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. 2025 Feb 27;31(2):311–328. doi: 10.1007/s12298-025-01555-9

Genome-wide identification and expression analysis of CYP450s under various stress treatment in Dendrobium huoshanense

Guohui Li 1, Xingen Zhang 1, Yuyue Li 1, Xilu Zhang 1, Muhammad Aamir Manzoor 2, Chuanbo Sun 1, Cheng Song 1, Min Zhang 1,✉
PMCID: PMC11890713  PMID: 40066459

Abstract

The cytochrome P450 monooxygenases (CYP450) are the largest enzyme family in plant metabolism, playing a key role in the biosynthesis of both primary and secondary metabolites. However, the CYP450 has not yet been systematically characterized in Dendrobium species. In this study, 193 DhCYP450 genes were identified in the genome of Dendrobium huoshanense through bioinformatics, and divided into 2 groups and 10 clans. Chromosome localization results revealed that DhCYP450 genes are distributed across 19 chromosomes. We identified eight common conserved motifs within the DhCYP450 family of D. huoshanense. Furthermore, prediction analysis of cis-acting elements in the promoter region indicated the presence of elements responsive to low temperature, drought, and hormones responsive elements in most DhCYP450 genomes. Quantitative real-time PCR (qRT-PCR) analysis demonstrated the experiments expression patterns of DhCYP450 genes in response to cold, drought treatment, and hormones, suggesting their involvement in abiotic stress responses and their role in Dendrobium growth. Overall, these results provide valuable insights into the functional dynamics of the DhCYP450 genes and highlight potential candidates for further study of their biological roles.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-025-01555-9.

Keywords: Dendrobium huoshanense, Cytochrome P450, Gene family, Expression pattern, Abiotic stress

Introduction

The cytochrome P450 monooxygenases (CYP450s, CYPs) are a family of heme-containing enzymes found in animals, plants, bacteria, and fungi. These enzymes belong to the monooxygenase class and are primarily involved in catalyzing reactions in membrane organelles such as the endoplasmic reticulum, mitochondria, and Golgi apparatus (Nelson 2013). The reactions catalyzed by CYP450 are diverse and complex, including hydroxylation, epoxidation, oxidation, dealkylation, decarboxylation, C–C bond cleavage, and various reduction reactions (Rasool and Mohamed. 2016; Xu et al. 2015). The reactions catalyzed by CYP450s are extremely diverse, but they are usually involved on the activation and cleavage of molecular oxygen, so they are called monooxygenases. CYP450 enzymes typically have a common catalytic center. CYP450s have three highly conserved residues, namely the heme binding domain FxxGxRxCxG, the electron transfer channel residue PERF, and the EXXR residue in the K helix (Šrejber et al. 2018). Moreover, distinct substrate recognition characteristics of its members, different CYP450 members often lead to their involvement in catalyzing diverse plant metabolic reactions.

As one of the largest enzyme protein gene families, the CYP450 superfamily genes vary among different plants (Manikandan and Nagini 2018). Researchers have found that plants have 127 CYP450 families, and terrestrial plants are divided into 11 family clusters. Among them, CYP51, CYP74, CYP97, CYP710, CYP711, CYP727, and CYP746 are single family clusters, while CYP71, CYP72, CYP85, and CYP86 are multi family clusters (Mnguni et al. 2020). CYP51, CYP74, CYP97, CYP710, and CYP86 are conserved in green algae and entire terrestrial plants, performing important catalytic reactions in metabolism (Hansen et al. 2021). The other four families, CYP71, CYP72, CYP85, and CYP86 have evolved through dense gene replication and diversity. Among them, three families seem to be unique to the Brassicaceae family: CYP705 (belonging to the CYP71 family), CYP708, and CYP702 (belonging to the CYP85 family), which seem to have co evolved to form terpenoid related pathways (Nelson et al. 2008; Field and Osbourn 2008). In evolutionary history, the CYP71 family has experienced continuous gene duplication events, leading to family expansion. Therefore, members of the CYP450 superfamily, such as CYP71A, CYP71B, CYP705, and CYP81, repeat their tandem arrangement on different chromosomes.

