Genome-wide identification of the ATL gene family and their expression analysis in sweetpotato and its two diploid relatives.

Genome-wide identification of the ATL gene family and their expression analysis in sweetpotato and its two diploid relatives - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. 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Learn more: PMC Disclaimer | PMC Copyright Notice BMC Plant Biol . 2026 Mar 7;26:678. doi: 10.1186/s12870-026-08501-1 Search in PMC Search in PubMed View in NLM Catalog Add to search Genome-wide identification of the ATL gene family and their expression analysis in sweetpotato and its two diploid relatives Ruitao Liu Ruitao Liu 1 Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis and Utilization, Ministry of Education, College of Agronomy & Biotechnology, China Agricultural University, Beijing, 100193 China Find articles by Ruitao Liu 1 , Yinghui Yang Yinghui Yang 1 Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis and Utilization, Ministry of Education, College of Agronomy & Biotechnology, China Agricultural University, Beijing, 100193 China Find articles by Yinghui Yang 1 , Shaopei Gao Shaopei Gao 1 Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis and Utilization, Ministry of Education, College of Agronomy & Biotechnology, China Agricultural University, Beijing, 100193 China Find articles by Shaopei Gao 1 , Huan Zhang Huan Zhang 1 Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis and Utilization, Ministry of Education, College of Agronomy & Biotechnology, China Agricultural University, Beijing, 100193 China Find articles by Huan Zhang 1 , Ning Zhao Ning Zhao 1 Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis and Utilization, Ministry of Education, College of Agronomy & Biotechnology, China Agricultural University, Beijing, 100193 China Find articles by Ning Zhao 1 , Shaozhen He Shaozhen He 1 Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis and Utilization, Ministry of Education, College of Agronomy & Biotechnology, China Agricultural University, Beijing, 100193 China Find articles by Shaozhen He 1 , Qingchang Liu Qingchang Liu 1 Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis and Utilization, Ministry of Education, College of Agronomy & Biotechnology, China Agricultural University, Beijing, 100193 China Find articles by Qingchang Liu 1 , Hong Zhai Hong Zhai 1 Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis and Utilization, Ministry of Education, College of Agronomy & Biotechnology, China Agricultural University, Beijing, 100193 China Find articles by Hong Zhai 1, ✉ Author information Article notes Copyright and License information 1 Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis and Utilization, Ministry of Education, College of Agronomy & Biotechnology, China Agricultural University, Beijing, 100193 China ✉ Corresponding author. Received 2025 Dec 5; Accepted 2026 Mar 2; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13081281  PMID: 41794661 Abstract Background Protein ubiquitination is a critical regulatory mechanism in plants, governing essential biological processes such as growth, development, and the response to stress by mediating the degradation of specific proteins. E3 ubiquitin ligases play a key role in this process. Among them, the Arabidopsis Tóxicos en Levadura (ATL) family of proteins functions as an E3 ubiquitin ligase and plays a critical role in plant growth, development, and stress responses. Sweetpotato ( Ipomoea batatas (L.) Lam.), a globally significant food and energy crop, has benefited from genome sequencing, enabling the comprehensive identification and functional analysis of the ATL gene family. Results In this study, we identified 85, 99, and 97 ATL genes in sweetpotato ( Ipomoea batatas , 2n = 6x = 90) and its two diploid relatives, Ipomoea trifida and Ipomoea triloba (both 2n = 2x = 30), respectively. Phylogenetic analysis revealed that these ATL genes were clustered into six distinct subgroups. We then systematically studied the ATL gene family, including its physicochemical properties, chromosomal localization, phylogenetic relationships, collinearity, gene structures, promoter cis -elements, protein-protein interaction network, and expression patterns. Conclusions Through a comprehensive genome-wide analysis of ATL genes in sweetpotato and its two diploid relatives, we identified a total of 281 ATL genes from sweetpotato, Ipomoea trifida , and Ipomoea triloba . Subsequently, we performed a systematic bioinformatic analysis of the ATL family. We further examined their expression patterns in response to hormonal and abiotic stress treatments. The analysis revealed that members of the ATL gene family play distinct and crucial roles in hormone signaling and abiotic stress responses within sweetpotato and its two diploid relatives. These findings lay the groundwork for further investigation into the abiotic stress response mechanisms of ATL genes in sweetpotato. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-026-08501-1. Keywords: Abiotic stress, ATL gene family, Expression pattern, Genome-wide identification, Sweetpotato Introduction In plants, ubiquitination is a crucial mechanism for protein regulation and a post-translational modification process that drives protein degradation. Through the selective degradation of proteins, it profoundly influences vital biological processes such as growth, development, and stress responses, and holds pivotal significance throughout the entire life cycle of plants [ 1 , 2 ]. Ubiquitin degradation requires the coordinated action of ubiquitin, the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2), the ubiquitin ligase (E3), and the entire 26S proteasome complex [ 3 , 4 ]. Under certain conditions, the target protein is polyubiquitinated and subsequently degraded by the 26S proteasome [ 5 ]. This process is orchestrated by E3 ubiquitin ligases, which confer specificity by bridging a particular E2 enzyme to its target protein substrate, ensuring precise ubiquitination [ 6 ]. Based on their mechanism of action and subunit composition, E3 ubiquitin ligases can be categorized into four principal classes: HECT, RING, U-box, and cullin-RING ligases (CRLs) [ 7 ]. Among them, RING-type E3 ubiquitin ligases possess a characteristic RING domain that binds two zinc ions in a conserved pattern involving seven cysteine residues and one histidine residue [ 8 ]. At present, it has been proven that the RING domain exists not only in single-subunit E3 ubiquitin ligases [ 9 , 10 ] but also in multi-subunit E3 ubiquitin ligases [ 11 , 12 ]. A common variant of the RING domain, in which the fifth cysteine residue is replaced by a second histidine residue, is known as the RING-H2 domain [ 13 ]. This variant is characteristic of the ATL family, a major group of plant-specific E3 ligases whose catalytic activity depends on this domain. ATL proteins play a key role in governing plant responses to various environmental stresses by catalyzing the ubiquitination of specific substrate proteins [ 14 ]. ATL family genes have been widely identified in plants, including 91, 121, 162, 96, 82, 92, 77, and 334 from Arabidopsis , rice, soybean, grapevine, tomato, pear, maize, and wheat, respectively [ 14 – 19 ]. So far, the ATL gene family has not been reported in sweetpotato. Extensive research indicates that ATL proteins are pivotal in various processes of plant growth and development. In alfalfa, the ATL gene MsRH2-1 affects plant height through the auxin signal transduction pathway [ 20 ]. In Arabidopsis , the degradation of ABT1 by ATL5 increases seed longevity [ 21 ]. Loss of ATL15 function results in sugar insensitivity, whereas ATL15 overexpression inhibits growth. These results demonstrate that ATL15 plays a pivotal role in regulating sugar responses [ 22 ]. The ATL54 gene is involved in the regulation of secondary cell wall biosynthesis and programmed cell death during lignification in Arabidopsis [ 23 ]. In rice, OsATL53 negatively regulates lignin accumulation in anthers and roots by suppressing OsCCR14 activity [ 24 ]. Under nitrogen induction, the E3 ubiquitin ligase EL5 regulates root development by counteracting cytokinin and superoxide