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Article

CaHY5 Mediates UV-B Induced Anthocyanin Biosynthesis in Purple Pepper

by
Xiang Zhang
1,†,
Yunrong Mo
1,†,
Huidan Zhou
1,
Mengjuan Li
1,
Hong Cheng
1,
Pingping Li
1,
Ruihao Zhang
1,
Yaoyao Huang
1,
Yanyan Wang
1,
Junqiang Xu
1,
Jingjing Liao
1,
Qiaoli Xie
2,
Kai Zhao
1,
Minghua Deng
1 and
Junheng Lv
1,*
1
Key Laboratory of Vegetable Biology of Yunnan Province, College of Landscape and Horticulture, Yunnan Agricultural University, Kunming 650201, China
2
College of Bioengineering, Chongqing University, Chongqing 400044, China
*
Author to whom correspondence should be addressed.
†
These authors have contributed equally to this work.
Agronomy 2025, 15(1), 28; https://doi.org/10.3390/agronomy15010028
Submission received: 7 November 2024 / Revised: 19 December 2024 / Accepted: 24 December 2024 / Published: 26 December 2024
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
Browse Figures
Versions Notes

Abstract

:
Anthocyanins are important flavonoid compounds in plants that are associated with the color formation and antioxidant activity of flowers, fruits, and other organs. Ultraviolet B radiation (UV-B) is one of the key environmental factors that influence anthocyanin accumulation in plants and HY5 is involved in plant photomorphogenesis. However, the molecular mechanism of the signal network of UV-B regulating anthocyanin biosynthesis in capsicum via HY5 remains unclear. In this study, we identified the transcription factor CaHY5, which mediates UV-B signaling, and demonstrated its regulatory role in anthocyanin biosynthesis in purple pepper (Capsicum annuum L.). The results showed that there were photoresponsive and hormone-responsive elements on the CaHY5 promoter that responded to UV-B, indoleacetic acid, salicylic acid, 6-benzyladenine, abscisic acid, and melatonin treatments. UV-B treatment induced the expression of CaHY5 and anthocyanin structural genes. CaHY5 gene-silenced positive plants showed different degrees of the yellowing phenomenon, which affected the expression of the anthocyanin biosynthesis structural gene. The expression levels of anthocyanin biosynthesis-related genes in CaHY5-silenced positive plants increased considerably. This study provides insights into the role of CaHY5 in UV-B-induced anthocyanin biosynthesis in purple pepper.

