003e \u003cb\u003egenes in maize\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGene ID of all \u003cem\u003eAPY\u003c/em\u003e genes in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e was collected previous study (Chowdhury et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), protein sequences of these APYs were downloaded from Ensemble Plants (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://plants.ensembl.org/\u003c/span\u003e\u003cspan address=\"http://plants.ensembl.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and aligned for constructing HMM model. Protein sequences for maize (\u003cem\u003eZea mays\u003c/em\u003e) were also collected from Ensemble Plants, and \u003cem\u003eZmAPY\u003c/em\u003e genes were identified using HMMsearch with default parameters. Proteins identified by HMMsearch that lacked conserved domains were removed after submission to SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://smart.embl.de/\u003c/span\u003e\u003cspan address=\"https://smart.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Subsequently, MEGA X was used for multiple sequence alignment and phylogenetic tree construction of the identified 18 protein sequences with those from rice and Arabidopsis thaliana's APY proteins. The tree was built using Neighbor-Joining (NJ) method, pair-wise deletion, and bootstrap value set to 1000. As a result, 16 genes were identified as \u003cem\u003eZmAPY\u003c/em\u003e genes based on the evolutionary tree and named as \u003cem\u003eZmAPY1\u003c/em\u003e -\u003cem\u003eZmAPY16\u003c/em\u003e, respectively. The Synteny analysis of \u003cem\u003eZmAPY\u003c/em\u003e genes was employed by MCScanX by using default parameter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePrediction of physicochemical properties of ZmAPY proteins\u003c/h2\u003e \u003cp\u003eTo obtain data such as the number of amino acids, molecular weight, isoelectric point, and average hydrophobicity index of the ZmAPY proteins, a physicochemical property analysis of ZmAPY protein sequences was employed by using an online tool ProtParam on ExPASy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Gene structure analysis was performed by GSDS2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gsds.gao-lab.org/\u003c/span\u003e\u003cspan address=\"https://gsds.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The subcellular localization of ZmAPY proteins were predicted by WoLF PSORT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp/\u003c/span\u003e\u003cspan address=\"https://wolfpsort.hgc.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and CELLO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cello.life.nctu.edu.tw/\u003c/span\u003e\u003cspan address=\"http://cello.life.nctu.edu.tw/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression analysis of\u003c/b\u003e \u003cb\u003eZmAPY\u003c/b\u003e \u003cb\u003egenes in different tissues\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe expression data for the \u003cem\u003eZmAPY\u003c/em\u003e genes were obtained from various tissues, including seedling, roots, leaves, seeds, shoot apical meristems, internodes, tassel, cob, coleoptite, pericarp and anthers. These data were sourced from the qTeller (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://qteller.maizegdb.org/\u003c/span\u003e\u003cspan address=\"https://qteller.maizegdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The FPKM values of the \u003cem\u003eZmAPY\u003c/em\u003e genes were used to draw a heat map by using HEMI (Deng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eUpstream transcription factor prediction of\u003c/b\u003e \u003cb\u003eZmAPY\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 2kb upstream sequences of each \u003cem\u003eZmAPY\u003c/em\u003e gene were obtained from the maize genome for analysis. The PlantTFdb (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://planttfdb.gao-lab.org/\u003c/span\u003e\u003cspan address=\"https://planttfdb.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and jbrowse on maizeGDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jbrowse.maizegdb.org/\u003c/span\u003e\u003cspan address=\"https://jbrowse.maizegdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used to analyze the retrieved sequences and identify. All predicted upstream transcription factors were visualized by using Gephi (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gephi.org/\u003c/span\u003e\u003cspan address=\"https://gephi.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Then expression correlation coefficients between potential upstream transcription factors and \u003cem\u003eZmAPY\u003c/em\u003e genes were calculated by using the expression profiles collected from qTeller.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of\u003c/b\u003e \u003cb\u003eZmAPY\u003c/b\u003e \u003cb\u003egenes expression in maize under abiotic stresses.