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. 2026 Jan 22;27:77. doi: 10.1186/s12864-025-12295-1

Identification and characterization of the class III peroxidase gene family in sea buckthorn (Hippophae rhamnoides subsp. sinensis Rousi) and its potential role in lignin biosynthesis

Jing Zhao 1,✉,#, Kai Li 2,#, Meng Zhao 1, Xuan Jiang 1, Xing Liu 1, Hongli Wang 3, Kun Sun 1,✉
PMCID: PMC12828949  PMID: 41572154

Abstract

Background

Class Ⅲ peroxidases (PODs) are widely involved in multiple physiological processes, including lignin biosynthesis and stress responses. However, there are no reports on the identification and function of POD family in Hippophae rhamnoides subsp. sinensis Rousi.

Results

In this study, we identified 71 non-redundant HrPODs. Based on phylogenetic analysis, these genes were classified into 7 subgroups. Then, we analyzed the conserved domains of HrPODs proteins and found that they contain highly conserved domain. We also investigated their expression patterns in three developmental stages of sea buckthorn fruit, and the results showed that most of them were highly expressed in early stage. The GO and KEGG functional enrichment analysis showed that they widely involved in oxidative stress response and phenylpropanoid biosynthesis, respectively. According to the Pearson correlation analysis of HrPODs expression with lignin content and peroxidase activity, we further screened five HrPODs that may be involved in fruit texture quality by regulating lignin biosynthesis during fruit development. Sea buckthorn is usually distributed in arid areas with higher salt and alkaline content, and our findings showed that the different degrees of drought, salt, or alkali treatment can also affect the expression of these genes and lignin content during the germination stage. These results will help us better investigate the role of HrPODs in lignin biosynthesis to provide certain theoretical foundation in ideal berry breeding and broad spectrum stress resistance in cultivated sea buckthorn.

Conclusion

In this study, the findings not only provide us with a comprehensive identification and analysis of HrPODs from a bioinformatics perspective, but also preliminary intimation that the POD family may be tightly associated with fruit texture quality and abiotic stress response. These results will help us better investigate the role of HrPODs in lingin biosynthesis, enrich the theoretical research on the formation of sea buckthorn texture quality and understand the roles of HrPODs in abiotic stress resistance in cultivated sea buckthorn.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-025-12295-1.

Keywords: Sea buckthorn, Class III peroxidases, Phylogenetic analysis, Expression pattern, Functional enrichment analysis, Lignin content, Abiotic stress response

Background

Peroxidases (EC number 1.11.1.x) are a large group of antioxidant enzymes, which extensively exist in animals, plants and microorganisms, and catalyze the oxidation of assorted substrates by reducing hydrogen peroxide [1]. Based on whether their protein structure contains heme, they have been classified into heme peroxidases and non-heme peroxidases [2, 3]. The latter can be further divided into class Ⅰ, Ⅱ and Ⅲ peroxidases [4, 5]. Among them, class Ⅲ peroxidases (EC number 1.11.1.7), as classical secretory plant peroxidases, was abbreviated as Prxs [6], PRXs [7], and PODs [8], and broadly presented in land plant [9]. Recently, many studies have chosen to use PODs as the abbreviation for class III peroxides [10–13]. In this study, we also prefer to use PODs as the abbreviation. Based on possessing the specialized function and catalytic property, they have been reported to participate in a wide range of physiological processes throughout the entire plant life cycle [14], such as fruit development [15], response to stresses [16, 17], seed germination [18], especially, cell wall lignification [19]. Lignification typically occurs during normal growth and stress responses [20, 21]. Lignin is the main components of the secondary cell wall and serves as the material basis for mechanical support and defense [22]. According to existing research findings, lignin could can be subdivided into four monomer types, the latter being the final products of phenylpropanoid biosynthesis pathway [23]. In this pathway, PODs, laccases and hydrogen peroxide directly participate in lignin biosynthesis together [24].

Numerous studies have shown that POD genes could regulate fruit quality by participating in lignin biosynthesis, especially fruit texture. Lignin plays multiple roles in the formation of fruit quality, and moderate lignification can reduce damage and enhance storage by promoting fruit texture [25]. Excessive lignification can lead to an increase in stone cells, significantly influencing fruit texture and consumer acceptance [26]. Under GA treatment, the expression of PpPOD1 in the pear was lower than that in the control, which was highly consistent with the changes in lignin content [27]. Four PbPRXs participate in lignin monomer synthesis, which promotes lignification of stone cells and influences the quality of pears [28]. Lignin deposition mediated by LAC/PRX leads to skin browning during the dehydration process of longan [29]. CgPRX24/41/65 plays a key role in the lignification of granular juice sacs during citrus fruit senescence [30]. Overexpression of FaPRX27 leads to the conversion of anthocyanins into lignin in strawberry fruits and promotes lignin deposition [31]. The “PuNAC21-PuDof2.5-PuPRX42 like/PuCCoAOMT1” module can effectively inhibit the development of stone cells in pear fruit under calcium ion induction, which may help improve pear fruit quality [32]. ZjbZIP33-ZjPRX1 positively regulates the formation of lignin in jujube seeds and can be an important candidate gene for optimizing stone fruit breeding [33].

