Functional characterization of StBBX19 and StBBX24 in UV-B-induced anthocyanin biosynthesis in pigmented potato

Abstract

BBX transcription factors can respond to UV-B signals, promote plant photomorphogenesis, and regulate plant anthocyanin biosynthesis. Based on transcriptome sequencing analysis, this study analyzed the transcription factors of StBBXs under UV-B treatment and identified two differentially expressed genes, StBBX19 and StBBX24. We explored the protein structures, promoters, and phylogenetics of StBBX19 and StBBX24, speculating that they respond to UV-B treatment and regulate anthocyanin biosynthesis. We constructed overexpression vectors, YFP, and bimolecular fluorescence complementation vectors for the transient transformation of tobacco. The results suggest that StBBX19 and StBBX24 may not function as direct regulators of anthocyanin biosynthesis, but rather enhance anthocyanin accumulation through interaction with StMYB308. Still, after interacting with StMYB308, purple spots appeared on tobacco leaves, suggesting that the interaction between StBBX19, StBBX24, and StMYB308 increases anthocyanin content in tobacco leaves. This study provides significant evidence for the adaptation mechanism of plants under UV-B treatment and offers new insights for molecular plant breeding of pigmented potatoes.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12870-026-09407-8.

Introduction

Ultraviolet-B (UV-B) radiation, a crucial component of the solar spectrum, serves as a pivotal environmental signal that regulates diverse aspects of plant growth and development, particularly through the modulation of secondary metabolism. As a protective mechanism against UV-induced damage, plants have evolved sophisticated regulatory networks to stimulate the biosynthesis of photoprotective compounds, with anthocyanins representing one of the most important classes of flavonoid pigments that confer antioxidative and UV-shielding properties [1, 2]. The biosynthesis of anthocyanins is intricately controlled by light signaling pathways, wherein UV-B perception triggers a cascade of transcriptional reprogramming events that ultimately lead to the accumulation of these protective pigments. The regulatory machinery governing anthocyanin biosynthesis primarily involves the coordinated action of several transcription factor families, including MYB, bHLH, WD40, and, more recently identified, BBX proteins. These factors often form multi-protein complexes that bind to promoters of structural genes in the anthocyanin pathway, enabling precise spatiotemporal control of pigment accumulation in response to environmental stimuli [3].

Among these, the BBX (B-box) family of zinc finger transcription factors has emerged as a critical component integrating light signaling with anthocyanin biosynthesis across plant species. BBX proteins contain one or two conserved B-box domains that facilitate protein-protein interactions and exhibit diverse functions in plant development and photomorphogenesis [4, 5]. In Arabidopsis thaliana, multiple BBX members participate in UV-B responses through intricate interactions with key light signaling components. BBX24 negatively regulates UV-B signaling by forming inactive heterodimers with ELONGATED HYPOCOTYL 5 (HY5), while BBX31 functions as a crucial signaling intermediate for both visible and UV-B light transduction, operating through HY5-independent pathways [2, 6–8]. The E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) mediates the light-dependent regulation of several BBX members, promoting the expression of BBX5 and BBX18 while suppressing BBX7 and BBX8 under UV-B conditions [2, 8].In fruit crops, BBX proteins demonstrate particularly significant roles in regulating light-induced anthocyanin accumulation. In apple, UV-B radiation promotes the binding of MdHY5 to the promoter of MdCOL11 (a BBX member), enhancing its expression and consequently increasing anthocyanin accumulation in fruit peel [9]. Conversely, MdBBX37 negatively regulates anthocyanin synthesis by interfering with the DNA-binding capacity of MdMYB1 and MdMYB9 to their target genes [10]. Additional apple BBX members, including MdCOL4, MdBBX20, and MdBBX22, have also been implicated in the regulation of anthocyanin biosynthesis [11–13]. Similarly, in Pyrus spp., PpBBX16 promotes anthocyanin accumulation by activating the expression of PpMYB10, whereas PpBBX18 and PpBBX21 act as negative regulators by competitively binding to the pear HY5 protein [14, 15]. A 14-nucleotide deletion in the coding region of PpBBX24 has been linked to the red skin trait in ‘Hongzaosu’ pear [16]. Notably, BBX proteins also play conserved roles in light-mediated pigmentation in ornamental species. In Petunia hybrida, BBX18, BBX24, and BBX25 respond to light and regulate anthocyanin accumulation [17]. In lilies, LvBBX24 enhances anthocyanin synthesis by upregulating the transcription of LvMYB5 [18]. Despite these advances in elucidating BBX functions across model plants and fruit crops, the specific roles and mechanisms of BBX proteins in UV-B-mediated anthocyanin regulation in potato (Solanum tuberosum L.) remain largely unknown. As the fourth most important food crop globally, potato is a critical agricultural commodity, especially pigmented varieties that accumulate substantial anthocyanins in their tubers [3, 19, 20]. These compounds contribute not only to tuber coloration but also to enhanced nutritional value and health benefits, including cancer prevention, anti-aging, and arteriosclerosis prevention [19].

