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Article

Genome-Wide Identification, Phylogenetic Analysis, and Expression Pattern of Polyamine Biosynthesis Gene Family in Pepper

by
Duo Lin
,
Xianqi Zhao
,
Qingshan Hu
,
Su Wang
,
Yan Zhang
and
Zijian Xu
*
College of Horticulture, Qingdao Agricultural University, Qingdao 266109, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(17), 8208; https://doi.org/10.3390/ijms26178208
Submission received: 16 July 2025 / Revised: 16 August 2025 / Accepted: 22 August 2025 / Published: 24 August 2025
(This article belongs to the Section Molecular Genetics and Genomics)
Browse Figures
Versions Notes

Abstract

Polyamines (PAs), including putrescine, spermidine, spermine, and thermospermine, play essential roles in plant growth, development, and responses to stress. However, the structure and function of PA biosynthetic genes in pepper remain poorly characterized. This study aimed to identify PA biosynthesis genes in the pepper genome using bioinformatics approaches and to assess their expression under various stress conditions. A total of 16 PA biosynthesis-related genes were identified, representing members of the arginine decarboxylase (ADC), ornithine decarboxylase (ODC), agmatine iminohydrolase (AIH), N-carbamoylputrescine amidohydrolase (CPA), S-adenosylmethionine decarboxylase (SAMDC), spermidine synthase (SPDS), spermine synthase (SPMS), and ACAULIS5 (ACL5) gene families. These genes encode proteins with an average molecular weight of approximately 40 kDa, primarily localized in the mitochondria and cytoplasm. Promoter analysis revealed multiple cis-acting elements associated with stress and phytohormone responsiveness. Gene expression was induced by various abiotic stresses, including saline-alkaline, drought, heat, cold, and hydrogen peroxide, as well as by phytohormones such as abscisic acid, ethylene, salicylic acid, auxin, and gibberellin. Overall, this study provides a comprehensive analysis of PA biosynthesis genes in pepper and highlights their potential roles in stress adaptation and hormone signalling, offering a foundation for further exploration of PA-mediated stress tolerance mechanisms.

1. Introduction

Polyamines (PAs) are a class of strongly biologically active, low molecular weight, aliphatic nitrogenous bases produced during biological metabolism. They were among the first key substances discovered in biochemical research [1]. PAs exhibit polycationic properties, typically enabling interactions with polyanionic substances, including nucleic acids, proteins, membrane phospholipids, and pectic polysaccharides, through hydrogen and ionic bonds. These interactions regulate cellular ion balance [2]. Furthermore, PAs enhance the structural stability of proteins, RNA, and DNA, reducing membrane-lipid phase transitions and maintaining membrane integrity [3]. Recent studies have shown that PAs also perform hormone-like functions, such as participating in signal transduction within plant cells and activating downstream responses by directly serving as “second messengers” or indirectly modulating the Ca2+ signalling system [4].
In higher plants, several types of PAs are present, with putrescine (Put), spermidine (Spd), and spermine (Spm) being the predominant forms. Additionally, small amounts of cadaverine, thermospermine (Tspm), norspermine (Nspm), and norspermidine (Nspd) have been identified [5]. There are three principal forms of PAs in plants: free, soluble conjugated, and insoluble bound. The accumulation of these forms is essential for plant responses to stress. However, the content and function of PAs are mainly regulated by the dynamics of their synthesis and catabolism [6]. The biosynthesis of PAs in plants primarily proceeds via the arginine (Arg) and ornithine (Orn) pathways. In one pathway, Arg is catalyzed by arginine decarboxylase (ADC) to form agmatine (Agm), which is further converted to Put through the sequential action of agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA). Alternatively, Orn generates Put via ornithine decarboxylase (ODC). The subsequent synthesis of Spd and Spm from Put requires aminopropyl groups donated by decarboxylated S-adenosylmethionine (dcSAM), produced through the action of S-adenosylmethionine synthase (SAM) and S-adenosylmethionine decarboxylase (SAMDC). In this process, spermidine synthase (SPDS) and spermine synthase (SPMS) serve as key catalytic enzymes [7]. ACAULIS5 (ACL5) encodes Tspm synthase, which catalyzes the synthesis of less abundant PAs, including Tspm and Nspm [8]. The accumulation of PAs also depends on catabolic pathways, primarily mediated by copper amine oxidase (CuAO) and polyamine oxidase (PAO). Put is degraded by CuAO, whereas the catabolism of Spd and Spm is regulated by PAO [9].
PAs contribute to various aspects of plant growth and development, including cell division and differentiation, root elongation, flower development, and fruit ripening [10]. Increasing evidence shows that PAs are closely associated with stress responses and regulate plant stress resistance. Early research established that potassium (K+) deficiency increases Put accumulation in plants, prompting further investigations into the association between PAs and stress tolerance [11]. Numerous recent studies have shown the role of PAs in plant responses to abiotic stresses, including oxidative, salinity, drought, high temperature, and low temperature stress [12,13]. Stress-induced increases in PA levels are generally accompanied by enhanced activity of PA biosynthetic enzymes and upregulation of biosynthetic genes [14]. For example, expression of ADC and ODC genes is associated with drought and salinity tolerance, with ADC activity elevated under salinity stress [15,16]. In rice seedlings, salinity and drought stress induce the expression of SAMDC1 [17]. In tea plant, the CsSPMS gene is rapidly upregulated under cold stress [18]. However, the dynamics of PA levels differ across plant species and stress conditions. For example, levels of Put, Spd, and Spm increase markedly during low temperature acclimation in citrus [19], whereas many plants exposed to salinity stress show decreased Put and increased Spd and Spm contents; the (Spd + Spm)/Put ratio rises with increasing salinity, thereby enhancing salt tolerance [20]. Although substantial evidence supports an association between PAs and plant stress responses, the differential regulation of PA biosynthesis pathways under various stresses remains largely unknown across plant species.
Pepper (Capsicum annuum L.) is a major crop in the Solanaceae family, originating in Central and South America and now widely cultivated worldwide. As a warm-season crop with shallow root systems, pepper is frequently subjected to abiotic stresses, including drought, salinity, and low temperature. These stresses hinder growth and development, and may even result in plant death. Previous multi-omics analyses identified six CaPAO genes in pepper, with CaPAO2 and CaPAO4 positively contributing to cold stress responses [21]. In addition, the CaSPDS gene, which participates in PA biosynthesis, was also shown to respond to cold stress [22]. However, comprehensive studies on other PA biosynthetic genes in pepper are lacking, particularly regarding their response to different stress conditions. Therefore, this study utilized the T2T pepper genome database to identify candidate PA biosynthetic genes, analyze their gene structures, promoter cis-elements, protein properties, chromosomal localization, and phylogenetic relationships. The expression patterns in various tissues, organs, and under different hormonal and abiotic stresses were also examined to provide a basis for elucidating PA function and its role in stress resistance in pepper.