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExpression profiles data of \u003cem\u003eZmAPY\u003c/em\u003e genes under well-water and drought treatment were collected from qTeller and previous study (Zhang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The FPKM / TPM values of the \u003cem\u003eZmAPY\u003c/em\u003e genes were used to draw a heat map by using HEMI. For qPCR validation, the FastPure Universal Plant Total RNA Isolation kit (Vazyme) was employed for RNA extraction. HiScript III qRT SuperMix for qPCR (Vazyme) was used for reverse transcription to synthesize cDNA. Specifc primers were designed for the 16 \u003cem\u003eZmAPY\u003c/em\u003e genes and utilized for qRT-PCR analysis. The qRT-PCR reactions were performed using the SYBR Green master mix (ChamQ SYBR qPCR Master Mix, Vazyme). Three independent replicates were performed for each treatment. The primers used are described in Table S4.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssociation analysis of\u003c/b\u003e \u003cb\u003eZmAPY\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAssociation analysis for \u003cem\u003eZmAPYs\u003c/em\u003e was performed by using a maize association mapping population containing 540 inbred lines in a previous study (Liu et\u0026ensp;al. 2017). Among 1,227,480 high-quality SNP data with minor allele frequency (MAF)\u0026thinsp;\u0026ge;\u0026thinsp;0.05, 502 SNPs were found in the gene region of all 16 \u003cem\u003eZmAPYs\u003c/em\u003e. maize agronomic traits and drought tolerance trait were also collected from previous studies (Yang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The MLM model were chosen to detect the SNPs significantly associated with maize agronomic traits and drought tolerance by using the TASSEL5.0 program (Bradbury et\u0026ensp;al. 2007).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of maize\u003c/b\u003e \u003cb\u003eAPY\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTotally, we identified 16 \u003cem\u003eZmAPY\u003c/em\u003e family members from the Ensembl Plant database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plants.ensembl.org/index.html\u003c/span\u003e\u003cspan address=\"https://plants.ensembl.org/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), named \u003cem\u003eZmAPY1\u003c/em\u003e-\u003cem\u003eZmAPY16\u003c/em\u003e (Table S1). Their physicochemical properties, including gene ID, protein size, molecular weight (MW), isoelectric point (pI), the grand averages of hydropathicity (GRAVY), instability index and and localization prediction, were characterized and shown in Table S1. The ZmAPY proteins varied in length from 81 to 701 amino acids, with molecular weights ranging from 8.74822 kDa to 76.9779 kDa. Isoelectric points ranged from 4.70 to 11.62 acidic. Most ZmAPY proteins were hydrophilic (GRAVY\u0026thinsp;\u0026lt;\u0026thinsp;0) except for ZmAPY6 and ZmAPY7, which were hydrophobic (GRAVY\u0026thinsp;\u0026gt;\u0026thinsp;0). The estimated instability index ranged from 30.91 to 72.05. Subcellular localization predictions indicated that nine ZmAPY proteins were likely localized in the chloroplast, four in the plasma membrane, three in the mitochondria, one in the extracellular space, and one in the nucleus (Table S1), indicating diverse functional roles for these \u003cem\u003eZmAPY\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003eTo explore the phylogenetic relationships of ZmAPYs with other species, a phylogenetic tree was constructed, incorporating 16 ZmAPYs, 7 AtAPYs, and 9 OsAPYs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; Table S2). The phylogenetic tree topology classified these APY proteins into three Groups: Group I, II, III. In addition, there are two APY-like proteins (ZmAPYL1, ZmAPYL2) in maize that do not belong to Group I-III. Therefore, we classify them as an outgroup (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Table S2). The expansion of APYs in maize, compared to AtAPYs and OsAPYs, suggests the potential importance of this gene family to regulate biological processes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDistribution and collinearity analysis of maize\u003c/b\u003e \u003cb\u003eAPY\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate features of the \u003cem\u003eZmAPYs\u003c/em\u003e gene family, we analyzed the chromosome distribution of each \u003cem\u003eZmAPY\u003c/em\u003e gene. Our investigation showed that the \u003cem\u003eZmAPY\u003c/em\u003e genes in the maize genome were unevenly distributed across all 9 chromosomes, with the exception of chromosome 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The number of \u003cem\u003eAPY\u003c/em\u003e genes varied on each chromosome. Specifically, there is a single APY gene located on chromosomes Chr5, Chr6, Chr7, and Chr9. Chromosomes Chr2, Chr3, and Chr10 each contain two APY genes, while chromosomes Chr1 and Chr4 each have three \u003cem\u003eAPY\u003c/em\u003e genes. Gene duplications play a crucial role in the expansion of gene