CYP450 is widely involved in the biosynthesis and metabolism of secondary metabolites, such as terpenes, plant hormones, phenylpropanoids, alkaloids, phenolic compounds, fatty acids, sterols, etc. (Zheng et al. 2019). In recent years, increasing the production of secondary metabolites in plants has become a focus through research on CYP450 enzymes. The enzymes encoded by members of the CYP450 single family cluster are relatively more conserved, and their biological functions in plants are also relatively basic. In 2020, Nett found that GsCYP75A109, GsCYP75A110, and GsCYP71FB1 from Jialan can promote the synthesis of colchicine (Nett et al. 2020). The CYP73A, CYP98A, and CYP84A family in plants catalyze three benzene ring hydroxylation steps in the biosynthesis of lignin monomers (Wolf et al. 2022). Yasumoto et al. cloned genes CYP716A44, CYP716A46, and CYP716E26 from tomatoes and performed heterologous expression in yeast. They found that CYP716A44 and CYP716A46 can oxidize, respectively α-Aromatic resin alcohol and β- the C-28 position of aromatic resin alcohol produces ursolic acid and oleanolic acid, which are catalyzed by CYP716E26 β-C-6, a type of aromatic resin alcohol β Hydroxylase, producing mandrake terpene diol (Yasumoto et al. 2017). Huang et al. cloned the CYP716A155 gene, along with RoCPR01 and optim AtLUP genes, from rosemary and transferred them into brewing yeast. They found that CYP716A155 not only oxidizes α-Aromatic resin alcohol β-Aromatic resin alcohol generates ursolic acid and oleanolic acid and can more efficiently oxidize lupin alcohol to produce betulinic acid (Huang et al. 2019).

Plant hormones are trace organic compounds that have the ability to regulate plant growth, development, and defense (Huang et al. 2017; Takeuchi et al. 2021). Jasmonic acid isoleucine, the active form of plant hormone jasmonic acid, plays a critical role in plant hormone signaling. The level of jasmonic acid isoleucine receptor is regulated by CYP450 hydroxylases through catabolism, thereby influencing jasmonic acid metabolism (Griffiths 2020). Li et al. found that CYP450 enzyme can also act as a negative regulator in plant immune response. For example, overexpression of CYP71Z2 in rice inhibits auxin accumulation and enhances plant resistance to leaf blight disease (Li et al. 2015). Additionally, research shown that OsCYP714D1, derived from rice, is involved in the biosynthesis of gibberellins. Overexpression of OsCYP714D1 in poplar increases salt tolerance by regulating gibberellins biosynthesis homeostasis (Gao et al. 2021).

Dendrobium huoshanense is a perennial epiphytic plants in the Orchidaceae family recognized as a traditional Chinese medicine species under national protection (Wang et al. 2021). It is mainly planted in Anhui and Hubei provinces in China, with diverse planting patterns and significant differences in the quality of medicinal materials from different regions and planting modes, and the main chemical components of D. huoshanense include polysaccharides, flavonoids, dibenzyl, alkaloids, etc. Previous studies have found that significant differences in the traits, accumulation of active ingredients, and efficacy of D. huoshanense under different cultivation methods (Yi et al. 2021; Li et al. 2021), which is also diversity in the types and quantities of microorganisms in the roots and stems (Chen et al. 2020).

Currently, studies have been reported the presence of CYP450 family members in plants such as Arabidopsis, soybeans, rice, grapes, tomatoes, and ginseng (Wei and Chen 2018; Wang et al. 2018; Vasav and Barvkar 2019). However, no systematic research has yet been conducted on the CYP450 family members in D. huoshanense, and it remains unclear which CYP450s enzymes are involved in the biosynthesis of TIAs in D. huoshanense. To address this, we used the whole genome data of D. huoshanense to screen and identify the members of the DhCYP450 family, and to perform standardized nomenclature and phylogenetic analysis. Additionally, we predicted cis-acting elements in the promoter region and analyzed expression patterns under different hormone treatments, as well as under drought and low-temperature stress. The goal of this study is to elucidate the biological processes regulated by members of the DhCYP450 family in plant development regulation and stress responses.

Materials and methods

Plant material and stress treatments

Huoshan Dendrobium tissue culture seedlings were obtained from West Anhui University and used for total RNA extraction. The cultivation conditions, as well as drought and low-temperature treatment protocols for D. huoshanense were consistent with the methods described in our previous work (Li et al. 2024). For MeJA, ABA, and SA treatments, 100 μM of each compound was applied after filtration through a 0.22 μM filter. All samples were collected at 1, 12, 24, 48, and 72 h after treatment.

