Genome-Wide Identification and Comprehensive Characterization of the Aux/IAA Gene Family in Cucurbita moschata and Its Response Analysis to Abiotic Stress

Genome-Wide Identification and Comprehensive Characterization of the Aux/IAA Gene Family in Cucurbita moschata and Its Response Analysis to Abiotic Stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-Wide Identification and Comprehensive Characterization of the Aux/IAA Gene Family in Cucurbita moschata and Its Response Analysis to Abiotic Stress Mengyao Sun, Jiayi Duan, Lina Jiang, Junguo Zhou, Yufei Zhai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8529495/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Background Auxin is a central phytohormone involved in regulating plant growth, development, and stress responses, with the Aux/IAA gene family functioning as an essential component of the auxin signaling pathway. To elucidate the genomic features and potential functions of the Aux/IAA gene family in pumpkin ( Cucurbita moschata ), we performed a genome-wide identification and systematic characterization. Results A total of 72 CmIAA genes were identified, encoding proteins ranging from 158 to 1275 amino acids with predicted isoelectric points of 4.57 ~ 9.81. These genes were unevenly distributed across 20 chromosomes, with Chr17 harboring the highest number, while no CmIAA genes were detected on Chr3. Phylogenetic analysis classified the genes into nine subgroups (Group Ⅰ~Ⅸ), with Groups Ⅰ, Ⅳ, and Ⅵ exhibiting notable expansion. Gene structure and conserved motif analyses revealed subgroup-specific motif compositions, with motif 1 representing the core conserved domain. Intraspecific collinearity analysis identified 54 segmentally duplicated gene pairs but no tandem duplication events, whereas interspecific synteny revealed extensive orthologous relationships between pumpkin and Cucurbita pepo , Cucurbita maxima , and Arabidopsis thaliana . Promoter analysis showed that CmIAA genes contain abundant cis-elements associated with light response, hormone regulation, development, and abiotic stress. Tissue expression analysis demonstrated that many CmIAA genes were highly expressed in roots and stems, Several CmIAA genes exhibited tissue-specific and stress-type-dependent expression patterns under salt and drought treatments. Conclusions Overall, this study provides the first comprehensive characterization of the Aux/IAA gene family in pumpkin, offering fundamental insights into their structural features and expression dynamics, and providing candidate genes for future functional studies and molecular breeding applications. Aux/IAA Cucurbita moschata gene family abiotic stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Background Auxin is one of the central phytohormones regulating plant growth and development, with essential roles in cell division, organogenesis, fruit development, and abiotic stress respons [ 1 , 2 ] . The Aux/IAA proteins constitute a family of short-lived, nucleus-localized transcriptional repressors that interact with auxin response factor (ARF) to modulate the expression of auxin-responsive genes [ 3 , 4 ] . Their rapid degradation via the SCF^TIR1/AFB–mediated ubiquitin–proteasome pathway enables plants to fine-tune auxin signaling in response to developmental cues and environmental stimuli [ 5 ] . With advances in genome sequencing, Aux/IAA gene families have been characterized in a wide range of plant species, showing considerable variation in family size—for example, 89 members identified in turnip [ 1 ] 119 in Brassica napus L [ 6 ] , 36 in Hordeum vulgare [ 7 ] , 31 in Oryza sativa [ 8 ] , and 25 in Solanum lycopersicum [ 9 ] . This variation is largely attributed to whole-genome and tandem duplication events, which have contributed to the functional diversification of Aux/IAA genes [ 10 ] . Accumulating evidence also indicates that Aux/IAA genes participate in multiple abiotic stress responses through crosstalk with various hormone pathways, thereby functioning as key nodes linking environmental signals with developmental regulation [ 11 , 12 ] . In Capsicum annuum , numerous Aux/IAA genes exhibit strong induction under salt and drought stress [ 13 ] ; in Solanum lycopersicum , several members show time-dependent expression following stress exposure [ 14 ] ; while overexpression of OsIAA18 significantly enhances drought and salt tolerance in Oryza sativa [ 15 , 16 ] . Stress-responsive Aux/IAA homologs have also been reported in Zea mays , Glycine max , and Cicer arietinum [ 17 , 18 ] , indicating their critical roles in coordinating auxin signaling with abiotic stress response [ 19 – 21 ] . Pumpkin ( Cucurbita moschata ), a globally significant economic crop in the Cucurbitaceae family, is highly valued not only for its rich nutritional content but also for its extensive root system and robust environmental adaptability. These traits make it a preferred rootstock for grafting crops like cucumber and watermelon [ 22 , 23 ] , playing a pivotal role in enhancing scion resistance to various stresses. However, pumpkin cultivation is frequently challenged by complex environmental constraints, particularly the combined effects of drought and salinity, which significantly inhibit plant growth and limit yield and quality. [ 24 , 25 ] . The Aux/IAA gene family, serving as core repressors in the auxin signaling pathway, plays a fundamental role in both plant development and stress responses. While genome-wide identifications of this family have been conducted in species such as tomato, spinach, and the closely related cucumber [ 26 , 27 ] , a systematic characterization of the Aux/IAA family in pumpkin remains absent. Therefore, a comprehensive genome-wide identification and functional analysis of the pumpkin Aux/IAA family is essential. Such a study is of great significance for unraveling the molecular mechanisms underlying stress resistance and facilitating the breeding of superior rootstock varieties. In this study, we systematically identified Aux/IAA genes in the pumpkin genome and performed an integrative analysis of their chromosomal distribution, phylogenetic relationships, conserved motifs, gene structures, cis-regulatory elements, tissue-specific expression patterns, and responses to abiotic stresses. This work provides a foundation for understanding the biological functions of CmIAA genes and offers valuable candidate genes for molecular breeding aimed at improving stress tolerance in pumpkin. 1. Results 1.1. Identification of Gene Family Members and Analysis of Physicochemical Properties A genome-wide systematic search identified a total of 72 Aux/IAA genes in pumpkin(C. moschata). These genes were sequentially named according to their physical positions on the chromosomes, with CmIAA1 representing the first identified locus, followed by CmIAA2, CmIAA3, and so forth. The basic physicochemical properties of CmIAA proteins were predicted using the ExPASy ProtParam tool. Detailed information for all 72 CmIAA proteins is summarized in Table 1. The predicted protein lengths varied widely, ranging from 158 to 1275 amino acids, with corresponding molecular weights of 17,788.55 ~ 141,556.39 Da. The theoretical isoelectric points (pI) ranged from 4.57 to 9.81. 1.2. Chromosomal localization of the CmIAA Gene Family Chromosomal localization analysis showed that the 72 CmIAA genes were unevenly distributed across the 20 chromosomes of pumpkin (Fig. 1 ). Among them, chromosome (Chr) 17 harbored the largest number of CmIAA genes (seven), followed by Chr1, Chr5, and Chr8, each containing six genes. Chromosomes Chr4, Chr6, Chr12, and Chr14 each contained five CmIAA genes, whereas only one CmIAA gene was detected on Chr13, Chr16, and Chr20. No CmIAA genes were identified on Chr3. Notably, although Chr4 is the longest chromosome in the pumpkin genome, it contained only five CmIAA members, indicating that chromosome length does not directly determine the distribution density of this gene family. Overall, most chromosomes harbored a moderate number of CmIAA genes (four to six), while a few chromosomes exhibited relatively high or low gene densities. In addition, a small gene cluster consisting of three CmIAA genes was identified on Chr1. 1.3. Phylogenetic Analysis of the CmIAA Gene Family To investigate the evolutionary relationships of the Aux/IAA gene family, a neighbor-joining (NJ) phylogenetic tree was constructed using Aux/IAA protein sequences from pumpkin, A rabidopsis thaliana , and O ryza sativa . A total of 132 Aux/IAA genes were classified into nine conserved subgroups (Groups Ⅰ~Ⅸ).(Fig. 2 a) CmIAA genes were distributed across all subgroups and were relatively more abundant in Groups Ⅰ, Ⅳ, and Ⅵ. In addition, certain subgroups exhibited species-specific clustering patterns: pumpkin Aux/IAA genes clustered more closely with those of A rabidopsis thaliana in Groups V, Ⅵ, and Ⅸ, whereas closer relationships between pumpkin and Oryza sativa were observed in Group Ⅶ. Based on the cross-species phylogenetic framework, a separate phylogenetic tree was further constructed for the 72 CmIAA genes to resolve their intra-specific evolutionary relationships in pumpkin. The results demonstrated that the clustering characteristics of pumpkin members were highly consistent with the aforementioned cross-species grouping, with the 72 CmIAA genes being explicitly and comprehensively categorized into the same nine subfamilies (Groups I–IX).(Fig. 2 b) Each subgroup exhibited distinct clustering features, where genes within the same clade displayed compact branches and shorter branch lengths, suggesting a high degree of sequence conservation and recent evolutionary diversification within these subgroups. Overall, phylogenetic analysis indicates that the Aux/IAA gene family in pumpkin is largely conserved, while certain subgroups have undergone relative expansion. These results provide a phylogenetic framework for subsequent analyses of gene structure, duplication patterns, and expression characteristics. 1.4. Conserved motif Distribution and Gene Structure Analysis of CmIAA Genes To further characterize the structural features of the CmIAA gene family, conserved motifs and exon-intron structures were analyzed in combination with phylogenetic grouping (Fig. 3 ). A total of 20 conserved motifs were identified among the 72 CmIAA proteins. Most CmIAA proteins contained motif 1, indicating that this motif represents a highly conserved core element of the Aux/IAA family. CmIAA genes within the same phylogenetic subgroup exhibited a high degree of structural consistency, showing similar numbers, types, and arrangements of conserved motifs. Several motifs displayed subgroup-specific distributions; for example, motifs 4, 5, 9, 15, and 19 were exclusively detected in Group I, whereas motif 13 was only present in Groups V and Ⅵ. In contrast, motifs 3 and 11 were absent in Group I but widely distributed among other subgroups. These results suggest that the Aux/IAA gene family combines overall conservation with subgroup-specific structural variation. Gene structure analysis further supported the observed motif distribution patterns. Most CmIAA genes contained two to four exons, particularly those in Groups Ⅳ, Ⅶ, Ⅷ, and Ⅸ. Although gene structures were generally similar within each subgroup, a few exceptions were observed. For instance, CmIAA 10 in Group Ⅵ and CmIAA 5 in Group Ⅱ exhibited exon-intron organizations that differed markedly from other members of their respective subgroups. These structural variations indicate that CmIAA genes may have undergone different degrees of sequence divergence during evolution. 