1. Introduction

Various tissues of purple pepper (Capsicum annuum L.) exhibit a purple or purplish-black color due to the substantial accumulation of anthocyanins in the tissues [1]. Using high-performance liquid chromatography (HPLC) and other approaches, Taylor [2] discovered that the leaves of purple pepper contain a high concentration of a single anthocyanin, delphinidin-3-p coumaroylrutinoside-5-glucoside. This component confers a distinctive phenotype on capsicum [3].
Anthocyanins fall under the category of flavonoid compounds. The fundamental framework of anthocyanins consists of two benzene rings linked by a C3 unit, namely, the 2-phenylbenzopyrane structure, featuring hydroxyl and methoxy groups at different positions. Based on their distinct substitution positions, plant anthocyanins can be classified into six types, namely delphinidin, peonidin, cyanidin, petunidin, malvidin, and pelargonidin [4,5,6]. In plants, anthocyanins are extensively distributed as secondary metabolites, which endow plants with blue, red, and purple hues, and have high ornamental and economic values. Anthocyanins not only attract pollinators and seed dispersal agents for reproduction, but also protect plants against various abiotic and biotic stresses [7,8,9].
Anthocyanins are predominantly accumulated in diverse plant organs, such as stems, leaves, flowers, and fruits, with the primary accumulation site being the vacuole [6,10]. Anthocyanins are also crucial pigments that confer various colors to plants. Phenylalanine serves as the direct precursor for anthocyanin biosynthesis. Anthocyanin uses phenylalanine as the substrate within plant cells and ultimately forms anthocyanin components through a succession of enzymatic reactions [11]. Genes influencing anthocyanin metabolism are categorized into structural and regulatory genes. The structural genes directly encode the enzymes necessary for the biosynthesis of anthocyanins, including PAL, CHS, CHI, F3H, F3′5′H, DFR, ANS, and UFGT [12]. Anthocyanin permeability enzyme, ANP and glutathione transporter enzyme, GS are also requisite for anthocyanin biosynthesis, transportation, and enrichment in vacuoles [13].
Presently, the regulatory genes associated with anthocyanin biosynthesis primarily include R2R3-MYB, BHLH, and WD40 transcription factors [14,15,16]. MYB transcription factors co-regulate the expression of related structural genes by interacting with bHLH and WD40 to form a MBW complex. According to Tang [17], CaANT1, CaANT2, CaAN1, and CaTTG1 are involved in anthocyanin accumulation in purple pepper and they can activate anthocyanin accumulation by forming new MBW transcription complexes. In addition to the MBW complex, transcription factors, such as PIF3, HY5, COP1, WRKY, WIP, MADS-box, NAC, and SPL are involved in transcriptional regulation of anthocyanin biosynthesis [18,19,20].
As one of the key environmental factors, light affects anthocyanin biosynthesis in plants [21]. The structural genes encoding enzymes associated with the anthocyanin biosynthetic pathway are predominantly regulated by light and their expression is upregulated under a high light intensity, but decreased or not expressed under low light or dark conditions [22,23]. Ultraviolet (UV) light, which is a component of the light spectrum, can induce anthocyanin biosynthesis in plants and because the ozone layer absorbs UV light below 280 nm, only UV-A and UV-B are of biological importance [24]. UV-B can directly promote anthocyanin accumulation by inducing the expression of plant anthocyanin biosynthesis structural genes, such as CHS, ANS, UFGT, among others [25,26]. UV-B can regulate phenylalanine ammonia-lyase (PAL) enzyme activity, affect anthocyanin biosynthesis in apple peels, and enhance apple coloring [27,28]. In A. thaliana, UV-B radiation can induce the expression of the CHS gene, promote the synthesis of phenylpropane substances, and enhance UV-B radiation absorption by plants, thereby reducing the damage caused by UV-B radiation to plants [29]. The expression of CHS and CHI genes involved in the flavonoid biosynthetic pathway is substantially upregulated in maize leaves treated with UV-B [30,31,32].
The transcription factor, HY5 belongs to the basic leucine zipper family (bZIP transcription factor), which is closely associated with plant photomorphogenesis. HY5 occurs in different states when plants are under different darkness and light conditions. HY5 is retained in the plant nucleus in the form of a small amount of phosphorylated HY5 under dark conditions, and COP1 interacts with and degrades HY5. COP1 is transferred to the cytoplasm when plants are exposed to light, leading to the accumulation of HY5 [33,34]. Furthermore, HY5 promotes photomorphogenesis and regulates CHS in anthocyanin biosynthesis. In A. thaliana, HY5 inhibits MYBL2 expression by controlling MYBD to promote anthocyanin accumulation [35].
In the UV-B signal transduction pathway, HY5 can directly bind to the anthocyanin synthase structural gene and cis-acting elements of the transcription factor promoter to regulate anthocyanin biosynthesis [36]. In eggplant (Solanum melongena), SmHY5 can directly bind to the G-box element in the promoter region of SmMYB35 gene to regulate the expression of SmMYB35 [37] (Li et al., 2022). HY5 has been shown to regulate anthocyanin biosynthesis through transcriptional activation of PAP1. HY5 can recognize and bind to the MYBL2 promoter, and inhibit its expression. miR858a inhibits the expression of MYBL2 and is a negative regulator of anthocyanin biosynthesis. HY5-MIR858a-MYBL2 complex regulates phytoanthocyanin biosynthesis through transcriptional and post-transcriptional regulation in response to light and other environmental factors [37]. UV-B induces BBX31 expression to promote flavonoid accumulation and HY5 can directly bind to the BBX31 promoter and regulate its transcription level [38]. Anthocyanin accumulation in eggplants is regulated through light-mediated interaction of CRY1/CRY2-COP with SmHY5 and SmMYB1. PIF3 and HY5 directly bind to different regions of the same anthocyanin biosynthesis gene promoter to activate it and positively regulate anthocyanin biosynthesis, but are not directly associated with photochrome-mediated signaling [39].
To investigate the regulation of genes associated with anthocyanin biosynthesis in purple pepper by UV-B and HY5, this study analyzed the mechanisms of the transcription factor, CaHY5 in regulating anthocyanin biosynthesis in purple pepper under different durations of UV-B treatment. In addition, the expression of HY5 and anthocyanin biosynthesis-related genes, and changes in anthocyanin content in purple pepper under different durations of UV-B treatment were evaluated.

2. Materials and Methods

2.1. Experimental Materials

The purple pepper (C. annuum L.) used in this study was obtained from the Key Laboratory of Vegetable Biology of Yunnan Province, College of Landscape and Horticulture, Yunnan Agricultural University, Kunming, China. Under high light intensity, the exposed part of purple pepper appeared purple or purplish-black. The experimental materials were seeded and planted in cultivation pots with a diameter of 24.6 cm and a depth of 16 cm in late April of the same year. The inflorescences were artificially pollinated after they unfolded and a follow-up experiment was performed after 25 days of fruit development.