Identification of the DhP450 gene family

The protein sequences of Arabidopsis thaliana AtCYP90B1 from the NCBI website (Genebank: CAB62435.1) were downloaded for the analysis. The sequences were submitted then  to Pfam database (http://pfam.xfam.org/) to retrieve the amino acid sequence of the CYP450 conserved domain (PF00067). The amino acid sequence of this conserved domain were used as the query sequence in Hmmer 3.0 software and and alignment search (E-value = 0.001) were performed  against the D. huoshanense genome database, identifying candidate sequences for the DhCYP450 family member.

Subsequently, performed multiple sequence alignment duplicate sequences were removed using ClustalW in MEGA7.0 software. Further Pfam and SMART database were used (http://smart.embl-heidelberg.de/) to eliminate sequences lacking the CYP450 conserved domains, and ultimately identified the DhCYP450 family. The amino acid length (aa), molecular weight (kDa), and isoelectric point (pI) of the DhCYP450 proteins were analyzed using ExPASy ProtParam software (https://web.expasy.org/protparam/). Finally, the prediction of the subcellular localization of DhCYP450 proteins were done using the previously described method (Li et al. 2024).

Construction of phylogenetic tree of DhCYP450 family members

The CYP450s amino acid sequence of CYP450s used to construct the evolutionary tree were first aligned using MUSCLE software (default parameters). The Neighbor-Joining (N-J) method in MEGA X software was then applied to construct the NJ evolutionary tree (Tamura et al. 2013).

Conserved motif analysis of the DhCYP450 family

We used MEME's online software (http://meme-suite.org/tools/meme) to perform conserved motif analysis. A conserved motif logo was then generated using (http://weblogo.berkeley.edu/logo.cgi) Draw a conservative motif map.

Prediction of cis-acting elements in the DhCYP450 member promoter

The nucleotide sequence of 1500 bp upstream of the start codon of DhCYP450s was retrieved from the D. huoshanense genome database as the presumed promoter region. The PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict the types and numbers of cis-acting elements in promoters.

Chromosome location and gene collinearity analysis

We used MapChart software to perform chromosome localization analysis of the DhCYP450 gene family. Additionally, whole genome collinearity analysis and gene replication event analysis were conducted using McscanX software with default parameters (Wang et al. 2012).

RNA extraction and qRT-PCR analysis

Total RNA was rapidly extracted from Huoshan Dendrobium leaves using the plant Total RNA Isolation Kit, following the manufacturer’s instructions Biotechnology (Shanghai) Co., Ltd. The qRT-PCR experiments were performed using the LightCycler 96 (Roche). All the primers used in the experiments are listed in Table S1. Gene expression levels were calculated by the 2−△△CT method (Green and Sambrook 2018). The experiment was conducted with three biological and technical replicates.

Results

Identifcation, protein property and chromosomal distribution analysis of DhCYP450 gene family

In this study, a total of 193 DhCYP450s sequences were obtained and randomly assigned the names DhCYP4501 to DhCYP450193 (Table S2). These sequences ranged in length from 120 amino acids (DhCYP45025) to 2118 amino acids (DhCYP45082), with an average length of 512 amino acids. The molecular weights of the proteins ranged from 13.5 to 236.1 kDa, and their isoelectric points ranged from 4.74 to 9.79. Subcellular localization prediction revealed that the 193 DhCYP450 proteins were predominantly localized in the chloroplast, with a few located in the cytoplasm, nucleus, and vacuoles. Only one protein (DhCYP450134) was found to be localized in the plasma membrane.

To understand the distribution of DhCYP450s, we investigated the localization of DhCYP450s on the chromosomal (Fig. 1). The chromosomal localization results indicated that the 19 chromosomes of D. huoshanense contain CYP450 genes, through their distribution across chromosomes is uneven. Notably, the chromosomes 06 and 18 have the highest number of DhCYP450 genes, totaling 14, followed closely by chromosomes 10, 01, and 11, which contain 13, 12, and 11 DhCYP450 genes, respectively. However, chromosomes 05 and 07 have the fewest DhCYP450 genes, with only two genes on each chromosome.

Fig. 1.

