Introduction

Cytochrome P450 monooxygenases (CYP450s), named after the absorption band at 450 nm exhibited by their carbon-monoxide-bound form [1], are a ubiquitous class of enzymes found in various organisms, including bacteria, plants, and humans [2]. The discovery of the first CYP450 gene occurred in rat liver microsomes [3, 4]. Subsequently, the X-ray crystal structure of CYP450 was obtained from bacterial CYP450cam [5]. As more CYP450 genes were identified, the subfamily of CYP450 genes expanded [6,7,8]. The CYP450 family is present in a wide range of organisms, including plants, insects, animals, fungi, bacteria, and viruses [9]. Numerous CYP450 genes, including approximately 16,000 in plants, have been identified across different organisms [10]. Gene structure analysis shows that members of this family all contain a conserved heme-binding domain with a sequence of FxxGxRxCxG [11]. Additional conserved domains are also present in CYP450 proteins. One such domain is the I-helix, which plays a role in both oxygen binding and catalysis on the distal side of the heme group. The sequence of the I-helix is characterized by the presence of A/G-X-E/D-T-T/S residues [12]. Furthermore, the PERF domain contains an arginine residue, while the K-helix consists of glutamate and arginine residues, forming the E-R-R triad. This triad is responsible for stabilizing the core structure of CYP450 enzymes [13]. Based on evolutionary relationships, plant CYP450s are divided into 11 families, with the smallest family number within each clan serving as its name [14]. The CYP450 genes in plants can generally be divided into A-type and non-A-type. Among them, CYP71 is A type, and other families are non-A type [15]. A considerable number of CYP450 proteins are involved in the biosynthesis and breakdown of diverse substances, encompassing plant hormones, secondary metabolites, and defense compounds [12]. Within this context, several CYP450 families, including CYP71, CYP85, and CYP72, are responsible for conducting oxidation and rearrangement reactions that contribute to the biosynthesis of diterpenes. These diterpenes serve as essential components in the production of hormones, pharmaceuticals, aroma compounds, and food ingredients [16].

In Arabidopsis, the CYP450 family ranks as the third-largest gene family and plays crucial roles in the synthesis of antioxidants, phytohormones, structural polymers, and defense-related compounds [17,18,19,20,21,22,23,24,25]. AtCYP88A3 and AtCYP88A4 play a role in gibberellin biosynthesis, and mutations in CYP88 lead to a dwarf phenotype in barley and maize [12]. Recent studies have focused on CYP450 genes involved in stress resistance and secondary metabolism, such as the gossypol biosynthesis pathway in cotton [26,27,28,29]. Additionally, CYP450 genes have been implicated in drought tolerance, exemplified by CYP86A2 in Arabidopsis and CsCYP75B1 in citrus [30, 31]. Cold stress can also induce the expression of CYP450 genes in perennial ryegrass, tall fescue, and roses [32, 33]. Furthermore, CYP450 genes participate in the biosynthesis of jasmonic acid (JA), as demonstrated by GmCYP82A3 in soybean and DzCYP72As in Dioscorea zingiberensis [34]. Multiple investigations have provided evidence supporting the involvement of CYP716A subfamily genes in regulating the biosynthetic pathway of triterpenoids. In the case of Artemisia annua, the genes CYP716A14v2 and OSC2 (a multifunctional oxidosqualene cyclase) are responsible for the production of triterpenoids, which serve as constituents of the wax layer of the cuticle that covers the aboveground parts of the plants. Researchers suggest that specialized triterpenoids may serve a protective function against both biotic and abiotic stresses in A. annua [35]. Similarly, in sweet basil, CYP716A52 and CYP716A53 catalyze C-28 oxidation to yield oleanolic acid and unsolid acid. These compounds aid in the plant’s defense mechanism against stress [36]. These investigations propose that CYP450 genes play a critical role in both plant growth and stress response. Previous investigations have successfully identified and carried out functional analysis on individual genes in the CYP450 family of sweet potatoes. For example, IbCYP73A1(IbC4H) enhances the ability of plants to scavenge reactive oxygen species under stress. IbCYP82D47 interacts with the carotenoid biosynthesis-related protein IbGGPPS12, increasing the content of carotenoids in transgenic sweet potatoes [37, 38]. However, a systematic identification and analysis of CYP450 family members in sweet potatoes has not yet been conducted.