Previous studies have established that light quality significantly influences flavonoid composition in potatoes, with UV and blue-violet light being most effective in inducing flavonoid synthesis [1, 2, 7], while appropriate UV-B doses can promote flavonoid accumulation without detrimental effects on growth [21, 22]. Although UV-B-induced anthocyanin synthesis has been documented in various species including black peanut peanut [23], apple [24], rice [25], and potatoes [26, 27], and certain potato MYB factors like StMYB308 have been shown to promote anthocyanin accumulation in heterologous systems [26], the specific involvement of BBX transcription factors in this process remains unknown. Therefore, to address this knowledge gap, two BBX transcription factors, StBBX19 and StBBX24, which were differentially expressed under UV-B treatment, were identified through transcriptome sequencing. Research has found that the transcriptional levels of these genes are generally correlated with total anthocyanin content, suggesting they may be involved in regulating anthocyanin biosynthesis in potatoes. This study provides a theoretical foundation for further elucidating the mechanisms by which BBX transcription factors modulate UV-B-induced anthocyanin synthesis in potatoes.

Materials and methods

Plant material and sample preparation

The study used two different local, pigmented potato varieties, Huaxinyangyu (H), which has purple skin, yellow flesh, and purple ring patterns, and Jianchuanhong (J), which has red skin, yellow flesh, and red ring patterns. Both tetraploid cultivars (Solanum tuberosum) were provided by the Potato Crop Research Institute of Yunnan Agricultural University. Potato tissue-cultured seedlings were potted in a pasteurized soil mixture and maintained in an artificial climate chamber (24° 03′ 36.90′′ N, 102° 42′′ 01.45′′ E). Established three UV-B treatment regimes: a control (CK, 0 KJ/m²) and two treatment groups that received supplemental UV-B radiation from FLB-5 A lamps (15 W, 280–320 nm): T1 (5 KJ/m², lamp positioned 80 cm above the canopy) and T2 (10 KJ/m², lamp positioned 40 cm above the canopy). The lamp height was adjusted periodically to maintain consistent irradiance relative to plant growth. Tubers were harvested at 48, 60, 72, and 84 days after UV-B treatment initiation. Notably, plants in the T2 group did not survive beyond 72 days; thus, no data were collected for the 84-day time point for this treatment. For each time point and treatment, three biological replicates were collected and immediately frozen in liquid nitrogen and stored at -80 °C for subsequent analysis.

Total anthocyanin content (TAC) was quantified via the pH differential method [28] and expressed as cyanidin-3-glucoside equivalents (mg/100 g fresh weight). Fresh potato tissue (0.5 g) was extracted twice with hydrochloric acid-ethanol solution (10 mL, then 5 mL) using ultrasonication (45 °C, 30 min) followed by centrifugation (8000 rpm, 10 min). The combined supernatant (1 mL) was reacted with both pH 1.0 and pH 4.5 buffers for 1 h. Absorbance at the maximum wavelength (A_max) and at 700 nm (A₇₀₀) was then recorded for both pH conditions. TAC was calculated using the standard formula: , Where A_max and A′_max represent the absorbance at the maximum wavelength for the solutions buffered at pH 1.0 and pH 4.5, respectively; A₇₀₀ and A′₇₀₀ are the absorbances at 700 nm for the respective pH buffers; MW is the molecular weight of cyanidin-3-glucoside (449.2 g/mol); DD is the dilution factor; V is the total volume of the extraction solution (in mL); ε is the molar extinction coefficient of cyanidin-3-glucoside (26,900 L·mol⁻¹·cm⁻¹); L is the path length of the cuvette (1 cm); and m_f is the fresh mass of the sampled potato (in grams). Send the period with the highest anthocyanin content for transcriptome analysis, with three replicates for each sample, for a total of six samples.