Fig. 1

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Distribution map of 193 DhCYP450 on 19 chromosomes. The scale is in megabases (Mb). Chromosome numbers are displayed on the above of each chromosome, while gene IDs are shown on the right

Evolutionary and functional analysis of DhCYP450 gene family

To further investigate the classification and phylogenetic relationships of DhCYP450 family members, we constructed an NJ composite evolutionary tree by combining 193 DhCYP450s with 93 DoCYP450s sequences from Orchidaceae (Fig. S1). The phylogenetic tree analysis revealed that the DhCYP450 proteins were divided into two main types, including A-type CYP450 and non-A-type CYP450. Notably, the analysis revealed that all subfamilies include the CYP450 family genes from both D. huoshanense and Dendrobium officinale, indicating that the CYP450 family of these two species share a common ancestor. This phenomenon lays the foundation for future investigation into the evolutionary relationship between DhCYP450s.

We constructed an interspecific phylogenetic tree using 235 AtCYP450s reported in Arabidopsis, and predicted the potential function of DhCYP450 protein through phylogenetic clustering (Fig. 2). According to the universally recognized classification standards for CYP450s, we systematically categorized a total of 428 CYPs into 10 clans. The CYP71 clan is regarded a special cluster with the most members, further divided into 16 gene families. Previous research reported that CYP78 family (also known as CYP78A) contributes to regulating developmental processes, e.g. in seed growth (Fang et al. 2012), but we did not find any CYP450 members in this gene family in Dendrobium, suggesting DhCYP450 might not regulate development in seed growth. In addition to the members of the CYP78 family, CYP77 (also known as CYP77A6) also links metabolism to a specific developmental process (Kawade et al. 2018). Therefore, we speculate that the DhCYP450122 in this class may also have similar functions. In addition, overexpressed AtCYP85A2 promotes vegetative and reproductive growth and development of Arabidopsis thaliana (Yeon et al. 2022), and DhCYP450191, 192 and 193 are clustered into one class and presumably have similar functions.

Fig. 2.

Fig. 2

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Phylogenetic tree of CYP450 family members from D. huoshanense and A. thaliana using neighbor joining method through MEGA 7.0 with 1000 bootstrap. The tree clustered with 193 DhCYP450s and 235 AtCYP450s. Each clan denoted by distinct colors

Gene duplication relationship and collinearity analysis of DhCYP450s

The study of gene duplication events and amplification is crucial for understanding the evolution and expansion of the CYP450 gene family in dendrobium. We analyzed segment duplications across different chromosomes (Fig. 3). The gene duplication analysis revealed that 33 pairs of genes are involved in tandem duplication, while 21 pairs of genes are collinear, both of which contribute to the evolutionary dynamic of Dendrobium plants. Furthermore, the complex event systems were most frequently observed on chromosomes 6, 10 and 18, which may explain the high number of DhCYP450 genes on these chromosomes.

Fig. 3.

Fig. 3

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Synteny of DhCYP450 homologous genes. Collinear blocks across the genome are depicted in gray, while duplicated DhCYP450 gene pairs are linked with red curves

Conserved motif analysis of DhCYP450 proteins

To further explore the characteristics of Dendrobium CYP450 family, we analyzed distribution of conservative domains in DhCYP450 proteins (Fig. 4A). Sixteen motifs were identified through the MEME database. These motifs are numbered from 1 to 16, with conserved motifs commonly found in DhCYP450 protein corresponding to the sequences represented in logos 1 to 8 (Fig. 4B). Based on the structural properties of the conserved domains of CYP450s, we identified several key features: the heme-binding site is present in Motif 1 (89.62% of DhCYP450), the K helix is found in Motif 2 (90.32% of DhCYP450), and the I helix is present in Motif 3 (86.78% of DhCYP450). Additionally, the PERF motif is encapsulated in Motif 8 (82.28% of DhCYP450). Interestingly, the remaining motifs show varying degrees of conservation, and members sharing the same motif may exhibit similar biological functions.

Fig. 4.

Fig. 4

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Schematic diagram of conserved motifs of DhCYP450 family in D. huoshanense. (A) Distribution map of conserved motifs of DhCYP450 proteins. (B) The sequence logos for 8 frequently occurring motifs indicating conserved amino acid residues. Protein length estimation is facilitated by the scale at the bottom