What’s more, the expression of POD genes and lignin biosynthesis also affect the plant’s response to stress. ZlPOD3 was up-regulated and the lignin significantly accumulated in the epidermis of the water bamboo shoot during cold storage [34]. Pichia galeiformis could trigger phenylpropanoid biosynthesis, promote the CsPODs expression, and increase the lignin content in postharvest citrus [35]. PRX17 directly targets AGL15 and regulates lignified tissue formation in Arabidopsis thaliana [36]. AtPrx72 plays an important role in lignin biosynthesis [37]. Overexpression of the ZmPRX1 increases maize seedling drought tolerance and redound root lignin accumulation [18]. Kim et al. found overexpression of IbPrx04 in tobacco could increase the hydrogen peroxide production and lignin content [38]. The down regulation of prxA3a expression in transgenic aspen altered lignin content [39]. OsPRX38 overexpression in A. thaliana could reduce arsenic accumulation due to root cell apoplastic lignification [40]. Liu et al. found OsPODs negatively regulated Cd uptake and accumulation, and promoted the lignin formation [41]. The ZjMYB44-ZjPOD51 module enhances the defense response of jujube to phytoplasma by increasing lignin content [42]. AtPRX62 and AtPRX69 could promote root hair growth at low temperature [43].

Sea buckthorn (Hippophae rhamnoides subsp. sinensis Rousi) is considered to be a kind of tree with great ecological benefits, and usually distributed in arid areas with higher salt and alkaline content. These stresses exists throughout the entire process from seed germination to fruit ripening [44–46]. Its mature fruit is rich in antioxidant components and has the quality characteristics of soft texture and delicate taste [47, 48]. According to the above research, the fruit quality are often closely related to PODs expression and lignification of the cell wall [25–33]. Meanwhile, PODs have be widely reported to participate in responding to plant stress by regulating the lignin content [34–43]. However, there are relatively few reports on the members of this gene family in sea buckthorn, which greatly limits the research on fruit quality and stress response based on PODs-lignin modules. With the rapid development of genome sequencing technology, the POD family members have been extensively identified and studied in numerous plant species, and their numbers exist significant difference. For example, 22 PODs in Dicranum scoparium [12], 47 in Vitis vinifera [49], 73 in A. thaliana [6], 90 in Betula pendula [10], 119 in Zea mays [50], 124 in Glycine max [51], 210 in Nicotiana tabacum [11]. Recently, the sea buckthorn genome has been sequenced, assembled and released [52], providing the possibility for identifying and analyzing this gene family. Therefore, we used bioinformatics methods to identify the HrPOD family members, screen potentially pivotal candidate HrPODs involved in lignin biosynthesis in sea buckthorn fruit, and further preliminary exploration into whether these genes are involved in stress response. This research not only lays the foundation for the functional study of HrPODs, but also help us further understand multiple roles of HrPODs in fruit texture quality and abiotic stress resistance.

Materials and methods

Plant materials, growth conditions and treatments

Based on the previous sampling standards [53], the three stages (30, 70, 120 days after anthesis, DAA) of Hippophae rhamnoides subsp. sinensis Rousi fruit samples, namely, Hrh_S2, Hrh_S4, Hrh_S6, were collected from a sea buckthorn natural hybrid zone located in Qilian County, Qinghai Province, China (38°15′ N latitude, 100°16′ E longitude). The voucher specimen is deposited in Herbarium of Northwest Normal University (NWTC), Lanzhou, China under the voucher number NWTC-HR- 2209 (contact: Jing Zhao, zhaojing@nwnu.edu.cn). The sampled fruits were identified as Hippophae rhamnoides subsp. sinensis Rousi by morphological comparison with published keys and the Flora of China. The voucher specimen has been authenticated by Prof. Kun Sun of Northwest Normal University. The fruit samples were frozen in liquid nitrogen, and then placed in the ultra-low-temperature refrigerator for further use, including transcriptome sequencing, determination of lignin content and peroxidase activity and quantitative real-time PCR analysis.

Then, we further preliminary exploration into whether the candidate HrPODs are involved in stress response. Due to the fact that wild sea buckthorn is often distributed in arid areas with higher salt and alkaline content, stresses exists throughout the entire process from seed germination to fruit ripening, and it is generally believed that these stress has a significant impact on the seed germination stage [46, 54], so we preferred to conduct a series of germination experiments treated with different concentration of PEG6000 (0, 5, 10, 15, 20%), NaCl (0, 0.05, 0.10, 0.15, 0.20 mol/L) or NaHCO3 (0, 0.005, 0.010, 0.015, 0.020 mol/L) solution, respectively, to assess the effects of different degrees of drought, salt and alkali stress on the expression of these HrPODs. The specific steps of seed germination experiment are as follows:

The seeds were naturally dried and fully sterilized. Every 20 seeds were placed in a petri dish with 2 layers of filter paper. Subsequently, different solution or distilled water were separately put into petri dishes, and ensured that the seeds were submerged in the solution. Then, the petri dishes were placed in a growth chamber at 25 °C, with 16 h of light/8 h of darkness cycle. The filter papers and different solution were renewed every two days, and all experiments lasted for 7 days. Ultimately, the germination rate of seeds was recorded, and all seeding samples were packaged and frozen in liquid nitrogen, and then placed in the ultra-low-temperature refrigerator for further analysis of HrPODs expression, determination of lignin content and peroxidase activity and quantitative real-time PCR analysis.

