Genome-wide identification and analysis of the ZCN gene family in maize (Zea mays L.)

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Yong Yang, Mengyang Wu, Jinjie Zhu, Zhaoxu Gao, Xiantao Qi, Chuanxiao Xie, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7400912/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background The development and growth of floral organs serve as a hallmark of the transition from vegetative to reproductive growth in plants. Phosphatidylethanolamine-binding proteins (PEBPs) play a critical regulatory role in this transition, additionally influencing the morphological structure of inflorescences. In maize ( Zea mays L.), PEBP proteins are encoded by the ZCN (Zea mays CENTRORADIALIS) gene family. However, the functional characteristics of this gene family remain poorly characterized. Results This study systematically identified and cloned a total of 25 ZCN gene family members. Phylogenetic analysis divided the ZCN gene family into four subclades: FT-like , TFL1-like , MFT-like , and PEBP-like . Comprehensive multi-dimensional analyses of gene structures, promoter cis-acting elements, tissue-specific and abiotic stress expression patterns of ZCN family members revealed their functional diversity, indicating that ZCN genes play crucial roles in maize growth and development. Subcellular localization results in maize protoplasts showed that ZCN7, ZCN8, ZCN17, ZCN18, ZCN21, ZCN24, and ZCN26 were exclusively localized in the nucleus. Additionally, nuclear localization signal sequences of ZCN17 , ZCN20 , and ZCN25 were identified as GRRYIR/GRRYR. Targeted editing of all ZCN members led to the generation of maize mutant lines with significantly increased tassel branch numbers and remarkably delayed flowering times under field conditions. Among these, ZCN3 , ZCN17 , ZCN20 , and ZCN25 were identified as key ZCN genes exhibiting significant regulatory effects on tassel branch number. Conclusions This study systematically identified 25 ZCN family members in the maize genome and comprehensively analyzed their gene structure, physicochemical properties, evolutionary relationships, expression patterns, and responses to abiotic stresses. Our results not only clarified the structural evolution and regulatory features of the ZCN gene family but also highlighted the functional roles of key genes. These findings have significantly advanced our understanding of the ZCN gene family in maize and provided genetic resources for maize molecular breeding. Maize ZCN Gene Family Genome-Wide Identification Gene Editing Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The development of floral organs mark a hallmark of the transition from vegetative to reproductive growth in plants. The PEBP (PHOSPHATIDYLETHANOLAMINE-BINDING PROTEIN) gene family encodes proteins with a highly conserved PEBP domain—so named for its ability to bind phosphatidylethanolamine [ 1 ]. This domain plays a critical regulatory role in the vegetative-to-reproductive transition and modulates diverse biological processes, including plant morphological traits [ 2 ], flowering time, and periodic gene expression patterns [ 3 ]. PEBP proteins are ubiquitously distributed across plant species [ 4 ]. In flowering plants, the PEBP gene family can be divided into three subclades, which are FLOWERING LOCUS T (FT)-like, TERMINAL FLOWER 1 (TFL1)-like, and MOTHER OF FT AND TFL1 (MFT)-like, based on phylogenetic evolution and functional divergence [ 5 ]. Members of the PEBP gene family play regulatory roles in diverse physiological processes in plants, including FT- like and TFL1 -like subclade genes participating in the regulation of flowering time [ 3 ], and MFT- like subclade genes involvement in seed dormancy and germination regulation [ 6 ]. In Arabidopsis thaliana , protein FT and TFL1 in leaves compete for binding to the bZIP transcription factor FD protein (FLOWERING LOCUS D), thereby regulating flowering [ 7 ]; rice FT-like genes RFT1 (Rice Flowering Locus T1) and Hd3a (Heading date-3a) also play inductive roles in flowering [ 8 ]; soybean GmMFT [ 9 ] and two cotton MFT genes [ 10 ] inhibit seed germination via ABA and GA pathways. Notably, unique PEBP-like subclade members have been identified in crops such as cotton and wheat, with their transcript expression showing tissue specificity. For example, wheat TaPEBP1 is highly abundant during young spike development, yet its functional mechanisms remain unclear. This subclade may represent a new evolutionary lineage of plants adapted to specialized agronomic environments (Zhang et al., 2016; Dong et al., 2020). In maize ( Zea mays L.), the genes responsible for encoding PEBP proteins are named ZCN (Zea mays CENTRORADIALIS). Their functions not only contain regulating flowering time and seed dormancy but also potentially involve plant adaptability to environmental changes and fine-tuned regulation of growth and development [ 13 ]. For example, (ⅰ) ZCN8 , a PEBP gene highly homologous to AtFT , interacts with DLF1 to promote ZMM4 expression, thereby inducing the transition from vegetative growth to reproductive growth [ 14 ]; a SNP variant site in the ZCN8 promoter region is significantly associated with maize flowering time, and this variant affects the binding affinity of ZmMADS1 to the ZCN8 promoter, thereby controlling flowering time [ 15 ]; (ⅱ) ZCN1 inhibits the activity of the ZCN8-DLF1 complex to suppress flowering, whereas ZCN2 and ZCN5 enhance the inductive effect of ZCN8 to positively regulate maize flowering [ 13 ]; (ⅲ) ZCN4 promotes cell division and positively regulates bract width [ 16 ]; (ⅳ) ZCN7 positively regulates maize drought tolerance—its overexpression shortens the anthesis-silking interval under drought, thereby increasing yield [ 17 ]; (ⅴ) ZCN9/10 negatively regulate seed vigor, participate in the abscisic acid (ABA) signaling pathway, and reduce seed tolerance to ABA [ 18 ]. Maize as a globally important food crop, has floral organ development that significantly influences both yield and quality. Despite the established importance of the ZCN gene family, a comprehensive analysis of its functions in maize remains lacking. This study employed bioinformatics approaches to identify members of the ZCN gene family in the maize genome, analyzed their phylogenetic evolution, structural characteristics, expression patterns, and provided a reference for elucidating the regulatory mechanisms of maize flower development and the biological functions of ZCN genes. Additionally, we explored the functional differentiation of the ZCN gene family in environmental adaptability. These findings lay a foundation for further investigating the functions of ZCN genes and their specific roles in maize growth and development. Methods Genome-wide Identification and Biochemical Property Analysis of the ZCN Gene Family in Maize Firstly, we retrieved Hidden Markov model (HMM) files of ZCN protein domains (PF01161) from the Pfam database ( http://pfam.xfam.org/ )[ 19 ]. Next, we used HMMER 3.4 to generate ZCN protein sequence files for maize. We then accessed the NCBI database ( https://www.ncbi.nlm.nih.gov/ ) to further identify protein domains associated with candidate genes and obtained the amino acid sequences of ZCN . Using ExPASy tools (ProtParam and ProtScale; https://web.expasy.org/protparam/ )[ 20 ], we analyzed the amino acid length, molecular weight, isoelectric point (pI), and other relevant properties of candidate ZCN gene family members. The potential nuclear localization signals (NLS) of proteins were predicted via DeepLoc 2.1 ( https://services.healthtech.dtu.dk/services/DeepLoc-2.1 ) [ 21 ]. Chromosome length and position data corresponding to ZCN gene information were visualized using TBtools software[ 22 , 23 ]. We downloaded PEBP protein sequences of Arabidopsis thaliana , rice, and wheat from the EnsemblPlants database ( https://plants.ensembl.org/index.html )[ 24 ]. Multiple sequence alignment and phylogenetic tree construction were performed using MEGA 11, and the phylogenetic tree was visualized using iTOL. Subsequently, we extracted 2000 bp of sequence upstream of the transcription start site (TSS) of ZCN family genes in maize using TBtools software and predicted cis-acting elements in the promoter regions of ZCN gene family members via the Plant CARE tool ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). Finally, we used qTeller ( https://qteller.maizegdb.org/ ) to investigate and collect data on ZCN gene expression levels in different maize tissues and their responses to various abiotic stress conditions. Subcellular Localization of the ZCN Gene Family in Maize The coding sequences (CDS) of all ZCN gene family members were inserted into a CUB vector containing enhanced green fluorescent protein (eGFP) to construct the plasmid CUB- ZCN -eGFP. Protoplasts were isolated from the leaves of maize B73 seedlings. A total of 100 µg of CUB -ZCN -eGFP plasmid was transfected into 1 mL of maize protoplasts (approximately 5×10⁵ cells/mL) using PEG4000-mediated transfection. After culturing the protoplasts in the dark at 25°C for 16 hours, fluorescence signals were observed using a ZEISS LSM900 microscope. sgRNA Design and Genome Editing To construct a CRISPR/Cas9 mutant library for the ZCN gene family, we designed 25 sgRNAs targeting ZCN genes using CRISPR-P 2.0 software ( http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR )[ 25 ]. The "tRNA-sgRNA-scaffold" fusion sequences were separately ligated into two vectors to construct gene editing plasmids for the ZCN gene family[ 26 ]. The constructed plasmids were sent to Weimi Biotechnology Co., Ltd. (Changzhou, China) for transformation of the maize inbred line KN5585. Field Phenotypic Statistics Field trials were conducted in maize fields under natural long-day conditions (Beijing) and natural short-day conditions (Sanya). The flowering time was statistically calculated as the number of days from seedling emergence to when > 50% of plants in a row reached anthesis, silking, and pollen shedding. Agronomic traits and yield of wild-type and mutant plants were investigated 15–20 days after pollination. Maize tassel branch number investigations involve counting primary, secondary, and total branches. Primary tassel branches are lateral branches originating from the main axis of the maize inflorescence; secondary branches are sub-branches generated on these lateral branches; and total branch number is the sum of primary and secondary branches[ 27 ]. Plant height was measured as the vertical distance from the ground to the tip of the tassel main axis; ear height was measured as the vertical distance from the ground to the attachment site of the first effective ear shank; the number of leaves above the ear was counted as the leaves persisting between the first effective ear and the tassel; the number of leaves below the ear was counted as the leaves persisting between the first effective ear and the first leaf above the ground; ear length was measured as the distance from the base to the tip of the ear after removing the shank; 100-grain weight was determined by weighing 100 randomly selected intact, disease-free grains; and each group was replicated three times. Statistical Analysis Significant differences were assessed using a two-tailed Student's t -test in Microsoft® Excel 2019. Results with a P -value < 0.05 were considered statistically significant. Figures were prepared using GraphPad Prism (v8.0). Results Genome-wide Identification and Physicochemical Property Analysis of the ZCN Gene Family A total of 25 members of the ZCN gene family were identified in the maize genome (Table 1 ), among which ZCN22 and ZCN23 were classified as pseudogenes, and two newly identified members— ZCN26 and a PEBP-like gene ZCNPEBP —were added. These 25 members exhibited a relatively dispersed pattern in the maize genome, being unequally distributed across 10 maize chromosomes, with no distribution on chromosome 1 (Fig. 1 a). Physicochemical properties revealed that the number of amino acids in ZCN proteins ranged from 172 (ZCN9, ZCN10) to 192 (ZCN7), their relative molecular masses ranged from 15.078 kDa (ZCN3) to 21.935 kDa (ZCN7), and their isoelectric points (pI) ranged from 5.52 (ZCNPEBP) to 9.94 (ZCN11). Except for ZCN11, the grand average of hydropathy (GRAVY) values of all other members were less than 0, indicating that they are all hydrophilic proteins. To gain a deeper understanding of the structure and function of ZCN proteins, the signal peptides of all ZCN proteins were predicted. The results showed that none of the ZCN proteins contained signal peptides, indicating that they are unlikely to be secreted via the classical secretory pathway. Table 1 Information of ZCN gene family in maize Gene name ID Chromosome Number of amino acid (aa) Molecular weight (KDa) Theoretical pI ZCN1 Zm00001d044704 3 173 19.60 8.47 ZCN2 Zm00001d050649 4 173 19.51 9.47 ZCN3 Zm00001d023419 10 173 19.61 9.82 ZCN4 Zm00001d003804 2 176 19.77 9.68 ZCN5 Zm00001d025346 10 173 19.64 9.01 ZCN6 Zm00001d052537 4 177 19.88 9.15 ZCN7 Zm00001d038725 6 192 21.93 9.13 ZCN8 Zm00001d010752 8 175 19.99 8.74 ZCN9 Zm00001d008446 8 172 18.88 9.73 ZCN10 Zm00001d040113 3 172 18.90 7.16 ZCN11 Zm00001d037439 6 180 19.19 9.94 ZCN12 Zm00001d043461 3 177 19.89 8.71 ZCN13 Zm00001d016253 5 184 20.36 8.46 ZCN14 Zm00001d008939 8 173 19.31 7.34 ZCN15 Zm00001d036242 6 177 19.77 8.05 ZCN16 Zm00001d017134 5 174 19.81 8.18 ZCN17 Zm00001d004630 2 178 19.94 8.68 ZCN18 Zm00001d006116 2 174 19.31 6.94 ZCN19 Zm00001d025737 10 175 19.66 8.21 ZCN20 Zm00001d024023 10 175 19.17 8.31 ZCN21 Zm00001d004010 2 187 20.63 7.62 ZCN24 Zm00001d021135 7 173 19.43 8.42 ZCN25 Zm00001d003226 2 174 19.65 8.65 ZCN26 Zm00001d046300 9 187 20.83 7.57 ZCNPEBP Zm00001d010586 8 174 18.73 5.52 Phylogenetic and Structural Analysis of the ZCN Gene Family To further clarify the evolutionary relationships of the maize ZCN gene family, we constructed a phylogenetic tree using full-length sequences of PEBP proteins from Arabidopsis thaliana (4), rice (19), and wheat (78) (Fig. 1 b). The results indicated that all PEBP proteins from these four species clustered into four distinct subclades: FT-like, TFL1-like, MFT-like, and PEBP-like, suggesting that these subclades existed prior to the divergence of monocotyledons and dicotyledons. Additionally, compared with Arabidopsis, ZCN proteins in maize were more closely related to PEBP proteins in rice and wheat, which is consistent with the evolutionary relationships among Arabidopsis, wheat, rice, and maize. Analyses of the conserved structures and exon-intron organizations of ZCN proteins were also performed (Fig. 1 c–d). Six conserved motifs were detected (Fig. 1 e), with motifs 1 and 6 almost universally present across all ZCN proteins. Exon-intron structure analysis further revealed high structural conservation in ZCN genes: members of each of the four subclades exhibited nearly identical numbers and distribution patterns of exons and introns, showing little difference from the Arabidopsis PEBP gene family. These structural conservations suggest that the ZCN gene family likely underwent strong purifying selection during evolution, with the precise splicing requirements of the PEBP functional domain driving the stable evolution of its gene structure. Regulatory and Expression Pattern Analysis of the ZCN Gene Family To further investigate the functions of ZCN genes, we analyzed the cis-acting elements within the 2000 bp promoter regions upstream of maize ZCN genes. As shown in Fig. 2 , these elements were broadly categorized into four classes based on their functions: developmental regulation, light signal response, hormone signal response, and abiotic stress signal response. Almost all ZCN genes contained light-responsive elements, which were diverse in type. Next were elements related to hormone and abiotic stress responses, while those associated with plant development were fewer in number but diverse in type, including elements for endosperm development regulation (GCN4 motif), seed germination regulation (RY-element), cell cycle regulation (MSA-like), meristem expression regulation (CAT-box), and circadian rhythm regulation (Circadian rhythm). We also analyzed the expression patterns of ZCN family members across different plant tissues and under abiotic stress conditions. Among the 25 maize ZCN genes, not all were highly expressed: ZCN6 and ZCN13 were expressed at extremely low levels, ZCN21 was undetectable in all tissues, and the remaining 22 genes were expressed at relatively high levels. Nearly all members were highly expressed in stems and leaves, consistent with previous reports that florigen genes are expressed in leaves and stems and transported to the apical meristem to exert their functions [ 14 ]. Interestingly, expression levels varied across organs: for example, ZCN3 was highly expressed in stems, meristems, and male flowers; ZCN25 was highly expressed in stems and male flowers; and ZCN9 , ZCN10 , ZCN11 , and ZCNPEBP were expressed at significantly higher levels in embryos and endosperms than other members. These findings indicate that the ZCN gene family is functionally diverse and plays an indispensable role during maize growth and development. Under five abiotic stress conditions (low temperature, high temperature, salt, UV radiation, and drought), the expression patterns of ZCN genes revealed inconsistent responses to stress among family members. Seven genes— ZCN2 , ZCN12 , ZCN13 , ZCN15 , ZCN17 , ZCN20 , and ZCN21 —were undetectable under all stresses, suggesting they are unlikely to be involved in maize’s response to abiotic stresses. In contrast, nearly all members were sensitive to drought stress, with ZCN4 , ZCN7 , ZCN14 , and ZCN16 exhibiting particularly high expression under drought. ZCN1 , ZCN25 , and ZCN26 were expressed under all stress conditions, implying they may possess the ability to respond to a broad spectrum of stresses. This could enable them to coordinate multiple stress signaling pathways and enhance plant adaptability to environmental challenges. Subcellular Localization of the ZCN Gene Family We performed subcellular localization of all ZCN gene family members in maize protoplasts to determine their expression sites. As shown in Fig. 3 a, green fluorescence signals of certain members (e.g., ZCN7 , ZCN8 , ZCN17 , ZCN18 , ZCN21 , ZCN24 , and ZCN26 ) were exclusively localized in the nucleus, whereas those of other family members were distributed across both the nucleus and cytoplasm. These results revealed significant functional differentiation within the ZCN gene family: most ZCN proteins ( ZCN1-6 , 9–16 , 19–20 , 25 , and ZCNPEBP ) exhibited dual localization in both the nucleus and cytoplasm. These proteins likely regulate gene expression dynamically via nucleocytoplasmic shuttling, responding