2. Results

2.1. Identification of PA Biosynthesis Genes in Pepper

PA biosynthetic genes in pepper (Capsicum annuum L.) were systematically identified using the HMMER model (Table 1). Two ADC genes (CaADC1 and SlADC2) and three ODC genes (CaODC1, CaODC2, and CaODC3) were detected in the telomere-to-telomere gapless genome. Among these, only CaADC2 contained introns in its genomic sequence (Figure 1). Two further genes responsible for Put biosynthesis, agmatine iminohydrolase (CaAIH, CaT2T12g01633) and N-carbamoylputrescine amidohydrolase (CaCPA, CaT2T11g00431), were also identified; CaAIH and CaCPA contained 10 and 7 introns, respectively (Figure 1).
In the Spd, Spm, and TSpm biosynthetic pathways, one spermidine synthase (CaSPDS), one spermine synthase (CaSPMS), and three ACL5 genes (CaACL5-1, CaACL5-2, and CaACL5-3) were identified. All these genes exhibited multiple introns, with CaSPMS and CaACL5-1 each containing 9 introns, and the remaining CaACL5 genes containing 6 introns. In addition, four SAMDC genes (CaSAMDC1, CaSAMDC2, CaSAMDC3, and CaSAMDC4) were identified, with only CaSAMDC2 containing 7 introns (Figure 1).
The physicochemical properties of the encoded proteins were analyzed using ProtParam. The results indicated that these proteins had an average sequence length of approximately 400 amino acids and a mean molecular weight of approximately 45 kDa (Table 2). CaAIH, CaCPA, CaSAMDC4, and three CaACL5 proteins were predicted to be unstable based on their instability indices (Table 2).

2.2. Cis-Acting Elements in the Promoters of PA Biosynthesis Genes in Pepper

Cis-acting elements play a critical role in transcriptional regulation through interactions with trans-regulatory factors [23]. To clarify the regulatory potential, 2000 bp promoter sequences of PA biosynthetic genes were extracted and analyzed for cis-acting elements. A total of 57 cis-acting elements were identified. Universal elements such as the TATA-box and CAAT-box were found in all promoters, whereas the remaining 55 cis-acting elements were classified into four categories: plant growth and development (9), light-responsive (19), hormone-responsive (15), and abiotic/biotic stress-related (12) (Table S2). To further evaluate the role of cis-acting elements under stress and hormonal treatments, the number of hormone- and stress-responsive elements in each promoter was determined (Figure 2). The 15 hormone-responsive elements included motifs related to ABA, jasmonic acid (JA), SA, GA, auxin, and ethylene signalling, such as AREB, as-1, TGA-element, GARE-motif, TCA-element, TGACG-motif, and ERE. The 12 stress-related elements included cis-acting elements that respond to low temperature, drought, and dehydration stresses, as well as elements that interacting with stress-related transcription factor, such as MYB, MYC, and W-box.

2.3. Phylogenetic Analysis of PA Biosynthesis Genes

To assess the evolutionary relationships among PA biosynthetic genes, protein sequences from Arabidopsis, tomato, and pepper were used to construct a phylogenetic tree. The analysis grouped PA biosynthetic proteins into seven major clades. The ODC group consisted of six members, and three from pepper and three from tomato—whereas Arabidopsis lacked ODC genes in its genome. Notably, SPDS and SPMS proteins clustered within the same clade (Figure 3). Pepper and tomato, both members of Solanaceae, showed close evolutionary proximity, resulting in their homologous proteins clustering together, in contrast to those from Arabidopsis. Alignment analysis further demonstrated that PA biosynthetic proteins from pepper possessed conserved regions similar to those in tomato and Arabidopsis (Figures S1–S8).