production, which in turn prevents meristem cell death and maintains root morphological stability [ 25 ]. In Populus tremula×Populus alba , PtaRHE1 can participate in the formation of phloem fibers and plays a regulatory role in secondary phloem fibers [ 26 ]. ATL proteins also play a key role in mediating plant responses to abiotic and biotic stresses. In Arabidopsis , overexpression of AtATL78 [ 27 ] and ATL61 [ 28 ] improves drought resistance. Exogenous expression of the sweetpotato IbATL38 gene in Arabidopsis significantly enhances salt tolerance [ 29 ]. ATL31 and ATL6 positively regulate Arabidopsis tolerance to salt stress independently of ABA signaling pathways [ 30 ]. In rice, OsATL38 negatively regulates cold tolerance by ubiquitinating the positive regulator OsGF14d [ 31 ]. In potato, StATL2 participates in ROS scavenging and negatively regulates cold tolerance [ 32 ]. In Arabidopsis , ATL9 is crucial for resistance to the fungal pathogen Golovinomyces cichoracearum [ 33 ]. The ATL12 is involved in the salicylic acid and jasmonic acid pathways, as well as NADPH oxidase-mediated defense response, thereby enhancing the resistance of Arabidopsis to Golovinomyces cichoracearum [ 34 ]. In pear, PbrATL18 enhances resistance to Colletotrichum fructicola by regulating the activity of CHI, PAL, CAT, and POD [ 17 ]. Heterologous overexpression of the OsBIRF1 gene in tobacco enhances resistance to tobacco mosaic virus and Pseudomonas syringae pv. tabaci [ 35 ]. Overexpression of the capsicum ATL gene CaRING1 in Arabidopsis increases resistance to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis [ 36 ]. Sweetpotato ( Ipomoea batatas (L.) Lam.) belongs to the Convolvulaceae family and is an important food and energy crop worldwide [ 37 ]. Ipomoea trifida and Ipomoea triloba are two diploid wild relatives of sweetpotato [ 38 ] that are crucial for expanding the genetic base, identifying beneficial genes, and improving crop traits [ 39 ]. However, the sweetpotato possesses 90 chromosomes and a large genome of approximately 2.76 Gb [ 40 ]. Its complex autohexaploid genetic background, coupled with the incompatibility in interspecific and intraspecific hybridization, significantly constrains the utilization of wild resources and parental selection in sweetpotato breeding [ 41 ]. In addition, resistance traits are often negatively correlated with some quality traits, which limits the application of conventional breeding. It is a feasible method to improve sweetpotato with genetic engineering by exploiting high-quality genes of stress resistance [ 42 ]. At present, gene family analyses are being used to identify beneficial genes related to growth, development, and stress resistance in sweetpotato [ 43 – 47 ]. The recent emergence of the genome of the sweetpotato, as well as the sequencing of the genomes of its two diploid relatives [ 38 ], has enabled genome-wide identification of gene families in sweetpotato. In this study, we identified the ATL gene family members in sweetpotato and its two diploid relatives, I. trifida and I. triloba . The physicochemical properties, phylogenetic relationship with Arabidopsis , collinearity, chromosomal localization, gene structures, promoter cis -elements, and protein interaction were analyzed. To further investigate the function of the ATL gene family in sweetpotato and its two diploid relatives, we analyzed their expression patterns across different tissues, under hormone treatments, and in response to stress using RNA-seq data and qRT-PCR validation. Results Identification of ATL genes in sweetpotato and its two diploid relatives To identify the members of the ATL family in sweetpotato and its two diploid relatives, we performed an HMMer search using the HMMer profile of ATL proteins. We then conducted a BLASTp search against the protein sequence databases of sweetpotato and its two diploid relatives, using the ATL protein sequences from Arabidopsis as the query (Table S1). The final identification revealed 85, 99, and 97 ATL genes in sweetpotato, I. trifida , and I. triloba , respectively. The genes were systematically named with the prefixes “ Ib ”, “ Itf ”, and “ Itb ” for those from sweetpotato ( I. batatas ), I. trifida , and I. triloba , respectively, based on their chromosomal positions. The physicochemical properties of the IbATL family genes are summarized in Table S2. The lengths of IbATL gene sequences range from 570 bp to 7,377 bp, with IbATL85 being the longest and IbATL23 the shortest. The predicted proteins range from 118 to 750 amino acids (aa) in length, with molecular weights (MW) between 13.04 and 82.97 kDa. Among them, IbATL12 (750 aa) is the longest protein, and IbATL37 (118 aa) is the shortest. The theoretical isoelectric points (pI) range from 4.55 to 10.39. In terms of protein stability, 81 IbATL proteins exhibited instability indices greater than 40, indicating that most are unstable; only four proteins have values below 40. Hydrophobicity analysis, as reflected by the GRAVY index, showed that 65 IbATL proteins are hydrophilic (GRAVY < 0), whereas the remaining 20 exhibit hydrophobic characteristics. The physicochemical properties of ATL genes from I. trifida and I. triloba are highly similar to those of sweetpotato (Table S2). Subcellular localization prediction indicated that most of these proteins are located in the nucleus (Table S2). Chromosomal location of ATL genes in sweetpotato and its two diploid relatives The IbATL , ItfATL , and ItbATL genes were mapped to the chromosomes of I. batatas , I. trifida , and I. triloba , respectively (Fig. 1 ). Chromosomal localization analysis showed that 85 IbATL s were distributed across 15 chromosomes of I. batatas . Among these, chromosome LG5 contained the largest number of IbATLs (12), whereas LG4 and LG9 contained the fewest, with two genes each (Fig. 1 a). Similarly, both the ItfATLs and ItbATLs were distributed across 15 chromosomes in I. trifida and I. triloba , respectively, exhibiting high similarity in both gene number and chromosomal positions (Fig. 1 b and Fig. 1 c). Overall, the chromosomal distribution of ATL genes showed a conserved pattern across sweetpotato and its two diploid relatives. Fig. 1. Open in a new tab Chromosomal localization and distribution of ATL genes in I. batatas ( a ), I. trifida ( b ), and I. triloba ( c ). The bars represent chromosomes, the chromosome numbers are displayed on the left side, and the gene names are displayed on the right side. The relative chromosomal localization of each ATL gene is marked on the black line of the right side and indicated by the unit Mbp Phylogenetic analysis of the ATL gene family in sweetpotato, I. trifida , I. triloba , and Arabidopsis To investigate the evolutionary relationships of ATL genes, we constructed a phylogenetic tree using the Neighbor-Joining (NJ) Method based on 364 full-length ATL amino acid sequences (85 from sweetpotato, 99 from I. trifida , 97 from I. triloba , and 83 from Arabidopsis ). Based on their phylogenetic relationships and conserved structural features, the ATL genes from I. batatas and its two diploid relatives were classified into six groups (Groups I-VI; Fig. 2 ). As shown in the phylogenetic tree, each group contained ATL genes from all four species, with the following distributions: Group I contained 26 IbATLs , 32 ItfATLs , 33 ItbATLs , and 34 AtATLs ; Group II had 8, 11, 11, and 14; Group III had 11, 11, 10, and 15; Group IV had 14, 18, 18, and 13; Group V had 24, 24, 22, and 3; and Group VI had 2, 3, 3, and 4, respectively. The analysis indicated that sweetpotato ATL genes are most closely related to those from I. trifida and I. triloba . Fig. 2. Open in a new tab Phylogenetic tree analysis of the ATL family in I. batatas , I. trifida , I. triloba , and Arabidopsis . A total of 364 ATLs were divided into six subgroups (Group I-Ⅵ). Different symbols represent ATLs from different species: the star for I. batatas (85 IbATLs ), the square for I. trifida (99 ItfATLs ), the check mark for I. triloba (97 ItbATLs ), and the triangle for A. thaliana (83 AtATLs ) Collinearity analysis of IbATL genes in sweetpotato and other species In order to investigate the evolutionary history of the IbATL gene