Sweet potato [Ipomoea batatas (L.)] is a vine plant belonging to the Convolvulaceae family. It is an important crop for food, feed, industrial raw materials, and new energy sources. Its edible enlarged storage root is a valuable source of nutrients and phytochemicals, making it widely cultivated [39]. These unique characteristics make it a staple food for humans, a feed source for animals, and a raw material source for the food and nonfood industries [40]. It is also used for the production of biofuels and alcohol [41]. With the development of sequencing technology, an increasing number of plant genomes and transcriptomes are being revealed, leading to a broader scope of research in this area. The sweet potato is one of the plants that has been extensively studied. In recent years, an increasing number of CYP450 family genes have been discovered in crops, such as rice, soybean, and chili pepper [7, 42, 43]. However, there have been few reports on this gene family in sweet potatoes. In this study, the whole genome, evolutionary relationships, chromosomal localization, collinearity relationships, and expression patterns of the sweet potato CYP450 gene family were identified and comprehensively analyzed using bioinformatics methods. The results provide a theoretical basis for understanding the functions of CYP450 genes in sweet potatoes and for molecular breeding of sweet potatoes.

Materials and methods

Identification and physicochemical properties of IbCYP gene family members

The genomic data of sweet potatoes were obtained from the Ipomoea Genome Hub website (https://ipomoea-genome.org/) [44]. For the downloaded protein sequences, BLAST was used to construct a local database. In contrast, the gene and protein sequences of the Arabidopsis CYP450 gene family were obtained from the Cytochrome P450 Homepage website (http://drnelson.uthsc.edu/CytochromeP450.html). A BLAST comparison of CYP450 gene family protein sequences between sweet potato and Arabidopsis was conducted. At the same time, a hidden Markov model of the typical CYP450 family protein structure was downloaded from the Pfam database (http://pfam.sanger.ac.uk) [45], and HMMER software was used to search for the protein sequences [46] containing the characteristic domains (PF00067). The candidate proteins identified through the previously mentioned methods were further analyzed using Snapgene software. Incomplete reading frame sequences and redundant sequences were manually eliminated. The remaining candidate protein domains were validated using Pfam and the Conserved Domain Database (CDD) online analysis tools [47]. Gene sequences that did not contain the CYP450 gene family domain or had incomplete CYP450 domains were removed from the analysis. Finally, 95 IbCYP genes were obtained and all the genes contained the FxxGxRxCxG characteristic domain. The coding sequence (CDS) and amino acid sequences of 95 IbCYP genes were corrected using the existing transcriptome sequencing results of sweet potato in our laboratory. The ExPASy ProtParam tool (http://web.ExPASy.org/protparam/) was used to predict protein physicochemical parameters [48]. Subcellular localization predictions were generated with BUSCA (http://busca.biocomp.unibo.it/) [49].

Gene structure and conserved motif analysis

The exon–intron structure information of the candidate IbCYP was predicted by the online website GSDS2.0 (http://gsds.gao-lab.org/) [50]. The MEME online website (http://meme-suite.org/tools/meme) was used to predict the conserved domains in IbCYP450 protein sequences [51]. For this analysis, the number of motifs to be identified was set to 10, while default settings were adopted for other parameters and the results were visualized using TBtools. MEGA11 software [52] was used to perform multiple sequence alignment of 95 IbCYP proteins, and visualization was achieved with GeneDoc software.

Phylogenetic analysis of IbCYP proteins

One representative member of each plant CYP450 family was used for alignment and phylogenetic analysis. Members with identified functions in a family were preferentially selected for phylogenetic analysis. ClustalW was employed for multiple sequence alignment of CYP450 protein sequences between sweet potato, Salvia miltiorrhiza, pepper, tobacco, and Arabidopsis. The phylogenetic tree was constructed using MEGA11 neighbor-joining (NJ) with 1000 bootstrap replicates [52].

Chromosomal localization, gene duplication, and synteny analysis

The locations of 95 IbCYP genes on chromosomes were obtained based on the information annotated for the sweet potato genome and analyzed through the Gene Location Visualization of TBtools [53]. Arabidopsis, tomato, pepper, maize, and rice downloaded from NCBI. Analysis of genome collinearity between sweet potatoes and these species was performed using MCScanX software [54]. Circos and Dual Synteny Plot in TBtools were used for visualized mapping of the collinear gene pairs [53].

Cis-acting element analysis of IbCYP genes

The upstream promoter region (2,000 bp) of the IbCYP genes was extracted using TBtools software and submitted to the PlantCARE website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [55], which identified the cis-regulatory elements in the IbCYP genes. Then, the TBtools software was used to visualize the cis-regulatory element Fig. [53].

Plant material and treatments

The sweet potato materials used in the experiment were obtained from the experimental field of the College of Coastal Agriculture, Guangdong Ocean University (21°15′N, 110°30′E).

