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Article

Genome-Wide Identification of the BBX Gene Family: StBBX17 Positively Regulates Cold Tolerance in Potato

by
Xiaobo Luo
1,2,3,†,
Luo Wang
2,4,†,
Feng Shen
5,
Yi Mei
5,
Degang Zhao
1,* and
Fei Li
2,*
1
Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Collaborative Innovation Center for Mountain Ecology & Agro-Bioengineering (CICMEAB), College of Life Sciences/Institute of Agro-Bioengineering, Guizhou University, Guiyang 550025, China
2
Guizhou Institute of Biotechnology, Guizhou Provincial Academy of Agricultural Sciences, Guiyang 550009, China
3
Guizhou Key Laboratory of Agriculture Biotechnology, Guiyang 550009, China
4
Ministry of Agriculture and Rural Affairs Key Laboratory of Crop Genetic Resources and Germplasm Innovation in Karst Region, Guiyang 550009, China
5
Jiangsu Coastal Area Institute of Agricultural Sciences, Yancheng 224002, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(10), 1167; https://doi.org/10.3390/horticulturae11101167
Submission received: 3 August 2025 / Revised: 21 September 2025 / Accepted: 22 September 2025 / Published: 1 October 2025
(This article belongs to the Section Biotic and Abiotic Stress)
Browse Figures
Versions Notes

Abstract

Potato is an important crop in the world and is rich in various nutrients. Common tetraploid potato is not tolerant of low temperatures and frost. Low-temperature stress severely affects the growth above-ground and the yield underground in potato. The BBX genes play an important role in the plant response to low-temperature stress. However, the molecular mechanism underlying the potato StBBX genes involved in cold stress response remains unclear. In the present study, 30 StBBX genes were identified in potato and divided into five groups. A total of 10 motifs and 10 cis-acting elements were obtained in all BBX proteins. All StBBX genes contained light responsive elements in the promoter, of which nine StBBX genes harbored low-temperature responsive elements. In total, 15 pairs of StBBX genes were identified in duplicated genomic regions. The gene expression patterns of all StBBXs were assessed in different tissues by transcriptome data. The qRT-PCR analysis indicated that six StBBX genes were significantly induced in response to cold stress. Subcellular localization suggested that the StBBX17 protein was localized in the nucleus. Compared with wild type (WT), the cold tolerance in StBBX17 overexpression lines was dramatically increased. After cold treatment, the StBBX17 overexpression lines displayed a less injured area of leaves and lower electrolyte leakage compared with the WT plants, demonstrating StBBX17 positively regulated cold tolerances in potato. These results indicate that StBBX genes have important functions under cold stress, providing a theoretical reference for the breeding of cold-resistant potato.