2.4. Chromosomal Localization and Duplication of PA Biosynthesis Genes in Pepper

Sixteen PA biosynthetic genes were mapped to ten chromosomes in the pepper genome, with only chromosomes 07 and 09 lacked target genes. The gene locations were as follows: CaSAMDC1 (Chr01), CaACL5-1 (Chr02), CaODC3 (Chr04), CaSAMDC2 (Chr05), CaSAMDC3 (Chr06), CaADC2 (Chr10), CaAIH (Chr12), CaACL5-3 and CaADC1 (Chr08), CaCPA, CaSPDS, and CaSAMDC4 (Chr11), and CaODC1, CaODC2, CaSPMS, and CaACL5-2 (Chr03) (Figure 4).
Collinearity analysis was performed to assess gene duplication events between pepper and two model species, tomato and Arabidopsis. Six pairs of PA biosynthetic genes were identified as duplicated between the pepper and Arabidopsis genomes, whilst twelve pairs were identified between pepper and tomato. Genes including CaSAMDC1, CaODC2, CaCPA, CaSPDS, and CaSAMDC4 in pepper were homologous in both model genomes across all duplication events. The CaSPDS gene in pepper showed two homologous sites in both Arabidopsis and tomato. Among all PA biosynthetic genes, only CaAIH was not associated with duplication events in the pepper and tomato genomes (Figure 5).

2.5. Organ-Specific Expression Patterns of Pepper PA Biosynthesis Genes

The specific expression patterns of genes provide insights into their functional roles in plant growth and development [24]. In this study, the organ-specific expression profiles of PA biosynthetic genes were assessed using RT-qPCR in pepper. The expression of PA biosynthetic gene in seeds was used as a reference to calculate relative expression levels across different tissues and organs. The results showed that CaADC1, CaODC3, and CaSAMDC4 genes displayed high expression in various tissues and organs (Figure 6H). Comparatively, CaADC1, CaODC3, CaCPA, CaSPMS, CaSAMDC1, CaSAMDC2, CaSAMDC4, CaACL5-1, and CaACL5-3 exhibited the highest expression in roots, whereas CaADC2, CaAIH, CaODC1, CaODC2, and CaSAMDC3 were predominantly expressed in mature leaves. Notably, CaSPDS and CaACL5-2 did not show significant expression in any of the examined tissues or organs.

2.6. Effect of Exogenous Phytohormones on PA Biosynthesis Gene Expression

Recent research indicates that the association between polyamines and phytohormones enhances plant tolerance to abiotic stress [25]. In this study, several hormones were applied exogenously to the leaves of pepper seedlings, and PA biosynthetic gene expression was measured. The results demonstrated that different hormones induced the expression of PA biosynthetic genes (Figure 7). The most pronounced induction of CaSAMDC3 expression was observed following auxin treatment, followed by CaACL5-2, with peaks at 6 h and 12 h. Additionally, genes in the CaODC family, CaSPDS, and CaSAMDC1 were also significantly upregulated (Figure 7A). Expression of the CaODC gene family was strongly induced by both gibberellin and salicylic acid treatments. CaCPA and CaAIH expression was significantly elevated by gibberellin, whereas CaSPDS expression was strongly induced by salicylic acid (Figure 7B,E). The expression of CaODC2, CaODC3, CaSAMDC1, CaSAMDC3, and CaACL5-2 was upregulated in response to exogenous ethylene and peaked at 24 h (Figure 7C). Furthermore, CaSPMS and the CaSAMDC gene family were induced by abscisic acid treatment (Figure 7D).

2.7. Differential Expression of PA Biosynthesis Genes During Abiotic Stress

The expression profiles of pepper PA biosynthetic genes under abiotic stress, including low temperature, PEG-6000, high temperature, NaCl, saline–alkaline, and H2O2 treatments, were investigated. The results showed that most PA biosynthetic genes were induced by low temperature, PEG-6000, NaCl, and saline–alkaline stresses, whereas only a subset was responsive to high temperature and H2O2 (Figure 8). Differential expression patterns were observed under temperature stress, with CaODC, CaSAMDC3, and CaACL5-3 genes induced by both low and high temperatures (Figure 8A,C). Interestingly, the expression of the CaCPA gene is induced only by temperature stress. Nearly all PA biosynthetic genes, except CaSAMDC2, were upregulated following PEG-6000, NaCl, and saline–alkaline treatments (Figure 8B,D,E). Among them, the three CaODC genes and CaSAMDC3 were induced under all abiotic stress conditions. These findings suggest that polyamines may contribute to the response to abiotic stress in pepper.