2.2. CaHY5 TFs Cloning and Bioinformatics Analysis

Total RNA was extracted from purple pepper using an RNA extraction kit (Huayueyang Biotech Co., Ltd., Beijing, China) and the first strand cDNA was synthesized using PrimeScript reverse transcription kit (Takara Bio Inc., Kusatsu, Japan). A novel rapid plant genomic DNA extraction kit (BioTeke Corporation, Beijing, China) was used to extract total DNA from purple pepper. Using cDNA as template, CaHY5 RT-PCR specific primers were designed using Primer Premier 6.0 (FOR: GGTGCTTATTCATTCCATCAG; REV: AATCAGAACAAGACAGTCCA) to clone CaHY5. The 2000 bp DNA sequences upstream of CaHY5 gene coding sequences in the Zunla genome database were used as the reference sequence. Primers were designed (FOR: ACAACGGGTATGGCAAATCTTA; REV: GGAAGCAGCCTGATGGAATG), cloned, and sequenced the upstream promoter sequence of CaHY5 using total DNA extracted from purple pepper as the template. The upstream cis-acting elements were predicted using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 6 December 2021)). The cis-acting elements were screened and sorted using Microsoft Excel 365 (Microsoft Corp., Redmond, WA, USA), and visualized using Tbtools. The cloning reaction system and procedures used in the study are described in detail in Supplementary Table S1. Bioinformatics analysis of CaHY5 gene and protein sequences was performed according to a previously described method [40].

2.3. Subcellular Localization of CaHY5

The pAN580-GFP vector was double-digested with XbaI and BamHI restriction enzymes (New England Biolabs, Ipswich, MA, USA), and the full-length CaHY5 gene sequence was used as the insertion fragment. The inserted fragment and linearized vector were recombined into a pCaHY5-AN580 recombinant vector using In-Fusion HD Cloning kit (Takara Bio Inc., Kusatsu, Japan), according to the manufacturer’s instructions. Subcellular localization of CaHY5 was analyzed according to a previously described method [40].

2.4. Hormone Treatments

The concentration of treated hormone was determined by reference to relevant literature and pre-experiment [41]. Aqueous solutions of indole-3-acetic acid (IAA, 10 μM), 6-benzyladenine (6-BA, 1 μM), melatonin (MT, 100 μM), salicylic acid (SA, 100 μM), and abscisic acid (ABA, 1 μM) were prepared(Sigma-Aldrich, MO, USA). Uniformly grown and sized purple pepper fruits were selected and 30 biological replicates were set for each treatment. Distilled water was used as the control. The plant hormone solutions of each concentration were evenly sprayed on the surface of purple pepper plants. Purple pepper fruit samples were collected at 0, 0.5, 1, 3, and 6 h. The treated and control fruits were stored at −80 °C after quick freezing in liquid nitrogen or used directly to conduct subsequent tests.

2.5. Light Treatments

After 25 days of fruit development, purple pepper plants were subjected to UV-B treatment by placing a UV-B lamp (313 nm, irradiance range was less than or equal to 0.8 W/m2) 30 cm away from the plants [42]. Under the same conditions, the purple pepper plants were placed in an incubator with 100% light to simulate white light and 0% light to simulate dark treatment. The temperature of the incubator was set to 28 °C for all treatments. The leaves, stems, and fruits of purple pepper plants treated with UV-B, white light, and dark conditions over the same period (25 days after flowering) were sampled at 0, 0.5, 1, 3, 6, 12, and 24 h, frozen in liquid nitrogen, and stored at −80 °C for subsequent analysis.

2.6. Anthocyanin Content Determination

Purple pepper samples (0.5 g) were mixed with liquid nitrogen and ground into powder in a mortar. Thereafter, 95% ethanol–1.5 mol·L−1 hydrochloric acid (85:15, v/v) mixture was added to the samples and ultrasonic extraction was performed for 30 min, followed by centrifugation at 4 °C and 5000 rpm for 5 min. The supernatant was collected as a liquid and used to determine anthocyanin content by HPLC (Agilent 1260; Agilent Technologies, Santa Clara, CA, USA), according to the method described by [43].

2.7. Analysis of Relative Gene Expression

The cDNA was synthesized from untreated purple pepper organs using the method described in the preceding section. Quantitative real-time (qRT-PCR) was performed using Hieff qPCR SYBR Green Master Mix kit (Yeasen, Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer’s instructions. qRT-PCR reaction was performed on QuantStudio5 and the relative gene expression was calculated using the 2−ΔΔCt method. The primers used are listed in Supplementary Table S2.

2.8. Virus-Induced Gene Silencing

For gene silencing, CaHY5 fragment was silenced using the SGN VIGS tool (https://vigs.solgenomics.net/ (accessed on 10 April 2021)). The pTRV2 plasmid was double-digested with EcoR1 and BAMH1 restriction enzymes (New England Biolabs, Ipswich, MA, USA), and the pTRV2::CaHY5 silencing plasmid was reconstructed. pTRV1, pTRV2::PDS, and pTRV2::CaHY5 were transformed into the Agrobacterium strain GV3101. GV3101 pTRV1 with an OD600 = 0.8 was mixed with pTRV2::HY5, pTRV2::PDS, and pTRV2::00 bacterial solutions in a ratio of 1:1. The bacterial solution was gradually injected on the back of the two purple pepper cotyledons after they unfolded using a syringe. The expression levels of CaHY5 and ABGs related genes were determined by qRT-PCR. The positive silenced plants were exposed to white light, darkness, and UV-B, and the expression levels of related genes were determined after 3 h of treatment. Three biological replicates were set for each test [40]. The primers used are listed in Supplementary Table S3.