to diverse external signals to maintain normal plant growth and development. In contrast, ZCN7 , ZCN8 , and other members specifically localized to the nucleus indicate their functions are entirely dependent on the nuclear environment. These findings provide direct subcellular-level evidence for functional partitioning of the maize ZCN gene family and suggest spatial segregation as a mechanism underlying their functional differentiation. We selected ZCN17 (nuclear localization) and ZCN20/ZCN25 (nucleocytoplasmic colocalization) from the family for NLS prediction and identified conserved monopartite NLS motifs (GRRYIR/GRRYR) in their protein sequences. Upon removal of these NLSs, the proteins were exclusively localized to the cytoplasm (Fig. 3 b–c). ZCN Genes Regulate Male Floral Organ Development and Reproductive Phase Transition in Maize Using the CRISPR/Cas9 system, we designed single guide RNAs (sgRNAs) targeting these 25 ZCN genes (Fig. 1 c, Appendix S1) and achieved systematic knockout of the ZCN gene family (Appendix S3), generating ZCN gene family knockout mutants. During the growth period, compared with the wild type (KN5585), we observed morphological and developmental differences in the male flowers of some mutants (Fig. 4 d-e). Sequencing confirmed that these mutants were quadruple homozygous mutants of ZCN3/17/20/25 (Fig. 4 b). These mutants exhibited increased tassel branch number, delayed male flowering time, with an average delay of 3.9 days in anthesis, 4.3 days in silking, and 6.9 days in pollen shedding (Fig. 4 g-h). At the T 3 generation, agronomic traits (including plant height) and yield of the quadruple mutants were investigated. Results showed that compared with the wild type, the mutants exhibited no significant changes in most agronomic traits except for an average increase of 2.3 cm in ear height (Fig. 4 c). Discussion Danilevskaya previously identified 23 maize PEBP genes, but this was limited by the absence of complete maize genomic sequences in the EST database at the time [ 13 ]. By leveraging newly released maize genomic databases alongside those of Arabidopsis thaliana, rice, and wheat (four species spanning monocots and dicots), we performed a more rigorous search. This led to the systematic identification and evolutionary analysis of the PEBP gene family, resulting in the discovery of two novel members: ZCN26 and ZCNPEBP . As a highly domesticated monocot crop, maize exhibits explosive expansion of its FT subfamily, which may be closely linked to unique traits in monocots such as sexual organ differentiation, photoperiod adaptation, and agronomic trait domestication. Meanwhile, the moderate expansion of the MFT and TFL1 subfamilies likely reflects their adaptive diversification in conserved functions like seed development and meristem activity. This divergent pattern underscores how the PEBP gene family balances conserved regulatory demands with environmental adaptability through functional differentiation and non-coding sequence-driven gene duplication. Our findings suggest that the ZCN gene family operates via a multi-dimensional regulatory network. Promoter element analysis revealed a high enrichment of light-responsive elements (33.4%), indicating that ZCN genes mediate photoperiod-dependent rhythmic expression through the phytochrome/cryptochrome system [ 28 ]—a conserved mechanism shared with AtFT/AtTFL1 in flowering regulation[ 29 ]. Concurrently, the co-distribution of hormone-responsive elements (e.g., TGA and ABRE) and stress-responsive elements (e.g., MYB and WRKY binding sites) suggests ZCN genes dynamically couple environmental adaptation with developmental programs by integrating ABA, ROS, and other signaling pathways [ 30 ]. Although development-related elements (e.g., GCN4, MSA-like) are less abundant, their spatiotemporal specificity implies roles in regulating endosperm development, seed germination, and cell cycle progression to influence plant growth. Tissue-specific analysis showed that highly expressed members in stems and leaves (e.g., ZCN3/25 ) may modulate reproductive development by regulating photoperiod responses or assimilate transport, while those enriched in embryos and endosperms (e.g., ZCN9–ZCN11 ) are likely involved in grain filling and embryonic differentiation. Notably, the global silencing of ZCN21 and low expression of ZCN6/ZCN13 may reflect functional redundancy within the family, potentially shaped by epigenetic regulation or post-translational modification. Non-biotic stress response analysis showed that broadly responsive genes (e.g., ZCN1/ZCN25/ZCN26 ) may integrate hormonal signals to adapt to stresses, whereas drought-specific activators (e.g., ZCN4/ZCN7/ZCN14 ) likely synergize with the DREB/CBF regulatory network to enhance drought tolerance [ 17 ]. These spatiotemporally specific expression patterns and hierarchical stress-response features provide novel targets for dissecting ZCN family modular regulation in maize environmental adaptation and for targeted gene editing in breeding. Subcellular localization experiments further revealed functional compartmentalization: 7 nucleus-localized members likely directly activate meristem proliferation-related target genes, while 18 nucleocytoplasmic co-localized members may integrate hormonal or environmental signals to form dynamic regulatory networks. This spatial divergence aligns with the coordination of cell proliferation and signal transduction required during inflorescence development (Kobayashi and Weigel, 2007), offering new insights into the molecular mechanisms underlying ZCN family coupling of environmental adaptation and development. In mutant populations derived from our ZCN gene family library, we identified homozygous mutant lines ko#1 and ko#2 carrying mutations in ZCN3/17/20/25 . These lines exhibited a 2–3-fold significant increase in secondary tassel branch number, accompanied by elevated primary branch counts. Given the vascular expression pattern of ZCN3 and ZCN25 (Fig. 2 c), we hypothesize they may antagonize florigen signaling to limit excessive secondary meristem proliferation—similar to OsRCN2/4 [ 31 ] and AhTFL1 [ 32 ]—rather than directly regulating initial inflorescence meristem (SAM) differentiation. Intriguingly, compound mutations in ZCN17/20/25 (FT subfamily members) caused significant flowering time delays in ko#1 and ko#2 across four environments (Fig. 4 g), consistent with the rice OsFTL1 mechanism (activating OsMADS14/15 via the FAC complex to promote heading; Wei et al., 2025) and the central role of Arabidopsis FT [ 29 ]. Promoter analysis further showed that ZCN17/20/25 are enriched in auxin, gibberellin, and jasmonic acid responsive elements, suggesting they integrate hormonal signals to regulate flowering time. Conclusion This study systematically identified 25 ZCN family members in the maize genome and comprehensively analyzed their gene structure, physicochemical properties, evolutionary relationships, expression patterns, and responses to abiotic stresses. Our results not only clarified the structural evolution and regulatory features of the ZCN gene family but also highlighted the functional roles of key genes. By generating specific knockout mutants, we obtained maize lines with drastically increased tassel branch numbers and significantly delayed anthesis/pollen shedding, identifying ZCN3 , ZCN17 , ZCN20 , and ZCN25 as critical regulators of tassel branching. These findings significantly advance our understanding of the ZCN gene family in maize. Additionally, the data and conclusions from this study provide essential theoretical foundations and genetic resources for maize molecular breeding, contributing to improved crop stability and resource use efficiency under climate change. Declarations Funding This research was supported by the National Key Research and Development Program of China (2023YFD1202901), the China Agriculture Research System of MOF and MARA (CARS-02-06), and the Biological Breeding-National Science and Technology Major Project (2022ZD04020). Authors’ Contributions Y.Y. and M.Y.W. conducted experiments, performed bioinformatics analyses, and drafted the manuscript. C.L.L. and G.S.Y. designed the experiments, analyzed the data, and revised the manuscript. J.J.Z. and Z.X.G. provided experimental methodologies. X.T.Q. and C.X.X. supervised the study and critically reviewed the manuscript. All authors read and approved the manuscript. 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Supplementary Files S2.xlsx S3.xlsx S1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 14 Oct, 2025 Reviewers agreed at journal 13 Oct, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviewers agreed at journal 06 Oct, 2025 Reviewers invited by journal 06 Oct, 2025 Editor invited by journal 06 Oct, 2025 Editor assigned by journal 02 Sep, 2025 Submission checks completed at journal 02 Sep, 2025 First submitted to journal 18 Aug, 2025 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. 