families (Konrad et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Segmental duplications lead to the presence of large repetitive chromosomal blocks in the genome and are often associated with chromosomal rearrangements and polyploid events (Lallemand et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Colinearity analysis indicated the occurrence of three segmental duplication events involving five \u003cem\u003eZmAPY\u003c/em\u003e genes across the maize genome, while no tandem duplications were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAPY\u003c/b\u003e \u003cb\u003egene structures and predicted protein motifs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eStructural differences in exon-intron arrangement serve as sources of gene family variation and species diversity, leading to alterations in gene expression and function. To investigate the conservation and diversity of gene structure within the maize APY gene family, the exons and introns of 16 \u003cem\u003eAPY\u003c/em\u003e genes were analyzed based on their coding sequences and genomic data. The number of exons varied among the APY genes, ranging from 1 to 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Most members contained 6\u0026ndash;10 exons, with two members in Group I (\u003cem\u003eZmAPY5\u003c/em\u003e and \u003cem\u003eZmAPY6\u003c/em\u003e) and one member in Group III (\u003cem\u003eZmAPY13\u003c/em\u003e) having only 1 and 2 exons, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The number of introns ranged from 0 to 9. Two transposon insertions of 9.56 kb and 33.57 kb were identified in the intron regions of \u003cem\u003eZmAPY5\u003c/em\u003e and \u003cem\u003eZmAPY10\u003c/em\u003e, respectively. \u003cem\u003eZmAPY\u003c/em\u003e genes with collinearity exhibited similar gene structures. Additionally, members within the same subgroup typically displayed similar motifs and lengths, suggesting functional similarities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Analysis of the protein sequences of all \u003cem\u003eZmAPY\u003c/em\u003e gene family members revealed a conserved GDA1_CD39 domain in all proteins, with varying numbers of transmembrane regions. Specifically, members of Group I had one transmembrane region, most members of Group II had two transmembrane regions, and family members of Group III had no transmembrane regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This structural variation may contribute to the functional distinctions observed among different subgroups.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression patterns of\u003c/b\u003e \u003cb\u003eAPY\u003c/b\u003e \u003cb\u003egenes in maize\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe investigation into tissue-specific gene expression patterns provides valuable insights into the potential biological roles of the \u003cem\u003eZmAPY\u003c/em\u003e genes. Analysis of the expression patterns within the \u003cem\u003eZmAPY\u003c/em\u003e gene family revealed distinct expression profiles among different members (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), highlighting their diverse functions. Specifically, \u003cem\u003eZmAPY8\u003c/em\u003e, \u003cem\u003eZmAPY9\u003c/em\u003e, and \u003cem\u003eZmAPY12\u003c/em\u003e exhibited specific expression in roots, indicating a potential role for these genes in root-related processes. On the other hand, \u003cem\u003eZmAPY15\u003c/em\u003e and \u003cem\u003eZmAPY16\u003c/em\u003e showed significantly higher expression levels in anthers compared to other tissues, suggesting their involvement in anther-related functions. Moreover, there was a notable trend of high expression of \u003cem\u003eZmAPY11\u003c/em\u003e in seeds and endosperm, implying a potential role in seed development and maturation. Conversely, minimal to no detectable expression was observed for \u003cem\u003eZmAPY3\u003c/em\u003e, \u003cem\u003eZmAPY4\u003c/em\u003e, \u003cem\u003eZmAPY6\u003c/em\u003e, and \u003cem\u003eZmAPY7\u003c/em\u003e across all tissues and organ. Interestingly, similar tissue-specific expression patterns were observed between collinear \u003cem\u003eZmAPY\u003c/em\u003e genes, such as \u003cem\u003eZmAPY1\u003c/em\u003e/\u003cem\u003eZmAPY2\u003c/em\u003e/\u003cem\u003eZmAPY5\u003c/em\u003e and \u003cem\u003eZmAPY15\u003c/em\u003e/\u003cem\u003eZmAPY16\u003c/em\u003e, suggesting potential functional conservation or shared regulatory mechanisms among these gene clusters. The differential tissue-specific expression patterns observed among the \u003cem\u003eZmAPY\u003c/em\u003e genes indicate their diverse biological roles and potential contributions to various developmental processes and physiological functions in maize.