2.9. Transient Expression of CaHY5 in Purple Pepper

Full-length cDNAs of CaHY5 were cloned into pCambia1300: GUS [43]. The Agrobacterium tumefaciens strain GV3101 harboring pCambia1300-CaHY5: GUS or pCambia1300-GUS (used as control) was cultured overnight in an induction medium and the infiltrated tissue was collected three days after inoculation for further analysis [43]. Agrobacterium tumefaciens strain GV3101 harboring each construct was injected on the back of the purple pepper leaves. The expression levels of CaHY5 and ABGs related genes were determined by qRT-PCR. The positive silenced plants were exposed to white light, darkness, and UV-B, and the expression levels of related genes were determined after 3 h of treatment. Three biological replicates were set for each test. The primers used are listed in Supplementary Table S3.

2.10. Statistical Analyses

Data collection and sorting were conducted using Microsoft Excel 365 (Microsoft Corp., Redmond, WA, USA). IBM SPSS Statistics 23.0 software was used to conduct Duncan’s multiple range tests for one-way ANOVA. Significant differences were indicated when p < 0.05. Least significant difference test was used to determine significant differences between treatments. Graphs were plotted using GraphPad Prism 9 (GraphPad Software Inc., La Jolla, CA, USA).

3. Results

3.1. Anthocyanin Content in Purple Pepper After Exposure to Different Light Treatments

After 24 h of UV-B continuous treatment, there was no significant difference in the phenotype of purple peppers. Large purple spots were observed on purple pepper leaves after 20 days of UV-B treatment. The color of purple pepper leaves after UV-B treatment was purple, while the color of purple pepper leaves exposed to the dark treatment was yellow (Figure 1). Delphinin was only detected in purple pepper plants exposed to UV-B and its content was 248.56 μg·g−1.

3.2. Expression Levels of Transcription Factors and Anthocyanin Biosynthesis Genes in Purple Pepper Fruits Under Different Light Treatments

The results showed that UV-B treatment had varying effects on purple pepper fruits when compared to white light and dark treatments (Figure 2). Based on gene expression, the transcription factors CaHY5 and CaMYB113 were clustered together with anthocyanin biosynthesis structural genes CaDFR, CaUFGT, CaF3H, CaC4H, and Ca4CL, with similar expression patterns being observed. The anthocyanin biosynthesis structural genes, CaPAL and CaF3′5′H exhibited high expression levels, especially under UV-B treatment.

3.3. CaHY5 Cloning and Bioinformatics Analysis

The CaHY5 gene in purple pepper was isolated by homologous cloning method. The gel electrophoresis of CaHY5 gene amplification is shown in Supplementary Figure S1. Sequencing of specific amplification products showed that the RT-PCR product was 788 bp in length, contained an open reading frame of 477 bp, and encoded 158 amino acid residues (Supplementary Figure S2). The HY5 gene sequence obtained was homologous to the HY5 sequence in the NCBI database (GeneID: 107838859); therefore, the HY5 in purple pepper was named CaHY5. The molecular formula of CaHY5 protein was C715H1216N240O251S4 and its molecular weight was 1.73 kDa. The CaHY5 protein had a theoretical isoelectric point of 9.69, was fat-soluble, hydrophilic, and unstable. Moreover, the CaHY5 protein had no signal peptide, no transmembrane structure, and had 24 phosphorylation sites, including 7 T-type, 17 S-type, and no Y-type phosphorylation site.
The transcription factor CaHY5 belongs to the leucine zipper or bZIP family. Their site for protein structure domain: (ENKRLKRLLRNRVSAQQARERKKAYLIDLEARVKELETKNAELEERLSTLQNENQMLRHILKNT) 86–149. The amino acid sequence of CaHY5 of purple pepper was compared by BLAST and 15 significant conserved motifs were obtained by performing the motif significance test (Figure 3A). Motifs 8, 3, 7, 5, 2, and 1 had six different conserved motifs, which are the predicted motifs in plants, and they influence the function of these proteins. Combined with the prediction results of CaHY5 protein domain, motifs 2 and 1 cover the bZIP domain, which is the specific motif for this type of protein.
The prediction results of the secondary structure of proteins showed that the CaHY5 protein consisted of 77 α helices (48.73%), 71 random crimps (44.94%), nine β corners (5.70%), and one extended chain (0.63%) (Supplementary Figure S2). The tertiary structure of the CaHY5 protein was predicted based on I-TASSER. The predicted CaHY5 ligand structure C value was 0.20, and the binding site residues were 96, 99, 100, and 103 (Supplementary Figure S2). A phylogenetic tree was constructed using the adjacency matrix method with a total of 51 HY5 amino acid sequences from nine families selected (Figure 3B). The results showed that CaHY5 had the farthest relationship with legumes (Fabaceae) and tobacco (Solanaceae), but had the closest relationship with capsicum (Solanaceae).