family in sweetpotato, we conducted a collinearity analysis of its genome. Collinearity analysis in sweetpotato revealed 35 homologous IbATL gene pairs, comprising 6 tandem and 29 segmental duplications. The expansion of the IbATL gene family in sweetpotato involved both tandem and segmental duplications, with segmental duplication being the main mechanism; this included six gene pairs attributed to ancient segmental duplication events (Fig. 3 a and Table S3). Notably, g20440 ( IbATL31 ), g30147 ( IbATL48 ), and g60216 ( IbATL84 ) each underwent two segmental duplications (Fig. 3 a). The Ka/Ks ratios for all duplicated IbATL gene pairs were less than one, ranging from 0.08 to 0.64, with a mean value of 0.30, indicating strong purifying selection (Fig. 3 a). Fig. 3. Open in a new tab Collinear analysis of ATL genes within and between species. Collinear relationships are marked by blue lines. ( a) Intragenomic collinearity of IbATLs in I. batatas . The lilac bars represent the I. batatas chromosomes. Unit: Mb. The bar plots depict the distributions of non-synonymous substitution rates (Ka), synonymous substitution rates (Ks), and the Ka/Ks ratio across the IbATL homologous genes. ( b) Interspecies collinearity of I. batatas , I. trifida , and I. triloba . The bars represent different species chromosomes, the orange bars represent I. triloba chromosomes, the lilac bars represent I. batatas chromosomes, and the blue bars represent I. trifida chromosomes. ( c) Collinearity analysis between I. batatas and Arabidopsis . Lilac bars represent I. batatas chromosomes; green bars represent Arabidopsis chromosomes. ( d) Collinearity analysis among I. batatas , rice ( Oryza sativa ), and Zea mays . Lilac bars represent I. batatas chromosomes; deep purple bars represent Oryza sativa chromosomes; tomato bars represent Zea mays chromosomes To further explore the evolutionary mechanisms of the IbATL gene family, we extended our collinearity analysis to include other species: the two diploid relatives of sweetpotato ( I. trifida and I. triloba ), the model dicot Arabidopsis , and the monocots rice ( Oryza sativa L.) and maize ( Zea mays ) (Fig. 3 b, Fig. 3 c, and Fig. 3 d), leveraging the fact that plant genomes exhibit a degree of sequence and structural conservation during evolution. The highest number of collinear ATL gene pairs was observed between sweetpotato and its two diploid relatives: 134 with I. trifida and 143 with I. triloba (Fig. 3 b and Table S3). These results indicate that the ATL gene family has been conserved throughout the evolution of sweetpotato. Furthermore, this evolutionary trajectory is supported by gene duplication events, with both segmental and tandem duplications playing key roles in the expansion and maintenance of the family. Between sweetpotato and Arabidopsis , we identified 69 collinear ATL gene pairs (Fig. 3 c and Table S3), indicating a high degree of sequence and structural conservation of this gene family across dicotyledons. By contrast, only 32 collinear gene pairs were identified with rice and maize (Fig. 3 d, and Table S3), which was significantly fewer than those identified with Arabidopsis . This reflects a marked divergence in the composition and evolutionary trajectory of the ATL gene family between sweetpotato and monocotyledons, a divergence that was likely driven by their distinct histories of genomic duplication and functional specialization. Despite this divergence, some ATL genes remain highly conserved, implying that they carry out essential, ancestral functions required by plants. Conserved motifs, domains, and gene structures of ATLs in sweetpotato and its two diploid relatives The architecture of the gene and the conformation of the protein are pivotal in determining protein functionality. To gain a better understanding of the ATL family, we performed an analysis of its conserved motifs and gene structures. Motif analysis revealed that the IbATL, ItfATL, and ItbATL proteins contain between one and ten distinct motifs. The distribution of these motifs showed a high degree of similarity across different phylogenetic groups (Fig. 4 and Fig. S1). Notably, Motif 1 was universally present across all groups, indicating that it is structurally indispensable and constitutes the core functional motif essential for ATL protein activity. However, most IbATL proteins in Group V lacked Motif 4. Conserved domain analysis revealed that all identified members of the IbATLs, ItfATLs, and ItbATLs contain the RING-H2 and RING_Ubox superfamily domains. These domains are hallmark structural features of ATL E3 ubiquitin ligases (Fig. 4 and Fig. S1). Collectively, these findings highlight the complex domain architecture and diverse motif distribution within the ATL gene family of sweetpotato and its two diploid relatives, suggesting potential functional diversification among its members. Fig. 4. Open in a new tab Conserved motifs and domains of IbATLs and exon-intron of IbATL genes of I. batatas . Motifs 1–10, CDS, UTR, and different subgroups are represented in different colors In addition, we analyzed the exon-intron structures of ATL genes. In sweetpotato, the number of exons varied from 1 to 11, and introns from 0 to 10 (Fig. 4 ). Over half of the IbATL genes were found to contain only one exon and no introns, while IbATL15 exhibited the highest number of exons (11) and introns (10). Similarly, in the two diploid relatives, most ATL genes also exhibited an intronless structure, consisting solely of exons (Fig. S1). Similar gene structures have been reported in other species, such as maize and wheat, indicating that the exon–intron organization of the ATL gene family is relatively conserved. Analysis of cis -elements in the promoters of ATLs in sweetpotato and its two diploid relatives Residing in gene promoters, cis -elements are critical for precise transcriptional control via interaction with trans-acting factors. This mechanism allows them to modulate key aspects of plant biology, including growth, development, and stress responses. To elucidate how this regulatory mechanism modulates key processes in sweetpotato and its two diploid relatives, we performed a systematic analysis of cis -elements in the promoter regions (2000 bp upstream) of their ATL genes using the PlantCARE database. Promoter analysis revealed that sweetpotato and its two diploid relatives possess highly similar categories of cis -elements. The major functional categories identified across all three species included elements responsive to light, phytohormones, stress, and development. The types and distribution of these elements were largely conserved, indicating a shared transcriptional regulatory potential within the ATL family (Fig. 5 and Fig. S2). Analysis of cis- elements in the promoter regions allowed us to broadly classify the elements into five functional categories. For focused biological interpretation, we excluded the ubiquitous core promoter cis- elements (e.g., TATA-box, CAAT-box) and performed an in-depth analysis of the remaining four categories associated with specific regulatory functions: development, light response, hormone response, and abiotic/biotic stress response (Fig. 5 ). Among these, light-responsive elements exhibited the greatest diversity, with Box 4 being the most frequent motif (231 occurrences). Hormone-responsive elements were the second most abundant, with the abscisic acid (ABA)-responsive element ABRE being the most prevalent (173 occurrences). Concurrently, methyl jasmonate-responsive elements (TGACG-motif and CGTCA-motif) were also abundant (111 occurrences each), indicating that IbATLs may be extensively involved in multiple hormone signaling pathways in sweetpotato. Furthermore, numerous abiotic/biotic stress-responsive cis -elements were identified, among which anaerobic response elements (ARE) were the most abundant (143), while development-related elements were relatively scarce (Fig. 5 ). Fig. 5. Open in a new tab Cis -elements and their frequencies in the 2000 bp promoter region upstream of IbATL genes. The number of each cis -element is indicated by the intensity of deep purple Notably, the promoters of IbATL genes showed a significant enrichment of cis- elements associated