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1","display":"","copyAsset":false,"role":"figure","size":5841256,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic and Structural Analysis of the \u003cem\u003eZCN\u003c/em\u003e Gene Family. (a) Chromosomal localization of the \u003cem\u003eZCN\u003c/em\u003e gene family. Maize chromosomes are distinguished by different colors, with short lines marking each gene. (b) Phylogenetic relationships of the \u003cem\u003ePEBP\u003c/em\u003e gene family across maize, rice, wheat, and Arabidopsis thaliana. Genes are divided into four subclades (\u003cem\u003eFT-like, TFL1-like, MFT-like\u003c/em\u003e, and \u003cem\u003ePEBP-like\u003c/em\u003e) by different color blocks. Brown squares, green diamonds, orange triangles, and red circles represent \u003cem\u003ePEBP\u003c/em\u003e genes from maize, rice, wheat, and A. thaliana, respectively. (c) Phylogenetic relationships of the \u003cem\u003ePEBP\u003c/em\u003e gene family between maize and A. thaliana. (d) Gene structure of maize \u003cem\u003eZCN\u003c/em\u003e genes. Coding sequences (CDS), untranslated regions (UTR), introns, and guide RNAs (sgRNAs) are represented by black/gray bars and black/red lines, respectively. Scale bar: 1 kb (black bar). (e) Distribution of PEBP protein motifs. A total of six motifs are displayed using colored boxes.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7400912/v1/8eaaf869ff9ebb797680eb94.png"},{"id":93807246,"identity":"cb9e04b9-38c5-47ab-9a6b-17315329fb78","added_by":"auto","created_at":"2025-10-17 18:32:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2621019,"visible":true,"origin":"","legend":"\u003cp\u003eRegulatory and Expression Pattern Analysis of the \u003cem\u003eZCN\u003c/em\u003e Gene Family. (a) Positional distribution of cis-regulatory elements in the promoter region of \u003cem\u003eZCN\u003c/em\u003e genes. Black lines indicate the promoter length of \u003cem\u003eZCN\u003c/em\u003e genes, and color-coded blocks represent cis-acting elements with distinct functions. (b) Quantitative heatmap of cis-regulatory elements in the promoter region of \u003cem\u003eZCN\u003c/em\u003e genes. All regulatory elements are classified into four categories based on function, with cis-elements of similar function represented by the same color. The size and color of bubbles are proportional to the number of regulatory elements. (c) Expression level analysis of \u003cem\u003eZCN\u003c/em\u003e genes in different tissues. From left to right, the tissues are root, stem, leaf, tassel, ear seed, meristem, embryo, and endosperm, respectively. (d) Expression level analysis of \u003cem\u003eZCN\u003c/em\u003e genes under different abiotic stresses. From left to right, the stresses are low temperature stress, high temperature stress, salt stress, high light stress, and drought stress, respectively. The size and color of bubbles are proportional to gene expression levels.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7400912/v1/c4ac417885c0f4f917748c99.png"},{"id":93807979,"identity":"00d81a6e-7edf-41af-bcda-ce21dcbb6bd0","added_by":"auto","created_at":"2025-10-17 18:48:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5583034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eZCN\u003c/em\u003e gene subcellular localization. (a) Subcellular localization of \u003cem\u003eZCN\u003c/em\u003e gene family members in maize protoplasts. (b) Prediction results of NLS signals in ZCN17, ZCN20, and ZCN25 proteins. (c) Subcellular localization results of ZCN17, ZCN20, and ZCN25 proteins in maize plastids after NLS removal. BF: Bright field. DAPI and eGFP: Fluorescence fields. Merge: Composite field. Nuclei were stained with DAPI (in blue). Scale bar: 20 μm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7400912/v1/5d1de1775854561baa3a9aff.png"},{"id":93807250,"identity":"c1fd6ff1-6f14-4be3-b978-802218b7c353","added_by":"auto","created_at":"2025-10-17 18:32:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2311005,"visible":true,"origin":"","legend":"\u003cp\u003eRegulation of Male Floral Organ Development and Reproductive Phase Transition by \u003cem\u003eZCN\u003c/em\u003e Genes in Maize. (a) Schematic diagram of the \u003cem\u003eZCN\u003c/em\u003e gene editing vector structure. The tandem region of the \"tRNA-sgRNA-scaffold\" fused sequence is highlighted with a red box. (b) Selected homozygous mutant materials. (c) Basic agronomic traits of the mutant materials. (d) Representative field-grown plants of the mutant materials. Scale bar: 20 cm. (e) Representative tassels of the mutant materials in the field. Scale bar: 5 cm. (f) Representative ears of the mutant materials in the field. Scale bar : 5 cm. (g) Field flowering time statistics of T\u003csub\u003e3\u003c/sub\u003e generation mutant materials. (h) Field statistics of tassel branch number in T\u003csub\u003e3\u003c/sub\u003e generation mutant materials.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7400912/v1/08c2523eb4622c0f40476fc4.png"},{"id":93809035,"identity":"04d4604a-b041-4086-9347-357d1124b25a","added_by":"auto","created_at":"2025-10-17 19:05:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12612437,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7400912/v1/423e94ae-a949-40c6-8e07-7277c3b4aa7a.pdf"},{"id":93807245,"identity":"ef5638aa-d1e8-4870-988f-5dfdd8810cc5","added_by":"auto","created_at":"2025-10-17 18:32:43","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11977,"visible":true,"origin":"","legend":"","description":"","filename":"S2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7400912/v1/bb8bc6f00eed32d860e13b4e.xlsx"},{"id":93807978,"identity":"8e61d076-1cc8-480c-b1a2-dad6cceab9ce","added_by":"auto","created_at":"2025-10-17 18:48:43","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12283,"visible":true,"origin":"","legend":"","description":"","filename":"S3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7400912/v1/02cf8c73cbe462661b36bb1c.xlsx"},{"id":93807247,"identity":"9a1dfb1e-6159-4e4f-ba00-cf65a331b45e","added_by":"auto","created_at":"2025-10-17 18:32:43","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14487,"visible":true,"origin":"","legend":"","description":"","filename":"S1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7400912/v1/a8fa26e0ad0e1c4400c25ea4.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification and analysis of the ZCN gene family in maize (Zea mays L.)","fulltext":[{"header":"Background","content":"\u003cp\u003eThe development of floral organs mark a hallmark of the transition from vegetative to reproductive growth in plants. The \u003cem\u003ePEBP\u003c/em\u003e (PHOSPHATIDYLETHANOLAMINE-BINDING PROTEIN) gene family encodes proteins with a highly conserved PEBP domain\u0026mdash;so named for its ability to bind phosphatidylethanolamine [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This domain plays a critical regulatory role in the vegetative-to-reproductive transition and modulates diverse biological processes, including plant morphological traits [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], flowering time, and periodic gene expression patterns [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. PEBP proteins are ubiquitously distributed across plant species [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn flowering plants, the \u003cem\u003ePEBP\u003c/em\u003e gene family can be divided into three subclades, which are FLOWERING LOCUS T (FT)-like, TERMINAL FLOWER 1 (TFL1)-like, and MOTHER OF FT AND TFL1 (MFT)-like, based on phylogenetic evolution and functional divergence [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Members of the \u003cem\u003ePEBP\u003c/em\u003e gene family play regulatory roles in diverse physiological processes in plants, including \u003cem\u003eFT-\u003c/em\u003elike and \u003cem\u003eTFL1\u003c/em\u003e-like subclade genes participating in the regulation of flowering time [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and \u003cem\u003eMFT-\u003c/em\u003elike subclade genes involvement in seed dormancy and germination regulation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, protein FT and TFL1 in leaves compete for binding to the bZIP transcription factor FD protein (FLOWERING LOCUS D), thereby regulating flowering [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]; rice FT-like genes \u003cem\u003eRFT1\u003c/em\u003e (Rice Flowering Locus T1) and \u003cem\u003eHd3a\u003c/em\u003e (Heading date-3a) also play inductive roles in flowering [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]; soybean \u003cem\u003eGmMFT\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and two cotton \u003cem\u003eMFT\u003c/em\u003e genes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] inhibit seed germination via ABA and GA pathways. Notably, unique \u003cem\u003ePEBP-like\u003c/em\u003e subclade members have been identified in crops such as cotton and wheat, with their transcript expression showing tissue specificity. For example, wheat \u003cem\u003eTaPEBP1\u003c/em\u003e is highly abundant during young spike development, yet its functional mechanisms remain unclear. This subclade may represent a new evolutionary lineage of plants adapted to specialized agronomic environments (Zhang et al., 2016; Dong et al., 2020).