1. Introduction

Low temperature is a key environmental factor that seriously limits plant growth, development, and geographical distribution. Plants have developed complex regulatory mechanisms to cope with low-temperature stress. Temperate plants with the ability of cold acclimation have the ability to acquire cold resistance through pre-exposure to low but nonfreezing temperatures [1,2]. The C-repeat-binding factors/dehydration-responsive element binding proteins (CBFs/DREB1s) signaling pathway, extensively explored in Arabidopsis, plays a crucial role in cold acclimation [3]. Many studies have shown that CBF proteins directly bind to the C-repeat element (CRT)/DRE cis-elements in the promoters of COLD-RESPONSIVE (COR) genes [3,4]. Several transcriptional factors (TFs) positively regulate CBF expression and increase plant freezing tolerance, including inducer of CBF expression 1 (ICE1) [5], calmodulin binding transcription activators (CAMTAs) [6], and BIN2 (BRASSINOSTEROID-INSENSITIVE2) [7]. However, some transcription factors suppress CBF expression and decrease cold resistance, including MYB15, MYB30, ETHYLENE INSENSITIVE 3 [8], phytochrome-interacting factor 3 (PIF3) [9], and PIF4 [10]. Previous studies found that CBF genes regulated only 10–20% of COR genes in Arabidopsis [11,12], indicating the other transcription factors participate in cold stress via CBF-independent pathways.
The B-box (BBX) proteins are zinc-finger transcription factors that possess one or two B-box domains at the N termini, and some members also contain a CCT domain or valine-proline motif at the C terminus [13]. A total of 32 BBX genes were identified and divided into five subfamilies based on their domain structures in Arabidopsis [14]. Numerous studies revealed that the conserved CCT domain is involved in nuclear transport and transcriptional regulation [15]. BBX domain-contained proteins play crucial roles in regulating photomorphogenesis [16], flowering [17], anthocyanin accumulation [18], cold stress [19,20], salt and drought stress [21], and Fusarium wilt [22]. BBX28 and BBX29 restrains ELONGATED HYPOCOTYL 5 (HY5) binds to the BBX30 and BBX31 promoters, forming a transcriptional feedback loop to fine-tune photomorphogenesis in Arabidopsis [23]. SlBBX20 and SlBBX21 bind to the SlHY5 promoter, activate its transcription and promote UV-B photomorphogenesis in tomato [16]. CmBBX8 binds to CmFTL1 promoter and promotes flowering in chrysanthemum [17]. PpBBX18 enhances anthocyanin biosynthesis, and PpBBX21 reduces anthocyanin accumulation in pear [18]. Overexpression of IbBBX24 significantly improves Fusarium wilt disease resistance and increases yields in sweet potato. IbBBX24 activates IbJAZ10 transcription and inhibits the transcription of IbMYC2, suggesting that IbBBX24 plays an important role in regulating JA biosynthesis and signaling in sweet potato [22]. In apple, MIEL1 (MYB30-Interacting E3 Ligase1) and JAZ (JAZMONATE ZIM-DOMAIN) proteins are co-involved in the regulation of cold stress by the BBX37-ICE1-CBF module [19]. Arabidopsis BBX7 and BBX8 positively regulate freezing tolerance through CRYPTOCHROME 2 (CRY2), photomorphogenic 1 (COP1), and HY5 [20]. Previous studies found that SlBBX17 positively regulated cold tolerance in tomato [24]. BBX29 negatively regulates cold tolerance in Arabidopsis by a CBF-independent pathway [25]. Silencing of SlBBX7, SlBBX9, and SlBBX20 reduces tomato cold tolerance [26]. SlBBX31 positively regulates cold tolerance in tomato, and SlHY5 can directly bind to the SlBBX31 promoter [27]. In potato, 30 StBBX members have been identified and characterized, and the expression profiles of StBBXs were performed under diurnal cycle, etiolation and de-etiolations [28]. In our previous studies, overexpression of StBBX14 in potato displayed less leaf damage and lower electrolyte leakage compared with the wild type under cold stress [29]. These studies have confirmed that the BBX gene family plays an important role in cold stress. However, the molecular functions of StBBXs involved in regulating potato response to cold stress remain unclear.
Potato (Solanum tuberosum L.) is one of the most important tuber crops worldwide. The common cultivated potato is not resistant to cold stress, which will affect the growth of potato shoots and the yield of potato. The potato originates in the harsh environment of the Andes mountains, where the potato germplasm resources are rich and possess many cold-resistant wild diploids [30]. The potato genome assembly sequences of the wild diploid Solanum commersonii with freezing tolerance were widely reported [31,32,33]. Many studies have reported that many cold resistance genes were identified and verified based on the Solanum commersoni genotype [34,35]. Overexpression of Solanum commersonii SAD1 (ScSAD1) in cultivated potato increase its tolerance to freezing [34]. Overexpression of ScGolS1 distinctly increases freezing tolerance and antioxidant activity in the potato cultivar ‘Atlantic’ [36]. Overexpression of ScUGT73B4 improves glycosylated flavonoid accumulation and increases antioxidant capacity, resulting in enhanced freezing tolerance in potato [35]. The Solanum acaule arginine decarboxylase gene ADC1 (SaADC1) transform in S. tuberosum cv. E3 increases freezing tolerance of potato in cold climates and enhances CBF gene expression [37]. SaCBL1-like (calcineurin B-like protein) improves potato freezing tolerance by activating the expression of CBF-regulon [30]. However, the molecular mechanism underlying potato response to cold stress remains largely unknown.
In this study, the evolutionary relationship, gene structure, chromosome location, and collinearity analysis of BBX family genes were investigated. The expression patterns of all identified StBBX genes were analyzed under cold stresses by qRT-PCR. To evaluate the StBBX17 function of resistance to cold stress, the StBBX17 gene was overexpressed in potato. This research would provide a foundation for further dissecting the evolutionary relationship and functional differentiation of StBBX family genes and exploring the molecular function of StBBX genes under cold stress in potato.