with abiotic stress responses. Over 50% of promoters contained the drought-responsive element (MBS), suggesting the involvement of these genes in the sweetpotato’s response to drought stress. Moreover, over 75% of promoters contained both ABA-responsive elements (ABRE) and anaerobic induction elements (ARE), suggesting that the ABA signaling pathway may be a key mechanism in regulating IbATL -mediated drought adaptation. Our analysis of cis -elements suggests that the IbATL gene family acts as a pivotal regulatory node that integrates light, hormonal, and abiotic stress signals to coordinate sweetpotato growth and adaptation to the environment (Fig. 5 ). The protein interaction network of IbATLs in sweetpotato In order to investigate the functions of the 85 IbATL family members in sweetpotato, we aligned the IbATL proteins with their Arabidopsis homologues using the STRING database, and then predicted the corresponding protein interaction networks (Fig. 6 ). Network analysis revealed that IbATL proteins are involved in various aspects of plant growth and development. Our analysis indicates that multiple IbATL proteins form highly interconnected core nodes, suggesting a pivotal role in protein ubiquitination processes. Among these core proteins in the interaction network is BOI, an E3 ubiquitin ligase involved in regulating responses to pathogens and abiotic stress by promoting the degradation of MYB108/BOI. RIN2, also an E3 ubiquitin ligase, interacts with RIN3 to positively regulate the RPM1- and RPS2-dependent hypersensitive response (HR). Similarly, RHA2B positively regulates ABA signaling during seed germination and early seedling development, as well as responses to salt and osmotic stress. It may act synergistically with RHA2A to modulate ABA signaling and drought responses. These interactions suggest that IbATL proteins play crucial roles in plant growth and development, as well as in responses to abiotic and biotic stresses. Fig. 6. Open in a new tab Protein interaction networks predicted of IbATLs. The networks were generated by comparing IbATL proteins with their homologous genes in Arabidopsis using the STRING database. The circles between nodes represent proteins, and the lines between nodes represent protein-to-protein interactions Tissue-specific expression analysis of ATLs in sweetpotato and its two diploid relatives To explore the biological functions of IbATLs in sweetpotato growth and development, we analyzed their expression levels using RNA-seq data from eight tissues of two varieties, ‘Xuzi3’ and ‘Yan252’. The results showed that IbATLs are expressed across all tissues but with distinct specificities. The expression patterns of IbATL genes were highly conserved between the two sweetpotato varieties, ‘Xuzi3’ and ‘Yan252’, showing consistent trends. Notably, most genes in Groups II and III exhibited relatively high transcript levels across tissues. Specifically, IbATL2 , -9 , -33 , -47 , and -65 were consistently highly expressed in most tissues, whereas IbATL16 , -18 , -37 , -41 , and -68 showed low or undetectable expression. This low, but detectable, constitutive expression may allow for rapid transcriptional activation under specific developmental cues or environmental stresses. Several genes demonstrated specific tissue preferences: IbATL9 and IbATL44 were highly expressed in roots, IbATL2 , -21 , -23 , and -73 in stems, and IbATL30 and IbATL46 in mature leaves (Fig. 7 ). These results suggest that IbATLs play important roles in sweetpotato growth and development. Fig. 7. Open in a new tab The expression analysis of IbATLs in fibrous root (FR), initial expanding root (IR), expanding root (ER), mature root (MR), stem, shoot, young leaf (YL), and mature leaf (ML) of ‘Xuzi3’ and ‘Yan252’. The log 2 (FPKM + 1) values are shown in the cells. The color bar shows the scale, ranging from 0.00 to 10.00. Different colors correspond to different magnitudes within this range We then analyzed their expression patterns in two diploid relatives, I. trifida and I. triloba , across six tissues (Fig. S3). The two diploid relatives exhibited distinct tissue-specific expression profiles. Several genes in I. trifida showed root-preferential expression (e.g., ItfATL5 , -17 , and -19 ), while a different set was highly expressed in stems (e.g., ItfATL6 , -12 , and -28 ). A notable number of genes were specifically expressed in floral tissues, with some highly expressed in flowers (e.g., ItfATL25 and ItfATL98 ) and others in both flowers and buds (e.g., ItfATL10 and ItfATL18 ). A similar tissue-specific pattern was observed in I. triloba , but with key differences in gene membership and tissue breadth. For example, ItbATL4 and ItbATL6 showed root-preferential expression. Compared to I. trifida , fewer stem-specific genes were observed (e.g., ItbATL16 and ItbATL19 ), while some genes showed leaf-specific expression (e.g., ItbATL42 and ItbATL45 ). ItbATL3 and ItbATL92 showed expression in floral tissues. These results demonstrate that the ATL gene family in the two diploid relatives exhibits complex and partially conserved tissue-specific regulation, highlighting its potential role in organ development. Expression analysis of ATLs under hormone treatments in sweetpotato and its two diploid relatives To investigate the response of IbATLs to plant hormone signaling, we analyzed the expression of IbATL genes in sweetpotato under hormone treatments by qRT-PCR. Nine IbATLs ( IbATL17 , -21 , -38 , -42 , -44 , -46 , -57 , -69 , -72 ) were selected for qRT‑PCR analysis based on the abundance of hormone- and stress‑responsive cis- elements in their promoters. The qRT-PCR analysis results showed that most genes were upregulated under these hormone treatments, except that IbATL21 , - 38 , -42 , and -44 were downregulated under methyl jasmonate (MeJA) induction, IbATL38 was downregulated under salicylic acid (SA) induction, and IbATL17 , - 42 , and -69 were downregulated under indole-3-acetic acid (IAA) induction (Fig. 8 ). The upregulated genes showed different expression patterns under different hormone treatments: Under MeJA treatment, ATL17 , -46 , -57 , and -69 showed the highest expression levels at 1 h, 1 h, 24 h, and 24 h post-treatment, respectively (Fig. 8 a). Under SA treatment, IbATL21 , -42 , -57 , -69 , and -72 were markedly upregulated at most time points (Fig. 8 b). Under ABA treatment, IbATL17 , -21 , -38 , -42 , -44 , -46 , and -72 were significantly upregulated, whereas IbATL57 was significantly downregulated at most time points (Fig. 8 c). Under ethephon (ETH) treatment, IbATL17 , -21 , -38 , -46 , and -69 were significantly upregulated, whereas IbATL42 , -44 , -57 , and -72 were significantly downregulated at most time points (Fig. 8 d). Under IAA treatment, IbATL57 was markedly upregulated, whereas IbATL21 , -44 , - 46 , and - 69 were significantly downregulated at most time points (Fig. 8 e). Under gibberellic acid (GA) treatment, IbATL17 , -38 , -44 , and -46 were markedly upregulated, whereas IbATL21 , -42 , -57 , -69 , and -72 were significantly downregulated at most time points (Fig. 8 f). These results collectively demonstrate that the IbATL family is widely involved in hormonal signaling in sweetpotato. Notably, the expression trends in response to MeJA, SA, and ABA strongly correlate with the promoter analysis, as the promoters of these genes are enriched with corresponding MeJA-, ABA-, and SA-responsive cis -elements (Fig. 8 and Fig. S2), thereby supporting the observed expression patterns at the transcriptional regulatory level. Fig. 8. Open in a new tab Expression analysis of IbATLs in response to different hormone treatments. The relative expression levels of IbATLs were determined by qRT-PCR in sweetpotato subjected to the following treatments: ( a ) methyl jasmonate (MeJA); ( b ) salicylic acid (SA); ( c ) abscisic acid (ABA); ( d ) ethephon (ETH); ( e ) indole-3-acetic acid (IAA); and ( f ) gibberellic acid (GA). The analysis was performed at various time points after different hormone treatments: 0, 1, 2, 3, 6, 12, 24, and 48 h. The error bars represent the standard error of the mean (SEM) from three biological replicates. Significant differences compared with the 0 h control were