After sweet potato seedlings were taken from the experimental field, the tuberous, pencil root, primary root, flower, fruit, and stem were covered with dry ice after being quickly frozen with liquid nitrogen. The tissues were then sent to Biomarker Technologies for total RNA extraction, library construction, and full-length transcriptome sequencing. Several strong branches grown consistently were selected and cultured with clean water for 10 days before being subjected to abiotic stress. During the development of adventitious roots in sweet potato shoots, the control group (CK) was maintained by continuing the culture with clean water. For salt stress treatment, the culture was continued with 200 mmol/L NaCl solution, and for drought stress treatment, 300 mmol/L mannitol solution was used. Each group was treated in triplicate with 3 branches per replicate. After 24 h of stress treatment, the primary roots, young stems, and leaves were taken and cooled by liquid nitrogen, covered with dry ice, and sent to Biomarker Technologies for transcriptomic sequencing.

Protein-protein interaction (PPI) network construction

Using the default parameters, the online STRING database (https://string-db.org/) [56] was utilized to predict and execute potential protein-protein interaction networks using IbCYP proteins based on known Arabidopsis homologs. Cytoscape (V3.10.0) was used to visualize the resulting network [57].

Transcriptome analysis

Five transcriptome bio project datasets were chosen for the IbCYP450 gene expression profile analysis. Two bio project datasets (PRJNA511028 for hormone, and PRJNA987163 for cold) were downloaded from the NCBI database. Another three were our in-house (unpublished) sweet potato heat treatment, salt treatment, and drought treatment. Among them, “Xushu 18” was for hormonal treatment, clod-tolerant “Liaohanshu 21” and clod-sensitive “Shenshu 28” for cold treatment, heat-tolerant “Guangshu 87” and heat-sensitive “Ziluolan” for heat treatment and salt-tolerant and drought-tolerant “Guangshu 87” for salt and drought treatment. The CYP450 expression was measured in fragments per kilobase of exon per million fragments mapped (FPKM). The heat maps of expression were constructed by TBtools software.

Quantitative analysis of candidate IbCYP genes

The sweet potato (I. batatas) cultivar “Jishu 26” was used for qRT-PCR analysis in this study. Sweet potato plants were cultivated in a field at the experimental field of Guangdong Ocean University, Guangdong, China. For tissue expression, the flower, leaf, stem, primary root, firewood root, and tuberous root tissues were sampled from 3-month-old “Jishu 26” planted in the field. For the abiotic stress treatments, the twigs about 30 cm in length from 3-month-old filed-grown “Jishu 26” were cultured in the Hoagland solution for 14 days to treat: for salt stress treatment, the twigs were cultured in the Hoagland solution with 0 and 200 mM NaCl. For drought stress treatments, the twigs were cultured in Hoagland solution with 0 and 300 mM mannitol. The primary root, stem, and leaf samples were collected at 0, 8, 16, and 24 h after the treatments.

For qRT-PCR analysis, the 10 µL total reaction quantity of each sample contained 1 µL cDNA template, 0.5 µL (10µmol L-1) forward and reverse gene-specific primers, 5 µL 2×SYBR Green qPCR mix and 3 µL ddH2O. The qRT‒PCR reaction was conducted using the Bio-Rad system with the following thermal cycle conditions: 3 min of pre-degeneration at 95 °C, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing at 60 °C for 30 s. The reaction was completed with a 5-second step at 65 °C and a cooling rate of 0.5 °C to reach 95 °C. Each sample was replicated 3 times, referring to Dingfa’s method [58] using the IbARF gene as an internal reference. We calculated relative transcript levels using the 2−ΔΔCT method.

Results

Identification of cytochrome CYP450 family genes in sweet potato

The 95 IbCYP genes are given new names according to the classification and naming principles of CYP450. The proteins produced by these genes have different lengths, with amino acids ranging from 381 (IbCYP712A1) to 873 (IbCYP82D47). Their weights also vary, from 42.39 kD (IbCYP712A1) to 98.35 kD (IbCYP82D47). The predicted isoelectric points of the proteins range from 5.52 (IbCYP712A1) to 9.45 (IbCYP76G3). Of these, 75 proteins have isoelectric points higher than 7, making them positively charged in acidic solutions. The proteins have different levels of hydrophilicity, ranging from − 0.384 (IbCYP704A1) to 0.075 (IbCYP78A2). Furthermore, according to BUSCA subcellular localization predictions, all IbCYP proteins are found in the endomembrane system (Table 1).