3. Discussion

PA biosynthetic pathways have been extensively studied in many higher plants, including tomato, sweet orange, and wheat [26,27,28]. In this study, we identified sixteen non-redundant PA biosynthetic genes in the telomere-to-telomere gapless genome of pepper. Although the PA biosynthetic pathway is evolutionarily conserved, the number and types of genes differ across species. For instance, ODC genes are absent in Arabidopsis [29], but different numbers of ODC genes are present in tomato and wheat [26,28]. In pepper, three ODC genes were identified; all are intron-free and predicted to be localized in mitochondria (Figure 1 and Table 1). Subcellular localization of biosynthetic proteins provides insight into both their spatial distribution and their functional roles within the cell [30]. However, the actual subcellular compartments of pepper PA biosynthetic proteins have not been experimentally verified, which limits understanding of polyamine function in pepper. In this study, only CaAIH protein was predicted to be extracellular, five proteins from the ADC and ODC families were predicted to be mitochondrial, and the other ten proteins were predicted to be cytoplasmic (Table 1). Comparisons with wheat and Arabidopsis showed that SPDS, SPMS, and ACL5-1 were cytoplasmic, whereas SAMDC proteins varied in predicted localization across species [4,26]. Although bioinformatic prediction is informative, experimental confirmation is needed to clarify the precise localization and biological functions of these proteins.
Phylogenetic analysis is an essential approach for elucidating the evolutionary relationships of genes across species [31]. Here, we compared the protein sequences of PA biosynthetic genes from pepper, Arabidopsis, and tomato. The phylogenetic tree grouped the proteins into seven clusters, with functionally equivalent proteins from different species clustering together (Figure 3). SPDS and SPMS proteins clustered in the same branch, consistent with their roles in catalyzing aminopropyl transfer to form Spd and Spm [32,33]. Their shared conserved domains likely underlie their inability to be resolved into separate branches in phylogenetic analysis. Although clustering of proteins with similar functions is apparent, the evolutionary diversification of PA biosynthetic gene families in pepper warrants further study. It is well established that gene and genome duplication events play a key role in the emergence of new gene functions and in the diversification of genetic systems [34,35]. Comparative syntenic mapping provides insight into the evolutionary history of gene families [36]. Our comparative analysis revealed that most PA biosynthetic genes in pepper are more closely related to those in tomato than to those in Arabidopsis (Figure 5), reflecting the shared evolutionary background of Solanaceae crops. However, none of the CaAIH or CaACL5 genes in pepper were syntenic with genes in tomato or Arabidopsis, suggesting a higher degree of evolutionary divergence in these gene families.
Transcriptional regulation of plant genes depends on interactions among cis-acting elements and trans-acting factors, most of which are located in the promoter region upstream of the gene. The promoter regulates gene transcription by facilitating precise binding of RNA polymerase, thereby ensuring accurate initiation of transcription [37]. Many studies have established the importance of PAs in regulating plant stress tolerance [25]. In this study, we analyzed the promoter regions of pepper PA biosynthetic genes and identified numerous cis-acting elements associated with abiotic stress and phytohormone response (Figure 2 and Table S2). Notably, all promoters contained MYC recognition sites—conserved cis-acting elements bound by MYC transcription factors—which are central mediators of plant responses to diverse stresses [38]. Additional elements, including MYB recognition sites and STRE motifs, were also widely present. MYB elements are recognized by MYB transcription factors, which play crucial roles in stress-responsive gene regulation in plants [39]. In trifoliate orange (Poncirus trifoliata L. Raf.), the MYB cis-element in the PtADC promoter of trifoliate orange interacts with PtsrMYB to enhance dehydration tolerance [40]. The STRE motif, originally identified as a binding site for the Msn2p/Msn4p activator in yeast [41], is also found in wheat PA gene promoters [26], although plant-specific binding proteins have yet to be identified. The promoter of pepper PA biosynthetic genes also contains phytohormone-responsive elements, including ABRE (ABA response), ERE (ethylene response), TGACG- and CGTCA-motifs (jasmonate response), and TCA-element (salicylic acid response). ABRE, in particular, is a key cis-element in ABA signalling and interacts with AREB/ABF transcription factors. Disruption of these factors, as in areb1/areb2/abf3 triple mutants, leads to reduced expression of stress-responsive genes and impaired dehydration tolerance [42]. In trifoliate orange, PtrABF2 directly binds the ABRE motif in the PtrADC promoter and enhances drought tolerance through increased Put accumulation [43]. The presence of multiple hormone-responsive elements indicates that PAs are integrated into broader hormone signalling networks. These findings suggest that the expression of pepper PA biosynthetic genes is coordinately regulated by abiotic stress and phytohormones.
Under adverse conditions, plants have evolved diverse adaptive strategies to ensure survival. Among these, the accumulation of PAs plays a key role in counteracting stress. Endogenous PAs, including Put, Spd, and Spm, are known to accumulate to varying degrees under stress, and their function is often integrated with phytohormone signalling pathways in response to environmental stressors [44,45]. The expression of PA biosynthetic genes is dynamically regulated by abiotic stresses and phytohormone signals, supporting plant adaptation through transcriptional control [42,46]. In Arabidopsis, twelve genes encode enzymes responsible for PA biosynthesis, including AtADC1/2, AtSAMDC1/2, AtSPDS1/2, AtSPMS, and AtACL5, all of which are strongly induced by at least one abiotic stress such as salt, drought, or cold [47]. Loss-of-function mutants for atadc1 and atadc2 exhibit reduced salt and cold tolerance [48], whereas the atspms/atacl5 double mutant displays diminished tolerance to salt and drought [49]. Similarly, in apple, SAMDC1 and SAMDC2 are mainly induced by salt and temperature stress [50], and in cucumber, SAMDC3 is upregulated by salt stress [51]. In pepper, CaSPDS is rapidly induced under low-temperature stress, and overexpression of CaSPDS in Arabidopsis enhances cold tolerance, antioxidant enzyme activities, Spd content, and the expression of cold-responsive genes [22]. In this study, low-temperature stress significantly upregulated not only CaSPDS but also CaODC1/2/3, CaSPMS, CaSAMDC1/3, and CaACL5-2. Reports have indicated that polyamine biosynthesis in plants primarily occurs through the arginine pathway, in which ADC, AIH, and CPA play important roles [29]. However, various abiotic stresses induce the expression of ODC genes in pepper, suggesting that the pepper PA biosynthesis genes are induced stress via the ornithine pathway. Moreover, the CaODC genes appear to be highly sensitive to osmotic stress, as they are significantly induced under PEG-6000, NaCl and saline-alkaline stress, which all share the common effect of altering the cellular osmotic potential. Similarly, the CaCPA gene is more sensitive to temperature stress and only induced by high- or low-temperature stress (Figure 8). Additional PA biosynthetic genes in pepper were induced by various phytohormone treatments (Figure 7). Most previous studies indicate that ABA strongly induces polyamine biosynthetic genes [52]. Although some PA biosynthetic genes in pepper were upregulated by ABA, a greater number showed pronounced expression under IAA and GA3 treatments. It has been reported that GAs positively regulate flowering, trichome formation, and reproductive development [53]. The CaODC3 gene is significantly induced under GA3 treatment, and also shows significant expression in flowers (Figure 6H), suggesting that PAs and GA may have crosstalk during flower development. However, the relationship of PAs and phytohormones still needs further study. Collectively, the results show that PA biosynthetic genes in pepper respond to a broad spectrum of abiotic stresses and phytohormones, highlighting their important role in enhancing plant stress tolerance.