determined using One-Way ANOVA followed by Dunnett’s test ( *P < 0.05, **P < 0.01) Furthermore, we analyzed the expression patterns of ItfATLs and ItbATLs in I. trifida and I. triloba using RNA-seq data from plants treated with IAA, BAP, GA, and ABA (Fig. S4). Significant differences were observed in the gene expression responses of I. trifida and I. triloba to these hormone treatments. The number of differentially expressed genes induced by the different hormone treatments in I. trifida was as follows: 2 for IAA, 11 for BAP, 9 for GA, and 13 for ABA (Fig. S4a). In I. triloba , IAA treatment yielded 5 differentially expressed genes (1 upregulated and 4 downregulated), BAP treatment yielded 7 (all downregulated), GA treatment yielded 8 (3 upregulated and 5 downregulated), and ABA treatment yielded 8 (5 upregulated and 3 downregulated) (Fig. S4b). These results suggest that different species exhibit distinct responses to identical hormone treatments in terms of the number of differentially expressed genes. Furthermore, the extent of gene expression regulation varies among hormones within the same species. These differences may be due to the genetic background, physiological characteristics, and evolutionary history of the species. Expression analysis of ATLs under abiotic stress in sweetpotato and its two diploid relatives To investigate the response of IbATLs to abiotic stress, we first analyzed RNA-seq data from PEG-treated Xushu 55 − 2 (Fig. 9 ). The expression analysis under PEG-induced drought stress revealed distinct response patterns among the IbATL groups. Genes from five of the six groups (Groups I–V) showed varying degrees of upregulation, while Group VI showed no clear induction. Notably, within Group V, a substantial number of IbATLs maintained relatively low expression levels, suggesting that members of this group may function primarily under other stress conditions or specific developmental cues. A subset of genes, including IbATL17 , -21 , -23 , -38 , -42 , -44 , -46 , -57 , -69 , -72 , -76 , and -77 , displayed sustained and significant upregulation across most time points following PEG treatment. These results collectively indicate that IbATLs play important roles in the drought response mechanism of sweetpotato. Fig. 9. Open in a new tab Expression analysis of IbATL genes in Xushu 55 − 2 under 30% PEG6000 treatment based on RNA-seq data. The log 2 (FPKM + 1) values are shown in the cell. The color bar shows the scale, ranging from 0.00 to 8.00. Different colors correspond to different magnitudes within this range Then, we analyzed RNA-seq data from I. trifida and I. triloba under drought, salt, cold, and heat stresses (Fig. S5). Drought treatment in I. trifida resulted in 9 upregulated and 6 downregulated ItfATL genes, while salt treatment showed 6 upregulated and 4 downregulated ItfATL genes. Cold treatment exhibited 13 upregulated and 24 downregulated genes, and the heat treatment showed 2 upregulated and 5 downregulated genes (Fig. S5a). In I. triloba , drought treatment resulted in 8 upregulated and 5 downregulated ItbATL genes; salt treatment showed 4 upregulated and 4 downregulated genes; cold treatment revealed 13 upregulated and 19 downregulated genes; heat treatment exhibited 4 upregulated and 3 downregulated genes (Fig. S5b). Further analysis revealed that no single gene in either species responded to all four stresses simultaneously, though some responded to two or three. Cold stress elicited the highest number of responsive genes among the stress types, with both I. trifida and I. triloba exhibiting relatively high total numbers of both upregulated and downregulated genes. The observed differences in the number and response pattern of genes to the same stress between I. trifida and I. triloba reflect the distinct stress tolerance strategies that have evolved in these species. Finally, we analyzed the expression patterns of the nine previously selected IbATL genes in response to abiotic stresses (PEG, NaCl, heat, and cold) using qRT-PCR (Fig. 10 ). Under these stresses, most IbATL genes were upregulated, with the exception of IbATL21 , -42 , -69 , and -72 , which were downregulated under cold stress. These stress-induced upregulated genes exhibited distinct expression patterns. Under PEG treatment, most IbATL genes were significantly induced, consistent with the transcriptome data. Among them, IbATL17 , -42 , and -72 were significantly upregulated, whereas IbATL69 was significantly downregulated at most time points (Fig. 10 a). Under NaCl treatment, IbATL17 and IbATL46 were significantly upregulated, whereas IbATL21 , -42 , -44 , -57 , -69 , and -72 were significantly downregulated at most time points (Fig. 10 b). Following heat treatment, IbATL42 , -44 , -46 , -69 , and -72 were markedly upregulated, whereas IbATL38 and IbATL57 were significantly downregulated at most time points (Fig. 10 c). Under cold treatment, IbATL38 and IbATL44 were significantly upregulated, while IbATL42 , - 46 , - 57 , - 69 , and - 72 were significantly downregulated at most time points (Fig. 10 d). These expression patterns under different stresses highlight the important and diversified functions of IbATL genes in mediating abiotic stress adaptation in sweetpotato. Fig. 10. Open in a new tab Expression analysis of IbATL genes under abiotic stress treatments. The relative expression levels of the IbATL genes were detected via qRT-PCR in sweetpotato subjected to the following treatments: ( a ) 30% PEG (simulate drought stress); ( b ) sodium chloride (simulate salt stress); ( c ) heat stress (37 °C); and ( d ) cold stress (4 °C). The expression levels were measured at the following time points: 0, 1, 2, 3, 6, 12, 24, and 48 h. The error bars represent the standard error of the mean (SEM) from three biological replicates. Significant differences compared to the 0 h control were determined using one-way ANOVA followed by Dunnett’s test ( *P < 0.05, **P < 0.01) Discussion Evolution of the ATL gene family in sweetpotato and its two diploid relatives Genome-wide analysis of the ATL gene family has been extensively conducted in numerous species. In Arabidopsis , rice, soybean, grapevine, tomato, pear, maize, and wheat, 91, 121, 162, 96, 82, 92, 77, and 334 ATL genes have been identified, respectively [ 14 – 19 ]. However, the ATL gene family in sweetpotato has not been reported previously. In this study, we obtained 85, 99, and 97 ATL genes from sweetpotato, I. trifida , and I. triloba , respectively (Table S1). Based on chromosomal localization, phylogenetic analysis, and gene collinearity analysis, we found that the number of ATL genes in sweetpotato is lower than that in its two diploid relatives (Fig. 1 , Fig. 2 , and Fig. 3 ). This discrepancy may be attributed to lineage-specific gene loss, chromosomal rearrangements, and functional streamlining of the ATL family during sweetpotato evolution, which likely reflects adaptive optimization following polyploidization [ 38 , 48 , 49 ]. Gene duplication events provide a key source of genetic novelty for plants to adapt to various environments by increasing the number of members within specific gene families [ 50 , 51 ]. Previous reports have indicated that 31 ATL genes in grapevine are located in homologous chromosomal regions derived from segmental or whole genome duplications [ 14 ]. In the ATL gene family of pear, a total of 49 pairs of duplicated genes were identified [ 17 ]. Numerous segmental duplication events exist in the wheat ATL gene family, in addition to syntenic relationships with other monocotyledonous and dicotyledonous plants such as Arabidopsis and maize [ 19 ]. In this study, collinearity analysis revealed the presence of 35 pairs of duplicated genes in the sweetpotato ATL gene family, indicating that segmental duplication is also an important evolutionary force driving the expansion of the ATL gene family in sweetpotato (Fig. 3 a). There were 134 collinear ATL gene pairs between sweetpotato and I. trifida , and 143 between sweetpotato and I. triloba (Fig. 3 b and Table S3), indicating that the ATL gene family has been conserved throughout the evolution of sweetpotato. Additionally, ten IbATL genes (IbATL 14 , -2 9 , - 31 , - 39 , - 40 , - 48 , - 52 , - 57 , - 62 , - 77 ) showed synteny in both monocots and dicots (Fig. 3 b, Fig. 3 c, and Fig. 3 d). The maintenance of synteny for these genes since before the monocot-dicot split strongly indicates that they have been subject to strong purifying selection and perform indispensable, core biological functions. Gene structure and genomic architecture play crucial roles in determining gene regulation and functional output [ 52 ]. In plants, the main characteristic of the ATL family is that it contains the RING-H2 domain [ 53 ]. Previous studies have shown that the RING-H2 domain of Arabidopsis AtATLs is the key domain for its function as an E3 ubiquitin ligase [ 13 , 14 , 21 ]. The gene structure of the IbATL family reveals a potential driver of its functional diversity. The near-absence of introns in most members, a feature consistent with previous reports [ 19 ], likely enables rapid transcriptional responses, a key step toward functional specialization. Moreover, the conserved presence of Motif1 in most proteins, with similar architectural patterns within phylogenetic groups, suggests a strong correlation between protein motif composition and functional specialization (Fig. 4 and Fig. S1). Promoter architecture underlies the multi-hormone responsiveness and stress adaptation of the ATL gene family in sweetpotato and its two diploid relatives Studies have shown that cis -elements in the promoter region are closely associated with genes involved in regulating plant growth, development, and abiotic stress responses [ 54 – 56 ]. In this study, we performed a predictive analysis of the promoter cis- elements of ATL genes in sweetpotato and its two diploid relatives (Fig. 5 and Fig. S2). Various light-responsive elements, such as Box 4 and G-box, were identified in the promoters of IbATL genes (Fig. 5 ). Previous studies have confirmed that Box 4 and G-box exist in the promoter regions of various light-inducible genes and participate in mediating light responses [ 57 , 58 ]. The ABA-responsive element ABRE is a binding site for bZIP transcription factors. Under drought stress, ABA activates SnRK2 kinases, which subsequently phosphorylate downstream transcription factors. These transcription factors then induce gene expression by binding to the ABRE element in the promoter regions of target genes, thereby enhancing plant drought tolerance [ 59 – 61 ]. In this study, the promoters of ATL genes in sweetpotato and its two diploid relatives contained a large number of ABRE elements (Fig. 5 and Fig. S2). This finding, coupled with the result that IbATLs were generally upregulated under ABA treatment (Fig. 8 c), strongly suggests that the ABA signaling pathway is a key mechanism regulating IbATL -mediated drought adaptation. Furthermore, the MeJA-responsive elements CGTCA-motif and TGACG-motif were also abundant (Fig. 5 ), and studies have reported that these elements participate in jasmonate signaling responses in various plants [ 62 ]. The presence of SA-responsive elements (such as the TCA-element) also indicates that IbATL genes may be involved in SA-mediated abiotic stress resistance [ 63 ]. The drought-responsive element MBS plays an important role in abiotic stress responses [ 64 ], and in sweetpotato, more than half of the IbATL genes contain this element (Fig. 5 ). Previous studies have reported that the promoters of wheat TaATLs contain many ABA- and MeJA-related response elements, indicating that TaATLs may participate in abiotic stress responses through ABA- and MeJA-mediated signaling pathways [ 19 ]. Similarly, the significant enrichment of MeJA- and ABA-responsive cis -elements in the promoters of ATL genes from sweetpotato and its diploid relatives suggests that they may also participate in abiotic stress responses through ABA- and MeJA-mediated signaling (Fig. 5 and Fig. S2). Protein-protein interaction network reveals that the IbATLs play a central role in the regulatory network of sweetpotato ubiquitination Members of the ATL family, functioning as RING-type E3 ubiquitin ligases, are core regulators of plant abiotic and biotic stress responses [ 14 , 17 ]. The ubiquitin-proteasome system (UPS) is a vital system in plants, and the protein ubiquitination it mediates is closely associated with plant stress responses [ 7 ]. In this study, we found that the sweetpotato protein interaction network is enriched with several functionally characterized E3 ligases, including BOI, which mediates biotic and abiotic stress responses through the degradation of MYB108 [ 65 ]; RIN2, which regulates the hypersensitive response [ 66 ]; and RHA2B, a positive regulator of ABA and osmotic stress responses [ 67 ]. This finding suggests that sweetpotato may coordinate plant responses to abiotic and biotic stresses through ubiquitin-mediated protein degradation (Fig. 6 ). Future experimental validation of these predicted interactions, particularly between IbATLs and their putative substrates in sweetpotato, will be essential for confirming their precise roles within this regulatory network and elucidating the molecular mechanism of this pathway. The tissue-specific expression of ATLs was different in sweetpotato, I. trifida , and I. triloba In plants, the expression patterns of the ATL family genes are often closely associated with their biological functions in specific tissues. Previous studies have shown that ATL family members participate in various tissue development and physiological processes: rice E3 ubiquitin ligase EL5 maintains cell viability in the root apical meristem and is crucial for root development [ 25 ]; members of the maize ZmATL gene family exhibit significant differential expression across different tissues, and a considerable number of members maintain high expression levels in kernels after pollination, suggesting their potential role in seed development [ 18 ]; in wheat, the expression level of TaATL1 decreases significantly during the transition from vegetative to reproductive growth, and overexpression of TaATL1 interferes with cell division and negatively regulates flowering time, indicating that such members may be involved in reproductive transition and meristem-related developmental regulation [ 68 ]. In this study, through tissue-specific expression profile analysis, we found that although ATL genes in sweetpotato and its two diploid relatives ( I. trifida and I. triloba ) are all expressed in roots, stems, leaves, and flowers, their expression trends differ significantly. In I. trifida and I. triloba , most ItfATL and ItbATL genes exhibit high expression in floral tissues, which may align with the potential function of ATLs in reproductive development in the two diploid relatives. In contrast, in sweetpotato, most IbATL genes show high expression in stems and roots (Fig. 7 and Fig. S3). This expression trend, shifting from reproductive tissues in diploids to vegetative storage tissues in sweetpotato, suggests that the regulatory network of ATL genes may have undergone functional remodeling during evolution to adapt to the unique storage root development requirements of sweetpotato. Hormonal response patterns of the ATL gene family in sweetpotato and its two diploid relatives Plant hormones are core signaling molecules that coordinate growth and development with stress responses, playing essential roles in defense reactions, growth regulation, and stress adaptation [ 69 – 71 ]. There is a close relationship between plant hormone signaling and ubiquitination processes. For instance, under high concentrations of MeJA, VvPUB8 ubiquitinates and degrades the VvbHLH93 transcription factor, thereby inhibiting the expression of anthocyanin biosynthesis-related genes in grapevine [ 72 ]. In Arabidopsis , SA treatment promotes the degradation of OTS1/OTS2 proteins while simultaneously enhancing the accumulation of SUMO1/2 conjugates; OTS1/2 form a feedback loop within the SA signaling pathway to modulate SA signal intensity [ 73 ]. PUB35 negatively regulates ABA signaling by mediating AFP1-dependent degradation of ABI5 [ 74 ]. Similarly, CKRW1/WAV3 subfamily E3 ubiquitin ligases target IAA32/34 for ubiquitination and degradation, a process inhibited by TMK1c-mediated phosphorylation; this antagonistic