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePrediction of upstream regulators of\u003c/b\u003e \u003cb\u003eAPY\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe analysis of variations in expression patterns among different \u003cem\u003eZmAPY\u003c/em\u003e genes has led to the identification of potential upstream regulators that may control \u003cem\u003eAPY\u003c/em\u003e gene transcription in maize. By utilizing planttfdb software and existing ChIP-seq data of 104 transcription factors (Tu et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), a total of 251 upstream regulators were predicted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table. S3). Subsequently, the correlation between the expression levels of these predicted regulators and \u003cem\u003eZmAPY\u003c/em\u003e gene expression was examined. Significantly, NACTF78, ZIM36, bZIP79, and CCHH26 displayed strong correlations with the transcription levels of \u003cem\u003eZmAPY2\u003c/em\u003e (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.51), \u003cem\u003eZmAPY5\u003c/em\u003e (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.56), \u003cem\u003eZmAPY9\u003c/em\u003e (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.67), and \u003cem\u003eZmAPY14\u003c/em\u003e (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.60), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Notably, NACTF78 has been previously reported to regulate Fe concentrations in maize kernels, potentially enabling the cultivation of maize varieties with both high yield and high Fe concentrations in their kernels using a molecular marker in the \u003cem\u003eNACTF78\u003c/em\u003e promoter (Yan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, V\u0026eacute;lez-Berm\u0026uacute;dez et al. reported that ZML2 (ZIM36) regulates wound-induced lignin genes in maize (V\u0026eacute;lez-Berm\u0026uacute;dez et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), while ZmTGA9-1 (bZIP79) has been shown to regulate male sterility in maize (Jiang et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The observed correlations between the expression levels of \u003cem\u003eZmAPY\u003c/em\u003e genes and these transcription factors suggest that \u003cem\u003eZmAPY\u003c/em\u003e genes may also play a role in regulating these biological processes, indicating a potential link between \u003cem\u003eAPY\u003c/em\u003e gene expression and the modulation of Fe concentrations, lignin gene regulation, and male sterility in maize. These findings provide valuable insights into the regulatory network involving \u003cem\u003eZmAPY\u003c/em\u003e genes and their upstream regulators in maize, shedding light on the diverse biological processes influenced by these genes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression analysis of\u003c/b\u003e \u003cb\u003eZmAPYs\u003c/b\u003e \u003cb\u003eunder drought, cold, heat stresses\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the potential role of \u003cem\u003eAPY\u003c/em\u003e genes in regulating maize abiotic stress responses, we analyzed the transcription levels of \u003cem\u003eAPY\u003c/em\u003e genes under drought, cold, and heat stress using RNA-seq data from the maize inbred line B73. Our findings revealed the transcription of the \u003cem\u003eZmAPY\u003c/em\u003e gene is responsive to drought, cold, and heat stress, displaying distinct response profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Subsequently, we validated the transcription of \u003cem\u003eZmAPY\u003c/em\u003e genes under drought, cold, and heat stress through qRT-PCR. The results revealed significant upregulation of 6 \u003cem\u003eZmAPY\u003c/em\u003e genes, \u003cem\u003eZmAPY1\u003c/em\u003e, \u003cem\u003eZmAPY2\u003c/em\u003e, \u003cem\u003eZmAPY8\u003c/em\u003e, \u003cem\u003eZmAPY13, ZmAPY14\u003c/em\u003e and \u003cem\u003eZmAPY15\u003c/em\u003e under severe drought (DT4), while the transcription of \u003cem\u003eZmAPY15\u003c/em\u003e was suppressed by drought (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Cold stress induced the transcription of \u003cem\u003eZmAPY1\u003c/em\u003e but inhibited the transcription of \u003cem\u003eZmAPY5\u003c/em\u003e, \u003cem\u003eZmAPY8\u003c/em\u003e, \u003cem\u003eZmAPY11\u003c/em\u003e, \u003cem\u003eZmAPY13\u003c/em\u003e and \u003cem\u003eZmAPY15\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Moreover, heat stress significantly inhibited the transcription of 7 \u003cem\u003eZmAPY\u003c/em\u003e genes, \u003cem\u003eZmAPY1\u003c/em\u003e, \u003cem\u003eZmAPY2\u003c/em\u003e, \u003cem\u003eZmAPY5\u003c/em\u003e, \u003cem\u003eZmAPY8\u003c/em\u003e, \u003cem\u003eZmAPY11\u003c/em\u003e, \u003cem\u003eZmAPY14\u003c/em\u003e, and \u003cem\u003eZmAPY16\u003c/em\u003e, while the transcription of \u003cem\u003eZmAPY15\u003c/em\u003e was induced by heat (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). It is noteworthy that some of the expression analysis results for the \u003cem\u003eZmAPY\u003c/em\u003e genes are absent from Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC due to their expression levels falling below the detection threshold of qRT-PCR. The stress-responsive expression