Identification and physicochemical properties analysis of HrPODs

The genome assembly information of sea buckthorn was downloaded from China National Gene Bank Database (https://db.cngb.org/search/project/CNP0001846/). Firstly, 73 AtPrx protein sequences were obtained from the TAIR database (https://www.arabidopsis.org/) and used as query sequences to search for potential HrPOD protein numbers in sea buckthorn genome database using local BLASTP (E-value ≤ 10− 5). Then, the Hidden Markov Model (HMM) profile of the POD (PF00141) was downloaded from the PFAM database (http://pfam.xfam.org/), and used as a query to search against for potential HrPODs in sea buckthorn genome database using HMMER 3.0 (E-value ≤ 10− 5) [55]. Finally, the candidate HrPOD protein sequences were subjected to NCBI Batch CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and SMART (http://smart.embl-heidelberg.de/) to further exclude incomplete sequences and confirm eventually identified HrPODs [56, 57]. The chromosomal location information of HrPODs was extracted from the GFF3 file and visualized using the MG2C website (http://mg2c.iask.in/mg2c_v2.1/index.html). Moreover, we used expasy online tool (https://web.expasy.org/protparam) to evaluate physicochemical properties with the default parameters. The subcellular localization was predicted by using the online website WoLF-PSORT (https://www.genscript.com/wolf-psort.html).

Phylogenetic analysis of HrPODs

All PODs from two species (sea buckthorn and A. thaliana) were aligned by using the MUSCLE program in MEGA software (version 7.0) [58], and all parameters were set to default values. Subsequently, the multiple alignment results were imported into the software to construct an phylogenetic tree using the neighbor-joining (NJ) method with 1000 bootstrap replications, and the phylogenetic tree was visualized by using the iTOL online tool (https://itol.embl.de).

Conserved domain, motif and gene structure analysis of HrPODs

The conserved domain of HrPODs was identified with help of NCBI Batch CDD website (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi), and the conserved motifs of HrPODs were identified by the using the online tool MEME (https://meme-suite.org/meme/tools/meme). Then, the gene structure file of HrPODs was extracted by using the software TBtools [59]. Subsequently, the conserved domains, motifs, and gene structure were integrated and visualized using the software TBtools [59].

Cis-acting element analysis of HrPODs

The 2000 bp sequence upstream of the initiation codon of HrPODs were extracted from genome information using the TBtools software [59], and the cis-acting elements of these HrPODs were analysised using the online tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Then, the results were visualized using the TBtools software [59].

Gene duplication, selection pressure and collinearity analysis of HrPODs

All HrPODs were mapped to the chromosomes of sea buckthorn using “Basic Circos” program of the Tbtools software with the default parameters. Then, the duplication pairs of HrPODs were identified using the “One Step MCScanX” program of the Tbtools software with the default parameters [59]. The selection pressure analysis of HrPODs was represented by the Ka/Ks ratio, and the KaKs_Calculator software was used to calculate the Ka/Ks ratio [60, 61]. The collinearity of HrPODs and AtPrxs was conducted using the “One Step MCScanX” program of the TBtools software with the default parameters [59].

Differential expression and functional enrichment analysis of HrPODs

The three developmental stages of sea buckthorn fruit samples were used for transcriptome analysis by Novogene Bioinformatics Technology Co., Ltd, and the expression patterns of these HrPODs were displayed the form of heatmap using Tbtools software with the default parameters [59]. The Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) functional enrichment analysis were conducted and visualized by using the online tool NovoMagic (https://magic.novogene.com/) and Origin 2021 software, respectively.

Correlation analysis of HrPODs with lignin content and peroxidase activity

Lignin content of the three stages of fruit samples was determined by a lignin content test kit (Beijing Solarbio, China, No. BC4200) according to the provided protocol. POD activity was measured using the guaiacol method [62]. Each sample had three biological replicates. Pearson’s correlation analysis of expression expression levels of HrPODs with lignin content and peroxidase enzyme activity was analyzed and visualized using the online tool OmicShare (https://www.genedenovo.com/). The correlation analysis between qRT-PCR result and RNA seq data was also analyzed and visualized using this online tool.

Quantitative real-time PCR (qRT-PCR) analysis

Total RNA was extracted from the fruit and seeding samples by using TIANGEN Polysaccharide Polyphenol Total RNA Extraction Kit (DP441, TIANGEN, China), and cDNA was generated using Takara PrimeScript RT Reagent Kit with gDNA Eraser (Code No. RR047A, Takara, Dalian). The five candidate HrPODs were selected and the primers were designed by the online tool Meinverse (https://meinverse.cn/), and the Hr18S1 was selected to serve as the internal reference gene. The detailed information of their primers was listed in Table S1. Each HrPOD has three biological replicates. qRT-PCR was performed on an QuantStudio 1 Plus (Thermo Fisher Scientific, USA), and the relative expression of these HrPODs was calculated by using 2−△△Ct [63].

Statistical analysis

The data were analyzed with SPSS 27.0 software (SPSS, Inc., Chicago, IL, USA) and represented as mean ± standard error (SE) of at least three measurements. One-way analysis of variance (ANOVA) and Duncan’s method were used to analyze the significance of differences between different samples, with the significance level set at 0.05 (P < 0.05). Different lowercase letters indicate significant differences.

Results

Identification and physicochemical analysis of HrPODs

In this study, a total of 71 non-redundant HrPOD family members were identified from sea buckthorn genomic information by BLASTP, HMMER, NCBI Batch CD and SMART analysis. For the convenience of subsequent analysis, these HrPODs were renamed as HrPOD1-HrPOD71 based on their physical position on the chromosome. Based on the genomic information of sea buckthorn, these HrPODs were unevenly distributed on sea buckthorn chromosomes 1 to 12. Notably, Chromosomes 1–8, and 11 contained more than three genes, whereas chromosomes 9, 10, and 12 contained only two genes (Fig.S1).