4. Materials and Methods

4.1. Plant Growth Conditions and Treatments

The pepper (Capsicum annuum L.) cultivar used in this study was ‘Jiaozhongyu’, produced by hybrid breeding of PP1351A × Z121. Pepper seeds were soaked in distilled water in a conical flask and germinated at 28 °C on a shaker. Germinated seeds were sown in 72-well plates filled with a substrate composed of peat, vermiculite, and perlite in a 3:1:1 (v/v/v) ratio. Seedlings were irrigated with Hoagland nutrient solution and grown in an artificial climate chamber under controlled conditions: 25 °C/18 °C (day/night), a 14 h light/10 h dark photoperiod, and a photosynthetic photon flux density (PPFD) of 250 μmol·m−2·s−1. Seedlings were transplanted into nutrient pots (7 cm diameter) once the third true leaf was fully expanded.
At the six-leaf and one-heart stage, seedlings were subjected to abiotic stress and phytohormone treatments. Abiotic stress treatments included irrigation with 200 mM saline-alkaline mixed solution (NaCl:Na2SO4:NaHCO3:Na2CO3 = 1:9:9:1, pH 8.90), 200 mM NaCl and 20% polyethylene glycol-6000 (PEG-6000), and foliar spraying with 100 mM hydrogen peroxide (H2O2), as well as high temperature (42 °C) and low temperature (4 °C) treatments. For phytohormone treatments, leaves were respectively sprayed with 100 μM abscisic acid (ABA), 100 μM indole-3-acetic acid (IAA), 100 μM ethylene (Eth), 100 μM salicylic acid (SA), or 100 μM gibberellin (GA). Leaves were collected at 0 h, 1 h, 6 h, and 12 h after treatment, and the 0 h was used as the control. Samples of roots, stems, leaves, flowers, fruits, and seeds were also collected. All samples were rapidly frozen in liquid nitrogen and stored at −80 °C.

4.2. Identification of PA Biosynthesis Genes

Gene and protein sequences of PA biosynthetic genes from Arabidopsis thaliana were obtained from The Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org, accessed on 1 April 2025). Protein sequences were used as queries for BLASTP searches against the pepper genome with an E-value threshold of <1e−0. Sequences from tomato (Solanum lycopersicum) were obtained from the Sol Genomics Network (SGN, https://solgenomics.net/, accessed on 1 April 2025). The pepper genome sequence was derived from the telomere-to-telomere gapless genome (PGDB, http://www.pepperbase.site/node/3, accessed on 1 July 2025) [54]. Candidate protein sequences were verified in Pfam (http://pfam.xfam.org/, accessed on 1 April 2025) and NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 1 April 2025).