patterns of \u003cem\u003eZmAPY\u003c/em\u003e genes suggest their potential regulatory roles in drought, cold, heat, and salt stress responses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eZmAPYs\u003c/b\u003e \u003cb\u003ewas associated with agronomic traits and drought resistance of maize\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further investigate the impact of \u003cem\u003eAPY\u003c/em\u003e on maize agronomic traits and drought resistance, we examined the relationship between SNPs in the \u003cem\u003eZmAPY\u003c/em\u003e gene region and 17 agronomic traits as well as drought phenotypes using the MLM model. Our analysis revealed a significant association between the SNP (chr9_124751424, CC/TT) in \u003cem\u003eZmAPY16\u003c/em\u003e and maize plant height (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequent analysis indicated notable differences not only in plant height but also in ear height, drought resistance, and \u003cem\u003eZmAPY16\u003c/em\u003e expression between the \"CC\" and \"TT\" genotypes. Plants with the \"TT\" allele, showing high \u003cem\u003eZmAPY6\u003c/em\u003e expression, exhibited greater plant height, ear height, and drought survival rates compared to those with the \"CC\" allele (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-E), suggesting a positive regulatory role of \u003cem\u003eZmAPY16\u003c/em\u003e in maize plant height, ear height, and drought resistance. Additionally, we observed a significant association between the SNP (chr1_54070081, CC/TT) in \u003cem\u003eZmAPY5\u003c/em\u003e and the spinemaking period in maize (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Plants with the \"TT\" allele and high \u003cem\u003eZmAPY5\u003c/em\u003e expression displayed a delayed spinemaking period and enhanced drought survival rates compared to plants with the \"CC\" allele and low \u003cem\u003eZmAPY5\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-I), indicating a negative regulation of maize spinemaking and a positive regulation of maize drought resistance by \u003cem\u003eZmAPY5\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic variation within\u003c/b\u003e \u003cb\u003eZmAPYs\u003c/b\u003e \u003cb\u003eregulate the content of drought-induced metabolites\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMetabolites, as small molecules that serve as the end products of metabolic processes and physiological pathways, are known to play crucial roles in plant drought resistance (Kim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Todaka et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These compounds can act as osmoprotectants, antioxidants, signaling molecules, and regulators of various stress-responsive pathways in plants (Nakabayashi et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Obata et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; F\u0026agrave;bregas et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Analyzing the genome-wide metabolite profiles of 385 maize natural inbred lines grown under well-watered and drought-stressed conditions (Zhang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e), we identified metabolite quantitative trait loci (mQTL) for 18 metabolites that co-located with the \u003cem\u003eZmAPY\u003c/em\u003e genes, indicating a potential relationship between \u003cem\u003eZmAPYs\u003c/em\u003e and the levels of these metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Further investigation into these metabolites revealed that four drought-induced metabolites were influenced by genetic variations within the \u003cem\u003eZmAPYs\u003c/em\u003e gene region. Specifically, genetic variations chr1.S_65348503 and chr1.S_65352471 within the \u003cem\u003eZmAPY15\u003c/em\u003e gene region were found to regulate the contents of metabolites PN_group_05106 and PN_group_17082, while another genetic variation chr1.S_65352471 within the \u003cem\u003eZmAPY11\u003c/em\u003e gene region was associated with the regulation of the metabolite PN_group_00505. Additionally, a genetic variation chr10.S_71730551 within the \u003cem\u003eZmAPY7\u003c/em\u003e gene region was linked to the regulation of the metabolite PN_group_11670 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). These findings suggest that \u003cem\u003eZmAPY\u003c/em\u003e genes may impact maize drought resistance by modulating the contents of these drought-induced metabolites. This insight highlights the potential role of \u003cem\u003eZmAPY\u003c/em\u003e genes in mediating maize response to drought stress through the regulation of key metabolites involved in stress adaptation and tolerance.