Genome-wide identification analysis of the StBBXs family in pigmented potatoes

The chromosome and specific chromosome position information of the BBX family of pigmented potatoes are sourced from transcriptome databases (not previously published). Draw a chromosome location map using the online software Map Gene to Chromosome (http://mg2c.iask.in/mg2c_v2.0/). Upload the amino acid sequences of all and StBBX family members to the online software MEME (http://meme-suite.org/tools/meme). Identify the conserved motif features of the StBBXs family in pigmented potatoes. Set the maximum number of motifs to 10, with other parameters set to default values. Visualize gene structure and conserved motifs using TBtools tools. Construct phylogenetic trees 34 of the potato StBBXs family and the Arabidopsis AtBBXs family using MEGA X software [29]. Use R language 4.3.2 to draw a volcano map of the potato StBBXs family, displaying differentially expressed genes under UV-B treatment.

Bioinformatics analysis of StBBX19 and StBBX24 genes

Using DNAMAN (https://www.lynnon.com/dnaman.html), the full-length CDS sequences cloned from two materials were compared, and a phylogenetic tree was constructed with MEGA X. Protein domains of StBBX19 and StBBX24 were analyzed using the online tool SMART (http://smart.embl-heidelberg.de/). The tertiary structures of the genes and proteins were predicted via SWISS-MODEL (https://swissmodel.expasy.org/). Additionally, cis-acting elements within the promoter regions were identified using Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Potato RNA Extraction and cDNA chain synthesis

Total RNA was extracted from the sample (Huaxinyangyu) using a Trizol assay kit (Invitrogen, Carlsbad, CA, USA). RNA purity and concentration were evaluated on an Agilent 2100 biological analyzer (Agilent Technologies, Palo Alto, CA, USA), and RNA integrity was checked by agarose gel electrophoresis without RNase. Take 2 µg of RNA sample and synthesize cDNA according to the instructions of the E v o M-MLV reverse transcription kit (Aikeli Biotechnology Co., Ltd.). Take 1 µL of cDNA and measure its purity on a Quawell nucleic acid concentration detector in the United States (A260: A280 > 1.8; A260: A230 < 2.0). Then, dilute it with enzyme-free water to a concentration of 100 ng/µ L and store it at -20 ℃. The extraction of tobacco leaf RNA and synthesis of cDNA are the same as above.

Fluorescence quantitative PCR analysis

Based on the CDS sequences of StBBX19 and StBBX24 genes obtained from transcriptome analysis, fluorescent quantitative PCR primers were designed using NCBI’s Primer Blast; potato StGAPDH is an internal reference gene (Table S1). Single-factor analysis of variance (ANOVA) was used to analyze data from three independent biological replicates, and significant differences were determined using Duncan’s multiple range test (p < 0.05) using SPSS 22.0 statistical software (SPSS Inc., Chicago, Illinois, USA). After the instantaneous expression, the tobacco NtGAPDH was used as the internal reference gene (Table S1), and the gene expression level was detected by fluorescence quantitative PCR, with the method being the same as that for potatoes.

Vector construction

Specific primers StBB19/24 F and StBB19/24 R were designed based on the cDNA sequence of the StBB19/24 gene (Table S1). The full-length sequence was amplified by PCR using cDNA derived from colored potato tubers. The 50 µL reaction mixture contained 25 µL of 2× Phanta Flash Master Mix, 2 µL of each primer, and 2 µL of cDNA template, made up to volume with ddH₂O. The PCR protocol comprised an initial denaturation at 98 °C for 0 s; 35 cycles of denaturation at 98 °C for 10 s, annealing at Tm for 5 s, and extension at 72 °C for 5 s/kb; followed by a final extension at 72 °C for 1 min. PCR products were separated on a 1% agarose gel run at 120 V for 30 min, and the target band was purified using the Tiangen gel recovery kit.