Subsequently, we predicted the physicochemical properties of these HrPODs, including number of amino acids, molecular weight, isoelectric point, instability index, aliphatic index and grand average of hydropathicity. The number of amino acids of HrPODs varied from 246 (HrPOD6) to 641 (HrPOD55), with an average number of 334.1. The molecular weights of HrPODs ranged from 27.87 kDa (HrPOD6) to 69.05 kDa (HrPOD55). The isoelectric points of HrPODs varied from 4.31 (HrPOD42) to 9.61 (HrPOD61). The instability index of HrPODs ranged from 22.2 (HrPOD43) to 62.6 (HrPOD46). The aliphatic index of HrPODs varied from 73.17 (HrPOD50) to 100.7 (HrPOD16). The grand average of hydropathicity of HrPODs varied from − 0.578 (HrPOD1) to 0.124 (HrPOD2). The detailed information of physicochemical properties prediction and sequence of these HrPODs was listed in Table S2. Meanwhile, we also predicted the subcellular localization and secondary structure characteristics of these HrPODs, and the detailed information was listed in Table S3.

Phylogenetic analysis of HrPODs

For further investigate the evolutionary relationships of HrPODs, we constructed an un-rooted neighbor-joining tree with the amino acid sequences of 71 HrPODs and 73 AtPrxs by using MEGA software. Based on the homology of AtPrxs, the phylogenetic tree was classified into 7 subgroups (Gr1-Gr7) in this study (Fig. 1) [6]. AtPrx27-HrPOD44 were grouped into subgroup 1 (Gr1); HrPOD21-AtPrx62 were grouped into subgroup 2 (Gr2); AtPrx47-HrPOD9 were grouped into subgroup 3 (Gr3); AtPrx17-HrPOD5 were grouped into subgroup 4 (Gr4); HrPOD25-HrPOD29 were grouped into subgroup 5 (Gr5); HrPOD14 and HrPOD63 were grouped with AtPrx12 and named subgroup 6 (Gr6); HrPOD46 and AtPrx48 were located on the same branch and named subgroup 7 (Gr7). The detailed information was listed in Table S4. We also constructed an un-rooted neighbor-joining tree with only 71 HrPODs, and the phylogenetic tree was also divided into 7 subgroups (Gr1-Gr7) (Fig.S2).

Fig. 1.

Fig. 1

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Phylogenetic tree and classification of the PODs between Hippophae rhamnoides subsp. sinensis Rousi and Arabidopsis thaliana. The POD protein sequences of the two species were used as input sequences, and the phylogenetic tree was constructed with the neighbor-joining method with 1000 bootstrap replicates using MEGA 7.0 software. The classification of these HrPODs was based on their homology with A. thaliana, with different groups marked with different colors

Conserved domain, motif and gene structure analysis of HrPODs

In this study, we collectively analyzed the conserved domain of 71 HrPOD proteins, and the results showed that all HrPODs contained a secretory-peroxidase domain (Fig. 2A). Meanwhile, a total of 10 conserved motifs (named motif 1–10) were identified and the length of these motifs ranged from 15 (motif 5, 7, 8, 9, 10) to 41 (motif 3). Most HrPODs contained 10 conserved motifs, except for ten HrPODs. Among them, HrPOD55 contained more than 10 motifs; HrPOD20 possessed two motif 8, but lacked motif 7; HrPOD4, HrPOD57, HrPOD30, HrPOD68, HrPOD23 lacked motif 7; HrPOD17 lacked motif 4; HrPOD21 lacked motif 8; HrPOD51 lacked motif 8, 2 and 9 (Fig. 2B). The detailed information of motifs was listed in Table S5.

Fig. 2.

Fig. 2

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Conserved structure, motif and gene structure analyses of 71 HrPODs. Conserved structure analysis of these HrPOD proteins, and all HrPODs contained the filled yellow box that represented secretory-peroxidase domain (A). Conserved motif analysis of HrPODs, and the majority of PODs contained the same type and quantity of motifs, with only a few PODs having significantly different types and quantities of motifs. The box filled with different colors represented different motif (B). Gene structure analysis of HrPODs, and the gene structure presented exhibits diversity due to differences in the distribution and quantity of introns and exons (C)

At the same time, the structure diversity of HrPODs showed that the number of exons varied from one to eight, and the number of introns varied from zero to six. Among them, 41 HrPODs contained four exons and three introns; 12 HrPODs contained three exons and two introns; 9 HrPODs contained two exons and one intron; 4 HrPODs (HrPOD29, HrPOD64, HrPOD24, HrPOD21) only contained one exon; 4 HrPODs (HrPOD1, HrPOD17, HrPOD45, HrPOD65) contained five exons and four introns; 1 HrPOD (HrPOD55) only contained eight exons and six introns (Fig. 2C).

Cis-acting element analysis of HrPODs

The promoter region of HrPODs contained many cis-acting elements, which could be categorized into four types: abiotic/biotic stress responsiveness, plant growth and development, phytohormone responsive and light-responsive. Among the four types, light responsiveness elements accounted for the largest proportion, particularly Box 4, G-Box and GT1-motif. These elements existed in many HrPODs, especially HrPOD64, HrPOD18, HrPOD40, HrPOD26, HrPOD1, HrPOD28, HrPOD62. The second largest types of elements was phytohormone responsiveness, such as ABRE (abscisic acid responsiveness), CGTCA-motif and TGACG-motif (MeJA-responsiveness). And these elements also existed in many HrPODs, especially HrPOD32, HrPOD2, HrPOD49, HrPOD36, HrPOD19, HrPOD18, HrPOD67. Among abiotic/biotic stress responsiveness types of elements, ARE (anaerobic induction element), MBS (MYB binding site involved in drought-inducibility) and TC-rich repeats (defence and stress responsive elements) existed in many HrPODs, especially HrPOD64, HrPOD18, HrPOD40, HrPOD26, HrPOD1, HrPOD28, HrPOD62 (Fig. 3). The detailed information was listed in Table S6.