\u003c/p\u003e\u003cp\u003eIn maize (\u003cem\u003eZea mays\u003c/em\u003e L.), the genes responsible for encoding PEBP proteins are named \u003cem\u003eZCN\u003c/em\u003e (Zea mays CENTRORADIALIS). Their functions not only contain regulating flowering time and seed dormancy but also potentially involve plant adaptability to environmental changes and fine-tuned regulation of growth and development [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. For example, (ⅰ) \u003cem\u003eZCN8\u003c/em\u003e, a \u003cem\u003ePEBP\u003c/em\u003e gene highly homologous to \u003cem\u003eAtFT\u003c/em\u003e, interacts with \u003cem\u003eDLF1\u003c/em\u003e to promote \u003cem\u003eZMM4\u003c/em\u003e expression, thereby inducing the transition from vegetative growth to reproductive growth [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]; a SNP variant site in the ZCN8 promoter region is significantly associated with maize flowering time, and this variant affects the binding affinity of ZmMADS1 to the ZCN8 promoter, thereby controlling flowering time [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]; (ⅱ) ZCN1 inhibits the activity of the ZCN8-DLF1 complex to suppress flowering, whereas ZCN2 and ZCN5 enhance the inductive effect of ZCN8 to positively regulate maize flowering [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; (ⅲ) ZCN4 promotes cell division and positively regulates bract width [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]; (ⅳ) \u003cem\u003eZCN7\u003c/em\u003e positively regulates maize drought tolerance\u0026mdash;its overexpression shortens the anthesis-silking interval under drought, thereby increasing yield [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]; (ⅴ) \u003cem\u003eZCN9/10\u003c/em\u003e negatively regulate seed vigor, participate in the abscisic acid (ABA) signaling pathway, and reduce seed tolerance to ABA [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMaize as a globally important food crop, has floral organ development that significantly influences both yield and quality. Despite the established importance of the \u003cem\u003eZCN\u003c/em\u003e gene family, a comprehensive analysis of its functions in maize remains lacking. This study employed bioinformatics approaches to identify members of the \u003cem\u003eZCN\u003c/em\u003e gene family in the maize genome, analyzed their phylogenetic evolution, structural characteristics, expression patterns, and provided a reference for elucidating the regulatory mechanisms of maize flower development and the biological functions of \u003cem\u003eZCN\u003c/em\u003e genes. Additionally, we explored the functional differentiation of the \u003cem\u003eZCN\u003c/em\u003e gene family in environmental adaptability. These findings lay a foundation for further investigating the functions of \u003cem\u003eZCN\u003c/em\u003e genes and their specific roles in maize growth and development.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eGenome-wide Identification and Biochemical Property Analysis of the\u003c/b\u003e \u003cb\u003eZCN\u003c/b\u003e \u003cb\u003eGene Family in Maize\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFirstly, we retrieved Hidden Markov model (HMM) files of ZCN protein domains (PF01161) from the Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Next, we used HMMER 3.4 to generate ZCN protein sequence files for maize. We then accessed the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to further identify protein domains associated with candidate genes and obtained the amino acid sequences of \u003cem\u003eZCN\u003c/em\u003e. Using ExPASy tools (ProtParam and ProtScale; \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)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], we analyzed the amino acid length, molecular weight, isoelectric point (pI), and other relevant properties of candidate \u003cem\u003eZCN\u003c/em\u003e gene family members. The potential nuclear localization signals (NLS) of proteins were predicted via DeepLoc 2.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/DeepLoc-2.1\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/DeepLoc-2.1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Chromosome length and position data corresponding to \u003cem\u003eZCN\u003c/em\u003e gene information were visualized using TBtools software[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. We downloaded PEBP protein sequences of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, rice, and wheat from the EnsemblPlants 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)[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Multiple sequence alignment and phylogenetic tree construction were performed using MEGA 11, and the phylogenetic tree was visualized using iTOL. Subsequently, we extracted 2000 bp of sequence upstream of the transcription start site (TSS) of \u003cem\u003eZCN\u003c/em\u003e family genes in maize using TBtools software and predicted cis-acting elements in the promoter regions of \u003cem\u003eZCN\u003c/em\u003e gene family members via the Plant CARE tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Finally, we used 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) to investigate and collect data on \u003cem\u003eZCN\u003c/em\u003e gene expression levels in different maize tissues and their responses to various abiotic stress conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSubcellular Localization of the\u003c/b\u003e \u003cb\u003eZCN\u003c/b\u003e \u003cb\u003eGene Family in Maize\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe coding sequences (CDS) of all \u003cem\u003eZCN\u003c/em\u003e gene family members were inserted into a CUB vector containing enhanced green fluorescent protein (eGFP) to construct the plasmid CUB-\u003cem\u003eZCN\u003c/em\u003e-eGFP. Protoplasts were isolated from the leaves of maize B73 seedlings. A total of 100 \u0026micro;g of CUB\u003cem\u003e-ZCN\u003c/em\u003e-eGFP plasmid was transfected into 1 mL of maize protoplasts (approximately 5\u0026times;10⁵ cells/mL) using PEG4000-mediated transfection. After culturing the protoplasts in the dark at 25\u0026deg;C for 16 hours, fluorescence signals were observed using a ZEISS LSM900 microscope.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003esgRNA Design and Genome Editing\u003c/h2\u003e\u003cp\u003eTo construct a CRISPR/Cas9 mutant library for the \u003cem\u003eZCN\u003c/em\u003e gene family, we designed 25 sgRNAs targeting \u003cem\u003eZCN\u003c/em\u003e genes using CRISPR-P 2.0 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR\u003c/span\u003e\u003cspan address=\"http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The \"tRNA-sgRNA-scaffold\" fusion sequences were separately ligated into two vectors to construct gene editing plasmids for the \u003cem\u003eZCN\u003c/em\u003e gene family[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The constructed plasmids were sent to Weimi Biotechnology Co., Ltd. (Changzhou, China) for transformation of the maize inbred line KN5585.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eField Phenotypic Statistics\u003c/h3\u003e\n\u003cp\u003eField trials were conducted in maize fields under natural long-day conditions (Beijing) and natural short-day conditions (Sanya). The flowering time was statistically calculated as the number of days from seedling emergence to when \u0026gt;\u0026thinsp;50% of plants in a row reached anthesis, silking, and pollen shedding. Agronomic traits and yield of wild-type and mutant plants were investigated 15\u0026ndash;20 days after pollination. Maize tassel branch number investigations involve counting primary, secondary, and total branches. Primary tassel branches are lateral branches originating from the main axis of the maize inflorescence; secondary branches are sub-branches generated on these lateral branches; and total branch number is the sum of primary and secondary branches[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Plant height was measured as the vertical distance from the ground to the tip of the tassel main axis; ear height was measured as the vertical distance from the ground to the attachment site of the first effective ear shank; the number of leaves above the ear was counted as the leaves persisting between the first effective ear and the tassel; the number of leaves below the ear was counted as the leaves persisting between the first effective ear and the first leaf above the ground; ear length was measured as the distance from the base to the tip of the ear after removing the shank; 100-grain weight was determined by weighing 100 randomly selected intact, disease-free grains; and each group was replicated three times.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eSignificant differences were assessed using a two-tailed Student's \u003cem\u003et\u003c/em\u003e-test in Microsoft\u0026reg; Excel 2019. Results with a \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Figures were prepared using GraphPad Prism (v8.0).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eGenome-wide Identification and Physicochemical Property Analysis of the ZCN Gene Family\u003c/h2\u003e\u003cp\u003eA total of 25 members of the \u003cem\u003eZCN\u003c/em\u003e gene family were identified in the maize genome (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), among which \u003cem\u003eZCN22\u003c/em\u003e and \u003cem\u003eZCN23\u003c/em\u003e were classified as pseudogenes, and two newly identified members\u0026mdash;\u003cem\u003eZCN26\u003c/em\u003e and a PEBP-like gene \u003cem\u003eZCNPEBP\u003c/em\u003e\u0026mdash;were added. These 25 members exhibited a relatively dispersed pattern in the maize genome, being unequally distributed across 10 maize chromosomes, with no distribution on chromosome 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Physicochemical properties revealed that the number of amino acids in ZCN proteins ranged from 172 (ZCN9, ZCN10) to 192 (ZCN7), their relative molecular masses ranged from 15.078 kDa (ZCN3) to 21.935 kDa (ZCN7), and their isoelectric points (pI) ranged from 5.52 (ZCNPEBP) to 9.94 (ZCN11). Except for ZCN11, the grand average of hydropathy (GRAVY) values of all other members were less than 0, indicating that they are all hydrophilic proteins. To gain a deeper understanding of the structure and function of ZCN proteins, the signal peptides of all ZCN proteins were predicted. The results showed that none of the ZCN proteins contained signal peptides, indicating that they are unlikely to be secreted via the classical secretory pathway.