Promoter cis-acting elements analysis of DhCYP450 genes

Plant cis-acting elements play a crucial role in the regulation of gene expression by influencing the transcription of nearby genes (Reilly et al. 2021). We conducted cis-acting element analysis on 1500 bp promoter region upstream of the start codon of DhCYP450s.The results revealed that the cis-acting elements in the DoCYP450s promoter region are involved in regulating various physiological processes including plant growth and development, stress response, hormone response, and transcription factor regulation (Figs. 5, S2). Among the cis-acting elements identified, those related to ethylene response and MeJA responses were more abundant, while fewer elements were associated with SA, and ABA responses. In addition, several stress-related were analyzed, including those involved in light response, low-temperature response, defense and stress, and heat response. The thermal response elements were present in a few genes, with a relatively small number. In addition, the DhCYP450s promoter region also contains a large number of elements that interact with MYB and MYC transcription factors. Furthermore, the DhCYP450 promoter regions contained numerous elements that interact with MYB and MYC transcription factors. Notably, most DhCYP450 promoter regions featured MBSI elements, which bind to MYB transcription factors and regulate flavonoid biosynthesis. These findings suggest that the DhCYP450 family in D. huoshanense plays a critical role in regulating growth, hormone signal transduction, and responses to environmental stress.

Fig. 5.

Fig. 5

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.The quantitative data and statistical enrichment analysis of cis-acting elements in 193 DhCYP450 upstream regions. As shown in the bar at the lower right corner, the quantitative data and statistical enrichment of cis-acting elements are expressed in different colors on the map

Expression patterns of DhCYP450s under drought and cold stress

To study the response of DhCYP450 gene family to drought and cold stress in Dendrobium. We have selected 12 candidate genes with high expression levels in Dendrobium leaves (Fig. S3) and analyzed their expression patterns in Dendrobium leaves (Figs. 6, 7). The results showed that the expression levels of most DhCYP450 genes significantly changed under drought stress compared to the control group (Figs. 6, S4). For example, the expression levels of DhCYP45016, and DhCYP450192 showed a sustained upward trend, reaching their maximum at 72 h, with increases of about 2.5 times, 6 times, respectively. However, DhCYP450160, and DhCYP450176 showed no differential expression compared to control on each time points. Additionally, DhCYP45064 and DhCYP45096 showed a trend of first increasing and then decreasing.

Fig. 6.

Fig. 6

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The expression profiling of 12 DhCYP450 genes under drought stress. The standard error (SE) represented by the error bars is based on three biological replicates. **Significant difference (P < 0.01), *significant difference at P < 0.05.

Fig. 7.

Fig. 7

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The expression profiling ofof 12 DhCYP450 genes under low-temperature stress (4 °C). The standard error (SE) represented by the error bars is based on three biological replicates. **Significant difference (P < 0.01), *significant difference at P < 0.05

Similar to the drought stress response, cold stress also induced specific expression patterns in the DhCYP450 genes. The expression levels of DhCYP45032, DhCYP45048, DhCYP45096 and DhCYP450128 showed a trend of first increasing and then decreasing, reaching their peak at 24 h, 48 h and 12 h, respectively. In contrast, the expression levels of DhCYP45016, DhCYP45064, DhCYP4050112 and DhCYP4050176 exhibited a sustained upward trend, increasing by approximately 4.3 times, 3.4 times, 8.2 times and 7.8 times compared to the control group, respectively. On the other hand, only DhCYP45080 showed a continuous downward trend (Figs. 7, S5). The qRT-PCR experiment results provide further support the involvement of the DhCYP450 gene in the response of Dendrobium to drought and cold stress.

Expression patterns of DhCYP450s under hormone stress

Next, we examined the differential expression of these 12 candidate genes under hormone treatment (MeJA, SA and ABA). Under MeJA treatment, the expression of DhCYP45032 was continuously downregulated throughout the treatment period, while the expression of DhCYP450128, DhCYP450144, and DhCYP450160 was upregulated (Figs. 8, S6). Beisdes, the expression levels of DhCYP45016, DhCYP45048, DhCYP45064, and DhCYP450112 first increased and then decreased. The expression peaks of DhCYP45096 and DhCYP40192 occurred at 24 h, with expression levels approximately 25-fold and 12.7-fold higher than at 0 h, respectively.

Fig. 8.

Fig. 8

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The expression profiling of 12 DhCYP450 genes under MeJA treatment. The standard error (SE) represented by the error bars is based on three biological replicates. **Significant difference (P < 0.01), *significant difference at P < 0.05

For ABA treatment, the expression profiling of 12 DhCYP450 genes increased to varying degrees (Figs. 9, S7). Among them, the expression levels of DhCYP450128 and DhCYP450192 continued to significantly increase, reaching their peak at 72 h. The expression levels were 2.7 and 30.1 times higher than those at 0 h, respectively. In the early stage of ABA treatment, the expression levels of DhCYP45016, DhCYP45096, and DhCYP450160 increased. However, it was found that the transcription levels of DhCYP45048 and DhCYP450122 were significantly inhibited. In addition, the expression levels of DhCYP45032, DhCYP45064, DhCYP450144, and DhCYP450176 were induced to varying extents, with DhCYP450176 showing a dramatic increased of approximately 48 times within 48 h.