1.5. Gene Duplication Patterns and Collinearity Analysis of the CmIAA Family To further investigate the evolutionary characteristics of the CmIAA gene family in pumpkin, both intra- and interspecific collinearity analyses were performed (Fig. 4 ). Among the 72 identified CmIAA genes, 54 were involved in segmental duplication events, whereas no tandem duplication events were detected, indicating that segmental duplication represents the primary mechanism driving the expansion of this gene family. Intragenomic collinearity analysis (Fig. 4 a) revealed that the most prominent syntenic relationships occurred between chromosomes 8 and 17. In addition, multiple segmentally duplicated CmIAA gene pairs were distributed across chromosomes 1, 5, and 12. Ka/Ks ratio analysis showed that all duplicated gene pairs exhibited values ranging from 0.0505 to 0.4093, which were consistently lower than 1. These results suggest that CmIAA genes have predominantly undergone strong purifying selection during evolution, thereby maintaining relatively conserved protein functions after duplication. Interspecific collinearity analysis further identified varying degrees of syntenic relationships between pumpkin and Arabidopsis thaliana , Cucurbita pepo , and Cucurbita maxima , with 49, 64, and 77 orthologous gene pairs detected, respectively (Fig. 4 b). Notably, the strongest collinearity was observed between pumpkin and C. pepo , including both one-to-one and one-to-many orthologous relationships, reflecting their close evolutionary relationship within the Cucurbitaceae family. Overall, segmental duplication appears to be the major force contributing to the expansion of the Aux/IAA gene family in pumpkin, while purifying selection has constrained sequence divergence among duplicated genes. Moreover, the high level of synteny observed among cucurbit species highlights the conservation of ancestral genomic features during Aux/IAA gene family evolution. 1.6. Promoter Analysis of CmIAA Genes To further elucidate the potential regulatory features of the Aux/IAA gene family in pumpkin during growth, development, and stress responses, cis-acting elements within the 2 kb upstream promoter regions of the CmIAA genes were systematically analyzed using the PlantCARE database (Fig. 5 ; Additional file 2). A total of 38 types of cis-acting elements were identified and classified into four major functional categories, including light responsiveness, hormone responsiveness, developmental regulation, and environmental stress responsiveness, showing substantial diversity and clear gene-specific distributions. Light-responsive elements were widely distributed across the promoters of most CmIAA genes, suggesting that the Aux/IAA gene family may be broadly regulated by light signals. Hormone-responsive elements were also abundant, whereas development-related elements occurred at relatively lower frequencies overall. Notably, several stress-responsive elements, including ARE, DRE, GC-motif, LTR, and TC-rich repeats, were significantly enriched in the promoter regions of genes such as CmIAA 69 and CmIAA 26. The presence of these elements suggests that these CmIAA genes may possess strong inducibility under abiotic stress conditions, such as drought, low temperature, and salt stress.Taken together, the high diversity and pronounced gene-specific distribution of cis-acting elements in CmIAA promoters highlight the potential complexity of regulatory mechanisms governing the Aux/IAA gene family. 1.7. Tissue-Specific Expression Profiles of CmIAA Genes To further characterize the expression profiles of the Aux/IAA gene family across different tissues in pumpkin, RNA-seq data were used to analyze the transcriptional levels of 72 CmIAA genes in roots, stems, leaves, and fruits (Fig. 6 ; Additional file 3). The results revealed pronounced tissue-specific expression patterns within the CmIAA gene family. Several CmIAA genes, including CmIAA 46, CmIAA 31, CmIAA 25, and CmIAA 55, exhibited relatively high expression levels in roots, suggesting a potential association with root-related physiological processes. Among these, some genes (e.g., CmIAA 46 and CmIAA 31) also maintained elevated expression levels in stems. In contrast, the majority of CmIAA genes showed low expression levels in leaves and fruits, with many genes being markedly downregulated in these tissues. Overall, roots and stems represent the primary tissues with high CmIAA expression, whereas leaves and fruits exhibit generally low expression levels. This tissue-specific expression pattern indicates that CmIAA genes may play distinct regulatory roles during the development of different pumpkin organs. 1.8. Expression Profiles of CmIAA Genes under Abiotic Stress Conditions To further elucidate the expression patterns of the CmIAA gene family in response to abiotic stresses in pumpkin, qRT-PCR were used to analyze the transcriptional changes of 72 CmIAA genes in leaves and roots under salt (NaCl) and drought (PEG) treatments (Fig. 7 ; Additional file 4). The results revealed pronounced tissue-specific and stress-specific expression patterns of CmIAA genes under stress conditions. In leaves,(Fig. 7 a) several CmIAA genes, including CmIAA 38 and CmIAA 46, were consistently upregulated under both NaCl and PEG treatments, whereas CmIAA 12 and CmIAA 69 were markedly downregulated under the two stress conditions. In roots,(Fig. 7 b) CmIAA 42 and CmIAA 61 were strongly induced by NaCl treatment but showed relatively weak responses to PEG stress. Distinct response preferences were observed among CmIAA genes under salt and drought stresses. For instance, CmIAA 46 in leaves responded sensitively to both stress treatments, whereas CmIAA 61 in roots primarily responded to salt stress. Collectively, these results indicate that the CmIAA gene family exhibits complex and finely regulated expression patterns under abiotic stress conditions, with different members displaying tissue- and stress-dependent transcriptional responses. 2. Discussion The Aux/IAA gene family constitutes a core regulatory component in the auxin signaling pathway and plays critical roles in plant growth, development, and responses to abiotic stresses [ 10 , 28 , 29 ] In this study, a total of 72 CmIAA genes were identified in the pumpkin ( C. moschata ) genome. Compared with model plant species such as Arabidopsis thaliana and Oryza sativa , the relatively larger size of the Aux/IAA family in pumpkin suggests that this gene family may have undergone lineage - specific expansion, potentially associated with genome duplication events during evolution [ 30 ] . Collinearity analysis indicated that segmental duplication and dispersed duplication events may have played major roles in the expansion of the CmIAA gene family [ 9 , 31 ] , a pattern consistent with that reported in Solanum lycopersicum ,C ucumis sativus , and members of the Brassicaceae family. In addition, the uneven chromosomal distribution of CmIAA genes may be related to historical genome rearrangements or local gene loss events, a phenomenon that has also been observed in Solanum lycopersicum and Brassica rapa [ 9 , 32 ] At the protein structural level, most CmIAA proteins retained the characteristic conserved motifs of the Aux/IAA family. The widespread presence of motif 1 further supports the high degree of structural conservation within this gene family [ 29 ] . Meanwhile, differences in motif composition and exon–intron organization were observed among different phylogenetic subgroups. For instance, CmIAA10 in Group VI contains an additional intron, suggesting that this gene may have accumulated limited structural variation on the basis of overall conservation, thereby increasing potential regulatory complexity [ 33 ] . Intraspecific phylogenetic analysis revealed that Groups Ⅰ, Ⅳ, and Ⅵ exhibited apparent expansion in pumpkin, implying that these subgroups may have undergone species-specific duplication and selection processes, which could contribute to response to specific ecological environments or developmental requirements [ 34 ] . Ka/Ks analysis showed that most homologous gene pairs were subjected to purifying selection, reflecting the functional conservation and importance of Aux/IAA proteins in auxin signal transduction [ 35 ] . Moreover, interspecific collinearity analysis demonstrated a high degree of synteny between pumpkin and Arabidopsis thaliana , Cucurbita pepo , and Cucurbita maxima , further indicating that the Aux/IAA gene family is generally conserved within the Cucurbitaceae lineage. Promoter cis-element analysis showed that the upstream regulatory regions of CmIAA genes are enriched with cis-acting elements related to light responsiveness, hormone signaling, and abiotic stress responses, suggesting that this gene family may be involved in multilayered transcriptional regulatory networks [ 36 ] . In particular, the presence of light-responsive elements indicates that certain CmIAA members may participate in the regulation of plant growth and development, a mechanism that has been validated for IAA9 in Solanum lycopersicum [ 37 ] . Moreover, co-expression network analysis revealed coordinated transcriptional changes between several CmIAA genes and key components of the auxin signaling pathway. This observation is consistent with the conserved Aux/IAA–ARF regulatory module reported across multiple plant species [ 38 ] , providing reliable candidate genes for subsequent functional studies. Tissue-specific expression analysis indicated that CmIAA genes displayed distinct expression patterns among different organs, with several members (e.g., CmIAA 46 and CmIAA 55) maintaining relatively high expression levels in roots and stems. This expression pattern is consistent with previous reports describing the involvement of Aux/IAA genes in root development in Glycine max and Zea mays [ 39 ] . From a crop improvement perspective, root system architecture is a key trait influencing plant stress tolerance, and genetic regulation of root-related genes has been shown to optimize root architecture in Oryza sativa [ 40 ] and Triticum aestivum [ 41 ] . In contrast, these genes generally exhibited lower expression levels in leaves and fruits, and may be activated only at specific developmental stages or under stress conditions, as previously