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInformation of \u003cem\u003eZCN\u003c/em\u003e gene family in maize\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChromosome\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNumber of \u003c/p\u003e\u003cp\u003eamino acid (aa)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMolecular weight \u003c/p\u003e\u003cp\u003e(KDa)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTheoretical\u003c/p\u003e\u003cp\u003epI\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d044704\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e173\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d050649\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e173\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d023419\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e173\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN4\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d003804\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e176\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN5\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d025346\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e173\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN6\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d052537\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e177\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN7\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d038725\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e21.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN8\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d010752\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.74\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN9\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d008446\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e172\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e18.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.73\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN10\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d040113\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e172\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e18.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN11\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d037439\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e180\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN12\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d043461\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e177\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN13\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d016253\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e184\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN14\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d008939\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e173\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN15\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d036242\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e177\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN16\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d017134\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN17\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d004630\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e178\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN18\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d006116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN19\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d025737\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN20\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d024023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN21\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d004010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e187\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN24\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d021135\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e173\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN25\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d003226\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCN26\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d046300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e187\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eZCNPEBP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZm00001d010586\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e18.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5.52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic and Structural Analysis of the\u003c/b\u003e \u003cb\u003eZCN\u003c/b\u003e \u003cb\u003eGene Family\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further clarify the evolutionary relationships of the maize \u003cem\u003eZCN\u003c/em\u003e gene family, we constructed a phylogenetic tree using full-length sequences of PEBP proteins from Arabidopsis thaliana (4), rice (19), and wheat (78) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The results indicated that all PEBP proteins from these four species clustered into four distinct subclades: FT-like, TFL1-like, MFT-like, and PEBP-like, suggesting that these subclades existed prior to the divergence of monocotyledons and dicotyledons. Additionally, compared with Arabidopsis, ZCN proteins in maize were more closely related to PEBP proteins in rice and wheat, which is consistent with the evolutionary relationships among Arabidopsis, wheat, rice, and maize. Analyses of the conserved structures and exon-intron organizations of ZCN proteins were also performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u0026ndash;d). Six conserved motifs were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), with motifs 1 and 6 almost universally present across all ZCN proteins. Exon-intron structure analysis further revealed high structural conservation in \u003cem\u003eZCN\u003c/em\u003e genes: members of each of the four subclades exhibited nearly identical numbers and distribution patterns of exons and introns, showing little difference from the Arabidopsis \u003cem\u003ePEBP\u003c/em\u003e gene family. These structural conservations suggest that the \u003cem\u003eZCN\u003c/em\u003e gene family likely underwent strong purifying selection during evolution, with the precise splicing requirements of the PEBP functional domain driving the stable evolution of its gene structure.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRegulatory and Expression Pattern Analysis of the\u003c/b\u003e \u003cb\u003eZCN\u003c/b\u003e \u003cb\u003eGene Family\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the functions of \u003cem\u003eZCN\u003c/em\u003e genes, we analyzed the cis-acting elements within the 2000 bp promoter regions upstream of maize \u003cem\u003eZCN\u003c/em\u003e genes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, these elements were broadly categorized into four classes based on their functions: developmental regulation, light signal response, hormone signal response, and abiotic stress signal response. Almost all \u003cem\u003eZCN\u003c/em\u003e genes contained light-responsive elements, which were diverse in type. Next were elements related to hormone and abiotic stress responses, while those associated with plant development were fewer in number but diverse in type, including elements for endosperm development regulation (GCN4 motif), seed germination regulation (RY-element), cell cycle regulation (MSA-like), meristem expression regulation (CAT-box), and circadian rhythm regulation (Circadian rhythm).\u003c/p\u003e\u003cp\u003eWe also analyzed the expression patterns of \u003cem\u003eZCN\u003c/em\u003e family members across different plant tissues and under abiotic stress conditions. Among the 25 maize \u003cem\u003eZCN\u003c/em\u003e genes, not all were highly expressed: \u003cem\u003eZCN6\u003c/em\u003e and \u003cem\u003eZCN13\u003c/em\u003e were expressed at extremely low levels, \u003cem\u003eZCN21\u003c/em\u003e was undetectable in all tissues, and the remaining 22 genes were expressed at relatively high levels. Nearly all members were highly expressed in stems and leaves, consistent with previous reports that florigen genes are expressed in leaves and stems and transported to the apical meristem to exert their functions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Interestingly, expression levels varied across organs: for example, \u003cem\u003eZCN3\u003c/em\u003e was highly expressed in stems, meristems, and male flowers; \u003cem\u003eZCN25\u003c/em\u003e was highly expressed in stems and male flowers; and \u003cem\u003eZCN9\u003c/em\u003e, \u003cem\u003eZCN10\u003c/em\u003e, \u003cem\u003eZCN11\u003c/em\u003e, and \u003cem\u003eZCNPEBP\u003c/em\u003e were expressed at significantly higher levels in embryos and endosperms than other members. These findings indicate that the \u003cem\u003eZCN\u003c/em\u003e gene family is functionally diverse and plays an indispensable role during maize growth and development.