Fig. 9.

Fig. 9

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The expression profiling of 12 DhCYP450 genes under ABA treatment. The standard error (SE) represented by the error bars is based on three biological replicates. **Significant difference (P < 0.01), *significant difference at P < 0.05

Under SA treatment, DhCYP45016, DhCYP45096, DhCYP450112, and DhCYP450192 were more significantly induced, exhibiting higher expression differences at each stage compared to the control group (Figs. 10, S8). Following SA treatment, the expression levels of DhCYP450144, DhCYP450160, and DhCYP450192 were initially upregulated, then decreased, and eventually increased. However, the expression levels of DhCYP45032 and DhCYP45080 were downregulated throughout the entire experimental period. Additionally, we observed that the expression levels DhCYP450128 and DhCYP45096 were downregulated throughout the experimental period. In summary, our experiments revealed distinct expression patterns of DhCYP450 gene in D. huoshanense under different stresses. We also speculate that the diversity of these expression patterns may be related to the functional of genes.

Fig. 10.

Fig. 10

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The expression profiling of 12 DhCYP450 genes under SA treatment. The standard error (SE) represented by the error bars is based on three biological replicates. **Significant difference (P < 0.01), *significant difference at P < 0.05

Discussion

The CYP450 family plays a crucial role in various biological processes, including in plant growth, development, and secondary metabolite biosynthesis (Vasav and Barvkar 2019; Zhang et al. 2023). As the largest enzyme family in plants, CYP450 family members exhibit diverse functions and have been identified across various plant genomes. However, there is currently limited understanding of how the DhCYP450 gene responds to biotic and abiotic stress, as well as its role in the growth and development of Dendrobium. The number of CYP450 genes identified in most plants ranges from 100 to 300. For example, the Arabidopsis contains 246 CYP450s (Nelson et al. 2004), maize (Zea mays) has 236 CYP450s (Li and Wei 2020), and Aquilaria agallocha has 136 CYP450s (Ankur et al. 2023). However, some plant genomes have been found to contain over 300 CYP450 genes, such as 356 CYP450s in rice (Oryza sativa) (Li and Wei 2020), and 331 CYP450s in millet (Foxtail millet) (Li et al. 2023). In this study, we identified a total of 193 members of the DhCYP450 gene family in the D. huoshanense genome, a relatively large number of DhCYP45 gene family in the D. huoshanense genome. The size of the CYP450 gene superfamily is often influenced by polyploid evolution. For example, heterohexaploid wheat has a particularly larger CYP450 family, with up to 1285 members (Li and Wei 2020). Interestingly, genome size does not seem to determine the quantity of CYP450. For example, cultivated peanuts have a genome size of 2.4 Gb and 589 AhCYP450s, whereas Arabidopsis has a much smaller genome (125 Mb), but contains 246 AtCYP450s. The diversity within the CYP450 family members includes variations in amino acid length, physicochemical properties, and other attributes, underscoring the functional diversity of the gene superfamily.

According to the gene family classification of D. officinale CYP450 genes, DhCYP450 genes were divided into 2 family clusters and 22 gene families (Fig. S1). The CYP71 family cluster contains the largest number of CYP450 gene members, which is consistent with previous research results that almost half of all P450 members in higher plants belong to the CYP71 family cluste (Nelson 2013). In addition, many plant-specific enzymes encoded by CYP450 genes are part of the CYP71 and are involved in the metabolism of secondary products. The CYP51, CYP74, CYP710 and other families were not identified in the CYP450 family of Dendrobium species, which may be related to gene deletions during evolution. In contrast, the increase in the number of DhCYP74 family members compared to DoCYP74 is likely due to gene duplication during evolutionary processes.