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eApyrase (APY) is widely existed in eukaryotes and is highly conserved throughout the evolution of eukaryotes. In plants, apyrase genes have been reported to regulate a variety of biological processes, including root hair development, stomatal movement, and defense responses (Clark et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, no apyrase genes have been identified in maize, and their functions remain unclear. In this study, through sequence alignments and phylogenetic analysis, we identified 16 apyrase genes in maize genome and further divided these genes into three subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The number of group I \u003cem\u003eZmAPY\u003c/em\u003e genes far exceeds the number of group I apyrase genes in \u003cem\u003eArabidopsis\u003c/em\u003e and rice that identified in a previous study (Clark et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Chowdhury et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Syntenic analysis revealed that most \u003cem\u003eZmAPY\u003c/em\u003e genes are of the dispersed repeat type, but segmental duplication also played a significant role in the expansion process of group I \u003cem\u003eZmAPY\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Meanwhile, genes with synteny show higher similarity in expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results suggest that \u003cem\u003eAPY\u003c/em\u003e genes in maize may have produced functional redundancy through expansion, and since apyrase plays an important regulatory role in plant growth and development and stress responses, the expansion and functional redundancy of gene subgroups generated by segmental and dispersed duplication may enhance the robustness of the maize regulatory network.\u003c/p\u003e \u003cp\u003eThe members of the \u003cem\u003eZmAPY\u003c/em\u003e genes are predicted to encode proteins ranging from 81 to 701 amino acid in length, and the PI ranging from 4.70 to 11.62 (Table S1). These results suggest that the differences among \u003cem\u003eZmAPY\u003c/em\u003e genes may be more pronounced than those in rice (in which OsAPY proteins ranging from 451 to 702 amino acid in length, and the PI ranging from 5.44 to 9.34) (Chowdhury et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The predicted gene structures of the \u003cem\u003eZmAPY\u003c/em\u003e genes contain one exon to ten exons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), similar to the gene structures in rice, with two to twelve exons (Chowdhury et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similar to OsAPY proteins, the \u003cem\u003eZmAPY\u003c/em\u003e proteins were predicted to localize to various cellular compartments, including the chloroplast, mitochondrial, plasma membrane, cytoplasm, nuclear and extracellular (Table S1). In summary, \u003cem\u003eZmAPY\u003c/em\u003e genes share similar characteristics with apyrase in rice, but exhibits greater diversity among family members, suggesting that although \u003cem\u003eZmAPY\u003c/em\u003e genes maintain functional conservation, they may also have undergone functional divergence.\u003c/p\u003e \u003cp\u003eTranscription factors are core elements that regulate transcriptional levels during various stages of plant life processes. Analysis of the promoter regions of all \u003cem\u003eZmAPY\u003c/em\u003e genes revealed the presence of diverse upstream transcription factors which may regulate \u003cem\u003eZmAPY\u003c/em\u003e genes expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Among the 251 potential upstream transcription factors, several genes have been reported to regulate maize growth and stress response progresses (Table S3). For example, WRKY48 / ZmWRKY40 was predicted to binding the promoter sequences of seven \u003cem\u003eZmAPY\u003c/em\u003e genes, overexpression \u003cem\u003eZmWRKY40\u003c/em\u003e could enhance drought tolerance in transgenic Arabidopsis by regulating stress-related genes, and the reactive oxygen species (ROS) content in transgenic lines was reduced compared with wild-type plants under drought stress (Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). ZmBES1/BZR1-5 was predicted to bind the promoter sequences of six \u003cem\u003eZmAPY\u003c/em\u003e genes, ZmBES1/BZR1-5 decreases ABA sensitivity and confers tolerance to osmotic stress in transgenic \u003cem\u003eArabidopsis.\u003c/em\u003e Meanwhile, ZmBES1/BZR1-5 can also positively regulates kernel size (Sun et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A bZIP transcription factor id1 was predicted to binding the promoter sequences of ten \u003cem\u003eZmAPY\u003c/em\u003e genes, previous study showed that id1 can regulate maize flowering time and floral inductive signals (Colasanti et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Muszynski et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). These results further suggested potential roles of \u003cem\u003eZmAPY\u003c/em\u003e genes in maize growth and stress response progresses.