\u003c/p\u003e\u003cp\u003eUnder five abiotic stress conditions (low temperature, high temperature, salt, UV radiation, and drought), the expression patterns of \u003cem\u003eZCN\u003c/em\u003e genes revealed inconsistent responses to stress among family members. Seven genes\u0026mdash;\u003cem\u003eZCN2\u003c/em\u003e, \u003cem\u003eZCN12\u003c/em\u003e, \u003cem\u003eZCN13\u003c/em\u003e, \u003cem\u003eZCN15\u003c/em\u003e, \u003cem\u003eZCN17\u003c/em\u003e, \u003cem\u003eZCN20\u003c/em\u003e, and \u003cem\u003eZCN21\u003c/em\u003e\u0026mdash;were undetectable under all stresses, suggesting they are unlikely to be involved in maize\u0026rsquo;s response to abiotic stresses. In contrast, nearly all members were sensitive to drought stress, with \u003cem\u003eZCN4\u003c/em\u003e, \u003cem\u003eZCN7\u003c/em\u003e, \u003cem\u003eZCN14\u003c/em\u003e, and \u003cem\u003eZCN16\u003c/em\u003e exhibiting particularly high expression under drought. \u003cem\u003eZCN1\u003c/em\u003e, \u003cem\u003eZCN25\u003c/em\u003e, and \u003cem\u003eZCN26\u003c/em\u003e were expressed under all stress conditions, implying they may possess the ability to respond to a broad spectrum of stresses. This could enable them to coordinate multiple stress signaling pathways and enhance plant adaptability to environmental challenges.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSubcellular Localization of the\u003c/b\u003e \u003cb\u003eZCN\u003c/b\u003e \u003cb\u003eGene Family\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe performed subcellular localization of all \u003cem\u003eZCN\u003c/em\u003e gene family members in maize protoplasts to determine their expression sites. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, green fluorescence signals of certain members (e.g., \u003cem\u003eZCN7\u003c/em\u003e, \u003cem\u003eZCN8\u003c/em\u003e, \u003cem\u003eZCN17\u003c/em\u003e, \u003cem\u003eZCN18\u003c/em\u003e, \u003cem\u003eZCN21\u003c/em\u003e, \u003cem\u003eZCN24\u003c/em\u003e, and \u003cem\u003eZCN26\u003c/em\u003e) were exclusively localized in the nucleus, whereas those of other family members were distributed across both the nucleus and cytoplasm. These results revealed significant functional differentiation within the \u003cem\u003eZCN\u003c/em\u003e gene family: most \u003cem\u003eZCN\u003c/em\u003e proteins (\u003cem\u003eZCN1-6\u003c/em\u003e, \u003cem\u003e9\u0026ndash;16\u003c/em\u003e, \u003cem\u003e19\u0026ndash;20\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e, and \u003cem\u003eZCNPEBP\u003c/em\u003e) exhibited dual localization in both the nucleus and cytoplasm. These proteins likely regulate gene expression dynamically via nucleocytoplasmic shuttling, responding to diverse external signals to maintain normal plant growth and development. In contrast, \u003cem\u003eZCN7\u003c/em\u003e, \u003cem\u003eZCN8\u003c/em\u003e, and other members specifically localized to the nucleus indicate their functions are entirely dependent on the nuclear environment. These findings provide direct subcellular-level evidence for functional partitioning of the maize \u003cem\u003eZCN\u003c/em\u003e gene family and suggest spatial segregation as a mechanism underlying their functional differentiation.\u003c/p\u003e\u003cp\u003eWe selected \u003cem\u003eZCN17\u003c/em\u003e (nuclear localization) and \u003cem\u003eZCN20/ZCN25\u003c/em\u003e (nucleocytoplasmic colocalization) from the family for NLS prediction and identified conserved monopartite NLS motifs (GRRYIR/GRRYR) in their protein sequences. Upon removal of these NLSs, the proteins were exclusively localized to the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u0026ndash;c).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eZCN\u003c/b\u003e \u003cb\u003eGenes Regulate Male Floral Organ Development and Reproductive Phase Transition in Maize\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing the CRISPR/Cas9 system, we designed single guide RNAs (sgRNAs) targeting these 25 \u003cem\u003eZCN\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Appendix S1) and achieved systematic knockout of the \u003cem\u003eZCN\u003c/em\u003e gene family (Appendix S3), generating \u003cem\u003eZCN\u003c/em\u003e gene family knockout mutants. During the growth period, compared with the wild type (KN5585), we observed morphological and developmental differences in the male flowers of some mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e). Sequencing confirmed that these mutants were quadruple homozygous mutants of \u003cem\u003eZCN3/17/20/25\u003c/em\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). These mutants exhibited increased tassel branch number, delayed male flowering time, with an average delay of 3.9 days in anthesis, 4.3 days in silking, and 6.9 days in pollen shedding (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-h). At the T\u003csub\u003e3\u003c/sub\u003e generation, agronomic traits (including plant height) and yield of the quadruple mutants were investigated. Results showed that compared with the wild type, the mutants exhibited no significant changes in most agronomic traits except for an average increase of 2.3 cm in ear height (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDanilevskaya previously identified 23 maize \u003cem\u003ePEBP\u003c/em\u003e genes, but this was limited by the absence of complete maize genomic sequences in the EST database at the time [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. By leveraging newly released maize genomic databases alongside those of Arabidopsis thaliana, rice, and wheat (four species spanning monocots and dicots), we performed a more rigorous search. This led to the systematic identification and evolutionary analysis of the \u003cem\u003ePEBP\u003c/em\u003e gene family, resulting in the discovery of two novel members: \u003cem\u003eZCN26\u003c/em\u003e and \u003cem\u003eZCNPEBP\u003c/em\u003e. As a highly domesticated monocot crop, maize exhibits explosive expansion of its FT subfamily, which may be closely linked to unique traits in monocots such as sexual organ differentiation, photoperiod adaptation, and agronomic trait domestication. Meanwhile, the moderate expansion of the MFT and TFL1 subfamilies likely reflects their adaptive diversification in conserved functions like seed development and meristem activity. This divergent pattern underscores how the \u003cem\u003ePEBP\u003c/em\u003e gene family balances conserved regulatory demands with environmental adaptability through functional differentiation and non-coding sequence-driven gene duplication.\u003c/p\u003e\u003cp\u003eOur findings suggest that the \u003cem\u003eZCN\u003c/em\u003e gene family operates via a multi-dimensional regulatory network. Promoter element analysis revealed a high enrichment of light-responsive elements (33.4%), indicating that \u003cem\u003eZCN\u003c/em\u003e genes mediate photoperiod-dependent rhythmic expression through the phytochrome/cryptochrome system [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u0026mdash;a conserved mechanism shared with \u003cem\u003eAtFT/AtTFL1\u003c/em\u003e in flowering regulation[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Concurrently, the co-distribution of hormone-responsive elements (e.g., TGA and ABRE) and stress-responsive elements (e.g., MYB and WRKY binding sites) suggests \u003cem\u003eZCN\u003c/em\u003e genes dynamically couple environmental adaptation with developmental programs by integrating ABA, ROS, and other signaling pathways [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although development-related elements (e.g., GCN4, MSA-like) are less abundant, their spatiotemporal specificity implies roles in regulating endosperm development, seed germination, and cell cycle progression to influence plant growth.\u003c/p\u003e\u003cp\u003eTissue-specific analysis showed that highly expressed members in stems and leaves (e.g., \u003cem\u003eZCN3/25\u003c/em\u003e) may modulate reproductive development by regulating photoperiod responses or assimilate transport, while those enriched in embryos and endosperms (e.g., \u003cem\u003eZCN9\u0026ndash;ZCN11\u003c/em\u003e) are likely involved in grain filling and embryonic differentiation. Notably, the global silencing of \u003cem\u003eZCN21\u003c/em\u003e and low expression of \u003cem\u003eZCN6/ZCN13\u003c/em\u003e may reflect functional redundancy within the family, potentially shaped by epigenetic regulation or post-translational modification. Non-biotic stress response analysis showed that broadly responsive genes (e.g., \u003cem\u003eZCN1/ZCN25/ZCN26\u003c/em\u003e) may integrate hormonal signals to adapt to stresses, whereas drought-specific activators (e.g., \u003cem\u003eZCN4/ZCN7/ZCN14\u003c/em\u003e) likely synergize with the DREB/CBF regulatory network to enhance drought tolerance [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These spatiotemporally specific expression patterns and hierarchical stress-response features provide novel targets for dissecting \u003cem\u003eZCN\u003c/em\u003e family modular regulation in maize environmental adaptation and for targeted gene editing in breeding. Subcellular localization experiments further revealed functional compartmentalization: 7 nucleus-localized members likely directly activate meristem proliferation-related target genes, while 18 nucleocytoplasmic co-localized members may integrate hormonal or environmental signals to form dynamic regulatory networks. This spatial divergence aligns with the coordination of cell proliferation and signal transduction required during inflorescence development (Kobayashi and Weigel, 2007), offering new insights into the molecular mechanisms underlying \u003cem\u003eZCN\u003c/em\u003e family coupling of environmental adaptation and development.