reported in Solanum lycopersicum and Capsicum annuum [ 13 , 14 ] . Under salt and drought stress conditions, CmIAA genes exhibited distinct stress- and tissue-dependent expression patterns, with individual members showing differential responsiveness to the two stress treatments. Similar transcriptional diversity has been reported in Oryza sativa [ 15 ] , Asparagus officinalis [ 36 ] and Medicago sativa [ 42 ] further supporting the important role of Aux/IAA genes in abiotic stress responses. Notably, several CmIAA members displayed stress-type-specific expression patterns, suggesting functional divergence within the gene family. Together with the enrichment of stress-responsive cis-elements, such as ABRE and MBS, in their promoter regions, these findings indicate that promoter engineering strategies aimed at stress-inducible expression of CmIAA genes may provide a potential avenue for improving stress tolerance in C. moschata [ 43 ] . In summary, this study performed a comprehensive genome-wide identification and characterization of the AUX/IAA gene family in pumpkin. Through integrated genomic, phylogenetic, and expression analyses, we elucidated the expansion mechanisms, structural conservation, and functional diversification of the Aux/IAA genes. While the family as a whole is under strong purifying selection, certain subfamilies appear to have developed adaptive functions in response to pumpkin-specific ecological niches and stress conditions, indicating conserved core roles alongside diversified regulatory functions. These results provide a solid foundation for the functional validation of key CmIAA genes, especially those associated with root and stem development and stress responses, and offer clear molecular targets for genetic engineering and breeding strategies aimed at enhancing stress tolerance and improving yield-related traits in pumpkin. 3. Conclusion In this study, a total of 72 CmIAA genes were identified from the pumpkin ( C. moschata ) genome. These genes are unevenly distributed across multiple chromosomes and were classified into several subgroups based on phylogenetic analysis. The CmIAA members exhibited both similarities and differences in conserved motifs, gene structures, and protein architectures, indicating structural conservation accompanied by diversification within the family. Promoter cis-acting element analysis suggested that CmIAA genes may be involved in light signal perception, hormone-mediated regulation, and responses to abiotic stresses. Furthermore, expression profiling revealed diverse tissue-specific expression patterns of CmIAA genes, and demonstrated that several members showed pronounced transcriptional responses to salt and drought stresses. Collectively, these findings provide an important theoretical foundation for further functional characterization of CmIAA genes in pumpkin growth, development, and stress response. 4. Materials and Methods 4.1. Plant Materials and Treatments The inbred line "360-3" of pumpkin ( Cucurbita moschata ) was provided by the Pumpkin Germplasm Resources Innovation and Utilization Team of the College of Horticulture and Landscape Architecture, Henan Institute of Science and Technology. Plump pumpkin seeds were selected for germination. Upon full cotyledon expansion, the seedlings were hydroponically cultured in ½ Hoagland’s nutrient solution for two weeks. Following a 24-h acclimation in distilled water, uniform-sized seedlings with consistent growth performance were subjected to drought and salt stress treatments, respectively. Specifically, drought stress was simulated by exposure to 20% polyethylene glycol 6000 (PEG6000) for 3 h, whereas salt stress was imposed using a 200 mM NaCl solution for 3 h. After stress treatments, root and leaf tissues were immediately harvested for total RNA extraction and subsequent qRT-PCR analysis. 4.2. Identification of CmIAA Family Members Aux/IAA protein sequences from Arabidopsis thaliana and Oryza sativa were retrieved using TBtools and employed as query templates. Homology-based searches were conducted against the pumpkin genome in the Cucurbit Genomics Database (CuGenDB) [ 44 ] using BLAST,. Redundant sequences were removed using a similarity threshold of 50% and an e-value cutoff of 1×10 − 5 . All non-redundant candidate proteins were subsequently verified for the presence of conserved Aux/IAA domains using NCBI-CDD and SMART [ 45 ] . Additional filtering steps were applied to eliminate any remaining redundancy, resulting in the final set of CmIAA family members. 4.3. Prediction of Physicochemical Properties of CmIAA Proteins The ExPASy ProtParam [ 46 ] tool was used to predict the basic physicochemical characteristics of each CmIAA protein, including amino acids number, molecular weight, and theoretical isoelectric point (pI). 4.4. Phylogenetic Tree Construction A phylogenetic tree was constructed using the Neighbor-Joining method with 1,000 bootstrap replicates in MEGA-X [ 47 ] , based on the amino acid sequences of the CmIAA proteins. The resulting tree was subsequently refined and visualized using the Interactive Tree of Life (iTOL) online platform [ 48 ] . 4.5. Collinearity Analysis Gene family files Cucurbita pepo , Cucurbita maxima and Arabidopsis thaliana were obtained from Ensembl Plants,and those for pumpkin were downloaded from CuGenDB [ 44 ] . Both interspecific synteny analysis among these species and intraspecific synteny analysis of pumpkin CmIAA genes were conducted via TBtools [ 49 ] . Furthermore, to assess selection pressure, Ka/Ks ratios between duplicated CmIAA gene pairs were determined using KaKs_Calculator 2.0. 4.6. Promoter Cis-Regulatory Element Analysis Coding sequences (CDS) and full-length genomic sequences of all 72 CmIAA genes were retrieved from CuGenDB [ 44 ] . A 2,000 bp upstream region from each gene was extracted using TBtools and analyzed for cis-regulatory elements using the PlantCARE [ 50 ] database. The results were graphically visualized with TBtools [ 49 ] . 4.7. Chromosomal Localization The chromosomal positions of all 72 CmIAA genes were obtained from CuGenDB [ 44 ] . Their distribution across chromosomes was mapped and visualized using TBtools [ 49 ] . 4.8. Gene Structure and Conserved motif Analysis Conserved motifs within the CmIAA protein sequences were identified using the MEME [ 51 ] online program. Gene structure information for all CmIAA members was obtained using the CSDS 2.0 web server. 4.9. Tissue Expression Pattern Analysis RNA-seq datasets of four tissue types (root, stem, leaf, and fruit) in pumpkin were downloaded from the CuGenDB database (PRJNA385310). Fragments per kilobase of transcript per million mapped reads (FPKM) values were calculated to estimate the expression levels of CmIAA genes. The expression data were log2 transformed, and heatmaps were constructed using TBtools [ 49 ] visualize the expression patterns across different tissues. 4.10. Rna Extraction And qRT-PCR Analysis of Abiotic Stress Treatment Expression profiles of CmIAA under abiotic stress conditions were analyzed via quantitative real-time PCR (qRT-PCR). Briefly, Total RNA was isolated from the collected samples using the Omini Plant RNA Kit (CWBIO, Beijing, China). For cDNA synthesis and qRT-PCR amplification, commercial reagent kits purchased from TaKaRa were utilized, with all reactions conducted on a Bio-Rad real-time fluorescence quantitative PCR system. The qRT-PCR reaction mixture (10 µL) consisted of 5 µL TB Green Mix, 0.4 µL each of the forward and reverse primers, 0.2 µL of cDNA template, and 4 µL of ddH 2 O. The thermal cycling conditions were as follows: an initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. A melting curve program (95°C for 5 s, 65°C for 5 s, and 95°C for 5 s) was performed to verify the specificity of the amplification products. The pumpkin Actin gene was utilized as an internal reference, and the relative expression levels were quantified using the 2 − ∆∆Ct method. The experiment was performed with three independent biological replicates. Primers for qRT-PCR were listed in Additional file 1. Declarations Ethics approval and consent to participate All plant materials used in this study, including pumpkin ( C.moschata ), were grown and collected according to institutional and national guidelines. The pumpkin cultivars used are common agricultural varieties and are not endangered species. All methods complied with relevant institutional, national, and international guidelines and legislation. Consent for publication NOT APPLICABLE Availability of data and materials Raw sequence was data of C.moschata was downloaded from the NCBI database using accession number PRJNA385310 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA385310/).RNA-seq data were derived from the Cucurbit Genomics Database (CuGenDB) using accession number PRJNA385310. (http:// cucurbitgenomics. org/ rnaseq/ pu/25). Alldatasets generated in this study are included in the published article/Additional Files. Wedsites used for analyses in this study are as follows:Cucurbit Genomics Database (http://cucurbitgenomics. org/ organism/9).TBtools (https://github.com/CJ-Chen/TBTools). SMART (http://smart.embl-heidelberg.de/). ExPASy (http://web.expasy. org/ prot param/). iTOL (https: //itol. embl.de/). Ensembl Plants (https://plants. ensembl.org/index.html). Plant CARE (http://bioinformatics.psb. ugent. be/webtools/plantcare/html/). MEME (https://meme-suite. org/meme/ doc/meme.html). SDS2.0 (https://gsds.gao-lab.org/Gsds_help.php). Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . Funding This research was supported by the Natural Science Foundation of Henan Province (Grant No. 252300420662). Authors’ contributions YZ and MS conceived and designed the project. MS, JD, and JZ performed the experiments and collected the plant materials. LJ and MS analyzed the data and performed the bioinformatic analysis. MS wrote the original draft. YZ, JD, and LJ revised the manuscript. All authors read and approved the final manuscript. Acknowledgements We are grateful to the providers of genomic and transcriptome data. Authors' information 1 Henan Institute of Science and Technology, Xinxiang 453000, Henan, P. R. China References Xu H, Liu Y, Zhang S, Shui D, Xia Z, Sun J. 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Int J Mol Sci. 2017;85(3–4):107–19. Zhang J, Li S, Gao X, Liu Y, Fu B. Genome-Wide Identification And Expression Pattern Analysis Of The Aux/IAA ( Auxin/Indole-3-Acetic Acid) Gene Family In Alfalfa ( Medicago Sativa ) And The Potential Functions Under Drought Stress. BMC Genomics. 2024;25(1):382–382. Huang X, Shad MA, Shu Y, Nong S, Li X, Wu S, Yang J, Rao MJ, Aslam MZ, Huang X, et al. Genome-Wide Analysis Of The Auxin/Indoleacetic Acid ( Aux/IAA ) Gene Family In Autopolyploid Sugarcane ( Saccharum Spontaneum) . Int J Mol Sci. 2024;25(13):7473–7473. Zheng Y, Wu S, Bai Y, Sun H, Jiao C, Guo S, Zhao K, Blanca J, Zhang Z, Huang S, et al. Cucurbit Genomics Database ( Cugendb ): a Central Portal For Comparative And Functional Genomics Of Cucurbit Crops . Nucleic Acids Res. 2018;47(D1):D1128–36. Letunic I, Khedkar S, Bork P. SMART: Recent Updates, New Developments And Status In 2020. Nucleic Acids Res. 2021;49(D1):D458–60. 