Table 1 Identification of IbCYP genes and analysis of physicochemical properties of proteins in sweet potato
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Motif compositions and gene structure of the IbCYP genes

According to the classification principles of the CYP450 family, we have divided the identified 95 IbCYP proteins into 5 families (Fig. 1A). After analyzing the CYP450 protein sequence of sweet potato using the MEME online tool, ten conserved motifs were predicted (Fig. 1B). Different IbCYP proteins showed variations in the number and distribution of these motifs. Each gene had between six and ten motifs, and all IbCYP proteins had a conserved heme-binding domain Motif 1. The C-terminal region of the IbCYP protein was highly conserved, with Motif 2 and Motif 3 commonly found in most proteins, while the N-terminal region was less conserved. The majority (86.7%) of A-type CYP450 proteins contained all ten motifs, while non-A-type CYP450 proteins typically had between six and nine motifs. Motif 10 was not found in the CYP97 or CYP72 families, and motif 5 was absent in the CYP86 family. This suggests that the IbCYP gene family shows both a high level of conservation and some differences. Different subfamilies have distinct types of motifs, which may be related to the various biological functions of genes within each subfamily.

Fig. 1
figure 1

Phylogenetic tree, conserved motif, and gene structure of the IbCYP family in sweet potato. A A neighbor-joining (NJ) phylogenetic tree of sweet potato protein with 1000 bootstrap replicates was constructed based on the full-length sequence in MEGA11. B Distribution of conservative motifs in IbCYP proteins with colored boxes representing motifs 1-10 and scale representing 50 amino acids. C The genetic structure of the IbCYP gene, including introns (black line), exons (yellow rectangle), and untranslated regions (UTRs, green rectangle), with the scale representing 1 kb

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We studied the structure of the coding sequences of all 95 IbCYP genes and found that the CYP450 gene family members of sweet potato had 1 to 15 coding sequences. The number of introns varied from 1 to 3, indicating that there was significant variation in the gene structure of IbCYP genes (Fig. 1C), the yellow box represents the coding sequence (CDS) of the CYP450 gene family members.

Conserved sequence alignment of the IbCYPs

Multiple sequence alignment was performed for 95 IbCYP proteins using ClustalW. The results showed that all the IbCYP proteins have a highly conserved heme-binding region at the C-terminal end. The vast majority of IbCYP proteins (95.7%) exhibit the presence of all three conserved domains (Fig. 2), namely the K-helix region (ExxR), the PERF motif (PERF), and the heme-binding region (FxxGxRxCxG).

Fig. 2
figure 2

Conserved domains of sweet potato IbCYP proteins. Alignment of conservative motifs generated by the MEME online website for the 3 protein domains

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Phylogenetic analysis of IbCYP proteins

We constructed a phylogenetic tree containing 201 CYP450 genes from five species (I. batatas, A. thaliana, C. annuum, N. tabacum, and S. miltiorrhiza) using MEGA software. We divided all of the CYP450 genes into one of two major clades: A type, which contained the CYP71 family, and non-A type, which contained 4 clans (Fig. 3).

Fig. 3
figure 3

Phylogenetic Trees of CYP450 Proteins for Sweet Potato, Capsicum, Salvia miltiorrhiza, tobacco, and Arabidopsis. Arabidopsis CYP450 protein sequences were downloaded from the Cytochrome P450 Homepage website. A phylogenetic tree was constructed by the neighbor-joining method based on MEGA11 with 1000 bootstrap replicates. The tree was divided into 5 families represented by outer rings with different colors, black circles, white circles, black triangles, gray triangles, and white triangles representing the Arabidopsis, sweet potato, tobacco, capsicum, and Salvia miltiorrhiza CYP450 proteins

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Chromosomal locations and gene duplication analysis

Gene duplication is recognized as a prominent driver in the evolutionary process of genomes and genetic systems [59]. Two main types of gene duplication, namely tandem and segmental duplication, can give rise to numerous gene families [60]. Tandem duplication occurs when multiple members of a gene family are found within the same intergenic region or neighboring intergenic regions [61, 62]. On the other hand, segmental duplication involves the duplication of multiple genes through polyploidy events, often followed by chromosomal rearrangements [63].

After analyzing the chromosomal localization, we found that the 95 IbCYP genes were spread across 15 chromosomes (Fig. 4). Chromosomes 1 (LG1), 6 (LG6), and 13 (LG13) had the highest number of genes, with nine IbCYP genes each. Chromosomes 5 (LG5), 7 (LG7), and 8 (LG8) followed closely with eight IbCYP genes each. Chromosomes 2 (LG2) and 3 (LG3) contained seven IbCYP genes. In contrast, chromosomes 10 (LG10) and 12 (LG12) had the fewest genes, with only two and three genes, respectively. Additionally, we identified 15 pairs of tandemly duplicated genes among the IbCYP genes. These genes were located close to each other on the chromosomes and formed clusters on the phylogenetic tree, indicating similar functions. The expansion of the gene family was mainly attributed to tandem duplication and segment duplication, as shown by the presence of 13 duplicate gene pairs distributed on different chromosomes through MCScanX collinearity analysis (Fig. 5). This suggests that tandem duplication and segment duplication played a role in the expansion of CYP450 genes.