\u003c/p\u003e\u003cp\u003eIn mutant populations derived from our \u003cem\u003eZCN\u003c/em\u003e gene family library, we identified homozygous mutant lines ko#1 and ko#2 carrying mutations in \u003cem\u003eZCN3/17/20/25\u003c/em\u003e. These lines exhibited a 2\u0026ndash;3-fold significant increase in secondary tassel branch number, accompanied by elevated primary branch counts. Given the vascular expression pattern of \u003cem\u003eZCN3\u003c/em\u003e and \u003cem\u003eZCN25\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), we hypothesize they may antagonize florigen signaling to limit excessive secondary meristem proliferation\u0026mdash;similar to \u003cem\u003eOsRCN2/4\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and \u003cem\u003eAhTFL1\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u0026mdash;rather than directly regulating initial inflorescence meristem (SAM) differentiation. Intriguingly, compound mutations in \u003cem\u003eZCN17/20/25\u003c/em\u003e (FT subfamily members) caused significant flowering time delays in ko#1 and ko#2 across four environments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg), consistent with the rice \u003cem\u003eOsFTL1\u003c/em\u003e mechanism (activating \u003cem\u003eOsMADS14/15\u003c/em\u003e via the FAC complex to promote heading; Wei et al., 2025) and the central role of Arabidopsis FT [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Promoter analysis further showed that \u003cem\u003eZCN17/20/25\u003c/em\u003e are enriched in auxin, gibberellin, and jasmonic acid responsive elements, suggesting they integrate hormonal signals to regulate flowering time.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study systematically identified 25 \u003cem\u003eZCN\u003c/em\u003e family members in the maize genome and comprehensively analyzed their gene structure, physicochemical properties, evolutionary relationships, expression patterns, and responses to abiotic stresses. Our results not only clarified the structural evolution and regulatory features of the \u003cem\u003eZCN\u003c/em\u003e gene family but also highlighted the functional roles of key genes. By generating specific knockout mutants, we obtained maize lines with drastically increased tassel branch numbers and significantly delayed anthesis/pollen shedding, identifying \u003cem\u003eZCN3\u003c/em\u003e, \u003cem\u003eZCN17\u003c/em\u003e, \u003cem\u003eZCN20\u003c/em\u003e, and \u003cem\u003eZCN25\u003c/em\u003e as critical regulators of tassel branching. These findings significantly advance our understanding of the \u003cem\u003eZCN\u003c/em\u003e gene family in maize. Additionally, the data and conclusions from this study provide essential theoretical foundations and genetic resources for maize molecular breeding, contributing to improved crop stability and resource use efficiency under climate change.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Key Research and Development Program of China (2023YFD1202901), the China Agriculture Research System of MOF and MARA (CARS-02-06), and the Biological Breeding-National Science and Technology Major Project (2022ZD04020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.Y. and M.Y.W. conducted experiments, performed bioinformatics analyses, and drafted the manuscript. C.L.L. and G.S.Y. designed the experiments, analyzed the data, and revised the manuscript. J.J.Z. and Z.X.G. provided experimental methodologies. \u0026nbsp;X.T.Q. and C.X.X. supervised the study and critically reviewed the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the experimental research on plants were comply with the Ethical Rules applicable to BMC Plant Biology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBernier I, Joll\u0026eacute;s P. Purification and characterization of a basic 23 kDa cytosolic protein from bovine brain. 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Cloning and functional analysis of \u003cem\u003eAhTFL1\u003c/em\u003e of cultivated peanuts (\u003cem\u003eArachis hypogaea \u003c/em\u003eL\u003cem\u003e.\u003c/em\u003e). Chin J Oil Crop Sci. 2023;45:728\u0026ndash;34. https://doi.org/10.19802/j.issn.1007-9084.2022130.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Maize, ZCN Gene Family, Genome-Wide Identification, Gene Editing","lastPublishedDoi":"10.21203/rs.3.rs-7400912/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7400912/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe development and growth of floral organs serve as a hallmark of the transition from vegetative to reproductive growth in plants. Phosphatidylethanolamine-binding proteins (PEBPs) play a critical regulatory role in this transition, additionally influencing the morphological structure of inflorescences. In maize (\u003cem\u003eZea mays\u003c/em\u003e L.), PEBP proteins are encoded by the ZCN (Zea mays CENTRORADIALIS) gene family. However, the functional characteristics of this gene family remain poorly characterized.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThis study systematically identified and cloned a total of 25 \u003cem\u003eZCN\u003c/em\u003e gene family members. Phylogenetic analysis divided the \u003cem\u003eZCN\u003c/em\u003e gene family into four subclades: \u003cem\u003eFT-like\u003c/em\u003e, \u003cem\u003eTFL1-like\u003c/em\u003e, \u003cem\u003eMFT-like\u003c/em\u003e, and \u003cem\u003ePEBP-like\u003c/em\u003e. Comprehensive multi-dimensional analyses of gene structures, promoter cis-acting elements, tissue-specific and abiotic stress expression patterns of \u003cem\u003eZCN\u003c/em\u003e family members revealed their functional diversity, indicating that \u003cem\u003eZCN\u003c/em\u003e genes play crucial roles in maize growth and development. Subcellular localization results in maize protoplasts showed that ZCN7, ZCN8, ZCN17, ZCN18, ZCN21, ZCN24, and ZCN26 were exclusively localized in the nucleus. Additionally, nuclear localization signal sequences of \u003cem\u003eZCN17\u003c/em\u003e, \u003cem\u003eZCN20\u003c/em\u003e, and \u003cem\u003eZCN25\u003c/em\u003e were identified as GRRYIR/GRRYR. Targeted editing of all \u003cem\u003eZCN\u003c/em\u003e members led to the generation of maize mutant lines with significantly increased tassel branch numbers and remarkably delayed flowering times under field conditions. Among these, \u003cem\u003eZCN3\u003c/em\u003e, \u003cem\u003eZCN17\u003c/em\u003e, \u003cem\u003eZCN20\u003c/em\u003e, and \u003cem\u003eZCN25\u003c/em\u003e were identified as key \u003cem\u003eZCN\u003c/em\u003e genes exhibiting significant regulatory effects on tassel branch number.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study systematically identified 25 \u003cem\u003eZCN\u003c/em\u003e family members in the maize genome and comprehensively analyzed their gene structure, physicochemical properties, evolutionary relationships, expression patterns, and responses to abiotic stresses. Our results not only clarified the structural evolution and regulatory features of the \u003cem\u003eZCN\u003c/em\u003e gene family but also highlighted the functional roles of key genes. These findings have significantly advanced our understanding of the \u003cem\u003eZCN\u003c/em\u003e gene family in maize and provided genetic resources for maize molecular breeding.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification and analysis of the ZCN gene family in maize (Zea mays L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 18:32:38","doi":"10.21203/rs.3.rs-7400912/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-10-14T14:14:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308486429529706018958030425266689542214","date":"2025-10-13T04:56:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333123589729456474318219884166713471556","date":"2025-10-08T12:30:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"70253180250267934596821237078031045092","date":"2025-10-06T14:00:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-06T12:04:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-06T07:41:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-02T13:53:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-02T13:53:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-08-18T14:46:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"687140bd-9e84-418e-8a2c-f99199c3128b","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-17T18:32:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-17 18:32:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7400912","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7400912","identity":"rs-7400912","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}