Extract plasmids from pEGC-YC155, pEGC-YN173, pHELLSGATE-YFP, and pCAMBIA2305.1 vectors by shaking, and use specific enzyme Xba I to cleave pEGC-YC155 and pEGC-YN173; Use Xba I and Kpn I to cleave the pHELLSGATE-YFP and pCAMBIA2305.1 plasmids, and recover the cleaved vectors. Using a homologous recombination reaction, the cloned StBBX19 and StBBX24 fragments were ligated into the digested pEGC-YC155, pEGC-YN173, pHELLSGATE-YFP, and pCAMBIA2305.1 vectors (the primers used in Table S1) and transformed into E. coli DH5α, incubated in Kan-resistant LB plates for 12 h at 37℃. After 12 h of incubation, single colonies were picked for PCR-positive detection, and three positive clones were selected and sent to Kinko’s for sequencing.

Subcellular localization

Transform the recombinant vector plasmids containing the target gene into Agrobacterium competent cells GV3101-P19 and culture them on YEB plates containing Kan and Rif resistance at 28 °C for 48–72 h. Select a single colony and add 10 ml of YEB medium containing Kan and Rif resistance to activate the Agrobacterium. After activation, extract 600 µl of the bacterial solution and add it to 30 ml of YEB medium with Kan and Rif resistance to prepare the working solution. When the OD value of the bacterial solution reaches 0.6–0.8, centrifuge it at 4000 rpm for 10 min to collect the bacteria. Adjust the OD to 1.0-1.2 using a tobacco buffer (100 µM AS, 1 M MES, 0.1 M MgCl₂), and incubate in the dark for 3 h. Next, aspirate the suspended bacterial solution using a 1 mL syringe and inject it into the back of 4-week-old safflower tobacco leaves. After 3–5 days, collect tissue samples from the injection site on the tobacco leaves for testing. The tobacco transient expression analysis method is the same as above.

The constructed YFP fusion expression vector was transiently transformed into Nicotiana benthamiana through Agrobacterium-mediated transformation. After being cultured in a dark and dark environment under average temperature and humidity conditions for 2 days, it was placed in an artificial climate box for 2 days of light culture. We carefully peeled off the stained tobacco leaf epidermis and put it on a glass slide with distilled water. The YFP signal was observed under a confocal laser microscope.

Double-molecule fluorescence complementary verification of interacting proteins

Recombinant vector plasmids containing different N-termini (StMYB308) and C-termini (StBBX19, StBBX24) of the target gene were transformed into Agrobacterium tumefaciens. The transformed Agrobacterium was mixed in a 1:1 ratio and injected into the tobacco leaves. After the injection, the leaves underwent a light and dark treatment for 1 day. The leaves were then cultured under light conditions for 2–3 days. Finally, the transient expression fluorescence signal in the tobacco leaves was observed using a laser confocal microscope.

Results and analysis

Acquisition, chromosome localization, and gene structure analysis of potato BBX gene family members

A total of 11 StBBX transcription factors were identified based on transcriptomic sequencing results (Table S2), designated as StBBX19, StBBX20, StBBX21, StBBX22, and StBBX24. Chromosomal localization of the identified StBBXs genes was performed based on the annotation information of the transcriptome (Figure S2A). Among the 12 chromosomes, chromosome 8 had the fewest genes, with only two genes present. Chromosome 6 had three genes, while chromosomes 3, 10, and 11 each contained four genes. The remaining chromosomes 1, 2, 4, 5, 7, 9, and 12 distributed five genes, indicating that the number of StBBX genes is not directly proportional to chromosome length. Additionally, the structural characteristics of the five StBBX genes were analyzed (Figure S2B), identifying 10 conserved motifs. All five StBBXs include motifs 1, 2, and 3, whereas StBBX24 includes only 4. The relative positions of the protein motifs are relatively conserved among the five StBBXs. Gene structure analysis showed that, apart from StBBX19, all StBBX genes feature two exon regions. Furthermore, all five StBBX genes contain coding sequence (CDS) regions (Figure S2B).