Fig. 4
figure 4

Locations of IbCYP genes on chromosomes. The basic unit indicated a chromosome length of 5.0 Mb. For each chromosome, the number is labeled on the upper side with red indicating a gene pair with tandem duplication

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Fig. 5
figure 5

Distribution and collinearity of the IbCYP gene family in the sweet potato genome. IbCYPs labeled with red had collinearity, while those labeled with black had no collinearity. The two rings in the middle represent the gene density of each chromosome. The gray background lines represent a collinear background and the red lines indicate a collinear relationship between IbCYP members

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Synteny analysis of IbCYP genes in sweet potato, pepper, tomato, rice, maize, and Arabidopsis

To better understand how the CYP450 family evolved in sweet potatoes compared to other species, we conducted an evolutionary relationship analysis of CYP450 genes. Specifically, we compared sweet potatoes with three dicotyledonous plants (Arabidopsis, tomato, and pepper) and two monocotyledonous plants (maize and rice) (Fig. 6). The analysis revealed that sweet potatoes shared 27 collinear genes with Arabidopsis, and 31, 26, 7, and 6 collinear genes with tomato, pepper, rice, and maize, respectively. These findings suggest that the IbCYPs in sweet potatoes have a close evolutionary relationship with the CYP450 genes in dicotyledonous plants, particularly with tomatoes and peppers from the Solanaceae family.

Fig. 6
figure 6

Collinearity analysis of CYP450 proteins in sweet potato among species. The species were Arabidopsis, tomato, pepper, maize, and rice. The red line represents the homologous CYP450 gene pair of the plant genome, and the gray line represents the collinear block of the plant genome

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Analysis of cis‑regulatory element distribution in IbCYP promoters

We extracted the genomic sequence of the IbCYP genes upstream region, specifically 2000 base pairs, to study their potential biological functions. This sequence was considered a hypothetical promoter sequence for cis-acting element analysis (Fig. 7). Our analysis revealed the presence of different types of cis-acting elements in the IbCYP gene family, which are associated with plant growth and development, hormone responses, and responses to abiotic stress. When examining the genes, it was found that 91 genes contained one to ten light response elements, 39 genes contained one to four auxin response elements, and 30 genes contained one to three gibberellin response elements.

Fig. 7
figure 7

Distribution of cis-acting elements of the IbCYP gene family in sweet potato. Distribution of cis-acting elements identified in the 2000 bp upstream promoter region of the sweet potato IbCYP gene

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Moreover, some genes contained cis-acting elements related to plant hormones and abiotic stress, such as MeJA-responsive elements, salicylic acid response elements, abscisic acid response elements, and elements responding to drought, hypoxia, and low temperature. Additionally, a few genes had cis-acting elements linked to plant secondary metabolism and growth development, involving zein metabolism regulation, flavonoid biosynthesis, endosperm and meristem expression, and phytochrome downregulation response elements. Two genes also contained wound-responsive elements. Overall, the promoter regions of these IbCYPs contained various types of cis-elements, indicating their potential involvement in diverse biological processes and regulatory pathways.

Transcript factors networks of IbCYP genes

Through analysis of potential transcription factors (TFs), it was found that a total of 687 TFs were identified in the IbCYP genes under salt stress, distributed among 56 different TF families, such as AP2/ERF-ERF, MYB, bHLH, NAC, WRKY, C2H2, bZIP, GRAS, and others (Fig. 8A). In the IbCYP genes under drought stress, a total of 478 TFs were identified, distributed among 48 different TF families, including AP2/ERF-ERF, MYB, NAC, bHLH, WRKY, C2H2, bZIP, HB-HD-ZIP, GRAS, LOB, and others (Fig. 8B). The analysis of TF quantity revealed that there are 346 common TFs shared between salt stress and drought stress (Fig. 8C).

Fig. 8
figure 8

The putative transcription factor regulatory network analysis of IbCYP genes. A Distribution of putative transcription factor regulatory networks of the IbCYP gene under salt stress. B Distribution of putative transcription factor regulatory networks of the IbCYP gene under drought stress. C Statistical analysis of the differences in putative transcription factor regulatory networks of the IbCYP gene under salt and drought stress

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Regulatory network in sweet potato

We used the STRING database to predict potential interactions among the IbCYP proteins (Fig. 9). There were 20 nodes in the IbCYP protein interaction network, each of which interacted with other nodes. Some proteins exhibited direct interactions, such as IbCYP706A2 and IbCYP94A2, whereas others exhibited more complex multigene interactions, such as IbCYP714E1 (Gas) and IbCYP79A1. Notably, IbCYP72A8 was predicted to be central nodes, radiating six connections to other genes.