In this study, we identified 8 common conserved motifs in the DhCYP450 proteins using MEME online software (Fig. 3A). Among them, Motif 1 serves as a conserved heme binding domain (FxxGxRxCxG), and both this motif and Motif 3 (transport channel residue PERF) are characteristic motifs of CYP450 (Li et al. 2007), and these two conserved motifs also exist in almost every identified DhCYP450 proteins. The motif composition of different members of the DhCYP450 protein family varies greatly, while the motif composition of DhCYP450 proteins in the same family is often the same, but the arrangement of their motifs often varies among different subfamilies. This phenomenon suggested that DhCYP450 from the same family may have similar functions, and some conserved non-CYP450 motifs may play an important role in the functional specificity of each family.

The cis-acting elements in the promoter are crucial for gene expression and developmental regulation in plants (Xu et al. 2023). Previous studies have reported that many plant CYP450 gene promoters contain MYB and MYC transcription factor binding elements, which are recognition sites for the ACGT core sequence and TGA box (Rudolf et al. 2017; Shen and Li 2023). These sites are involved in regulating plant defense, and the response of each CYP450 gene to various stresses is strictly controlled (Zhang et al. 2021). In the current study, various cis-acting elements have been discovered in the DhCYP450s (Figs. 5, S2), including MBS, ARE, ABRE, TATC-box, and TCA-element, and these elements are related to hormone responses, low temperature, and drought stress. It is worth noting that almost all DhCYP450 members contain three or more cis-elements. Therefore, our analysis along with previous studies, indicates that the DhCYP450 gene is involved in transcriptional regulation of plant growth and stress response (Singh et al. 2021; Hansen et al. 2021; Li et al. 2023).

Analyzing gene expression patterns based on gene expression profiles is crucial for exploring gene functions. Previous studies have reported that plant CYP450s play important roles in various biochemical pathways and in various biological processes, including development and stress response (Xu et al. 2015; Bathe and Tissier 2019). For example, the biosynthesis and catabolism of brassinolides (BRs) required the involvement of CYP450s, including CYP500A, CYP90B, CYP90C, CYP90D (Ohnishi et al. 2012). Interestingly, AtCYP703A2, AtCYP86C3, OsCYP704B2, and OsCYP703A3 catalyzed the hydroxylation of mid-chain and long-chain fatty acids during xenobiotic pollen synthesis (Xu et al. 2014). Similarly, the ABA hydroxylase genes CYP707A1, CYP94C1, and CYP94B3 in tobacco are signifcantly upregulated under drought stress, indicating the likely roles of these genes in drought stress tolerance (Rabara et al. 2015).

Furthermore, previous studies have shown that the expression of plant CYP450 genes undergoes varying degrees of changes under drought, low temperature, and other hormone effects (Li et al. 2023; Shen and Li 2023). However, there have been no reports on the response of DhCYP450 genes to drought, low temperature, and hormones in plants. To elucidate the differential expression patterns of CYP450 genes in Dendrobium under different stresses, we conducted qRT-PCR experiments under drought, low temperature, and different hormone treatments (MeJA, ABA and SA) (Figs. 6, 7, 8, 9, 10). We found that drought, low temperature, and different hormone treatments can induce partial expression of DhCYP450, significantly upregulating its transcription level. In Changchun flowers, CrG10H is also induced by MeJA and ABA and positively regulates the biosynthesis of TIAs in Changchun flowers (Wang et al. 2010; Wei 2010). In addition, CtCYP82G24 OE lines demonstrated an increased levels of anthocyanins and favonoids accumulation at 24 h of MeJA treatment in Carthamus tinctorius (Wang et al. 2023). Therefore, we speculate that CYP450 protein may play an important role in the MeJA hormone regulatory network in JA signal transduction, regulating the expression of downstream genes of JA by binding to transcription factors, ultimately promoting the accumulation of plant metabolites.

Although there have been no reports on the expression regulation of G10H by SA, most of the DhCYP450 promoter regions have identified SA responsive cis-acting element TCA element, and experiments have shown that SA can significantly induce transcription of DhCYP450160, DhCYP450176, and DhCYP450192. These findings suggest that different CYP450 genes in Dendrobium may have different functions in various biological processes, including biotic and abiotic stress responses as well as hormone signaling pathways. In the future, further studies are imperative to elucidate the precise regulatory inffuence of these hormones on CYP450 transcription levels in D. huoshanense. In addition, we also hope to explore the effect of exogenous hormones on the regulation of Dendrobium CYP450 expression levels, in order to determine whether the growth and development of Dendrobium can be altered by regulating exogenous hormones.