3.4. Subcellular Localization of CaHY5 in Purple Pepper

The pCaHY5-AN580-GFP recombinant vector (Figure 4A) was transferred into the protoplasts of A. thaliana using an empty pAN580-GFP vector as a control. According to the results, pAN580-GFP exhibited green fluorescence in the protoplast nucleus, cytoplasm, and cell membrane of A. thaliana, while the fusion protein pCaHY5-AN580-GFP exhibited green fluorescence in the protoplast nucleus (Figure 4B), indicating that the CaHY5 protein was located in the nucleus.

3.5. Upstream Cis-Element Analysis of CaHY5 and Heat Map of CaHY5 Expression Under Different Exogenous Factors

The upstream promoter of CaHY5 gene in purple pepper was cloned using total DNA of purple pepper as template, and its length was 1935 bp after sequencing (Supplementary Figure S3). PlantCARE prediction results showed that the CaHY5 promoter of purple pepper differed from that of Zunla. CaHY5 in purple pepper had defense against stress and hormone response cis-elements, such as ABA and SA (Figure 5A), indicating that CaHY5 was regulated by stress and plant hormones. To verify whether different plant hormones affect CaHY5 expression in purple pepper, water was used as a control in this study and quantitative fluorescence PCR was used to determine the expression level of CaHY5 under five different plant hormones (IAA, SA, 6-BA, ABA, and MT). The results showed that IAA, SA, 6-BA, ABA, and MT affected CaHY5 expression after 0.5–6 h of plant hormone treatments and the expression patterns of CaHY5 varied under different plant hormone treatments. CaHY5 expression was considerably affected by IAA and MT treatments, with its expression reaching a peak 1 h after IAA treatment. CaHY5 expression was substantially upregulated under MT treatment, although its expression after 1 h of treatment was low (Figure 5B).

3.6. Downregulation of CaHY5 Decreases Genes of Anthocyanin Biogenesis Pathway Expression

The anthocyanin biogenesis pathway and virus-induced gene silencing (VIGS) vector are shown in Figure 6A,B. The results showed that the CaPDS-silenced purple pepper plants at the cotyledon stage were bleached 21 days after inoculation (Figure 6C), indicating that the silencing system was precise. The leaves of CaHY5-silenced plants exhibited different degrees of bleaching, although the change in leaf color was weaker than that of CaPDS-silenced plants. The qPCR was used to determine the expression levels of CaHY5 gene and anthocyanin biosynthesis-related genes in CaHY5-silenced plants. The results showed that expression level of the CaHY5 gene was downregulated to varying degrees. CaHY5 expression in CaHY5-silenced plants was significantly different from that in control and pTRV2::00 plants, indicating that CaHY5 gene silencing was successful. The expression levels of all anthocyanin biosynthesis-related genes were downregulated to varying degrees and differences in their expression levels were significant (p < 0.05). Among the anthocyanin biosynthesis-related genes, CaHY5 had the greatest effect on CaF3H gene after silencing and its expression level was only 0.41% when compared to that of the control (p < 0.05) (Figure 6D).
Regarding the effect of light on gene expression, the results showed that anthocyanin biosynthesis-related genes in purple pepper plants exposed to different light conditions were upregulated to varying degrees (Figure 6E). The expression level of the CaHY5 gene was significantly higher in positive silenced plants exposed to white light and UV-B treatments than that in untreated silenced plants (p < 0.05), indicating that white light and UV-B induced CaHY5 gene expression, with UV-B exerting the strongest effect (p < 0.05). The expression of the CaCOP1 gene was upregulated to a certain extent under dark and UV-B treatments. White light had the greatest effect on CaMYB113, followed by UV-B treatment (p < 0.05). UV-B treatment exerted the strongest effect on structural genes associated with anthocyanin biosynthesis (p < 0.05), with the expression levels of CaPAL, CaC4H, and Ca4CL increasing substantially. However, the expression levels of CaCHS, CaF3H, and CaF3′5′H were slightly increased (p < 0.05).

3.7. Transient Overexpression of CaHY5 in Purple Pepper

GUS staining results showed that both 35S:GUS and 35S:CaHY5-GUS leaves stained blue (Figure 7A), indicating successful CaHY5-GUS expression. The results of qPCR showed that the expression level of CaHY5 in purple pepper leaves treated with 35S: CaHY5-GUS increased significantly by 111-fold that in leaves treated with 35S:GUS (p < 0.05) (Figure 7B). The transcription factor CaMYB113 was upregulated together with CaC4H, CaF3H, CaF3′5′H, CaDFR, and CaANS to varying degrees. Overexpression of CaHY5 had a significant effect on the expression of CaF3H and CaDFR genes (p < 0.05). The results suggested that CaHY5 regulated anthocyanin biosynthesis in purple pepper by regulating MYB113 and anthocyanin biosynthesis genes.