\u003c/p\u003e \u003cp\u003eMaize is a world major food crop, and its yield is seriously threatened by abiotic stresses (Lesk et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Although a large number of abiotic stress response genes and loci have been identified in recent years through genetic and molecular biology techniques, due to limited understanding of the plant abiotic stress response system, only a few genes have been successfully applied in commercialization (Zhang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, it remains essential to further elucidate the abiotic stress response system of maize. Previous studies have shown that the extracellular ATP (eATP) level rises when plants are subjected to stress, leading to the production of reactive oxygen species (ROS) and cell death. Apyrase can regulate the production of ROS by hydrolyzing eATP, and participate in various stress response processes in plants (Deng et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). By analyzing the transcriptomic data from previous studies, we investigated the expression patterns of \u003cem\u003eZmAPY\u003c/em\u003e genes under different abiotic stresses (Makarevitch et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The results showed that seven \u003cem\u003eZmAPY\u003c/em\u003e genes responded to drought stress, six to cold stress, and eight to heat stress, and the stress responsive \u003cem\u003eZmAPY\u003c/em\u003e genes were further confirmed by qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results indicated that, like other plants, \u003cem\u003eZmAPY\u003c/em\u003e genes could also be involved in regulating maize's abiotic stress response. Of course, more researches are required to clearly elucidate the function of \u003cem\u003eZmAPY\u003c/em\u003e genes in maize abiotic stress responses.\u003c/p\u003e \u003cp\u003eNatural variations significantly affect maize agronomic traits and stress resistance, and numerous elite alleles have been identified through genetic approaches which significantly accelerated the breeding process (Xiao et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). To explore the potential application value of \u003cem\u003eZmAPY\u003c/em\u003e genes and their genetic variations in maize breeding, we collected drought tolerance and agronomic traits data of maize association panels published in previous study (Zhang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and utilized high-density SNP markers to perform association analyses on the \u003cem\u003eZmAPY\u003c/em\u003e gene and its flanking regions. The result indicated that natural variations in \u003cem\u003eZmAPY16\u003c/em\u003e gene and flanking region were significantly associated with maize plant height (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), further analysis revealed that these variations were also correlated with the \u003cem\u003eZmAPY16\u003c/em\u003e expression and maize drought tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-E). Additionally, we found that \u003cem\u003eZmAPY5\u003c/em\u003e was significantly associated with maize silking time (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), and associated variations were also correlated with the \u003cem\u003eZmAPY5\u003c/em\u003e expression and maize drought tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-I). These findings suggest that variations of \u003cem\u003eZmAPY\u003c/em\u003e gene expression may influence maize agronomic traits and drought tolerance. Furthermore, since many metabolic pathways involve energy conversion, while apyrase can affect the levels of ATP/ADP. To investigate the effects of apyrase on maize metabolites, we performed co-localization analysis between \u003cem\u003eZmAPY\u003c/em\u003e variations and previously reported metabolite regulation sites (Zhang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e), which revealed that seven \u003cem\u003eZmAPY\u003c/em\u003e genes were co-localized with 18 metabolite regulation sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Some of these metabolites showed significant content change under drought stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Although the functions of these metabolites are not well understood at present, these results suggest that \u003cem\u003eZmAPY\u003c/em\u003e genes and their genetic variations may participate in maize drought response by affecting the levels of drought-responsive metabolites. In summary, we revealed the variations of \u003cem\u003eZmAPY\u003c/em\u003e gene expression among maize populations, which were associated with multiple important maize traits. These results indicate that \u003cem\u003eZmAPYs\u003c/em\u003e and their genetic variations may have potential applications in maize breeding.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Key Technologies Research and Development Program, China (2022YFE0100500), The National Natural Science Foundation of China (31971954, 32061143031).