2. Materials and Methods

2.1. Identification and Phylogenetic Analysis of BBX Gene Family

The genome sequences were retrieved from the potato genome database (DM6.1). The conserved domain of BBX proteins (PF00643) was downloaded from the Pfam database (http://pfam.xfam.org/; accessed on 28 August 2022) to search StBBX candidate sequences. The HMMER 3.0 software with an E-value cut-off of 0.01 was used to obtain candidate StBBXs [38]. The BBX domain was verified using SMART (http://smart.embl-heidelberg.de/; accessed on 28 August 2022) and Pfam [39]. The MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/; accessed on 28 August 2022) software was employed to construct multiple alignments of the predicted protein sequences in the potato, tomato and Arabidopsis [40]. The MEGA 11 v11.0.13 software was used to construct unrooted phylogenetic trees using the neighbor-joining method with 1000 bootstrap replicates [41]. The names of StBBXs were generated based on the evolutionary relationship between Arabidopsis and tomato.

2.2. Sequence Analysis of StBBXs

The exons and introns of StBBX gene structures were analyzed using Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/; accessed on 1 September 2022). The cis-acting elements of StBBX promoters with 2000 bp regions upstream of the CDS were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 3 September 2022) [42].

2.3. Synteny Analysis and Chromosomal Localization of StBBXs

Multiple Collinearity Scan toolkit (MCScanX) was employed to identify StBBX gene sequence repetition events [43]. The intra-species paralogous pairs of the protein sequence were identified by BLASTP, and the parameters were set as follows: (1) alignment significance: E_VALUE (default: 1 × 10−5), (2) MATCH_SCORE: final score (default: 50), (3) MATCH_SIZE: number of genes required to call a collinear block (default: 5) and the maximum gaps (default: 25) [44]. The relative positions of StBBX genes on potato chromosomes were located by EVOLVIEW (http://www.evolgenius.info/evolview/; accessed on 31 August 2022).

2.4. qRT-PCR Analysis

The potato variety ‘Desiree’ were cultured on MS medium containing 3% sucrose and 0.8% agar at pH 5.8. The 4-week-old plants were transplanted into a controlled environment chamber with a 16 h light/8 h dark cycle at a temperature of 23 °C/21 °C (light/dark). The 4-week-old plants of ‘Desiree’ was subjected to 2 °C for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h. Total RNA in the leaves of ‘Desiree’ was isolated with different cold treatment times by MiNiBEST Universal RNA Extraction kit (TaKaRa, Beijing, China). The reverse transcription was performed with StarScript II RT Mix (GeneStar, Beijing, China). The real-time fluorescence quantification was performed on a CFX96 detection system (Bio-Rad, Hercules, CA, USA). The qRT-PCR reaction contained 1 µL of cDNA, 0.5 µL of forward and reverse primer, 10 µL of qPCR Master Mix and 8 µL of ddH2O. β-tubulin was selected as internal reference gene. The relative gene expression was calculated using the Ct (2−∆∆Ct) method. The experiment was performed with three biological replicates. All primers are listed in Supplementary Table S1.

2.5. Subcellular Localization and Plant Transformation

The coding sequence (CDS) of StBBX17 was cloned into the PBWA (V)-GFP vector. The empty-GFP vector was used as negative control. All plasmids were transferred into Agrobacterium tumefaciens GV3101, and then injected into N. benthamiana. The DAPI marker was used to identify nuclei. The GFP fluorescence was observed by a laser confocal microscope (Nikon ECLIPSE Ti2) at excitation wavelength of 488 nm.
The CDS sequence of StBBX17 was isolated from potato cDNA with specific primers. The PCR products and the vector pFGC1008 with the promoter of CaMV35S were digested with Kpn I and Sac I, and ligated into pFGC1008 vector through homologous recombination. The plasmid was transformed into Agrobacterium tumefaciens GV3101 using the electric shock method. The confirmed plasmids were transformed into the cold sensitive variety “Desiree”. The potato miniature tubers were used to perform genetic transformation followed the previous methods [34]. All positively transgenic plants regenerated from tissue culture were validated by PCR amplification with genomic DNA primers. The gene expression of StBBX17 was confirmed in the transgenic lines by qRT-PCR. The transgenic potato was planted in the greenhouse, and the wild type with the same conditions was used as a control. All the plants (30 days old) were used to perform cold treatment at −1 °C for 14 h and carry out phenotype investigation.

2.6. Frost Resistance Determination

Five-week-old potato seedlings were either exposed to cold stress at 4 °C or kept under normal conditions. Potato leaves were placed in test tubes with ice chips, and then transferred into a freezing bath with an initial temperature of 0 °C. Test tubes were cooled in increment of 0.5 °C every 30 min until −10 °C was reached. After the test tubes reached the experimental temperature, they were taken out of the freezing bath and put on ice overnight. The samples were gradually thawed and immersed in 25 mL deionized water, and then the samples were used to measure electrical conductivity (R1). The samples were autoclaved at 121 °C for 15 min and cooled for 24 h, then the total conductivity (R2) was measured. Relative electrolytic leakage was calculated as (R1/R2) × 100%, following a previously described method [45].