Conclusions

This study comprehensively analyzed the CYP450 gene superfamily in Dendrobium huoshanense and identified a total of 193 DhCYP450s. We examined the distribution, classification, evolution, and amplification of the DhCYP450 superfamily through phylogenetic analysis, chromosome localization, conserved domains, promoter features, and collinearity analysis. Additionally, our qRT-PCR analysis of 12 DhCYP450 genes from different subfamilies confirmed that the DhCYP450 genome is widely involved in the plant’s biotic and abiotic stress, including drought, low temperature, and varying concentrations of hormones. In summary, our findings provide valuable insights into the function of CYP450 genes in Dendrobium. These results ofer novel insights into the molecular mechanisms that underlie stress responses in Dendrobium plants and could have practical implications for breeding stress-tolerant Dendrobium cultivars.

Supplementary Information

Below is the link to the electronic supplementary material.

Fig. S1 (4.7MB, jpg)

Phylogenetic tree of CYP450 family members from D. huoshanense and D. officinale using neighbor joining method through MEGA 7.0 with 1000 bootstrap. The tree clustered with 193 DhCYP450s and 93 DoCYP450s. Each clan denoted by distinct colors. (JPG 4819 KB)

Fig. S2 (252.5KB, jpg)

Pie charts of different sizes indicated the ratio of each function in each category, respectively. (JPG 252 KB)

Fig. S3 (615.5KB, jpg)

The expression profiling of DhCYP450 genes in the leaves of D. huoshanense. Randomly select 24 DhCYP450 genes from 22 subfamilies. The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 615 KB)

Fig. S4 (1MB, jpg)

The expression profiling of 12 DhCYP450 genes under drought stress. The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 1027 KB)

Fig. S5 (1MB, jpg)

The expression profiling of 12 DhCYP450 genes under low-temperature stress (4 °C). The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 1025 KB)

Fig. S6 (1,022KB, jpg)

The expression profiling of 12 DhCYP450 genes under MeJA treatment. The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 1022 KB)

Fig. S7 (1MB, jpg)

The expression profiling of 12 DhCYP450 genes under ABA treatment. The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 1036 KB)

Fig. S8 (1,022.1KB, jpg)

The expression profiling of 12 DhCYP450 genes under SA treatment. The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 1022 KB)

Supplementary file9 (DOC 26 KB) (26KB, doc)
Supplementary file10 (DOC 486 KB) (486.1KB, doc)

Funding

Anhui Provincial University Research Projects (Grand Number 2023AH052637), Horizontal Project—Development and Industrialization of Huangjing Series Noodles (Grand Number 0045022037), Innovation and Entrepreneurship Training Program for College Students of West Anhui University (S202410376078).

Declarations

Conflict of interest

No potential conflict of interest was reported by the authors.

Footnotes

Publisher's Note

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 (4.7MB, jpg)

Phylogenetic tree of CYP450 family members from D. huoshanense and D. officinale using neighbor joining method through MEGA 7.0 with 1000 bootstrap. The tree clustered with 193 DhCYP450s and 93 DoCYP450s. Each clan denoted by distinct colors. (JPG 4819 KB)

Fig. S2 (252.5KB, jpg)

Pie charts of different sizes indicated the ratio of each function in each category, respectively. (JPG 252 KB)

Fig. S3 (615.5KB, jpg)

The expression profiling of DhCYP450 genes in the leaves of D. huoshanense. Randomly select 24 DhCYP450 genes from 22 subfamilies. The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 615 KB)

Fig. S4 (1MB, jpg)

The expression profiling of 12 DhCYP450 genes under drought stress. The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 1027 KB)

Fig. S5 (1MB, jpg)

The expression profiling of 12 DhCYP450 genes under low-temperature stress (4 °C). The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 1025 KB)

Fig. S6 (1,022KB, jpg)

The expression profiling of 12 DhCYP450 genes under MeJA treatment. The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 1022 KB)

Fig. S7 (1MB, jpg)

The expression profiling of 12 DhCYP450 genes under ABA treatment. The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 1036 KB)

Fig. S8 (1,022.1KB, jpg)

The expression profiling of 12 DhCYP450 genes under SA treatment. The heatmap was generated by TBtools software according to the RNA-seq database. Log2-based-fold changes were used to create a heatmap. As shown in the bar at the lower right corner, the gene transcription level is expressed in different colors on the map. (JPG 1022 KB)

Supplementary file9 (DOC 26 KB) (26KB, doc)
Supplementary file10 (DOC 486 KB) (486.1KB, doc)

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