Fig. 9
figure 9

rotein–protein interaction (PPI) network of significant genes in sweet potato. Nodes represent proteins, central nodes are indicated in red, and black lines indicate interactions between nodes. The darker the color, the more important the protein in the interaction network

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Expression patterns of IbCYP genes in sweet potato

We analyzed the expression patterns of the IbCYP genes in various plant tissues using transcriptome data. The expression levels were measured as fragments per kilobase of exon model per million mapped fragments (FPKM). Our findings indicate that 95 IbCYP genes showed significant differences in expression patterns across different tissues (Fig. 10). Specifically, three genes exhibited high expression levels in tuber roots, 13 genes in leaves, 14 genes in flowers, and 9 genes in fruits.

Fig. 10
figure 10

Expression heatmap of the IbCYP gene in different tissues of sweet potato. Red and blue indicate the intensity of genes in the heatmap: the more intense the red color is, the higher the gene expression level, while the more intense the blue color is, the lower the gene expression level

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We also examined the expression of IbCYP genes under salt and drought stress conditions in sweet potato tissues (Fig. 11). In tissues exposed to salt and drought stress, the expression levels of all 95 IbCYP genes showed significant differences. Specifically, under salt stress, 21 genes were upregulated and 28 genes were downregulated in root tissues. In stem tissues, 15 genes were upregulated and 11 genes were downregulated, while in leaf tissues, 5 genes were upregulated and 10 genes were downregulated. Under drought stress, 9 genes were upregulated 22 genes were downregulated in root tissues, 20 genes were upregulated and 4 genes were downregulated in stem tissues, 13 genes were upregulated and 11 genes were downregulated in leaf tissues (Table 2). These findings suggest that IbCYP genes have distinct expression patterns under salt and drought stress conditions. Overall, most of the genes responded to different stress conditions.

Fig. 11
figure 11

Expression heatmap of IbCYP in root, stem, and leaf tissues of sweet potato under salt and drought stress. Red and blue indicate the intensity of genes in the heatmap: the more intense the red color is, the higher the gene expression level, while the more intense the blue color is, the lower the gene expression level

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Table 2 The regulation of the IbCYP gene under salt and drought stresses
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The expression of 95 IbCYP genes was detected under high-temperature stress, and we focused on two specifically highly expressed genes, including IbCYP82G1 in “Ziluolan” fibrous roots and IbCYP78A1 in “Guangshu 87” roots (Fig. 12A). These two genes may be related to the heat tolerance of sweet potatoes. Similarly, the expression of 95 IbCYP genes was detected under cold stress (Fig. 12B). In “Shenshu 28”, after 3 h of cold stress, IbCYP82G1 and IbCYP82G3 were highly expressed, but after 24 h of cold stress, the expression levels of these two genes decreased, while IbCYP707A1 showed specific high expression. In “Liaohanshu 21”, after 3 h of cold stress, seven genes including IbCYP82G7 were highly expressed, but after 24 h of cold stress, the expression levels of these seven genes decreased, while IbCYP82D47 and IbCYP82G4 were highly expressed.

Fig. 12
figure 12

A Gene expression patterns of IbCYP genes under heat stress as determined by RNA-seq. F: fibrous roots; T: tuberous roots; Z: heat-sensitive “Ziluolan”; G: heat-tolerant “Guangshu 87”. B Gene expression patterns of IbCYP genes under cold stress as determined by RNA-seq. ss: clod-sensitive “Shenshu 28”, lhs: cold-tolerant “Liaohanshu 21”

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The expression profiles of 95 IbCYP genes were identified in three distinct tissues using the “Xushu 18” RNA-seq data obtained from the NCBI database (PRJNA511028) (Fig. 13). In fibrous roots, IbCYP736A3, IbCYP736A2, and IbCYP72A4 were highly expressed after ABA treatment, while IbCYP76G3 and IbCYP712A1 were highly expressed after MeJA treatment. In stems, IbCYP82G1 showed specific high expression after ABA treatment, IbCYP76C1 was highly expressed after SA treatment, and IbCYP82F1 showed specific high expression after MeJA treatment.