4.3. Gene Structure, Subcellular Localization and Protein Physicochemical Properties Analysis

Coding sequences (CDS), corresponding genomic DNA, and protein sequences of pepper PA biosynthetic genes were extracted from local genomic data using HMMER 3.0 [55]. Exon-intron structures were analyzed using the Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn, accessed on 3 April 2025) [56]. Protein secondary structures were predicted with the SOPMA method (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html, accessed on 6 April 2025) [57]. Subcellular localization was predicted with Euk-mPLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/, accessed on 3 April 2025) [58]. Physicochemical properties, including instability index, aliphatic index, isoelectric point, and molecular mass, were calculated using ExPaSy (http://web.expasy.org, accessed on 3 April 2025).

4.4. Cis-Element Prediction for PA Biosynthesis Gene Promoters

Promoter sequences (2 kb upstream of the ATG) for each PA biosynthetic gene were extracted from the PGDB and submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 6 April 2025) for cis-element analysis. Visualization of results was performed using TBtools (v1.098745, Guangzhou, China) [59].

4.5. Sequence Alignment and Phylogenetic Analysis

Multiple sequence alignments of PA biosynthetic proteins were performed using ClustalW. Phylogenetic trees were constructed using the neighbour-joining method in MEGA 7.0 software, with 1000 bootstrap replications, the Poisson model, and pairwise deletion of gaps [49].

4.6. Chromosomal Location and Duplication Analysis

Annotations and chromosomal locations of pepper PA biosynthetic genes were obtained from the local genome data according to gene ID. To visualize the location of the genes on the chromosomes using TBtools software (v1.098745, Guangzhou, China). The chromosomal locations of genes in tomato and Arabidopsis were identified from SGN website (https://solgenomics.net/, accessed on 7 April 2025) and the TAIR website (http://www.arabidopsis.org, accessed on 1 April 2025), respectively, and a collinearity graph of pepper, tomato and Arabidopsis was drawn using TBtools software (v1.098745, Guangzhou, China) [59].

4.7. Total RNA Extraction and Gene Expression Analysis

Total RNA was extracted using the PLANTpure Plant RNA Kit (Aidlab, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized with HiScript IV All-in-One Ultra RT SuperMix for qPCR (Vazyme, Nanjing, China). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was conducted using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) on an ABI QuantStudio 5 (Applied Biosystems, Waltham, MA, USA). Relative gene expression was calculated using the 2−ΔΔCt method [60], with CaActin (Gene ID: CaT2T05g00422) as the internal control. PCR primers were designed using Primer Premier 5 and are listed in Table S1.

5. Conclusions

This study identified sixteen PA biosynthesis-related genes in the pepper genome, representing key enzymes responsible for the biosynthesis of Put, Spd, Spm, and Tspm. Gene structure, subcellular localization, phylogenetic relationships, and cis-acting regulatory elements were predicted using bioinformatics analyses, and expression profiles were quantitatively examined across different organs and under various abiotic stress and phytohormone treatments. The results demonstrate that pepper PA biosynthetic genes exhibit both evolutionary conservation and diversity, and play important roles in response to abiotic stresses and phytohormones. These findings provide new perspectives for understanding the biological functions of PAs in plant stress adaptation and the screened stress-responsive genes can be applied to molecular breeding for stress-tolerant pepper plants through molecular genetic markers or genetic transformation technology, which offer a foundation for the functional studies of PAs and efforts to improve stress resistance in pepper.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26178208/s1.

Author Contributions

Z.X. and D.L. designed the experiments; X.Z., Q.H., S.W., and Y.Z. performed the experiments and analyzed the data; Z.X. was mainly responsible for writing the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the Key R&D Program of Shandong Province (2024LZGC013), Science & Technology for Public Benefit Demonstration Project of Qingdao (25-1-5-xdny-13-nsh), Sci-Tech Innovation Action Plan for Rural Vitalization of Shandong Province (2024TZXD019), Vegetable Industrial Technology System of Shandong Province (SDAIT0507) and Natural Science Foundation of Qingdao (24-4-4-zrjj-27-jch).