Fig. 3.

Fig. 3

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Cis-acting elements analysis of 71 HrPODs. The 2000 bp sequence upstream of the initiation codon of HrPODs were used to cis-acting elements analysis, and the boxes of different colors at the bottom of the heatmap represented different types of potential signal responses. The number in each small box in the heatmap represented the number of corresponding cis-acting elements

Gene duplication, collinearity and selection pressure analysis of HrPODs

In this study, 31 duplicated gene pairs of HrPODs were found in sea buckthorn genome, and the detailed information was listed in Table S7. Based on the above gene duplication analysis, 30 possible gene pairs were segmental duplication, and just one gene pair (HrPOD31-HrPOD32) was tandem duplication, which implied that segmental duplication was the main driving force responsible for the formation of HrPOD gene family (Fig. 4A).

Fig. 4.

Fig. 4

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Gene duplication, collinearity and selection pressure analysis of HrPODs. Gene duplication analysis of HrPODs in the sea buckthorn genome, and the duplicated genes were linked by red lines (A). Syntenic relationship analysis between HrPODs and AtPrxs, and syntenic gene pairs were linked by red lines (B). The selection pressure analysis of HrPODs were visualized with a three-dimensional scatter plot of Ka/Ks. Each red sphere represented one HrPOD, and its corresponding values on the three axes of three-dimensional space represented Ka, Ks, and Ka/Ks values (C)

To further better study how the POD gene family evolved in sea buckthorn compared to other species, the collinearity analysis between sea buckthorn (HrPODs) and A. thaliana (AtPrxs) was established. The results showed that sea buckthorn shared 52 syntenic gene pairs with A. thaliana, with a higher number of POD syntenic gene pairs found on chromosome 2, 3, 4 of sea buckthorn (Fig. 4B). However, no POD syntenic gene pairs were found on chromosomes 12 in sea buckthorn, and the detailed information was listed in Table S8. These results suggested that the HrPODs in sea buckthorn may have a close homology with the AtPrxs in A. thaliana.

Furthermore, the selection pressure analysis of coding sequences could be represented by the ratio of the non-synonymous mutation rate (Ka) to the synonymous mutation rate (Ks). In this study, the Ka/Ks ratio of 26/31 HrPOD gene pairs were calculated, ranged from 0.093834179 (HrPOD5-HrPOD59) to 0.360740473 (HrPOD9-HrPOD53), and the detailed information was listed in Table S9. We also conducted a three-dimensional scatter plot of Ka/Ks analysis (Fig. 4C).

Differential expression and functional enrichment analysis of HrPODs

In this study, RNA-seq data was used to investigate the expression pattern of HrPODs in three development stages of sea buckthorn fruits. As shown in Figs. 5A and 43 HrPODs displayed high expression in early stage (Hrh_S2); 11 HrPOD genes were highly expressed in middle stage (Hrh_S4); 6 HrPOD genes were found to be highly expressed in late stage (Hrh_S6); 11 HrPOD genes were not expressed in any stage, and the detailed information was listed in Table S10.

Fig. 5.

Fig. 5

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Differential expression and functional enrichment analysis of HrPODs. Expression profiles of three fruit developmental stages based on RNA-seq data was analysis and visualized (A). Gene Ontology enrichment analysis of HrPODs. The light purple bar chart represented “Biological Process”, and the light orange bar chart represented “Molecular Function” (B). KEGG enrichment analysis of HrPODs (C-D)

In order to further explore the biological functions of the HrPODs, we performed GO and KEGG functional enrichment analysis. A total of 7 GO terms were considered to be significantly enriched and these terms were divided into two groups. Among them, “Response to oxidative stress” and “Response to stress” terms belonged to “Biological Process”, and “Peroxidase activity”, “Oxidoreductase activity, acting on peroxide as acceptor”, “Antioxidant activity”, “Heme binding” and “Tetrapyrrole binding” terms belonged to “Molecular Function” (Fig. 5B). Accordingly, we found that all HrPODs participated in response to stress and antioxidant, and the detailed information was listed in Table S11. Then, just “Phenylpropanoid biosynthesis” term was significantly enriched by KEGG functional enrichment analysis, and 36/71 HrPODs were annotated to this term (Fig. 5C). Among of them, most of HrPODs are highly expressed in the Hrh_S2 stage (Fig. 5D). The detailed information was listed in Table S12.

Correlation analysis of HrPOD genes expression with lignin content and peroxidase activity

By further analyzing the biosynthesis pathway of phenylpropanoid, we found that these HrPODs were annotated to participate in lignin biosynthesis (Fig. 6). Then, we aimed to further screen for potential HrPODs involved in lignin biosynthesis.

Fig. 6.