Phylogenetic analysis and multi-sequence alignment of the StBBX protein

Transcriptome analysis revealed that under different UV-B treatments, the expression levels of StBBX21 and StBBX24 in the tubers of Huaxinyangyu were highest at T1 (5 KJ/m²). These results suggest that UV-B may promote the expression of StBBX19, StBBX21, and StBBX24. The identified StBBX proteins were compared with BBX transcription factors from Arabidopsis and tomato using multiple sequence alignment, and an evolutionary tree was constructed (Fig. 1A, Table S3). The results of the evolutionary tree indicate that the cloned StBBX19 and StBBX24 genes fall within the fourth subfamily and are associated with the regulation of anthocyanin synthesis. We created a volcano plot to identify five differentially expressed genes of StBBX under CK and UV-B treatments. StBBX19 and StBBX24 showed upregulation among these genes, while StBBX20 was downregulated. There was no significant difference in expression between StBBX21 and StBBX22 (Fig. 1B). Based on these findings, it is speculated that StBBX19 and StBBX24 may regulate anthocyanin synthesis in pigmented potatoes when exposed to UV-B conditions.

Fig. 1.

A Phylogenetic tree of the StBBX family in potatoes, Arabidopsis thaliana, and tomatoes. Ⅰ: Subfamily I regulates photomorphogenesis and flowering; Ⅱ: Subfamily II regulates light signal transduction; Ⅲ: Subfamily III regulates flowering time and stress responses; Ⅳ: Subfamily IV regulates anthocyanin synthesis under UV-B conditions; Ⅴ: Subfamily V regulates growth and development. The two target genes identified in this study, StBBX19 and StBBX24, are highlighted in red. B Differential expression of StBBXs under T1 treatment at 72 days

Analysis of sequence and structural features of StBBX19 and StBBX24

Analysis of the BOX domains of StBBX19 and StBBX24 proteins revealed that both possess two BBOX domains (Figure S3A). The three-dimensional structure of proteins is closely related to their functions. Analysis of the three-dimensional structures of StBBX19 and StBBX24 proteins revealed that they are two helix angle helix structures consistent with the predicted results of the BBOX functional domain (Figure S3B). Multiple sequence alignment was performed between StBBX19 and StBBX24 and between BBX19 and BBX24 from other species, and the analysis results showed that the alignment consistency was above 90% (Figure S3C).

Promoter analysis of StBBX19 and StBBX24 genes

Through PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ), the website predicts its promoter cis-acting elements, and the analysis results show that in addition to the essential promoter structural elements, StBBX19 and StBBX24 also contain photoresponsive elements and MYB binding sites (Figure S4). Therefore, we speculate that StBBX19 and StBBX24 may interact with MYB to participate in light response.

Changes in gene expression in the biosynthesis pathway of anthocyanins in pigmented potatoes under UV-B treatment

Naringin is the core metabolite of the flavonoid pathway, which can be converted into dihydroxynaringenin through F3H. Dihydroxynaringenin can be converted into flavonol through FLS and then into anthocyanins through special enzymes such as F3’H, F3’5’H, DFR, etc. (Fig. 2). Based on the transcriptome, the expression levels of relevant structural genes in the color potato anthocyanin biosynthetic pathway were analyzed under UV-B treatment. PAL, C4H, 4CL, CHS, CHI, F3H, F3’5’H, F3’H, DFR, ANS, UFGT/3GT, GST, and other enzymes are involved in the biosynthesis pathway of anthocyanins. Naringin is synthesized from phenylalanine through the phenylpropanol pathway, with PAL, C4H, 4CL, and CHS being key rate-limiting enzymes that catalyze the production of naringin chalcone by coumaroyl CoA ligase and chalcone synthase, and then catalyzed by chalcone isomerase to produce naringin. Under UV-B treatment, the expression levels of most genes were higher than the control, and CHI was significantly higher than the control, indicating a high naringin content.

Fig. 2.