Acknowledgments

We thank Zengwen Liang (Weifang University, Weifang, China) for providing ‘Jiaozhongyu’ pepper as the experimental material.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Gene structure of the pepper polyamine biosynthesis genes mapped using GSDS website. The yellow blocks indicate the coding sequence (CDS), the blue blocks represent the region upstream or downstream of the genes, and the black lines indicate the introns. The lengths of the DNA sequences are indicated by the scale bar.
Figure 1. Gene structure of the pepper polyamine biosynthesis genes mapped using GSDS website. The yellow blocks indicate the coding sequence (CDS), the blue blocks represent the region upstream or downstream of the genes, and the black lines indicate the introns. The lengths of the DNA sequences are indicated by the scale bar.
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Figure 2. Analysis of cis-acting elements in the promoter of polyamine biosynthesis genes. The promoter sequences were identified as 2000 bp upstream of ATG to analyses cis-acting elements. The gradient colors in the grid represent the number of cis-acting elements in the promoter of polyamine synthesis genes. The multicolor histogram indicates the number of different categories of cis-acting elements in these genes.
Figure 2. Analysis of cis-acting elements in the promoter of polyamine biosynthesis genes. The promoter sequences were identified as 2000 bp upstream of ATG to analyses cis-acting elements. The gradient colors in the grid represent the number of cis-acting elements in the promoter of polyamine synthesis genes. The multicolor histogram indicates the number of different categories of cis-acting elements in these genes.
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Figure 3. Phylogenetic analysis of polyamine biosynthesis proteins. The protein sequences of polyamine biosynthesis were extracted from pepper (Ca, green font), tomato (Sl, red font) and Arabidopsis (At, blue font). Multiple sequence alignments were generated with the ClustalW, and the phylogenetic tree was constructed using neighbor-joining method (NJ). ADC, arginine decarboxylase; AIH, agmatine iminohydrolase; CPA, N-carbamoylputrescine amidohydrolase; ODC, ornithine decarboxylase; SAMDC, S-adenosylmethionine decarboxylase; SPDS, spermidine synthase; SPMS, spermine synthase; ACL5, ACAULIS5.
Figure 3. Phylogenetic analysis of polyamine biosynthesis proteins. The protein sequences of polyamine biosynthesis were extracted from pepper (Ca, green font), tomato (Sl, red font) and Arabidopsis (At, blue font). Multiple sequence alignments were generated with the ClustalW, and the phylogenetic tree was constructed using neighbor-joining method (NJ). ADC, arginine decarboxylase; AIH, agmatine iminohydrolase; CPA, N-carbamoylputrescine amidohydrolase; ODC, ornithine decarboxylase; SAMDC, S-adenosylmethionine decarboxylase; SPDS, spermidine synthase; SPMS, spermine synthase; ACL5, ACAULIS5.
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Figure 4. Chromosomal locations of polyamine biosynthesis genes. Chromosomal mapping was based on the physical position in 12 pepper chromosomes. The polyamine biosynthesis genes marked in red. The yellow numbers represent chromosome numbers. Blue to red on the chromosome indicates gene density. The scale on the left is in megabytes (Mb).
Figure 4. Chromosomal locations of polyamine biosynthesis genes. Chromosomal mapping was based on the physical position in 12 pepper chromosomes. The polyamine biosynthesis genes marked in red. The yellow numbers represent chromosome numbers. Blue to red on the chromosome indicates gene density. The scale on the left is in megabytes (Mb).
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Figure 5. Collinear analysis of polyamine biosynthesis genes among pepper (Capsicum annuun), tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). The segmental duplicated genes are connected by blue lines in different species. Chromosome numbers are located above or below the chromosomes.
Figure 5. Collinear analysis of polyamine biosynthesis genes among pepper (Capsicum annuun), tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). The segmental duplicated genes are connected by blue lines in different species. Chromosome numbers are located above or below the chromosomes.
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Figure 6. The different tissues and organs of pepper, as well as the expression profiles of polyamine biosynthesis genes. (A–G) The different tissues and organs of ‘Ruila Jiaozhongyu’ pepper are (A) seeds, (B) flowers, (C) fruits, (D) young leaves, (E) stems, (F) mature leaves, and (G) roots. (H) The heatmap indicates the expression patterns of polyamine biosynthesis genes in various tissues and organs of peppers. CaACTIN was used as an internal control and the relative expression levels of genes were based on the gene expression of seed. Values represent the averages of three independent measurements.
Figure 6. The different tissues and organs of pepper, as well as the expression profiles of polyamine biosynthesis genes. (A–G) The different tissues and organs of ‘Ruila Jiaozhongyu’ pepper are (A) seeds, (B) flowers, (C) fruits, (D) young leaves, (E) stems, (F) mature leaves, and (G) roots. (H) The heatmap indicates the expression patterns of polyamine biosynthesis genes in various tissues and organs of peppers. CaACTIN was used as an internal control and the relative expression levels of genes were based on the gene expression of seed. Values represent the averages of three independent measurements.