Fig. 6

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Phenylpropanoid biosynthesis pathway. The internal information of four different colored boxes represents four pathways for lignin monomer synthesis, and the words marked in red represented the POD enzyme and lignin monomers that are of particular concern in this study

Firstly, we determined POD activity of Hrh_S2, Hrh_S4 and Hrh_S6, and the activity results were 147.55, 99.46 and 12.18 U·g− 1min− 1 FW, respectively (Fig. 7A). Secondly, we measured lignin content of Hrh_S2, Hrh_S4 and Hrh_S6, and the content results were 102.21, 82.13 and 35.59 mg·g− 1 DW, respectively (Fig. 7B). The detailed information was listed in Table S13. Then, we performed a Pearson correlation analysis of 36 HrPODs expression level with lignin content and POD activity. The results showed that HrPOD27, HrPOD44, HrPOD57, HrPOD63 expression level exhibited highly positive correlation with lignin content and peroxidase activity, and the correlation coefficients were all higher than 0.9. While HrPOD22 expression level presented highly negative correlation with lignin content and peroxidase activity, and its correlation coefficient was below − 0.9 (Fig. 7C). The detailed information was listed in Table S14.

Fig. 7.

Fig. 7

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Correlation analysis of HrPODs expression with lignin content and peroxidase activity. Determination of POD activity at the three fruit developmental stages, and the activity showed a decreasing trend (A). Determination of lignin content at the three fruit developmental stages, and the content also showed a decreasing trend (B). Person correlation analysis of 36 HrPODs expression with lignin content and peroxidase activity (C). The qRT-PCR analysis of the candidate HrPODs at three fruit developmental stages (D-H). Expression verification of the candidate HrPODs based on correlation analysis qRT-PCR and RNA-seq data (I). Different lowercase letters indicate significant differences in POD activity, lignin content and gene expression level at three fruit developmental stages (p < 0.05; one-way ANOVA)

Subsequently, the expression patterns of these HrPODs obtained by the RNA-seq data were assessed by qRT-PCR (Fig. 7D-H). Notably, HrPOD27, HrPOD44, HrPOD57, HrPOD63 evinced higher expression levels in Hrh_S2 compared to Hrh_S4 and Hrh_S6. Conversely, HrPOD22 was predominantly expressed in Hrh_S6, with least detectable expression in Hrh_S2. The correlation analysis between qRT-PCR result and RNA seq data showed that the expression pattern of these HrPODs were highly consistent with their expression patterns in the RNA-seq data (Fig. 7I). The detailed information was listed in Table S15.

Expression analysis of the candidate HrPODs under different abiotic stresses

Firstly, the germination rate of sea buckthorn gradually decreased with the deepening of degrees of different abiotic stress, but the changes in POD activity and lignin content showed different trends. The detailed information was presented in Figure S3. In addition, we simultaneously measured the expression of candidate HrPODs. The expression of HrPODs showed a trend of first increasing and then decreasing with the increase of PEG solution concentration, among which the HrPODs had the highest expression levels at 10% PEG content. Then, except for HrPOD63, the expression of HrPODs displayed a downward trend with the increase of NaCl solution concentration, and HrPOD63 had the highest expression levels at 0.05 mol/L NaCl solution. Finally, the expression of HrPODs showed a trend of first increasing and then decreasing with the increase of NaHCO3 solution concentration, among which the HrPODs had the highest expression levels at 0.0015 mol/L NaHCO3 solution, except for HrPOD57 (Fig. 8).

Fig. 8.

Fig. 8

Open in a new tab

Expression analysis of the candidate HrPODs under different stress treatments. Analysis of POD gene expression during the germination stage of sea buckthorn seeds under different degrees of drought stress (A-E), salt stress (F-J) and alkaline stress (K-O), respectively. Different lowercase letters indicate significant differences in gene expression level after treatment (p < 0.05; one-way ANOVA)

Discussion

Class III peroxidase genes (PODs) have been widely reported to participate in miscellaneous physiological processes, such as fruit development [15], response to stresses [16, 17], seed germination [18], especially, cell wall lignification [19]. Lignification typically occurs during normal growth and stress responses [20, 21]. The synthesis of lignin is usually completed in phenylpropanoid biosynthesis pathway, and PODs, laccases and hydrogen peroxide jointly participate in lignin synthesis together [23, 24]. Many studies have found that that PODs could regulate fruit quality [25–33] and plant’s response to stress [34–43] by involving in lignin biosynthesis. It is not difficult to prove that PODs play an important role in the growth and development of many plants. Sea buckthorn has been considered as an ecological and economic plant [44–48]. However, there are relatively few reports on the members of this gene family in sea buckthorn, including genome-wide identification, expression analysis, and potential roles in fruit quality and stress response.

The genome-wide identification and characteristic analysis of the HrPODs

In the past decade, the POD family members have been widely characterized and studied in many plant species [10–12]. However, there are currently no reports on the whole genome identification and analysis of this gene family in sea buckthorn. In this study, a total of 71 HrPODs were identified in sea buckthorn genome. The number of HrPODs in this study was similar to that in (A) thaliana (73) [6], D. carota (75) [64], higher than that in of D. scoparium (22) [12], V. vinifera (47) [49], but lower than that in (B) pendula (90) [10], N. tabacum (210) [11], G. max (124) [50], which may indicate that different species have undergone diverse dynamics of gene duplication and loss in evolution. Based on the genomic information of sea buckthorn, 71 HrPODs were unevenly distributed on chromosomes 1 to 12, and a vast majority of chromosomes contained more than two HrPODs, whereas others contained only two genes, which is consistent with V. vinifera [49], G. max [50], D. carota [64] and (C) lanatus [65]. In this study, the phylogenetic tree of HrPODs was also classified into 7 subgroups (Gr1-Gr7) based on the homology of AtPODs (Fig. 1A) [49], which is similar to A. thaliana [6] and (D) carota [64].