4. Discussion

4.1. CaHY5 Gene Is Regulated by Several Factors

As key transcription factors involved in photomorphogenesis, 15 cis-elements related to photoresponse, as well as plant hormone and anaerobic responses were identified at 2000 bp upstream of the HY5 gene in purple pepper. In addition to participating in photomorphogenesis, HY5 facilitates the regulation of plant hormones during the growth of purple pepper. According to previous studies, HY5 is involved in the signaling pathways of gibberellins, cytokinins, and other plant hormones [44]. The CaHY5 gene is a key factor influencing responses to abiotic stress, reactive oxygen species, and other signaling pathways. In this study, the expression of CaHY5 gene was upregulated to varying degrees under different exogenous factors, suggesting that it regulated hormone signaling and response in purple pepper. In addition, the results showed that MT had the greatest effect on the up-regulation of CaHY5 expression, indicating that CaHY5 gene was involved in MT signal response.

4.2. Effect of Different Light Conditions on Anthocyanin Biosynthesis in Purple Pepper

Light is one of the key environmental factors affecting plant growth and development. HY5, a member of the ZIP transcription factor family, regulates plant growth and development, as well as pigment accumulation in a light-dependent manner [44,45]. The results of this study revealed significant differences in the expression patterns of CaHY5 and CaCOP1 genes in purple pepper under white light and dark treatments. Specifically, the expression levels of CaHY5 and CaCOP1 genes under UV-B treatment, particularly in purple pepper fruits increased considerably. The results indicate that CaHY5 and CaCOP1 genes are involved in regulating the response of purple pepper to UV-B.
With regard to structural genes involved in anthocyanin biosynthesis, the expression levels of CaPAL, CaCHI, CaF3′5′H, and CaANS were significantly upregulated by 122.3~2183.1-fold under white light treatment. The expression levels of CaPAL, CaCHS, CaCHI, CaF3′5′H, CaANS, and other genes were substantially affected by UV-B treatment, with their expression levels being upregulated by 158.2~6271.6-fold. Studies have shown that almost all genes involved in anthocyanin biosynthesis are affected by light [46]. However, UV-B can regulate PAL enzyme activity and directly induce the expression of CHS, CHI, ANS and other anthocyanin biosynthesis genes in plants, which is consistent with the results of this study [25,26,30,31]. Therefore, structural genes involved in anthocyanin biosynthesis in purple pepper can be regulated indirectly by the transcription factor, CaHY5 or directly in response to UV-B. Although the phenotypic changes in purple pepper were not evident after 24 h of continuous treatment, large areas of purple plaques were observed on the surfaces of leaves after 20 days of UV-B treatment. The color of purple pepper leaves turned purple after UV-B treatment. However, the color of purple pepper leaves turned yellow after dark treatment (Figure 1). The results suggested that UV-B treatment induced anthocyanin biosynthesis and accumulation, but the accumulation occurred after a certain duration of time.
Notably, some anthocyanin biosynthesis genes were also upregulated to varying degrees under dark conditions, but the overall upregulation was minimal [3,6]. Zhou [47] found that the predominant anthocyanin in pepper was delphinidin. A small amount of delphinidin (3-O-glucoside) and a substantially low amount of pelargonidin (3-O-galactoside) are still synthesized. Furthermore, pelargonidin has been shown to accumulate only under dark conditions. DFR and F3′5′H genes regulate pelargonidin biosynthesis in larkspur flower (Delphinium sp.) [48]. In this study, CaDFR and CaF3′5′H and their downstream synthetic genes were up-regulated to varying degrees in the leaves and fruits of dark treated purple pepper. It is shown that different types of anthocyanins such as anthocyanins under light and dark treatment, although the content is low, but still accumulate.
The bZIP transcription factor, HY5 is a major hub for optical signal cascades under visible and UV-B light [49]. The results of this study showed that the CaHY5-silenced plants exhibited varying degrees of yellowing and white spots on leaves. The expression levels of CaHY5, CaCOP1, and capsaicin biosynthesis genes were not downregulated, whereas those of CaC4H, CaCHS, CaF3H, CaF3′5′H, and CaDFR were substantially downregulated. In A. thaliana, HY5 and LZF1 regulated anthocyanin and chlorophyll accumulation, and played a positive regulatory role in the deyelization process in A. thaliana [21,50]. Therefore, the change in leaf color observed in CaHY5-silenced plants could be because of the decreased expression of anthocyanin and chlorophyll biosynthesis-related genes. In addition, CaPDS gene expression decreased after CaHY5 silencing (Figure 6). PDS is a key gene involved in carotenoid biosynthesis.

4.3. UV-B Induces Expression of Anthocyanin Biosynthesis Genes to Promote Anthocyanin Accumulation

Previous studies have shown that UV-B can directly promote anthocyanin accumulation by inducing the expression of CHS, ANS, UFGT, and other structural genes involved in anthocyanin biosynthesis [25,26]. Under UV-B treatment, CaPAL, CaC4H, and Ca4CL were responsive to anthocyanin biosynthesis genes and the expression of CaCHS, CaF3H, and CaF3′5′H was upregulated. The results suggested that UV-B directly induced the expression of structural genes associated with anthocyanin biosynthesis in purple pepper, which is consistent with the findings of previous studies.