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Nucleic Acids Res. 2015;43(W1):W39–49. Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx Additionalfile1.xlsx Additionalfile2.xlsx Additionalfile3.xlsx Additionalfile4.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 13 May, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviews received at journal 19 Apr, 2026 Reviewers agreed at journal 04 Apr, 2026 Reviewers agreed at journal 02 Apr, 2026 Reviewers agreed at journal 05 Mar, 2026 Reviewers agreed at journal 10 Feb, 2026 Reviewers agreed at journal 05 Feb, 2026 Reviewers invited by journal 21 Jan, 2026 Editor assigned by journal 18 Jan, 2026 Editor invited by journal 16 Jan, 2026 Submission checks completed at journal 14 Jan, 2026 First submitted to journal 14 Jan, 2026 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8529495","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":579964386,"identity":"a6547d11-8bc9-40ce-90b0-a67cd3939899","order_by":0,"name":"Mengyao Sun","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Mengyao","middleName":"","lastName":"Sun","suffix":""},{"id":579964387,"identity":"22d87985-5bb7-45b9-8c50-1b46a380614d","order_by":1,"name":"Jiayi Duan","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Duan","suffix":""},{"id":579964388,"identity":"14cbbac5-4c00-4212-a07a-9743cc3d2f64","order_by":2,"name":"Lina Jiang","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Lina","middleName":"","lastName":"Jiang","suffix":""},{"id":579964389,"identity":"e7c36441-ead5-4d92-85ca-95a008483d26","order_by":3,"name":"Junguo Zhou","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Junguo","middleName":"","lastName":"Zhou","suffix":""},{"id":579964390,"identity":"eec4422c-ba64-4ef4-976f-184664918a56","order_by":4,"name":"Yufei Zhai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIie3PsWrDMBCA4VMF50XJfKFgvYKMlj6OTSBZTCkUSobQqBg89QECNelDFDLLBDKFzF3drh0MXTpkaJQti+wxEP2b4D5JBxAKXWIEYAFiwOjF2HZGsexJNKDY1PVyd6cT04OAI0CT8WZQzjJ3gzf5VjTHSRUPIVd2sKKUGd58fXoIq7bKEY2wU3a0pvsIUOvcQziljhyykr0qm6zpkRmBtz6CNG3dK4uSC2WzijJjO4ig/PSxFBFTW5sehCh/qKu9SkrBj2JLOik6dpHL6Uf786SkfG+K38P8OZZR0Xz7iOtGnB15x7iL/fUYCoVCoSvuH4JZTR70DKYAAAAAAElFTkSuQmCC","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Yufei","middleName":"","lastName":"Zhai","suffix":""}],"badges":[],"createdAt":"2026-01-06 09:39:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8529495/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8529495/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101397855,"identity":"a6544545-9a18-48de-b5a4-a6f07f5d0572","added_by":"auto","created_at":"2026-01-29 09:37:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1309056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromosomal distribution of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCmIAA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Gene in C. moschata\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/636c3e0656c642a87bd364b0.png"},{"id":101322607,"identity":"79bc9efb-79db-4c8b-b2af-dae662767c41","added_by":"auto","created_at":"2026-01-28 13:13:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15717200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of Aux/IAA proteins.\u003c/strong\u003e(a) Phylogenetic relationships of Aux/IAA proteins from A\u003cem\u003erabidopsis thaliana,\u003c/em\u003eO\u003cem\u003eryza sativa, \u003c/em\u003eand C. moschata\u003cem\u003e.\u003c/em\u003e(b) Phylogenetic relationships of Aux/IAA proteins in C. moschata.The phylogenetic trees were constructed using the neighbor-joining (NJ) method implemented in MEGA-X software. Bootstrap analysis was performed with 1000 replicates to assess branch reliability. The nine subgroups (Group I–IX) are indicated by different colored sectors.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/93e7194cb22582ccce60df15.png"},{"id":101322601,"identity":"6fa857cf-3eae-4148-8ee8-70af1d85bb25","added_by":"auto","created_at":"2026-01-28 13:13:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7231954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic grouping, conserved motif composition, and gene structure of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCmIAA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in C. moschata. \u003c/strong\u003e(a) panel shows the conserved motifs of \u003cem\u003eCmIAA\u003c/em\u003eproteins identified by MEME, with different colors representing distinct motifs. (b) panel illustrates the exon-intron structures of \u003cem\u003eCmIAA\u003c/em\u003e genes, where yellow boxes indicate coding sequences (CDSs), green boxes represent untranslated regions (UTRs), and black lines denote introns. All \u003cem\u003eCmIAA\u003c/em\u003egenes are arranged according to their positions in the protein-based phylogenetic tree.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/29f54131fcdf4c649f830462.png"},{"id":101322603,"identity":"c564be17-ca88-4d28-8e3e-5548df450132","added_by":"auto","created_at":"2026-01-28 13:13:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":19177072,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynteny analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCmIAA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes within C. moschata and among different plant species.\u003c/strong\u003e (a) represents intraspecies synteny of \u003cem\u003eCmIAA\u003c/em\u003e genes in C. moschata, where the colored outer blocks indicate chromosomes and red lines denote segmentally duplicated \u003cem\u003eCmIAA\u003c/em\u003egene pairs. (b) show interspecies synteny of \u003cem\u003eAux/IAA\u003c/em\u003e genes between C. moschata and Arabidopsis thaliana (top), Cucurbita pepo (middle), and Cucurbita maxima (bottom), respectively. Colored lines indicate orthologous gene pairs.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/8e0424537985638e4e870964.png"},{"id":101322609,"identity":"8b47b124-bc00-4a12-86dc-113161d3f1df","added_by":"auto","created_at":"2026-01-28 13:13:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6609807,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCis-acting element analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCmIAA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene promoters.\u003c/strong\u003eThe heatmap shows the distribution and frequency of cis-acting regulatory elements identified in the 2 kb upstream promoter regions of \u003cem\u003eCmIAA\u003c/em\u003e genes. The color scale represents the number of each cis-element detected in individual promoters.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/7abceb532060b6003e1dfc24.png"},{"id":101322610,"identity":"715f879e-3d92-4700-a4fc-a07e3ad89162","added_by":"auto","created_at":"2026-01-28 13:13:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36883925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTissue-specific expression profiles of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCmIAA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in pumpkin.\u003c/strong\u003eThe circular heatmap illustrates the expression patterns of \u003cem\u003eCmIAA\u003c/em\u003e genes in root, stem, leaf, and fruit tissues based on RNA-seq data. Expression levels were normalized and log\u003csub\u003e2 \u003c/sub\u003etransformed.\u003c/p\u003e","description":"","filename":"Figure6..png","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/a82192a21259416e38a6b293.png"},{"id":101322604,"identity":"8863ce70-8d5a-4178-9054-217983ec0320","added_by":"auto","created_at":"2026-01-28 13:13:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":23707044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns of CmIAA genes in leaf and root under abiotic stress conditions.\u003c/strong\u003e (a) Circular heatmap illustrating the expression profiles of\u003cem\u003e CmIAA\u003c/em\u003e genes in leaf tissues under control (CK), salt (NaCl), and drought (PEG) treatments. (b) Circular heatmap illustrating the expression profiles of \u003cem\u003eCmIAA\u003c/em\u003e genes in root tissues under the same abiotic stress conditions. Expression levels were normalized and log₂-transformed. Color gradients from blue to red indicate low to high expression levels, respectively.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/502de08b8d80d785086a25f0.png"},{"id":101399054,"identity":"b3f82b3e-a726-4c15-ad59-de431c145340","added_by":"auto","created_at":"2026-01-29 09:51:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":98223143,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/f6eaab3d-3397-4232-bab3-3e4f913cf8cf.pdf"},{"id":101322600,"identity":"86952381-064e-40d1-b2de-6515309553a2","added_by":"auto","created_at":"2026-01-28 13:13:42","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":34178,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/6fc4d2767cb2e1690406bf8c.xlsx"},{"id":101322602,"identity":"46e132a1-b2b5-42c5-beb3-19141d4cd67f","added_by":"auto","created_at":"2026-01-28 13:13:42","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13347,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/5cc7bc89b0c899c45f4e3039.xlsx"},{"id":101322606,"identity":"0d4d02e1-56ee-4dd8-ad92-95d20b7783da","added_by":"auto","created_at":"2026-01-28 13:13:42","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21454,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/a4ed182944d0001b582f889c.xlsx"},{"id":101322605,"identity":"fd163b3d-9331-4996-8678-092baa3b5a00","added_by":"auto","created_at":"2026-01-28 13:13:42","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14955,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/82ec54191cf58500041436ae.xlsx"},{"id":101322608,"identity":"904f4baa-0867-4eb0-abc6-9210f3d50321","added_by":"auto","created_at":"2026-01-28 13:13:43","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":21226,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8529495/v1/6d382588fe34828097fe0666.