All HrPODs contained a secretory-peroxidase domain, which belonged to class III of the plant heme-dependent peroxidase superfamily [66]. Most HrPODs contained 10 conserved motifs, motif 1, 2, 3, 3, 4 and 6 were annotated as the haem peroxidase superfamily. Especially, the motif 2 and motif 3 separately contained a peroxidase active site and a peroxidase haem-ligand binding site, which is similar to Prunus persica [13]. It is known that introns are usually existed in the genome [67], which could ensure that the corresponding genes undergo faster differentiation and pseudogenification during plant evolution [68]. In this study, the gene structure of HrPODs displayed diversity, and more than half of HrPODs contained four exons and three introns. Many researches find that introns can affect the transcript levels of upstream genes by changing the rate of transcription, transcript stability, and nuclear export [69, 70]. Therefore, this gene structure analysis may be helpful for further research on the expression of the specific HrPODs. Cis-acting elements in promoter regions typically bind to transcription factors to precisely initiate gene expression [71, 72]. To figure out the potential functions of cis-acting elements in HrPODs, we extracted and analyzed the upstream 2000 bp regions of these HrPODs, and the predicted results indicated that the HrPOD family might be widely involved in response to phytohormone signaling transduction and abiotic/biotic stress.

Gene duplication, including tandem duplication, segmental duplication, and whole genome duplication, were widely recognized as the main driving force responsible for the formation of gene families [73]. In this study, 30 possible gene pairs were segmental duplication, and just one gene pair was tandem duplication, which implied that segmental duplication was the main driving force responsible for the formation of HrPOD gene family. This result is analogous to that found in the N. tabacum [11], G. max [50], for which segmental duplication has also been reported as more eminent than tandem duplication. However, the result is in contrast with the expansion pattern in B. pendula [10], V. vinifera [49], D. carota [64]. Theoretically, Ka/Ks < 1 indicates negative selection, Ka/Ks = 1 indicates neutral selection and Ka/Ks >1 indicates positive selection [60]. In this study, we found that the Ka/Ks ratio of 26 HrPOD gene pairs were calculated, and ranged from 0.093834179 to 0.360740473, which implied that negative selection might played an important role in the evolution of the POD gene family in sea buckthorn.

The expression pattern and functional enrichment analysis of the HrPODs

Transcriptome sequencing can more comprehensively reveal the transcriptional changes of all members of the HrPODs at different tissues or developments stages. In this study, we preferred to prioritize studying the potential role of PODs-lignin modules in fruit quality, so we chose fruit materials from different developmental stages for transcriptome sequencing. RNA seq data showed a sharp decrease in the expression levels of 43 HrPODs, which may be closely related to the softening of fruit texture during development. However, 11 HrPODs were not expressed in any of the three stages, indicating that these HrPODs exhibit tissue-specific expression, and may express in other tissues, such as roots, stems, leaves, and flowers. The GO and KEGG enrichment analysis can further perform preliminary functional classification and metabolic pathway localization of HrPODs, respectively. The GO analysis indicated that all HrPODs were significantly enriched in ‘Response to stress”, “Peroxidase activity” and “Antioxidant activity” terms, which fully reflected the main functional characteristics of this type of gene. This result was similar to C. lanatus [65] and Pyrus. Bretschenedri [74]. The KEGG enrichment analysis indicated that HrPODs were mainly significantly enriched in “Phenylpropanoid biosynthesis” terms, which was also similar to C. lanatus [65] and Pyrus. Bretschenedri [74].

The potential roles of the HrPODs in fruit quality

By analyzing the biosynthesis pathway of phenylpropanoid, 36 HrPODs were annotated to participate in lignin synthesis. According to many research reports, the fruit quality, especially texture, are usually closely related to specific PODs expression and lignification of the cell wall [25–33]. In order to further screen the HrPODs may potentially influence the sea buckthorn fruit quality participate in lignin biosynthesis, we performed a Pearson correlation analysis to screen the potential HrPODs based on the correlation analysis of HrPODs expression with lignin content and POD activity. The lignin content and POD activity both showed a sharp decrease, and the results of Pearson correlation analysis showed that HrPOD27, HrPOD44, HrPOD57, HrPOD63 expression level exhibited significantly positive correlation with lignin content and peroxidase activity, and HrPOD22 expression level presented highly negative correlation with lignin content and peroxidase activity, which may be closely related to the softening of fruit texture during fruit development. Which of these HrPODs actually regulates fruit lignin synthesis and softening, and affects fruit texture, requires further detailed and in-depth functional research. These results will help us better investigate the role of HrPODs in lingin biosynthesis, enrich the theoretical research on the formation of fruit texture quality in sea buckthorn.