The biosynthetic pathway of anthocyanins in pigmented potatoes under UV-B treatment. Based on the average FPKM of three biological replicates, gene expression is displayed in the heatmap, with purple indicating high expression and yellow indicating low expression. PAL, phenylalanine ammonia lyase; C4H, cinnamic 4-hydroxylase; 4CL, 4-coumaroyl CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3’5’H, flavonoid 3’5 ‘hydroxylase; F3’H, flavonoid 3 ‘hydroxylase; DFR, dihydroflavonol-4-reductase; ANS, anthocyanin synthase; Anthocyanin reductase; UFGT/3GT, flavonoid 3-O-glucosyltransferase; GST and glutathione S-transferase. (See table S4 for its homologous genes)

Changes in expression levels of StBBX19 and StBBX24 genes under different UV-B treatments

As previously reported by our group [29], UV-B treatment significantly promoted anthocyanin accumulation in pigmented potato plants. Building on this foundation, the present study further investigates the underlying molecular mechanisms. Under different UV-B treatments, we observed that both Jianchuanhong and Huaxinyangyu exhibited the highest anthocyanin content under T1 treatment (Fig. 3A). Correspondingly, the expression level of StBBX24 was also highest under T1 in both varieties. In contrast, the expression level of StBBX19 varied between varieties but did not show significant differences (Fig. 3B). These results suggest that UV-B may regulate the expression of StBBX24.We further performed a correlation analysis between anthocyanin content at the peak accumulation period (72 days) and the expression of StBBX19 and StBBX24. In Jianchuanhong, under T1 treatment, the expression level of StBBX19 was positively correlated with anthocyanin content, while under T2 treatment, StBBX24 expression showed a positive correlation with anthocyanin content (Figure S5A). In Huaxinyangyu, anthocyanin content under both T1 and T2 treatments generally exhibited positive correlations with the expression levels of StBBX19 and StBBX24 genes (Figure S5B).

Fig. 3.

A Anthocyanin extracts from different periods and treatments. B The gene expression levels of StBBX19 and StBBX24 under different treatments at the highest anthocyanin content (72 days). According to Duncan’s test, different letters represent significant differences (p < 0.05). The error bar represents the mean ± SE. C Correlation analysis between anthocyanin content in Jianchuanhong and StBBX19 and StBBX24. D Correlation analysis between anthocyanin content in Huaxinyangyu and StBBX19 and StBBX24

Subcellular Localization of StBBX19 and StBBX24 Genes

As shown in Fig. 4, fluorescent signals were found on the nucleus, indicating that StBBX19 and StBBX24 are located in the nucleus and function within the nucleus.

Fig. 4.

Subcellular localization of StBBX19 and StBBX24. Bright-field: Bright field view; Fluorescence: Fluorescence signal channel; Merge: Fluorescence signal and bright field superposition (200⋅)

Double-molecule fluorescence complementary verification of interacting proteins

The observed YFP fluorescence signals (Fig. 5) preliminarily suggest a potential interaction between StBBX19, StBBX24, and StMYB308. However, to confirm this interaction, further validation using in vitro assays such as yeast two-hybrid is required in the future.

Fig. 5.

BiFC verification of StBBX19 and StBBX24 with StMYB308. Bright-field: Bright field view; Fluorescence: Fluorescence signal channel; Merge: Fluorescence signal and bright field superposition (200⋅)

Transient conversion analysis of tobacco

As shown in Fig. 6A, we found that the interaction between StBBX19, StBBX24, and StMYB308 was associated with visible anthocyanin accumulation in tobacco leaves. Purple spots appeared on tobacco leaves after the interactions of StBBX19 and StBBX24 with StMYB308. Extract its RNA and reverse it to cDNA for qRT-PCR identification of its gene expression level and the expression level of tobacco endogenous gene NtAN1b. In addition, the interaction between StBBX19, StBBX24, and StMYB308 can significantly increase their gene expression levels, and the expression level of tobacco endogenous gene NtAN1b increased after the interaction between StBBX19 and StBBX24 and StMYB308, respectively (Fig. 6B).

Fig. 6.

A Transgenic tobacco leaves. B qRT-PCR identification of transgenic tobacco gene expression levels (according to Duncan’s test, different letters represent significant differences (p < 0.05)). The error bar represents the mean ± SE

Discussion

BBX transcription factors are key components in plant photomorphogenesis and light signaling, and their role in anthocyanin biosynthesis has been extensively demonstrated across multiple species [30, 31]. Anthocyanin biosynthesis is coordinately regulated by the MBW protein complex, composed of MYB, bHLH, and WD40 transcription factors, which also participates in the broader flavonoid biosynthetic pathway [32]. While research in pear has demonstrated that PyBBX18 mediates UV-B-induced anthocyanin accumulation [30], the biological functions of BBX members in solanaceous crops, particularly within potato storage organs, remain poorly characterized.