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-Wide Identification and Comprehensive Characterization of the Aux/IAA Gene Family in Cucurbita moschata and Its Response Analysis to Abiotic Stress","fulltext":[{"header":"1. Background","content":"\u003cp\u003eAuxin is one of the central phytohormones regulating plant growth and development, with essential roles in cell division, organogenesis, fruit development, and abiotic stress respons\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The Aux/IAA proteins constitute a family of short-lived, nucleus-localized transcriptional repressors that interact with auxin response factor (ARF) to modulate the expression of auxin-responsive genes\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Their rapid degradation via the SCF^TIR1/AFB\u0026ndash;mediated ubiquitin\u0026ndash;proteasome pathway enables plants to fine-tune auxin signaling in response to developmental cues and environmental stimuli \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWith advances in genome sequencing, \u003cem\u003eAux/IAA\u003c/em\u003e gene families have been characterized in a wide range of plant species, showing considerable variation in family size\u0026mdash;for example, 89 members identified in turnip\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e 119 in \u003cem\u003eBrassica napus\u003c/em\u003e L\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, 36 in \u003cem\u003eHordeum vulgare\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, 31 in \u003cem\u003eOryza sativa\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, and 25 in \u003cem\u003eSolanum lycopersicum\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. This variation is largely attributed to whole-genome and tandem duplication events, which have contributed to the functional diversification of \u003cem\u003eAux/IAA\u003c/em\u003e genes\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAccumulating evidence also indicates that \u003cem\u003eAux/IAA\u003c/em\u003e genes participate in multiple abiotic stress responses through crosstalk with various hormone pathways, thereby functioning as key nodes linking environmental signals with developmental regulation\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eCapsicum annuum\u003c/em\u003e, numerous \u003cem\u003eAux/IAA\u003c/em\u003e genes exhibit strong induction under salt and drought stress \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e; in \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, several members show time-dependent expression following stress exposure\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e; while overexpression of OsIAA18 significantly enhances drought and salt tolerance in \u003cem\u003eOryza sativa\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Stress-responsive \u003cem\u003eAux/IAA\u003c/em\u003e homologs have also been reported in \u003cem\u003eZea mays\u003c/em\u003e, \u003cem\u003eGlycine max\u003c/em\u003e, and \u003cem\u003eCicer arietinum\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, indicating their critical roles in coordinating auxin signaling with abiotic stress response\u003csup\u003e[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePumpkin (\u003cem\u003eCucurbita moschata\u003c/em\u003e), a globally significant economic crop in the Cucurbitaceae family, is highly valued not only for its rich nutritional content but also for its extensive root system and robust environmental adaptability. These traits make it a preferred rootstock for grafting crops like cucumber and watermelon\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, playing a pivotal role in enhancing scion resistance to various stresses. However, pumpkin cultivation is frequently challenged by complex environmental constraints, particularly the combined effects of drought and salinity, which significantly inhibit plant growth and limit yield and quality.\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The Aux/IAA gene family, serving as core repressors in the auxin signaling pathway, plays a fundamental role in both plant development and stress responses. While genome-wide identifications of this family have been conducted in species such as tomato, spinach, and the closely related cucumber\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, a systematic characterization of the Aux/IAA family in pumpkin remains absent. Therefore, a comprehensive genome-wide identification and functional analysis of the pumpkin Aux/IAA family is essential. Such a study is of great significance for unraveling the molecular mechanisms underlying stress resistance and facilitating the breeding of superior rootstock varieties.\u003c/p\u003e \u003cp\u003eIn this study, we systematically identified \u003cem\u003eAux/IAA\u003c/em\u003e genes in the pumpkin genome and performed an integrative analysis of their chromosomal distribution, phylogenetic relationships, conserved motifs, gene structures, cis-regulatory elements, tissue-specific expression patterns, and responses to abiotic stresses. This work provides a foundation for understanding the biological functions of \u003cem\u003eCmIAA\u003c/em\u003e genes and offers valuable candidate genes for molecular breeding aimed at improving stress tolerance in pumpkin.\u003c/p\u003e"},{"header":"1. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.1. Identification of Gene Family Members and Analysis of Physicochemical Properties\u003c/h2\u003e \u003cp\u003eA genome-wide systematic search identified a total of 72 \u003cem\u003eAux/IAA\u003c/em\u003e genes in pumpkin(C. moschata). These genes were sequentially named according to their physical positions on the chromosomes, with CmIAA1 representing the first identified locus, followed by CmIAA2, CmIAA3, and so forth. The basic physicochemical properties of CmIAA proteins were predicted using the ExPASy ProtParam tool. Detailed information for all 72 CmIAA proteins is summarized in Table\u0026nbsp;1. The predicted protein lengths varied widely, ranging from 158 to 1275 amino acids, with corresponding molecular weights of 17,788.55\u0026thinsp;~\u0026thinsp;141,556.39 Da. The theoretical isoelectric points (pI) ranged from 4.57 to 9.81.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1.2. Chromosomal localization of the \u003cem\u003eCmIAA\u003c/em\u003e Gene Family\u003c/h2\u003e \u003cp\u003eChromosomal localization analysis showed that the 72 \u003cem\u003eCmIAA\u003c/em\u003e genes were unevenly distributed across the 20 chromosomes of pumpkin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among them, chromosome (Chr) 17 harbored the largest number of \u003cem\u003eCmIAA\u003c/em\u003e genes (seven), followed by Chr1, Chr5, and Chr8, each containing six genes. Chromosomes Chr4, Chr6, Chr12, and Chr14 each contained five \u003cem\u003eCmIAA\u003c/em\u003e genes, whereas only one \u003cem\u003eCmIAA\u003c/em\u003e gene was detected on Chr13, Chr16, and Chr20. No \u003cem\u003eCmIAA\u003c/em\u003e genes were identified on Chr3. Notably, although Chr4 is the longest chromosome in the pumpkin genome, it contained only five \u003cem\u003eCmIAA\u003c/em\u003e members, indicating that chromosome length does not directly determine the distribution density of this gene family.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, most chromosomes harbored a moderate number of \u003cem\u003eCmIAA\u003c/em\u003e genes (four to six), while a few chromosomes exhibited relatively high or low gene densities. In addition, a small gene cluster consisting of three \u003cem\u003eCmIAA\u003c/em\u003e genes was identified on Chr1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e1.3. Phylogenetic Analysis of the \u003cem\u003eCmIAA\u003c/em\u003e Gene Family\u003c/h2\u003e \u003cp\u003eTo investigate the evolutionary relationships of the \u003cem\u003eAux/IAA\u003c/em\u003e gene family, a neighbor-joining (NJ) phylogenetic tree was constructed using Aux/IAA protein sequences from pumpkin, A\u003cem\u003erabidopsis thaliana\u003c/em\u003e, and O\u003cem\u003eryza sativa\u003c/em\u003e. A total of 132 \u003cem\u003eAux/IAA\u003c/em\u003e genes were classified into nine conserved subgroups (Groups Ⅰ~Ⅸ).(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) \u003cem\u003eCmIAA\u003c/em\u003e genes were distributed across all subgroups and were relatively more abundant in Groups Ⅰ, Ⅳ, and Ⅵ. In addition, certain subgroups exhibited species-specific clustering patterns: pumpkin \u003cem\u003eAux/IAA\u003c/em\u003e genes clustered more closely with those of A\u003cem\u003erabidopsis thaliana\u003c/em\u003e in Groups V, Ⅵ, and Ⅸ, whereas closer relationships between pumpkin and \u003cem\u003eOryza sativa\u003c/em\u003e were observed in Group Ⅶ.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the cross-species phylogenetic framework, a separate phylogenetic tree was further constructed for the 72 \u003cem\u003eCmIAA\u003c/em\u003e genes to resolve their intra-specific evolutionary relationships in pumpkin. The results demonstrated that the clustering characteristics of pumpkin members were highly consistent with the aforementioned cross-species grouping, with the 72 \u003cem\u003eCmIAA\u003c/em\u003e genes being explicitly and comprehensively categorized into the same nine subfamilies (Groups I\u0026ndash;IX).(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) Each subgroup exhibited distinct clustering features, where genes within the same clade displayed compact branches and shorter branch lengths, suggesting a high degree of sequence conservation and recent evolutionary diversification within these subgroups.\u003c/p\u003e \u003cp\u003eOverall, phylogenetic analysis indicates that the \u003cem\u003eAux/IAA\u003c/em\u003e gene family in pumpkin is largely conserved, while certain subgroups have undergone relative expansion. These results provide a phylogenetic framework for subsequent analyses of gene structure, duplication patterns, and expression characteristics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e1.4. Conserved motif Distribution and Gene Structure Analysis of \u003cem\u003eCmIAA\u003c/em\u003e Genes\u003c/h2\u003e \u003cp\u003eTo further characterize the structural features of the \u003cem\u003eCmIAA\u003c/em\u003e gene family, conserved motifs and exon-intron structures were analyzed in combination with phylogenetic grouping (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A total of 20 conserved motifs were identified among the 72 CmIAA proteins. Most CmIAA proteins contained motif 1, indicating that this motif represents a highly conserved core element of the \u003cem\u003eAux/IAA\u003c/em\u003e family. \u003cem\u003eCmIAA\u003c/em\u003e genes within the same phylogenetic subgroup exhibited a high degree of structural consistency, showing similar numbers, types, and arrangements of conserved motifs. Several motifs displayed subgroup-specific distributions; for example, motifs 4, 5, 9, 15, and 19 were exclusively detected in Group I, whereas motif 13 was only present in Groups V and Ⅵ. In contrast, motifs 3 and 11 were absent in Group I but widely distributed among other subgroups. These results suggest that the \u003cem\u003eAux/IAA\u003c/em\u003e gene family combines overall conservation with subgroup-specific structural variation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGene structure analysis further supported the observed motif distribution patterns. Most \u003cem\u003eCmIAA\u003c/em\u003e genes contained two to four exons, particularly those in Groups Ⅳ, Ⅶ, Ⅷ, and Ⅸ. Although gene structures were generally similar within each subgroup, a few exceptions were observed. For instance, \u003cem\u003eCmIAA\u003c/em\u003e10 in Group Ⅵ and \u003cem\u003eCmIAA\u003c/em\u003e5 in Group Ⅱ exhibited exon-intron organizations that differed markedly from other members of their respective subgroups. These structural variations indicate that \u003cem\u003eCmIAA\u003c/em\u003e genes may have undergone different degrees of sequence divergence during evolution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e1.5. Gene Duplication Patterns and Collinearity Analysis of the \u003cem\u003eCmIAA\u003c/em\u003e Family\u003c/h2\u003e \u003cp\u003eTo further investigate the evolutionary characteristics of the \u003cem\u003eCmIAA\u003c/em\u003e gene family in pumpkin, both intra- and interspecific collinearity analyses were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Among the 72 identified \u003cem\u003eCmIAA\u003c/em\u003e genes, 54 were involved in segmental duplication events, whereas no tandem duplication events were detected, indicating that segmental duplication represents the primary mechanism driving the expansion of this gene family.