4.4. CaHY5 Directly Regulates CaF3H Expression to Promote Anthocyanin Accumulation in Purple Pepper

Previous studies have shown that the transcription factor, HY5 can directly recognize and bind upstream cis-elements of structural genes associated with anthocyanin biosynthesis in tomato (Solanum lycopersicum), such as G-box and ACGT elements in CHS1, CHS2, and DFR promoters [51]. An analysis of the upstream promoter of capsaicin biosynthesis-related genes revealed that CaHY5 regulated capsaicin biosynthesis by recognizing and binding to the G-box and ACGT elements in the upstream promoter region of capsaicin biosynthesis-related genes. CaHY5 silencing had the greatest effect on the CaF3H gene in CaHY5-silenced plants. Analysis of cis-elements in the upstream promoter region of the CaHY5 gene revealed the presence of G-box cis-elements upstream of the CaF3H gene. Therefore, anthocyanin biosynthesis genes in purple pepper was directly regulated by UV-B and indirectly regulated by CaHY5.
The intensity of UV-B radiation is very important for plants, and appropriate radiation dose can promote the establishment of plant photomorphology and improve plant tolerance to adversity, while excessive radiation dose will lead to plant damage [52]. However, due to the decrease of ozone level, the dose of UV-B radiation in the environment is increasing year by year, so it has great potential threat to the growth and development of plants [53]. Therefore, this study explains how purple pepper responds to UV-B and promotes anthocyanin biosynthesis, which has certain guiding significance in pepper production. Meanwhile, because purple pepper has high ornamental value, research on its anthocyanin biosynthesis will help us better understand its regulatory mechanism and help to cultivate purple pepper varieties.

5. Conclusions

This study showed that anthocyanin biosynthesis in purple pepper under UV-B treatment was regulated through various mechanisms. UV-B directly regulated structural genes involved in anthocyanin biosynthesis. The transcription factor CaHY5 also indirectly regulated the expression of anthocyanin biosynthesis-related genes, such as CaCHS, CaF3H, CaF3′5′H, thereby enhancing anthocyanin accumulation in purple pepper.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15010028/s1, Figure S1: CaHY5 gel electrophoresis; Figure S2: CaHY5 protein structure; Figure S3: Gel electrophoresis of CaHY5 gene promoter clone; Table S1: PCR reaction system and procedure; Table S2: The primer table of qRT-PCR; Table S3: Recombinant primers for plasmid construction.

Author Contributions

Conceptualization, X.Z. and H.Z.; Data curation, X.Z. and H.Z.; Formal analysis, X.Z. and H.Z.; Investigation, Y.M. and M.L.; Methodology, H.C.; Project administration, P.L., R.Z., Y.H. and Y.W.; Resources, J.X. and Q.X.; Software, K.Z.; Writing-original draft, X.Z., H.Z., Y.M., M.L. and H.C.; Visualization, Q.X., K.Z., M.D. and J.L. (Jingjing Liao); Writing–review & editing, Q.X., K.Z., M.D. and J.L. (Junheng Lv). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Science and Technology Projects in Yunnan Province (202205AR070001, 202402AE090012, 202204BI090004, 202202AE090031) and the Joint Project of Basic Agricultural Research in Yunnan Province (202101BD070001-005, 202101BD070001-079, 202301BD070001-016).