The potential roles of the HrPODs in stress response

The wild sea buckthorn is distributed in arid areas with higher salt and alkaline content, stresses usually exists throughout the entire process from seed germination to fruit ripening, and it is generally believed that these stress has a significant impact on the seed germination stage [46, 54], so we preferred to conduct plenty of germination experiments treated with different chemical solutions, respectively, to assess the effects of different degrees of drought, salt and alkali stress on the expression of these candidate HrPODs. In this study, the germination showed downward trends with the deepening of degrees of different abiotic stress, but the change in POD activity, lignin content and HrPODs expression displayed different trends. Firstly, we found that all candidate HrPODs had the highest expression levels at 10% PEG content. Then HrPOD63 had the highest expression levels at 0.05 mol/L NaCl solution. Finally, HrPOD22, HrPOD27 and HrPOD44 had the highest expression levels at 0.0015 mol/L NaHCO3 solution. Our expression analysis results indicated that HrPODs had a certain degree of stress resistance to drought, salt, or alkali accompanied by collaborative adjustment of lignin content and POD activity. In recent years, there have been many similar research reports on other species. Overexpression of the ZmPRX1 increases maize seedling drought tolerance and redound root lignin accumulation [18]. MsPOD220 expression showed a significant fluctuation trend with the deepening of degrees of salt, drought or cold stress [46, 75]. TaPRX-2 A in T. aestivum showed higher resistance to salt stress compared with wild type [75, 76]. Aleem et al. reported that the overexpression of GsPOD40 exhibited positive response to drought stress [50, 51]. Heterologous overexpression of PlPOD45 enhanced resistance to high-temperature stress and increased POD activity [76, 77]. In short, the expression patterns of the candidate HrPODs and lignin content under different stresses were varied and complicated, which may be related to the functional diversification. These results provide important insights for us to further understand the roles of HrPODs in abiotic stress resistance.

Taken together, the above findings not only provide us with a comprehensive identification and analysis of HrPODs from a bioinformatics perspective, but also preliminary intimation that the POD family may be tightly associated with fruit texture quality and abiotic stress response. These results will help us better investigate the role of HrPODs in lingin biosynthesis, enrich the theoretical research on the formation of sea buckthorn texture quality, understand the roles of HrPODs in abiotic stress resistance, and provide molecular design strategies for broad spectrum stress resistance and fresh berry breeding in cultivated sea buckthorn.

Conclusion

In conclusion, HrPODs were comprehensively identified and analyzed in sea buckthorn genome. A total of 71 non-redundant HrPOD members were identified, and classified into seven subgroups based on the phylogenetic relationship with A. thaliana. And then the physicochemical properties, chromosomal location, conserved domain, motif, gene structure, cis acting element, gene duplication, selection pressure and collinearity analysis were further conducted. Subsequently, the differential expression and functional enrichment analysis indicated that 36/71 HrPODs might be involved in phenylpropanoid biosynthesis, especially lignin biosynthesis. Furthermore, we screened five candidate HrPODs that may participate in lignin biosynthesis with the help of correlation analysis of HrPODs expression with POD activity and lignin content. The lignin content and POD activity both showed a sharp decrease, and the results of Pearson correlation analysis showed that HrPOD27, HrPOD44, HrPOD57, HrPOD63 expression level exhibited significantly positive correlation with lignin content and peroxidase activity, and HrPOD22 expression level presented highly negative correlation with lignin content and peroxidase activity, which may be closely related to the softening of fruit texture during fruit development. At the same time, the expression of these HrPODs and lignin content presented different trends with the deepening of degrees of different abiotic stress. These results will help us better investigate the role of HrPODs in lingin biosynthesis, enrich the theoretical research on the formation of sea buckthorn texture quality and understand the roles of HrPODs in abiotic stress resistance in cultivated sea buckthorn.

Supplementary Information

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Acknowledgements

Not applicable.

Abbreviations

POD

Class III peroxidase

DAA

Days after anthesis

PEG

Polyethylene glycol

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

qRT-PCR

Quantitative real-time PCR

Authors’ contributions

Jing Zhao and Kun Sun conceived and designed the study. Kai Li and Meng Zhao performed the experiments and bioinformatics analyses. Xuan Jiang and Xing Liu collected samples and carried out physiological assays. Hongli Wang assisted in qRT-PCR validation. Kai Li drafted the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32400190, 32060060), Gansu Key Research and Development Program (25YFFA061), and Northwest Normal University Graduate Research Grants (2023KYZZ-S176, 2024CXZX-LXS171).

Data availability

All basic data supporting the findings of this study are included within the article and its supplementary information files. The genome assembly of Hippophae rhamnoides subsp. sinensis Rousi is publicly available at China National Gene Bank (CNGB) CNP0001846 (https://db.cngb.org/search/project/CNP0001846). The protein and CDS sequences of the HrPODs have been clearly provided in Table S2. The RNA seq data is currently not shared, and is part of ongoing research. We will gradually release it in the future. If necessary, please contact the corresponding author according to reasonable requirements. The gene expression data of the HrPODs has been provided in Table S10.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jing Zhao and Kai Li contributed equally to this work.

Contributor Information

Jing Zhao, Email: zhaojing@nwnu.edu.cn.

Kun Sun, Email: kunsun@nwnu.edu.cn.

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Associated Data

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Supplementary Materials

Supplementary Material 1. (266.7KB, png)
Supplementary Material 2. (842.1KB, png)
Supplementary Material 3. (564.5KB, png)
Supplementary Material 4. (9.8KB, xlsx)
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Data Availability Statement

All basic data supporting the findings of this study are included within the article and its supplementary information files. The genome assembly of Hippophae rhamnoides subsp. sinensis Rousi is publicly available at China National Gene Bank (CNGB) CNP0001846 (https://db.cngb.org/search/project/CNP0001846). The protein and CDS sequences of the HrPODs have been clearly provided in Table S2. The RNA seq data is currently not shared, and is part of ongoing research. We will gradually release it in the future. If necessary, please contact the corresponding author according to reasonable requirements. The gene expression data of the HrPODs has been provided in Table S10.

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