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIntragenomic collinearity analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) revealed that the most prominent syntenic relationships occurred between chromosomes 8 and 17. In addition, multiple segmentally duplicated \u003cem\u003eCmIAA\u003c/em\u003e gene pairs were distributed across chromosomes 1, 5, and 12. Ka/Ks ratio analysis showed that all duplicated gene pairs exhibited values ranging from 0.0505 to 0.4093, which were consistently lower than 1. These results suggest that \u003cem\u003eCmIAA\u003c/em\u003e genes have predominantly undergone strong purifying selection during evolution, thereby maintaining relatively conserved protein functions after duplication.\u003c/p\u003e \u003cp\u003eInterspecific collinearity analysis further identified varying degrees of syntenic relationships between pumpkin and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eCucurbita pepo\u003c/em\u003e, and \u003cem\u003eCucurbita maxima\u003c/em\u003e, with 49, 64, and 77 orthologous gene pairs detected, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Notably, the strongest collinearity was observed between pumpkin and \u003cem\u003eC. pepo\u003c/em\u003e, including both one-to-one and one-to-many orthologous relationships, reflecting their close evolutionary relationship within the Cucurbitaceae family.\u003c/p\u003e \u003cp\u003eOverall, segmental duplication appears to be the major force contributing to the expansion of the \u003cem\u003eAux/IAA\u003c/em\u003e gene family in pumpkin, while purifying selection has constrained sequence divergence among duplicated genes. Moreover, the high level of synteny observed among cucurbit species highlights the conservation of ancestral genomic features during \u003cem\u003eAux/IAA\u003c/em\u003e gene family evolution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e1.6. Promoter Analysis of \u003cem\u003eCmIAA\u003c/em\u003e Genes\u003c/h2\u003e \u003cp\u003eTo further elucidate the potential regulatory features of the \u003cem\u003eAux/IAA\u003c/em\u003e gene family in pumpkin during growth, development, and stress responses, cis-acting elements within the 2 kb upstream promoter regions of the \u003cem\u003eCmIAA\u003c/em\u003e genes were systematically analyzed using the PlantCARE database (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Additional file 2). A total of 38 types of cis-acting elements were identified and classified into four major functional categories, including light responsiveness, hormone responsiveness, developmental regulation, and environmental stress responsiveness, showing substantial diversity and clear gene-specific distributions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLight-responsive elements were widely distributed across the promoters of most \u003cem\u003eCmIAA\u003c/em\u003e genes, suggesting that the \u003cem\u003eAux/IAA\u003c/em\u003e gene family may be broadly regulated by light signals. Hormone-responsive elements were also abundant, whereas development-related elements occurred at relatively lower frequencies overall.\u003c/p\u003e \u003cp\u003eNotably, several stress-responsive elements, including ARE, DRE, GC-motif, LTR, and TC-rich repeats, were significantly enriched in the promoter regions of genes such as \u003cem\u003eCmIAA\u003c/em\u003e69 and \u003cem\u003eCmIAA\u003c/em\u003e26. The presence of these elements suggests that these \u003cem\u003eCmIAA\u003c/em\u003e genes may possess strong inducibility under abiotic stress conditions, such as drought, low temperature, and salt stress.Taken together, the high diversity and pronounced gene-specific distribution of cis-acting elements in \u003cem\u003eCmIAA\u003c/em\u003e promoters highlight the potential complexity of regulatory mechanisms governing the \u003cem\u003eAux/IAA\u003c/em\u003e gene family.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e1.7. Tissue-Specific Expression Profiles of \u003cem\u003eCmIAA\u003c/em\u003e Genes\u003c/h2\u003e \u003cp\u003eTo further characterize the expression profiles of the \u003cem\u003eAux/IAA\u003c/em\u003e gene family across different tissues in pumpkin, RNA-seq data were used to analyze the transcriptional levels of 72 \u003cem\u003eCmIAA\u003c/em\u003e genes in roots, stems, leaves, and fruits (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Additional file 3). The results revealed pronounced tissue-specific expression patterns within the \u003cem\u003eCmIAA\u003c/em\u003e gene family. Several \u003cem\u003eCmIAA\u003c/em\u003e genes, including \u003cem\u003eCmIAA\u003c/em\u003e46, \u003cem\u003eCmIAA\u003c/em\u003e31, \u003cem\u003eCmIAA\u003c/em\u003e25, and \u003cem\u003eCmIAA\u003c/em\u003e55, exhibited relatively high expression levels in roots, suggesting a potential association with root-related physiological processes. Among these, some genes (e.g., \u003cem\u003eCmIAA\u003c/em\u003e46 and \u003cem\u003eCmIAA\u003c/em\u003e31) also maintained elevated expression levels in stems. In contrast, the majority of \u003cem\u003eCmIAA\u003c/em\u003e genes showed low expression levels in leaves and fruits, with many genes being markedly downregulated in these tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, roots and stems represent the primary tissues with high \u003cem\u003eCmIAA\u003c/em\u003e expression, whereas leaves and fruits exhibit generally low expression levels. This tissue-specific expression pattern indicates that \u003cem\u003eCmIAA\u003c/em\u003e genes may play distinct regulatory roles during the development of different pumpkin organs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e1.8. Expression Profiles of \u003cem\u003eCmIAA\u003c/em\u003e Genes under Abiotic Stress Conditions\u003c/h2\u003e \u003cp\u003eTo further elucidate the expression patterns of the \u003cem\u003eCmIAA\u003c/em\u003e gene family in response to abiotic stresses in pumpkin, qRT-PCR were used to analyze the transcriptional changes of 72 \u003cem\u003eCmIAA\u003c/em\u003e genes in leaves and roots under salt (NaCl) and drought (PEG) treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Additional file 4). The results revealed pronounced tissue-specific and stress-specific expression patterns of \u003cem\u003eCmIAA\u003c/em\u003e genes under stress conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn leaves,(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea) several \u003cem\u003eCmIAA\u003c/em\u003e genes, including \u003cem\u003eCmIAA\u003c/em\u003e38 and \u003cem\u003eCmIAA\u003c/em\u003e46, were consistently upregulated under both NaCl and PEG treatments, whereas \u003cem\u003eCmIAA\u003c/em\u003e12 and \u003cem\u003eCmIAA\u003c/em\u003e69 were markedly downregulated under the two stress conditions. In roots,(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) \u003cem\u003eCmIAA\u003c/em\u003e42 and \u003cem\u003eCmIAA\u003c/em\u003e61 were strongly induced by NaCl treatment but showed relatively weak responses to PEG stress.\u003c/p\u003e \u003cp\u003eDistinct response preferences were observed among \u003cem\u003eCmIAA\u003c/em\u003e genes under salt and drought stresses. For instance, \u003cem\u003eCmIAA\u003c/em\u003e46 in leaves responded sensitively to both stress treatments, whereas \u003cem\u003eCmIAA\u003c/em\u003e61 in roots primarily responded to salt stress. Collectively, these results indicate that the \u003cem\u003eCmIAA\u003c/em\u003e gene family exhibits complex and finely regulated expression patterns under abiotic stress conditions, with different members displaying tissue- and stress-dependent transcriptional responses.\u003c/p\u003e \u003c/div\u003e"},{"header":"2. Discussion","content":"\u003cp\u003eThe \u003cem\u003eAux/IAA\u003c/em\u003e gene family constitutes a core regulatory component in the auxin signaling pathway and plays critical roles in plant growth, development, and responses to abiotic stresses\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e In this study, a total of 72 \u003cem\u003eCmIAA\u003c/em\u003e genes were identified in the pumpkin (\u003cem\u003eC. moschata\u003c/em\u003e) genome. Compared with model plant species such as \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e, the relatively larger size of the \u003cem\u003eAux/IAA\u003c/em\u003e family in pumpkin suggests that this gene family may have undergone lineage - specific expansion, potentially associated with genome duplication events during evolution\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCollinearity analysis indicated that segmental duplication and dispersed duplication events may have played major roles in the expansion of the \u003cem\u003eCmIAA\u003c/em\u003e gene family\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, a pattern consistent with that reported in \u003cem\u003eSolanum lycopersicum\u003c/em\u003e,C\u003cem\u003eucumis sativus\u003c/em\u003e, and members of the Brassicaceae family. In addition, the uneven chromosomal distribution of \u003cem\u003eCmIAA\u003c/em\u003e genes may be related to historical genome rearrangements or local gene loss events, a phenomenon that has also been observed in Solanum lycopersicum and Brassica rapa\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAt the protein structural level, most CmIAA proteins retained the characteristic conserved motifs of the \u003cem\u003eAux/IAA\u003c/em\u003e family. The widespread presence of motif 1 further supports the high degree of structural conservation within this gene family\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, differences in motif composition and exon\u0026ndash;intron organization were observed among different phylogenetic subgroups. For instance, CmIAA10 in Group VI contains an additional intron, suggesting that this gene may have accumulated limited structural variation on the basis of overall conservation, thereby increasing potential regulatory complexity\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eIntraspecific phylogenetic analysis revealed that Groups Ⅰ, Ⅳ, and Ⅵ exhibited apparent expansion in pumpkin, implying that these subgroups may have undergone species-specific duplication and selection processes, which could contribute to response to specific ecological environments or developmental requirements\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Ka/Ks analysis showed that most homologous gene pairs were subjected to purifying selection, reflecting the functional conservation and importance of \u003cem\u003eAux/IAA\u003c/em\u003e proteins in auxin signal transduction\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Moreover, interspecific collinearity analysis demonstrated a high degree of synteny between pumpkin and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eCucurbita pepo\u003c/em\u003e, and \u003cem\u003eCucurbita maxima\u003c/em\u003e, further indicating that the \u003cem\u003eAux/IAA\u003c/em\u003e gene family is generally conserved within the Cucurbitaceae lineage.