Data Availability Statement

All relevant data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effects of different treatments on leaf phenotype of purple pepper. The leaves in the figure show the phenotype of pepper leaves under different treatments. The bottom pair of the leaf should visualize the color block and hexadecimal color code of the leaf color.
Figure 1. Effects of different treatments on leaf phenotype of purple pepper. The leaves in the figure show the phenotype of pepper leaves under different treatments. The bottom pair of the leaf should visualize the color block and hexadecimal color code of the leaf color.
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Figure 2. Heat map clustering of TFs and ABGs in pepper under different light conditions.
Figure 2. Heat map clustering of TFs and ABGs in pepper under different light conditions.
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Figure 3. Phylogenic tree of HY5 transcription factors (TFs)and motif analysis of amino acids in HY5 of different species. (A) Phylogenetic tree comprising CaHY5 in purple pepper and 50 HY5 TFs in plant. (B) Motif analysis of HY5 protein in purple pepper and 50 other plants.
Figure 3. Phylogenic tree of HY5 transcription factors (TFs)and motif analysis of amino acids in HY5 of different species. (A) Phylogenetic tree comprising CaHY5 in purple pepper and 50 HY5 TFs in plant. (B) Motif analysis of HY5 protein in purple pepper and 50 other plants.
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Figure 4. Subcellular localization of CaHY5 in purple pepper. (A) It was the schematic diagram of pCAHY5-AN580-GFP vector construction. (B) Green fluorescent protein (GFP) signals indicated that CaHY5 was localized to nucleus. GFP fluorescence signals were detected using a Zeiss lsm710 confocal laser scanning microscope.
Figure 4. Subcellular localization of CaHY5 in purple pepper. (A) It was the schematic diagram of pCAHY5-AN580-GFP vector construction. (B) Green fluorescent protein (GFP) signals indicated that CaHY5 was localized to nucleus. GFP fluorescence signals were detected using a Zeiss lsm710 confocal laser scanning microscope.
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Figure 5. Upstream cis-element analysis of CaHY5 and heat map of CaHY5 expression under different exogenous factors. (A) Cis-element analysis of 2000 bp upstream of CaHY5 in purple pepper. (B) The expression levels of CaHY5 TFs after 0.5–6 h treatment with IAA, SA, 6BA, ABA, and MT.
Figure 5. Upstream cis-element analysis of CaHY5 and heat map of CaHY5 expression under different exogenous factors. (A) Cis-element analysis of 2000 bp upstream of CaHY5 in purple pepper. (B) The expression levels of CaHY5 TFs after 0.5–6 h treatment with IAA, SA, 6BA, ABA, and MT.
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Figure 6. Anthocyanin biosynthetic pathway, VIGS vector, silenced plant phenotype and related gene expression level. (A) Schematic diagram of anthocyanin biosynthesis pathway. (B) VIGS vector construction diagram. (C) Pepper’s phenotype of VIGS. CK is pepper without inoculation; pTRV2::00 was inoculated with pTRV2 empty vector strain. pTRV2:: CaHY5 was a silent plant of CaHY5. (D) Expression levels of CaHY5 TFs and anthocyanin biosynthesis-related genes after CaHY5 silencing. (E) Effects of different light conditions on genes related to anthocyanin biosynthesis in silenced pepper. The same letters represent no significant difference (p ≥ 0.05), while different letters represent significant difference (p < 0.05).
Figure 6. Anthocyanin biosynthetic pathway, VIGS vector, silenced plant phenotype and related gene expression level. (A) Schematic diagram of anthocyanin biosynthesis pathway. (B) VIGS vector construction diagram. (C) Pepper’s phenotype of VIGS. CK is pepper without inoculation; pTRV2::00 was inoculated with pTRV2 empty vector strain. pTRV2:: CaHY5 was a silent plant of CaHY5. (D) Expression levels of CaHY5 TFs and anthocyanin biosynthesis-related genes after CaHY5 silencing. (E) Effects of different light conditions on genes related to anthocyanin biosynthesis in silenced pepper. The same letters represent no significant difference (p ≥ 0.05), while different letters represent significant difference (p < 0.05).
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Figure 7. GUS staining of pepper tissues and related gene expression level. (A) Staining of 35S:GUS and 35S:CaHY5-GUS pepper tissues. (B) Expression levels of TFs and ABGs after CaHY5 transient overexpression. The same letters represent no significant difference (p ≥ 0.05), while different letters represent a significant difference (p < 0.05).
Figure 7. GUS staining of pepper tissues and related gene expression level. (A) Staining of 35S:GUS and 35S:CaHY5-GUS pepper tissues. (B) Expression levels of TFs and ABGs after CaHY5 transient overexpression. The same letters represent no significant difference (p ≥ 0.05), while different letters represent a significant difference (p < 0.05).
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MDPI and ACS Style

Zhang, X.; Mo, Y.; Zhou, H.; Li, M.; Cheng, H.; Li, P.; Zhang, R.; Huang, Y.; Wang, Y.; Xu, J.; et al. CaHY5 Mediates UV-B Induced Anthocyanin Biosynthesis in Purple Pepper. Agronomy 2025, 15, 28. https://doi.org/10.3390/agronomy15010028

AMA Style

Zhang X, Mo Y, Zhou H, Li M, Cheng H, Li P, Zhang R, Huang Y, Wang Y, Xu J, et al. CaHY5 Mediates UV-B Induced Anthocyanin Biosynthesis in Purple Pepper. Agronomy. 2025; 15(1):28. https://doi.org/10.3390/agronomy15010028

Chicago/Turabian Style

Zhang, Xiang, Yunrong Mo, Huidan Zhou, Mengjuan Li, Hong Cheng, Pingping Li, Ruihao Zhang, Yaoyao Huang, Yanyan Wang, Junqiang Xu, and et al. 2025. "CaHY5 Mediates UV-B Induced Anthocyanin Biosynthesis in Purple Pepper" Agronomy 15, no. 1: 28. https://doi.org/10.3390/agronomy15010028

APA Style

Zhang, X., Mo, Y., Zhou, H., Li, M., Cheng, H., Li, P., Zhang, R., Huang, Y., Wang, Y., Xu, J., Liao, J., Xie, Q., Zhao, K., Deng, M., & Lv, J. (2025). CaHY5 Mediates UV-B Induced Anthocyanin Biosynthesis in Purple Pepper. Agronomy, 15(1), 28. https://doi.org/10.3390/agronomy15010028

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