Fig. 13
figure 13

Expression analysis of IbCYP genes in fibrous roots (FR), stems and leaves of sweet potato under hormones treatment as determined by RNA-seq

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Quantitative analysis of IbCYP genes in different tissues

To confirm the accuracy of the transcriptome data, we selected 11 genes that showed significant expression differences and performed qRT-PCR analysis (Fig. 14). The results of the expression analysis of IbCYP genes in different parts of sweet potato were consistent with the transcriptome data. In general, the expression of these IbCYP genes was primarily detected in the pencil roots and leaves of sweet potatoes. Additionally, there were noticeable differences in the expression of these IbCYP genes among different parts. Notably, IbCYP82G2 exhibited the highest expression level in the tuber, while IbCYP82G7 showed the highest expression level in the primary root. This suggests that genes belonging to the same subfamily in the CYP450 family may have diverse functions.

Fig. 14
figure 14

Expression patterns of 11 IbCYP genes in different tissues. The x-axes represent different tissues including primary root, pencil root, tuber root, stem, leaf, and flower; the y-axes indicate the relative expression of IbCYP genes. The different letters of a, b, c, d, and e indicate significant differences at p < 0.05, as determined by one-way ANOVA with SPSS single-factor tests

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Quantitative analysis of IbCYP genes under abiotic stresses

To evaluate the expression of IbCYP genes in different tissues of sweet potato under various stress conditions, we utilized a technique called quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of sweet potato were examined after subjecting them to different durations of stress (Fig. 15). The results showed that exposure to salt and drought stress caused an increase in IbCYP gene expression in different parts of sweet potato. Specifically, under both stress conditions, most IbCYP genes in the primary root initially showed an increase in expression followed by a decrease, indicating a consistent pattern of expression. However, the expression of these IbCYP genes in stems showed the opposite trend. In the case of sweet potato leaves, a more complex pattern of expression was observed. For instance, IbCYP736A2 displayed a gradual decrease in expression under salt stress but showed an initial increase followed by a decrease under drought stress. Additionally, it is important to note that the highest expression level of IbCYP76C2 in roots was 3230 times higher than that in the control group after 16 h of salt stress, and 2844 times higher after 8 h of drought stress. Similarly, the highest expression level of IbCYP82G7 in roots was 242 times higher than that in the control group after 16 h of salt stress and 177 times higher after 8 h of drought stress.

Fig. 15
figure 15

Changes in the expression levels of 11 IbCYP genes in different tissues under salt and drought treatments. The different letters of a, b, c, and d indicate significant differences at p < 0.05, as determined by one-way ANOVA with SPSS single-factor tests. A Expression of 11 genes in primary roots under salt and drought stress. B Expression of 11 genes in stems under salt and drought stress. C Expression of 11 genes in leaves under salt and drought stress

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Discussion

Plant CYP450 genes are involved in the catalysis of multiple reactions, including growth, development, and secondary metabolite biosynthesis pathways [64,65,66,67]. With the development of gene sequencing technology, an increasing number of plant genomes have been deciphered, and the identification of the CYP450 family is expanding to more plants, such as tea tree, foxtail millet, citrus, and peanut, etc. [68,69,70,71,72]. Due to gene duplication and divergence events, higher plants possess a large number of CYP450 genes. In this study, we employed more rigorous criteria to identify CYP450 genes in sweet potatoes. Our methodology included local BLAST, HMMER, CDD, and conserved motif analysis. To enhance accuracy, we refined the coding sequence (CDS) and amino acid sequences of 95 IbCYP genes using existing transcriptome sequencing data from our laboratory. In terms of gene structure, sweet potato genes show a wide range of gene lengths and significant differences in the number of exons. This may be related to the abundant biological functions carried out by the CYP450 superfamily. The sweet potato genome encompasses a total of 95 CYP450 genes, providing a valuable genetic foundation for further investigations into their functions.

In sweet potatoes, the predicted 95 CYP450 genes are classified into 5 gene families, namely, the CYP71 family, CYP72 family, CYP85 family, CYP86 family, and CYP97 family, totaling 31 subfamilies. Among them, 60 (63.1%) genes belonging to the CYP71 clan were classified as type A, while the rest were classified as nontype A. In contrast, more members of the CYP450 gene family have been identified in other crops. For example, 326 CYP450 family members were identified in rice, 332 CYP450 genes in soybean, and 478 CYP450 genes in pepper [7, 42, 43]. This could be attributed to the fact that sweet potato’s two genomes are too similar. In the future, there may be additional discoveries of CYP450 family members in sweet potatoes. Conducting a collinearity analysis within a specific species provides insights into the homology of genes across different chromosomes. Through chromosome localization and collinearity analysis, it was confirmed that IbCYP, a specific gene, consists of 28 duplicated gene pairs. Among these pairs, 15 were classified as tandem duplications, while the remaining 13 were classified as segmental duplications. Thus, it is speculated that the expansion of the CYP450 gene family in the evolutionary process was primarily driven by segmental duplications, with tandem duplications playing a supplementary role. In addition, a collinearity analysis was performed among different species to explore gene evolution and genetic relationships. The evolutionary relationship between sweet potato and other species was examined based on family genes using a collinear analysis. The results revealed that the CYP450 family genes of sweet potato were more closely related to other Solanales plants, such as tomato and pepper. Specifically, 31 and 26 collinear gene pairs were identified in tomato and pepper, respectively. However, the genetic relationship between sweet potato and gramineous plants, such as maize and rice, was found to be less significant, as only a few collinear gene pairs were observed. These findings align with the results obtained from the genetic relationship analysis.