\u003c/p\u003e \u003cp\u003ePromoter cis-element analysis showed that the upstream regulatory regions of \u003cem\u003eCmIAA\u003c/em\u003e genes are enriched with cis-acting elements related to light responsiveness, hormone signaling, and abiotic stress responses, suggesting that this gene family may be involved in multilayered transcriptional regulatory networks\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. In particular, the presence of light-responsive elements indicates that certain \u003cem\u003eCmIAA\u003c/em\u003e members may participate in the regulation of plant growth and development, a mechanism that has been validated for IAA9 in \u003cem\u003eSolanum lycopersicum\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Moreover, co-expression network analysis revealed coordinated transcriptional changes between several \u003cem\u003eCmIAA\u003c/em\u003e genes and key components of the auxin signaling pathway. This observation is consistent with the conserved Aux/IAA\u0026ndash;ARF regulatory module reported across multiple plant species\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, providing reliable candidate genes for subsequent functional studies.\u003c/p\u003e \u003cp\u003eTissue-specific expression analysis indicated that \u003cem\u003eCmIAA\u003c/em\u003e genes displayed distinct expression patterns among different organs, with several members (e.g., \u003cem\u003eCmIAA\u003c/em\u003e46 and \u003cem\u003eCmIAA\u003c/em\u003e55) maintaining relatively high expression levels in roots and stems. This expression pattern is consistent with previous reports describing the involvement of Aux/IAA genes in root development in \u003cem\u003eGlycine max\u003c/em\u003e and \u003cem\u003eZea mays\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. From a crop improvement perspective, root system architecture is a key trait influencing plant stress tolerance, and genetic regulation of root-related genes has been shown to optimize root architecture in \u003cem\u003eOryza sativa\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e and \u003cem\u003eTriticum aestivum\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. In contrast, these genes generally exhibited lower expression levels in leaves and fruits, and may be activated only at specific developmental stages or under stress conditions, as previously reported in \u003cem\u003eSolanum lycopersicum and Capsicum annuum\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnder salt and drought stress conditions, \u003cem\u003eCmIAA\u003c/em\u003e genes exhibited distinct stress- and tissue-dependent expression patterns, with individual members showing differential responsiveness to the two stress treatments. Similar transcriptional diversity has been reported in \u003cem\u003eOryza sativa\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eAsparagus officinalis\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e and \u003cem\u003eMedicago sativa\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003efurther supporting the important role of \u003cem\u003eAux/IAA\u003c/em\u003e genes in abiotic stress responses. Notably, several \u003cem\u003eCmIAA\u003c/em\u003e members displayed stress-type-specific expression patterns, suggesting functional divergence within the gene family. Together with the enrichment of stress-responsive cis-elements, such as ABRE and MBS, in their promoter regions, these findings indicate that promoter engineering strategies aimed at stress-inducible expression of \u003cem\u003eCmIAA\u003c/em\u003e genes may provide a potential avenue for improving stress tolerance in \u003cem\u003eC. moschata\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, this study performed a comprehensive genome-wide identification and characterization of the \u003cem\u003eAUX/IAA\u003c/em\u003e gene family in pumpkin. Through integrated genomic, phylogenetic, and expression analyses, we elucidated the expansion mechanisms, structural conservation, and functional diversification of the \u003cem\u003eAux/IAA\u003c/em\u003e genes. While the family as a whole is under strong purifying selection, certain subfamilies appear to have developed adaptive functions in response to pumpkin-specific ecological niches and stress conditions, indicating conserved core roles alongside diversified regulatory functions. These results provide a solid foundation for the functional validation of key \u003cem\u003eCmIAA\u003c/em\u003e genes, especially those associated with root and stem development and stress responses, and offer clear molecular targets for genetic engineering and breeding strategies aimed at enhancing stress tolerance and improving yield-related traits in pumpkin.\u003c/p\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eIn this study, a total of 72 \u003cem\u003eCmIAA\u003c/em\u003e genes were identified from the pumpkin (\u003cem\u003eC. moschata\u003c/em\u003e) genome. These genes are unevenly distributed across multiple chromosomes and were classified into several subgroups based on phylogenetic analysis. The \u003cem\u003eCmIAA\u003c/em\u003e members exhibited both similarities and differences in conserved motifs, gene structures, and protein architectures, indicating structural conservation accompanied by diversification within the family. Promoter cis-acting element analysis suggested that \u003cem\u003eCmIAA\u003c/em\u003e genes may be involved in light signal perception, hormone-mediated regulation, and responses to abiotic stresses. Furthermore, expression profiling revealed diverse tissue-specific expression patterns of \u003cem\u003eCmIAA\u003c/em\u003e genes, and demonstrated that several members showed pronounced transcriptional responses to salt and drought stresses. Collectively, these findings provide an important theoretical foundation for further functional characterization of \u003cem\u003eCmIAA\u003c/em\u003e genes in pumpkin growth, development, and stress response.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Plant Materials and Treatments\u003c/h2\u003e \u003cp\u003eThe inbred line \"360-3\" of pumpkin (\u003cem\u003eCucurbita moschata\u003c/em\u003e) was provided by the Pumpkin Germplasm Resources Innovation and Utilization Team of the College of Horticulture and Landscape Architecture, Henan Institute of Science and Technology. Plump pumpkin seeds were selected for germination. Upon full cotyledon expansion, the seedlings were hydroponically cultured in \u0026frac12; Hoagland\u0026rsquo;s nutrient solution for two weeks. Following a 24-h acclimation in distilled water, uniform-sized seedlings with consistent growth performance were subjected to drought and salt stress treatments, respectively. Specifically, drought stress was simulated by exposure to 20% polyethylene glycol 6000 (PEG6000) for 3 h, whereas salt stress was imposed using a 200 mM NaCl solution for 3 h. After stress treatments, root and leaf tissues were immediately harvested for total RNA extraction and subsequent qRT-PCR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Identification of \u003cem\u003eCmIAA\u003c/em\u003e Family Members\u003c/h2\u003e \u003cp\u003eAux/IAA protein sequences from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e were retrieved using TBtools and employed as query templates. Homology-based searches were conducted against the pumpkin genome in the Cucurbit Genomics Database (CuGenDB)\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e using BLAST,. Redundant sequences were removed using a similarity threshold of 50% and an e-value cutoff of 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. All non-redundant candidate proteins were subsequently verified for the presence of conserved Aux/IAA domains using NCBI-CDD and SMART\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Additional filtering steps were applied to eliminate any remaining redundancy, resulting in the final set of \u003cem\u003eCmIAA\u003c/em\u003e family members.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Prediction of Physicochemical Properties of CmIAA Proteins\u003c/h2\u003e \u003cp\u003eThe ExPASy ProtParam\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e tool was used to predict the basic physicochemical characteristics of each CmIAA protein, including amino acids number, molecular weight, and theoretical isoelectric point (pI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Phylogenetic Tree Construction\u003c/h2\u003e \u003cp\u003eA phylogenetic tree was constructed using the Neighbor-Joining method with 1,000 bootstrap replicates in MEGA-X\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e, based on the amino acid sequences of the CmIAA proteins. The resulting tree was subsequently refined and visualized using the Interactive Tree of Life (iTOL) online platform\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Collinearity Analysis\u003c/h2\u003e \u003cp\u003eGene family files \u003cem\u003eCucurbita pepo\u003c/em\u003e, \u003cem\u003eCucurbita maxima\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e were obtained from Ensembl Plants,and those for pumpkin were downloaded from CuGenDB\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Both interspecific synteny analysis among these species and intraspecific synteny analysis of pumpkin \u003cem\u003eCmIAA\u003c/em\u003e genes were conducted via TBtools\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Furthermore, to assess selection pressure, Ka/Ks ratios between duplicated CmIAA gene pairs were determined using KaKs_Calculator 2.