regulation fine-tunes IAA32/34 stability to control apical hook cell elongation [ 75 ]. In this study, qRT-PCR analysis demonstrated that IbATL17 , - 21 , - 38 , - 42 , - 44 , - 46 , - 57 , - 69 , and, - 72 were upregulated under the six hormone treatments (MeJA, SA, ABA, ETH, IAA, and GA), although several exceptions were noted: IbATL21 , -38 , -42 , and -44 were downregulated by MeJA; IbATL38 by SA; and IbATL17 , -42 , and -69 by IAA. Furthermore, these upregulated genes exhibited distinct expression patterns in response to different hormone treatments (Fig. 8 ). These results demonstrate that the IbATL gene family is broadly involved in the hormonal signaling networks of sweetpotato. Considering previous reports that GmRFP1 , a soybean ATL gene involved in ABA signaling, is upregulated by ABA and salt [ 76 ], we speculate that IbATLs may also play key roles in sweetpotato stress adaptation via similar ABA-dependent mechanisms. Furthermore, under IAA, BAP, GA, or ABA treatments, the number of induced ATL genes was lower in the two diploid relatives I. trifida and I. triloba , compared to sweetpotato, and there were marked differences in the number of differentially expressed genes induced by each hormone (Fig. S4). This suggests that the ATL genes in sweetpotato and its two diploid relatives may have undergone extensive functional diversification, enabling broad and distinct responses under multi-hormonal conditions. Differences in the functions of ATLs in sweetpotato, I. trifida , and I. triloba under abiotic stress In plants, ATL proteins play a role in abiotic stress responses through hormonal regulation and ubiquitination. In rice, the OsATL38 protein ubiquitinates the 14-3-3 protein OsGF14d, a positive regulator of cold tolerance, thereby attenuating resistance to cold stress [ 31 ]. GmRFP1 , a member of the soybean ATL gene family, is involved in ABA signal transduction and participates in plant stress responses. GmRFP1 expression is upregulated under ABA and salt stress, but downregulated under low temperature and drought stress, suggesting that it plays a significant role in the soybean response to these stresses [ 76 ]. Overexpression of the IbATL38 gene in Arabidopsis significantly enhances salt tolerance, accompanied by the upregulation of stress-responsive genes and a marked decrease in reactive oxygen species (H₂O₂) content [ 29 ]. In Arabidopsis , transgenic plants overexpressing the pear PbrATL18 gene exhibit stronger drought tolerance [ 17 ]. Under high temperatures, heat stress suppresses the expression and stability of the PpATL52 E3 ubiquitin ligase protein, leading to the accumulation of the PpHsfB2a protein. This, in turn, inhibits the transcription of PpHY5L , which is a key transcription factor in anthocyanin synthesis, ultimately reducing anthocyanin accumulation in pear fruit [ 77 ]. In this study, we investigated the differential expression of IbATLs under PEG stress using RNA-seq data from the sweetpotato line Xushu 55 − 2. The results showed that some IbATLs exhibited significant differences in expression levels (Fig. 9 ). Furthermore, using qRT-PCR, we found that most of the nine selected IbATL genes (which were induced by hormone and PEG treatments) were upregulated in response to abiotic stresses (PEG, NaCl, heat, and cold), although IbATL21 , -42 , -69 , and -72 were specifically downregulated under cold stress (Fig. 10 ). Moreover, these upregulated genes exhibited distinct expression patterns under different stress conditions. These genes could serve as candidate genes for stress tolerance. The expression patterns of ATLs in I. trifida and I. triloba differ significantly from those in sweetpotato under abiotic stress (Fig. S5). This discrepancy may be related to the complex evolution of sweetpotato, whereby these genes may have been altered or lost during the domestication of cultivated varieties [ 78 ]. Promoters, which are located upstream of genes, are key regulatory regions that control gene expression [ 79 ]. The differences in the number of abiotic stress-responsive elements between sweetpotato and its two diploid relatives might explain the more precise differences in ATL gene expression in sweetpotato compared with its two diploid relatives. Materials and methods Plant materials and treatments The highly drought-tolerant sweetpotato cultivar ‘Shangshu 19’ was used for gene expression analysis. Sweetpotato explant stem segments were placed in a 27 ± 1 °C environment and cultured in Hoagland’s liquid medium under 13 h of cool white light illumination and 11 h of darkness. To analyze expression under hormone treatments and abiotic stress conditions, sweetpotato stem segments grown in Hoagland’s solution were transferred to Hoagland’s medium supplemented with 100 µM MeJA, 100 µM SA, 100 µM ABA, 100 µM ETH, 100 µM IAA, 100 µM GA, 30% PEG, 200 mM NaCl, or subjected to heat (37 °C) or cold (4 °C). Samples were collected at 0, 1, 2, 3, 6, 12, 24, and 48 h post-treatment. Each treatment comprised three biological replicates, with three samples used per replicate. Identification of ATLs In order to obtain the sequences of the ATL gene family containing a clear RING-H2 zinc finger domain in sweetpotato, genome and annotation files were obtained from the Ipomoea Genome Hub ( https://sweetpotato.com/ ) and the Sweetpotato Genomics Resource ( http://sweetpotato.uga.edu/ ). The Arabidopsis ATL protein sequences were downloaded from the NCBI ( https://www.ncbi.nlm.nih.gov/ ) and TAIR ( https://www.arabidopsis.org/ ) databases and were then used for homology comparison against the sweetpotato ( I. batatas ), I. trifida , and I. triloba genomes to identify corresponding homologs. Then, the Hidden Markov Model (HMM) profile for the RING-H2 zinc finger domain (PF12678) was downloaded from the InterPro database ( https://www.ebi.ac.uk/interpro/ ) [ 80 ], and performed an HMM search using the Simple HMM Search tool in TBtools v.2.034 [ 81 ]. Subsequently, we combined the results from the two aforementioned sections. The conserved domain structures of all ATL protein sequences were predicted and verified using the SMART website ( https://smart.embl.de/ ) and the Conserved Domains Database (CDD) ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ) [ 82 ]. Analysis of the physical and chemical properties of ATL proteins The physicochemical properties of ATL proteins were analyzed using the ExPASy ProtParam tool ( https://web.expasy.org/protparam/ ). The subcellular localization of ATL proteins was predicted using the Cell-PLoc 2.0 Server ( http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ ) [ 83 ]. Chromosome localization of ATLs IbATLs , ItfATLs , and ItbATLs were separately mapped to the I. batatas , I. trifida , and I. triloba chromosomes based on the chromosomal locations provided in the Ipomoea Genome Hub ( https://sweetpotato.com/ ) and Sweetpotato Genomics Resource ( http://sweetpotato.uga.edu/ ). The visualization was generated using the TBtools v2.034 [ 81 ]. Phylogenetic analysis of ATLs Building on previous reports of Arabidopsis ATL genes, we used the RING-H2 conserved domain as the screening criterion and applied the NCBI CDD search, together with SMART conserved domain identification, to filter the previously reported genes, ultimately obtaining 83 Arabidopsis ATL genes for phylogenetic tree construction. The amino acid sequences of ATL proteins from Arabidopsis thaliana ( At ), I. batatas ( Ib ), I. trifida ( Itf ), and I. triloba ( Itb ) were aligned using ClustalW in MEGA11.0 software [ 84 ]. The specific identifiers for all sequences used in this analysis were provided in Table S1. Following alignment, a phylogenetic tree was constructed using MEGA11.0 [ 84 ] with the Neighbor-Joining method, and branch support was assessed by 1000 bootstrap replicates [ 45 ]. The phylogenetic tree was visualized and annotated using Evolview ( https://evolgenius.info//evolview-v2/#login ) [ 85 ]. Collinearity analysis of ATLs The genome and GFF3 annotation files for sweetpotato ( I. batatas ), its diploid relatives ( I. trifida and I. triloba ), Arabidopsis thaliana , rice ( Oryza sativa ), and maize ( Zea mays ) were downloaded from the Ipomoea Genome Hub ( https://sweetpotato.com/ ), the Sweetpotato Genomics Resource ( http://sweetpotato.uga.edu/ ), the Ensembl Genome Database ( https://asia.ensembl.org/index.html ), and the TAIR database ( https://www.arabidopsis.org/ ), respectively. Subsequently, collinear gene pairs among these species were predicted using the One Step MCScanX tool in TBtools v2.034, and the results were visualized within the same software [ 81 ]. Gene pairs located on the same chromosome were classified as tandem duplications, whereas those on different chromosomes were defined as segmental duplications [ 86 ]. To evaluate evolutionary pressure and divergence time among homologous genes, a Ka/Ks analysis was performed on the IbATL gene pairs. The Ka and Ks substitution rates were calculated using the “Simple Ka/Ks Calculator” in TBtools v.2.034 [ 81 ]. The Ka/Ks ratio was then used to infer selection pressure, with values less than one indicating purifying selection [ 86 ]. Conserved motifs and domains identification and gene structures analysis of ATLs The conserved motifs of ATL proteins were analyzed using MEME ( https://meme-suite.org/meme/tools/meme ), where the maximum number of motif parameters was set to 10 [ 87 ]. Conserved domains were identified using the Conserved Domains Database (CDD) ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ) and visualized with TBtools v2.034 [ 81 ]. To analyze the exon-intron structures of ATL genes, the sweetpotato genome annotation file was downloaded from the Ipomoea Genome Hub ( https://sweetpotato.com/ ). The Exon-Intron structure analysis of ATL genes was performed with TBtools v2.034 [ 81 ]. The visualization of motifs, domains, and gene structures was completed by TBtools v2.034 [ 81 ]. Promoter analysis The 2000 bp upstream sequences of the IbATL gene coding regions were extracted from the sweetpotato genome using GXF Sequence Extract in TBtools v.2.034 and analyzed with PlantCARE ( https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) under default parameters to identify cis -elements [ 81 , 88 ]. After statistical analysis, the results were visualized by plotting a heatmap using the “HeatMap tool” in TBtools v.2.034 [ 81 ]. Protein interaction network of IbATLs The protein interaction network of IbATLs was predicted using STRING ( https://cn.string-db.org/ ; medium confidence, 0.400) based on Arabidopsis orthologs. The protein-protein interaction network and node network diagrams were constructed using Cytoscape v3.10.0 (Institute for Systems Biology, Seattle, WA, USA) [ 89 ]. Transcriptome analysis The RNA-seq data for ItbATLs and ItfATLs from I. triloba and I. trifida were downloaded from the Sweetpotato Genomics Resource ( http://sweetpotato.uga.edu/ ). For I. batatas , the expression data for IbATLs in cultivars ‘Xuzi3’ and ‘Yan252’ were obtained from the China National Center for Bioinformation ( https://ngdc.cncb.ac.cn/gsa/ ) under accession number CRA000606 [ 90 ], while the data for sweetpotato line Xushu 55 − 2 were obtained from related research [ 91 ]. Following alignment of the raw reads to the genome with STAR [ 92 ], read counts were generated by featureCounts [ 93 ] and analyzed for differential expression using DESeq2 [ 94 ], after which log 2 (FPKM + 1) values were visualized as a heatmap in TBtools v2.034 [ 81 ]. The qRT-PCR analysis of IbATLs We performed an integrated analysis of promoter cis -elements and their association with transcriptomic expression. Based on these findings, we selected nine IbATLs ( IbATL17 , - 21 , - 38 , - 42 , - 44 , - 46 , - 57 , - 69 , and - 72 ) that exhibited significant differential expression at various time points in the PEG-treated sweetpotato line Xushu55-2 transcriptome for further validation by qRT-PCR. The primers used for qRT-PCR were listed in Table S4. Total RNA was extracted using the TRIzol method (Invitrogen, Carlsbad, CA, USA), after which a real-time PCR analysis was performed using a reaction mixture containing first-strand cDNA, primer mix, and SYBR Green Real-Time PCR Master Mix (TaKaRa Bio, Dalian, China; catalogue no. DRR037A), with a final volume of 20 µL. The sweetpotato ubiquitin 10 gene ( IbUBQ10 ) was used as an internal control (Table S4). Relative gene expression levels were quantified using the comparative CT method. qRT-PCR was performed on a 7500 Real-Time PCR instrument (Applied Biosystems, Foster City, CA, USA) using the SYBR detection protocol. The relative gene expression levels from qRT-PCR analysis were presented as column charts (bar graphs). This graphical representation was used to concisely compare expression patterns across the tested conditions. Conclusions In this study, we identified 85, 99, and 97 ATL gene family members in sweetpotato, I. trifida , and I. triloba , respectively. A systematic analysis of the ATL gene family was conducted, encompassing the examination of physicochemical properties, chromosomal localization, phylogenetic relationships, collinearity, gene structures, promoter cis -elements, and protein-protein interaction network. We then investigated the expression patterns of ATLs under hormonal treatments and abiotic stress conditions using qRT-PCR and RNA-seq data. Our findings reveal distinct roles for sweetpotato and its two diploid relatives in responding to abiotic stresses. This study provides crucial insights into the structure and function of ATL genes in sweetpotato and its two diploid relatives. Supplementary Information 12870_2026_8501_MOESM1_ESM.zip (5MB, zip) Additional file 1: Fig. S1. Distribution of conserved motifs and gene structures of ATL genes from I. trifida and I. triloba . Fig. S2. Analysis of cis -elements in the promoters of ATL genes from sweetpotato and its diploid relatives. Fig. S3. Tissue-specific expression analysis of ItfATLs and ItbATLs in I. trifida and I. triloba. Fig. S4. Hormone-induced expression analysis of ItfATLs and ItbATLs in I. trifida and I. triloba . Fig. S5. Abiotic stress-induced expression analysis of ItfATLs and ItbATLs in I. trifida and I. triloba . 12870_2026_8501_MOESM2_ESM.zip (80.2KB, zip) Additional file 2: Table S1. Nomenclature and corresponding numbers of the ATL family genes. Table S2. Physicochemical properties of the ATL family genes in sweetpotato, I. trifida and I. triloba . Table S3. One-to-one orthologous genes. Table S4. Primers used in this study for qRT-PCR analysis. Acknowledgements We thank the Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs (China Agricultural University) for providing the experimental facilities. Authors’ contributions R.L. and H.Zhai conceived and designed the study. R.L. and Y.Y. performed the experiments and analyzed the data. S.G., H.Zhang, N.Z., S.H., Q.L. and H.Zhai contributed to funding acquisition and supervised the project. The manuscript was drafted by R.L. and revised by Y.Y. and H.Zhai. All authors have read and approved the final manuscript. Funding This research was funded by the National Natural Science Foundation of China (32472120), the earmarked fund for CARS-10-Sweetpotato and the 2115 Talent Development Program of China Agricultural University. Data availability All data in this study are available in this article or supplementary information. 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Supplementary Materials 12870_2026_8501_MOESM1_ESM.zip (5MB, zip) Additional file 1: Fig. S1. Distribution of conserved motifs and gene structures of ATL genes from I. trifida and I. triloba . Fig. S2. Analysis of cis -elements in the promoters of ATL genes from sweetpotato and its diploid relatives. Fig. S3. Tissue-specific expression analysis of ItfATLs and ItbATLs in I. trifida and I. triloba. Fig. S4. Hormone-induced expression analysis of ItfATLs and ItbATLs in I. trifida and I. triloba . Fig. S5. Abiotic stress-induced expression analysis of ItfATLs and ItbATLs in I. trifida and I. triloba . 12870_2026_8501_MOESM2_ESM.zip (80.2KB, zip) Additional file 2: Table S1. Nomenclature and corresponding numbers of the ATL family genes. Table S2. Physicochemical properties of the ATL family genes in sweetpotato, I. trifida and I. triloba . Table S3. One-to-one orthologous genes. Table S4. Primers used in this study for qRT-PCR analysis. 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