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Figure 7. The expression profiles of pepper polyamine biosynthesis genes by exogenous phytohormones. (A–E) represent the expression of polyamine biosynthesis genes in pepper leaves for six different phytohormones treatments. IAA: 100 μM indole-3-acetic acid; GA3: 100 μM gibberellin; ETH: 100 μM ethylene; ABA: 100 μM abscisic acid; SA: 100 μM salicylic acid. CaACTIN was used as an internal control and the relative expression levels of genes were based on 0 h. Values represent the averages of three independent measurements.
Figure 7. The expression profiles of pepper polyamine biosynthesis genes by exogenous phytohormones. (A–E) represent the expression of polyamine biosynthesis genes in pepper leaves for six different phytohormones treatments. IAA: 100 μM indole-3-acetic acid; GA3: 100 μM gibberellin; ETH: 100 μM ethylene; ABA: 100 μM abscisic acid; SA: 100 μM salicylic acid. CaACTIN was used as an internal control and the relative expression levels of genes were based on 0 h. Values represent the averages of three independent measurements.
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Figure 8. The expression profiles of pepper polyamine biosynthesis genes during abiotic stress. (A–F) represent the expression of polyamine biosynthesis genes in pepper leaves for six different stress treatments. Low-temperature: 4 °C; PEG600: 20% polyethylene glycol-6000; High-temperature: 42 °C; NaCl: 200 μM NaCl; Saline-alkaline: 200 mM saline-alkaline solution; H2O2: 100 mM hydrogen peroxide. CaACTIN was used as an internal control and the relative expression levels of genes were based on 0 h. Values represent the averages of three independent measurements.
Figure 8. The expression profiles of pepper polyamine biosynthesis genes during abiotic stress. (A–F) represent the expression of polyamine biosynthesis genes in pepper leaves for six different stress treatments. Low-temperature: 4 °C; PEG600: 20% polyethylene glycol-6000; High-temperature: 42 °C; NaCl: 200 μM NaCl; Saline-alkaline: 200 mM saline-alkaline solution; H2O2: 100 mM hydrogen peroxide. CaACTIN was used as an internal control and the relative expression levels of genes were based on 0 h. Values represent the averages of three independent measurements.
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Table 1. The polyamine biosynthesis genes in pepper.
Table 1. The polyamine biosynthesis genes in pepper.
Gene ID from PGDBNamePositionCoding Sequences (bp)IntronSubcellular Localization
CaT2T08g03073CaADC1Chr08: 184132402~18413457621750Mitochondrion.
CaT2T10g00793CaADC2Chr10: 100641424~10064519612964Mitochondrion.
CaT2T12g01633CaAIHChr12: 128776924~128789031155710Extracell. Mitochondrion.
CaT2T11g00431CaCPAChr11: 10439223~104441418467Cytoplasm.
CaT2T03g01685CaODC1Chr03: 30721970~3072312310860Mitochondrion.
CaT2T03g01687CaODC2Chr03: 30758389~3075959112030Mitochondrion.
CaT2T04g00108CaODC3Chr04: 1757160~175846713080Mitochondrion.
CaT2T01g03927CaSAMDC1Chr01: 227578235~22757924510110Cytoplasm.
CaT2T05g00570CaSAMDC2Chr05: 19555986~1956495124697Cytoplasm.
CaT2T06g02607CaSAMDC3Chr06: 73468151~7346915510050Cytoplasm. Extracell.
CaT2T11g02366CaSAMDC4Chr11: 268736228~26873731010830Cytoplasm. Extracell. Nucleus.
CaT2T11g01658CaSPDSChr11: 210985086~21098983611047Cytoplasm. Nucleus.
CaT2T03g02219CaSPMSChr03: 42757075~4276451210449Cytoplasm.
CaT2T02g00038CaACL5-1Chr02: 4353693~436037110329Cytoplasm. Nucleus.
CaT2T03g04212CaACL5-2Chr03: 259804076~2598083207566Cytoplasm.
CaT2T08g00347CaACL5-3Chr08: 74094704~741000576546Cytoplasm.
Table 2. The physicochemical properties of the polyamine biosynthesis proteins in pepper.
Table 2. The physicochemical properties of the polyamine biosynthesis proteins in pepper.
NameNumber of Amino Acid (aa)Molecular Weight (kDa)Theoretical pIInstability IndexAliphatic IndexGrand Average of Hydropathicity
CaADC172478.155.0843.6488.23−0.053
CaADC243148.026.3248.5097.59−0.058
CaAIH51856.445.5430.8690.62−0.117
CaCPA28131.495.7832.5078.15−0.334
CaODC136140.007.6540.4195.93−0.060
CaODC240043.895.8441.0094.570.025
CaODC343546.945.5940.7185.290.007
CaSAMDC133637.935.4044.6079.14−0.061
CaSAMDC282292.007.7942.5177.53−0.360
CaSAMDC333437.515.7344.6077.90−0.082
CaSAMDC436039.385.2839.5982.89−0.099
CaSPDS36740.345.0951.2087.85−0.142
CaSPMS34738.665.1743.9287.09−0.165
CaACL5-134339.085.3134.0084.14−0.278
CaACL5-225128.445.2533.2781.16−0.414
CaACL5-321724.635.4029.1077.24−0.327
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Lin, D.; Zhao, X.; Hu, Q.; Wang, S.; Zhang, Y.; Xu, Z. Genome-Wide Identification, Phylogenetic Analysis, and Expression Pattern of Polyamine Biosynthesis Gene Family in Pepper. Int. J. Mol. Sci. 2025, 26, 8208. https://doi.org/10.3390/ijms26178208

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Lin D, Zhao X, Hu Q, Wang S, Zhang Y, Xu Z. Genome-Wide Identification, Phylogenetic Analysis, and Expression Pattern of Polyamine Biosynthesis Gene Family in Pepper. International Journal of Molecular Sciences. 2025; 26(17):8208. https://doi.org/10.3390/ijms26178208

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Lin, Duo, Xianqi Zhao, Qingshan Hu, Su Wang, Yan Zhang, and Zijian Xu. 2025. "Genome-Wide Identification, Phylogenetic Analysis, and Expression Pattern of Polyamine Biosynthesis Gene Family in Pepper" International Journal of Molecular Sciences 26, no. 17: 8208. https://doi.org/10.3390/ijms26178208

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Lin, D., Zhao, X., Hu, Q., Wang, S., Zhang, Y., & Xu, Z. (2025). Genome-Wide Identification, Phylogenetic Analysis, and Expression Pattern of Polyamine Biosynthesis Gene Family in Pepper. International Journal of Molecular Sciences, 26(17), 8208. https://doi.org/10.3390/ijms26178208

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