The cis-acting elements of promoters play a vital role in the regulation of gene expression. In this study, we verified that the IbCYP promoter region of sweet potato contained several elements related to the hormone regulation pathway. Among them, light responsiveness, auxin responsiveness, and gibberellin responsiveness were detected in most genes. Therefore, we inferred that light, gibberellin, and auxin may influence IbCYP gene expression, thereby affecting the growth and development of sweet potatoes. In Arabidopsis, CYP714A1 and CYP714A2 may function in the early stages of the GA biosynthetic pathway [73]. These drought-inducing elements were detected in 10 sweet potato genes. The results from the heatmap analysis revealed that IbCYP701A1 and IbCYP96A3 were highly expressed under drought stress. These findings indicated that IbCYP gene expression was regulated by cis-elements related to plant development and abiotic stress tolerance.

The expression pattern of genes reflects their functions to a certain extent. Therefore, in this study, we analyzed the expression patterns of the IbCYP genes. In sweet potatoes, the CYP71 clan exhibits specific expression in multiple tissues. For example, IbCYP71D5 is specifically expressed in primary roots; IbCYP71D8 is specifically expressed in stems; and CYP76C2 in Arabidopsis has been found to respond to leaf senescence and aging in cell cultures, which are related to cellular deterioration and eventual cell death [74]. Similarly, IbCYP76C2 was detected to exhibit leaf-specific expression in sweet potato. It is speculated that it may also play a similar role in sweet potato leaves; IbCYP77B1 is specifically expressed in the flowers, and IbCYP71D7 is specifically expressed in the fruits. The monogenic family CYP97 clan consists of two genes, namely IbCYP97A1 and IbCYP97B1. This is similar to the case of pepper (3 genes) [42]. These two genes are specifically expressed in the leaves and stems of sweet potato, indicating a potential relationship with the growth and development of sweet potato stems and leaves. IbCYP77A1 shows high expression in both flowers and fruits of sweet potatoes, which is similar to the role of CYP77A4 in Arabidopsis thaliana [75], where it is involved in the development of cotyledons. This suggests that IbCYP77A1 may play a role in the reproductive development of sweet potatoes. Under environmental stress, IbCYP genes also play a regulatory role. Usually, the root system is the first part to be affected by environmental stress. As the heatmap shows, the IbCYP genes were generally highly expressed in the primary root under different stresses. In sweet potatoes, a total of 34 IbCYP genes are upregulated in response to salt and drought stress. In response to cold and heat stress, IbCYP450 genes also show a response. IbCYP82G1, IbCYP82G3, and IbCYP707A1 exhibit different levels of response under cold stress. Under heat stress, both IbCYP82G1 and IbCYP78A1 are highly expressed, but the expression patterns of these two genes may vary among different varieties, possibly due to intervarietal differences. The IbCYP450 genes also show varying degrees of response to plant hormones, which corresponds to the presence of multiple plant hormone response elements in the promoter cis-acting elements of IbCYP450 genes. These findings have also been corroborated in foxtail millet. However, the response of CYP450 genes to low temperature, salt stress, and plant hormones differs between foxtail millet and sweet potato, which is likely due to genetic differences between the two species [68]. It has been reported that the CYP86 clan has a positive regulatory effect on the plant immune system [76]. CYP86 clan genes were expressed in the roots and leaves and were related to drought and salt tolerance.

Conclusion

We identified 95 IbCYP genes in the sweet potato genome, which were classified into 5 families and 31 subfamilies. Our evolutionary analysis of the CYP450 superfamily in sweet potato showed that the IbCYP genes have undergone frequent duplication and functional diversification. This will help us understand the complex agronomic traits and evolutionary processes of sweet potatoes. Additionally, we observed species- or family-specific expansions of the CYP450 superfamily, which may explain species divergence events. Expression analysis revealed the diversified expression patterns of CYP450 genes in sweet potatoes, which were expressed in different tissues, under various abiotic stress conditions, and in response to plant hormones. This indicates the functional diversity and regulation of CYP450 genes in sweet potatoes. The results of this study provide a solid foundation for further exploring the molecular evolution mechanisms and potential functions of the CYP450 gene family in sweet potatoes.