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhenhua He and Jie Zhang contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHubei Key Laboratory of Food Crop Germplasm and Genetic Improvement \u0026amp; Key Laboratory of Crop Molecular Breeding, Ministry of Agriculture and Rural Affairs, Food Crops Institute, Hubei Academy of Agricultural Sciences, Wuhan 430064, China\u003c/p\u003e\n\u003cp\u003eZhenhua He, Haitao Jia, Shilong Zhang \u0026amp; Xiaopeng Sun\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJie Zhang, Hui Zhang \u0026amp; Mingqiu Dai\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHubei Hongshan Laboratory, Wuhan 430070, China.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJie Zhang, Xiaopeng Sun, Hui Zhang \u0026amp; Mingqiu Dai\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMD, HZ, ZH and XS conceived and designed the research. ZH and JZ performed the experiments and collected the data. HZ and XS supervised the experiments. HZ and XS wrote the manuscript. MD modified manuscript. HJ and SZ polished the manuscript and images. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Mingqiu Dai or Hui Zhang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that these experiments comply with the ethical standards in China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026apos;s Note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlam I, Lee DG, Kim KH, Park CH, Sharmin SA, Lee H, Oh KW, Yun BW, Lee BH (2010) Proteome analysis of soybean roots under waterlogging stress at an early vegetative stage. 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Int J Mol Sci 22:9892\u003c/span\u003e\u003c/li\u003e\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-breeding","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"molb","sideBox":"Learn more about [Molecular Breeding](https://www.springer.com/journal/11032)","snPcode":"11032","submissionUrl":"https://submission.nature.com/new-submission/11032/3","title":"Molecular Breeding","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"maize, apyrase, abiotic stress response, association analysis, metabolic","lastPublishedDoi":"10.21203/rs.3.rs-4222821/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4222821/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eApyrase is a class of enzyme that catalyzes the hydrolysis of nucleoside triphosphates/diphosphates (NTP/NDP), which widely involved in regulation of plant growth and stress responses. However, apyrase family genes in maize have not been identified, and their characteristics and functions are largely unknown. In this study, we identified 16 apyrases (named as \u003cem\u003eZmAPY-ZmAPY16\u003c/em\u003e) in maize genome, and analyzed their phylogenetic relationships, gene structures, chromosomal distribution, upstream regulatory transcription factors and expression patterns. Analysis of the transcriptome database unveiled tissue-specific and abiotic stress-responsive expression of \u003cem\u003eZmAPY\u003c/em\u003e genes in maize. qPCR analysis further confirmed their responsiveness to drought, heat, and cold stresses. Association analyses indicated that variations of \u003cem\u003eZmAPY\u003c/em\u003e genes may regulate maize agronomic traits and drought responses. Our findings shed light on the molecular characteristics and evolutionary history of maize apyrase genes, highlighting their roles in various biological processes and stress responses. This study forms a basis for further exploration of apyrase functions in maize.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification and analyses of ZmAPY genes reveal their roles involved in maize development and abiotic stress responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-15 11:59:01","doi":"10.21203/rs.3.rs-4222821/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-11T03:39:30+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-11T03:31:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-11T00:50:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Breeding","date":"2024-04-10T03:20:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-breeding","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"molb","sideBox":"Learn more about [Molecular Breeding](https://www.springer.com/journal/11032)","snPcode":"11032","submissionUrl":"https://submission.nature.com/new-submission/11032/3","title":"Molecular Breeding","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c4cc9596-f2be-4cc0-a719-1850f647d1bc","owner":[],"postedDate":"April 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-14T00:40:15+00:00","versionOfRecord":{"articleIdentity":"rs-4222821","link":"https://doi.org/10.1007/s11032-024-01474-9","journal":{"identity":"molecular-breeding","isVorOnly":false,"title":"Molecular Breeding"},"publishedOn":"2024-05-01 00:40:15","publishedOnDateReadable":"May 1st, 2024"},"versionCreatedAt":"2024-04-15 11:59:01","video":"","vorDoi":"10.1007/s11032-024-01474-9","vorDoiUrl":"https://doi.org/10.1007/s11032-024-01474-9","workflowStages":[]},"version":"v1","identity":"rs-4222821","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4222821","identity":"rs-4222821","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}