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.6. Promoter Cis-Regulatory Element Analysis\u003c/h2\u003e \u003cp\u003eCoding sequences (CDS) and full-length genomic sequences of all 72 \u003cem\u003eCmIAA\u003c/em\u003e genes were retrieved from CuGenDB\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. A 2,000 bp upstream region from each gene was extracted using TBtools and analyzed for cis-regulatory elements using the PlantCARE\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e database. The results were graphically visualized with TBtools\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.7. Chromosomal Localization\u003c/h2\u003e \u003cp\u003eThe chromosomal positions of all 72 \u003cem\u003eCmIAA\u003c/em\u003e genes were obtained from CuGenDB\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Their distribution across chromosomes was mapped and visualized using TBtools\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.8. Gene Structure and Conserved motif Analysis\u003c/h2\u003e \u003cp\u003eConserved motifs within the CmIAA protein sequences were identified using the MEME\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e online program. Gene structure information for all CmIAA members was obtained using the CSDS 2.0 web server.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.9. Tissue Expression Pattern Analysis\u003c/h2\u003e \u003cp\u003eRNA-seq datasets of four tissue types (root, stem, leaf, and fruit) in pumpkin were downloaded from the CuGenDB database (PRJNA385310). Fragments per kilobase of transcript per million mapped reads (FPKM) values were calculated to estimate the expression levels of CmIAA genes. The expression data were log2 transformed, and heatmaps were constructed using TBtools\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e visualize the expression patterns across different tissues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.10. Rna Extraction And qRT-PCR Analysis of Abiotic Stress Treatment\u003c/h2\u003e \u003cp\u003eExpression profiles of CmIAA under abiotic stress conditions were analyzed via quantitative real-time PCR (qRT-PCR). Briefly, Total RNA was isolated from the collected samples using the Omini Plant RNA Kit (CWBIO, Beijing, China). For cDNA synthesis and qRT-PCR amplification, commercial reagent kits purchased from TaKaRa were utilized, with all reactions conducted on a Bio-Rad real-time fluorescence quantitative PCR system. The qRT-PCR reaction mixture (10 \u0026micro;L) consisted of 5 \u0026micro;L TB Green Mix, 0.4 \u0026micro;L each of the forward and reverse primers, 0.2 \u0026micro;L of cDNA template, and 4 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO. The thermal cycling conditions were as follows: an initial denaturation at 95\u0026deg;C for 30 s, followed by 40 cycles of 95\u0026deg;C for 15 s and 60\u0026deg;C for 30 s. A melting curve program (95\u0026deg;C for 5 s, 65\u0026deg;C for 5 s, and 95\u0026deg;C for 5 s) was performed to verify the specificity of the amplification products. The pumpkin Actin gene was utilized as an internal reference, and the relative expression levels were quantified using the 2\u003csup\u003e\u0026minus;\u003cem\u003e∆∆Ct\u003c/em\u003e\u003c/sup\u003e method. The experiment was performed with three independent biological replicates. Primers for qRT-PCR were listed in Additional file 1.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll plant materials used in this study, including pumpkin (\u003cem\u003eC.moschata\u003c/em\u003e), were grown and collected according to institutional and national guidelines. The pumpkin cultivars used are common agricultural varieties and are not endangered species. All methods complied with relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNOT APPLICABLE\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw sequence was data of \u003cem\u003eC.moschata\u0026nbsp;\u003c/em\u003ewas downloaded from the NCBI database using accession number PRJNA385310 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA385310/).RNA-seq data were derived from the Cucurbit Genomics Database (CuGenDB) using accession number PRJNA385310. (http:// cucurbitgenomics. org/ rnaseq/ pu/25). Alldatasets generated in this study are included in the published article/Additional Files. Wedsites used for analyses in this study are as follows:Cucurbit Genomics Database (http://cucurbitgenomics. org/ organism/9).TBtools (https://github.com/CJ-Chen/TBTools). SMART (http://smart.embl-heidelberg.de/). ExPASy (http://web.expasy. org/ prot param/). iTOL (https: //itol. embl.de/). Ensembl Plants (https://plants. ensembl.org/index.html). Plant CARE (http://bioinformatics.psb. ugent. be/webtools/plantcare/html/). MEME (https://meme-suite. org/meme/ doc/meme.html). SDS2.0 (https://gsds.gao-lab.org/Gsds_help.php).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential \u0026nbsp;conflict of interest\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Natural Science Foundation of Henan Province (Grant No. 252300420662).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYZ and MS conceived and designed the project. MS, JD, and JZ performed the experiments and collected the plant materials. LJ and MS analyzed the data and performed the bioinformatic analysis. MS wrote the original draft. YZ, JD, and LJ revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the providers of genomic and transcriptome data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eHenan Institute of Science and Technology, Xinxiang 453000, Henan, P. R. China\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXu H, Liu Y, Zhang S, Shui D, Xia Z, Sun J. Genome-Wide Identification And Expression Analysis Of The \u003cem\u003eAux/IAA\u003c/em\u003e Gene Family In Turnip (\u003cem\u003eBrassica Rapa Ssp. Rapa\u003c/em\u003e). BMC Plant Biol. 2023;23(1):342.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu Y, He H, Wang P, Ma Z, Mao J, Chen B. 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Interactive Tree Of Life (iTOL) v4: Recent Updates And New Developments. Nucleic Acids Res. 2019;47(W1):W256\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, Liu Y, Feng J, Chen H, He Y, et al. TBtools-II: A one for all, all for one bioinformatics Platform For Biological Big-Data Mining. Mol Plant. 2023;16(11):1733\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLescot M, D\u0026eacute;hais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouz\u0026eacute; P, Rombauts S. PlantCARE, a Database Of Plant Cis-Acting Regulatory Elements And a Portal To Tools For In Silico Analysis Of Promoter Sequences. Nucleic Acids Res. 2002;30(1):325\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBailey TL, Johnson J, Grant CE, Noble WS. The MEME Suite. Nucleic Acids Res. 2015;43(W1):W39\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\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":"Aux/IAA, Cucurbita moschata, gene family, abiotic stress","lastPublishedDoi":"10.21203/rs.3.rs-8529495/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8529495/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAuxin is a central phytohormone involved in regulating plant growth, development, and stress responses, with the \u003cem\u003eAux/IAA\u003c/em\u003e gene family functioning as an essential component of the auxin signaling pathway. To elucidate the genomic features and potential functions of the \u003cem\u003eAux/IAA\u003c/em\u003e gene family in pumpkin (\u003cem\u003eCucurbita moschata\u003c/em\u003e), we performed a genome-wide identification and systematic characterization.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 72 \u003cem\u003eCmIAA\u003c/em\u003e genes were identified, encoding proteins ranging from 158 to 1275 amino acids with predicted isoelectric points of 4.57\u0026thinsp;~\u0026thinsp;9.81. These genes were unevenly distributed across 20 chromosomes, with Chr17 harboring the highest number, while no \u003cem\u003eCmIAA\u003c/em\u003e genes were detected on Chr3. Phylogenetic analysis classified the genes into nine subgroups (Group Ⅰ~Ⅸ), with Groups Ⅰ, Ⅳ, and Ⅵ exhibiting notable expansion. Gene structure and conserved motif analyses revealed subgroup-specific motif compositions, with motif 1 representing the core conserved domain. Intraspecific collinearity analysis identified 54 segmentally duplicated gene pairs but no tandem duplication events, whereas interspecific synteny revealed extensive orthologous relationships between pumpkin and \u003cem\u003eCucurbita pepo\u003c/em\u003e, \u003cem\u003eCucurbita maxima\u003c/em\u003e, and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Promoter analysis showed that \u003cem\u003eCmIAA\u003c/em\u003e genes contain abundant cis-elements associated with light response, hormone regulation, development, and abiotic stress. Tissue expression analysis demonstrated that many \u003cem\u003eCmIAA\u003c/em\u003e genes were highly expressed in roots and stems, Several \u003cem\u003eCmIAA\u003c/em\u003e genes exhibited tissue-specific and stress-type-dependent expression patterns under salt and drought treatments.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOverall, this study provides the first comprehensive characterization of the \u003cem\u003eAux/IAA\u003c/em\u003e gene family in pumpkin, offering fundamental insights into their structural features and expression dynamics, and providing candidate genes for future functional studies and molecular breeding applications.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Identification and Comprehensive Characterization of the Aux/IAA Gene Family in Cucurbita moschata and Its Response Analysis to Abiotic Stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 13:13:24","doi":"10.21203/rs.3.rs-8529495/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-13T06:42:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69253156186092898243088995499325839374","date":"2026-04-20T15:08:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-19T11:14:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316538476833558200192938963189473986340","date":"2026-04-05T02:11:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77128963298728732730806059550803391769","date":"2026-04-02T11:12:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"105583607230556503591303390597668206569","date":"2026-03-05T06:36:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162450975383734122717385099272067204866","date":"2026-02-10T10:06:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63628684068708930351023878463726936815","date":"2026-02-05T16:30:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-22T04:22:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T03:31:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-16T05:34:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-14T06:51:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-01-14T06:36:40+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":"a1019102-460a-4500-9b33-136ef1c021ad","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-13T06:42:32+00:00","index":96,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-28T13:13:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 13:13:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8529495","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8529495","identity":"rs-8529495","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}