This study identified StBBX19 and StBBX24 as phylogenetically conserved that cluster with BBX proteins known to participate in anthocyanin synthesis in Arabidopsis, tomato, and apple [30, 32, 33]. This finding strongly suggests these genes are involved in light-regulated pigmentation in potatoes, filling a gap in this field. Functional analysis further revealed complex regulatory networks. StBBX19 and StBBX24 exhibited differential expression patterns across potato varieties and UV-B treatments, indicating their expression may be finely tuned by genotype background and signaling dynamics, potentially underpinning variations in anthocyanin accumulation. Co-transfection experiments provided direct evidence of synergistic interaction: co-expression of StBBX19, StBBX24, and StMYB308 effectively activated the endogenous anthocyanin synthesis pathway in tobacco, manifested as pigment accumulation and upregulation of NtAN1b expression. This suggests these three proteins may form a synergistic transcriptional module jointly regulating anthocyanin biosynthesis. Promoter analysis revealed the absence of typical light-responsive MYB binding sites in the StBBX24 promoter region, suggesting StMYB308 is unlikely to directly regulate its transcription. In contrast, BiFC experiments confirmed direct protein interactions between StBBX19/StBBX24 and StMYB308. We therefore speculate that StBBX19 and StBBX24 primarily interact with StMYB308 through post-translational mechanisms, potentially regulating anthocyanin synthesis by stabilizing the MBW complex, enhancing its DNA-binding capacity, or promoting its targeted localization. This mechanism is conserved with the PpBBX16–PpHY5 interaction in pear [34] and the EsMYB90–EsBBX24 partnership in Arabidopsis thaliana [35].

It should be noted that the current transcriptomic analysis in this study primarily focused on genes related to the anthocyanin biosynthesis pathway, aiming to efficiently screen candidate regulatory factors. A comprehensive enrichment analysis of all differentially expressed genes will be an important direction for future work. Future research plans include deepening the exploration of transcriptome data to systematically decipher the UV-B response network; further validating protein interactions through Co-IP, Y2H, and other experiments; and constructing transgenic potato materials to confirm the relevant mechanisms at the plant level. In summary, this study demonstrates that StBBX19 and StBBX24 are positive regulators of UV-B-induced anthocyanin synthesis in potatoes, likely acting through physical interaction with StMYB308. These findings provide new insights into the molecular mechanisms underlying light-mediated pigmentation in potatoes.

Conclusions

In conclusion, this study highlights the role of BBX transcription factors, specifically StBBX24, in responding to UV-B signals and their potential involvement in regulating anthocyanin biosynthesis in plants. StBBX19 expression varied between varieties but was not significantly induced by UV-B, suggesting it may not be a key regulator in this context. Through transcriptome sequencing, protein structure analysis, and functional validation via transient transformation in tobacco, we demonstrated that while StBBX19 and StBBX24 may not directly regulate anthocyanin biosynthesis, their interaction with StMYB308 significantly enhances anthocyanin accumulation in tobacco leaves. These findings provide valuable insights into the molecular mechanisms underlying plant adaptation to UV-B stress and offer a foundation for future research in molecular breeding, particularly for developing pigmented potato varieties with improved UV-B tolerance and enhanced anthocyanin production.

Authors’ contributions

QJ D. performed the experimental work, XY L. conducted formal data analysis and interpretation, J L. and JP X. designed and supervised the study, QJ D. wrote the original draft preparation. JP X. wrote the review and edited it. All authors have read and agreed to the definitive version of the manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (No. 32060500 and 32460477), the Yunnan Agricultural Fundamental Research Joint Projects (Grant No. 202301BD070001-135).

Data availability

All the raw transcriptomic sequencing data were uploaded to the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/). BioProject: PRJNA1026324. Released on June 23, 2026.

Declarations

Ethics approval and consent to participate

Not applicable. None of the species used in this study is endangered or protected; all plants were grown in greenhouses, and all experiments on these plants comply with all relevant guidelines and regulations. All plant materials were provided by Yunnan Agricultural University.

Consent for publication

All the authors read and approved the final manuscript. We declare that it has not been published elsewhere and that it has not been submitted simultaneously for publication elsewhere.

Competing interests

The authors declare no competing interests.