3. Results

3.1. Identification and Phylogenetic Analysis of StBBX Genes in Potato

A total of 30 StBBX candidate family members were obtained from potato genome (Supplementary Table S2) by HMMER software. To understand the evolutionary relationships and differences in gene functions among this gene family in three different species (potato, Arabidopsis and tomato), a phylogenetic tree was constructed after multiple alignment of amino acid sequences of 30 potato BBX (StBBX) genes, 32 Arabidopsis BBX (AtBBX) genes and 31 Solanum lycopersicum BBX (SlBBX) genes. These potato BBX members were named StBBX1 to StBBX31 based on the phylogenetic analysis results (Supplementary Table S3). The BBX gene family members of the three species were divided into five groups in the evolutionary tree (Figure 1). Group 1 contained five StBBXs, six AtBBXs and six SlBBXs. Group 2 contained 7 StBBXs, 9 AtBBXs and 7 SlBBXs. Group 3 comprised 3 StBBXs, 4 AtBBXs and 3 SlBBXs. Group 4 contained 8 BBX genes from each of the three species. Group 5 possessed 7 StBBXs, 5 AtBBXs, and 7 SlBBXs.

3.2. StBBX Gene Structure and Motif Analysis

To understand the conserved structure of BBX proteins, the amino acid sequences of potato BBX family members were investigated by MEME website (https://meme-suite.org/meme/tools/meme, accessed on 3 September 2022) [46]. Motif analysis showed that 10 motifs were identified in the 30 BBX proteins (Figure 2, Supplementary Figure S1). Motif 4 and motif 2 represented the main types in the five groups. Motif 1, motif 3 and motif 7 constituted most of the B-box domains and were distributed in three different groups. Motif 5 and motif 10 only existed in Group 2 and Group 3 subgroups, respectively. Motif 6 and motif 8 were only distributed in Group 4. The B-box1 domain existed in all BBX proteins (Figure 2). The B-box2 domain mainly existed in Group 1, Group 2 and Group 4. The CCT domain was mainly distributed in Group 1, Group 2 and Group 3.

3.3. Promoter Element Prediction for StBBX Gene Family

Cis-acting element analysis detected 10 cis-acting elements in the promoter of StBBX genes. All StBBX genes contained light responsive elements in the promoter (Supplementary Table S4). Hormone regulation related to salicylic acid responsive (9 StBBX genes), auxin responsive (12 StBBX genes), MeJA responsive (14 StBBX genes), abscisic acid responsive (20 StBBX genes) and gibberellin responsive (22 StBBX genes) elements were identified. In all, 22 StBBX genes contained cis-acting regulatory elements involved in anaerobic induction, and 13 StBBX genes contained elements related to defense and stress responsive elements. A total of nine genes harbored low-temperature responsive and drought responsive elements, respectively, which might have played important roles in cold and drought stress.

3.4. Chromosome Location of StBBX Genes

The results for chromosomal locations showed that 30 StBBX genes were located on twelve chromosomes (Figure 3A). The number of StBBX genes in each chromosome was varied. Chromosome 12 comprised the largest number of genes, of which StBBX05 and StBBX20 were located on the front end, and StBBX23, StBBX28, StBBX16 and StBBX07 were located on the tail end. Five StBBX genes were distributed on chromosome 07. Four StBBX genes were distributed on chromosome 02 and 05. Chromosome 04 contained three StBBX genes. Chromosomes 01 and 06 contained two StBBX genes. Chromosomes 03, 08, 09 and 10 possessed only one StBBX gene.

3.5. Synteny Analysis of the StBBX Gene Family

To further investigate the origin and evolution of the BBX gene family, tomato and Arabidopsis were selected for synteny analysis by BLASTP comparison of BBX homologous sequences. In total, 52 pairs of orthologous BBXs containing 26 SlBBX and 29 StBBX genes between tomato and potato were identified. A total of 47 pairs of orthologous BBXs containing 23 AtBBX genes in Arabidopsis and 25 StBBX genes in potato were obtained (Figure 3B; Supplementary Table S5).
Gene duplication events play essential roles in generating new functional genes and promoting the evolution of species. Whole genome duplication (WGD)/segmental duplication and tandem duplication events among StBBX genes were assessed by MCScanX. In total, 15 WGD/segmental duplication events were identified (Supplementary Table S6, Figure S2). It was found that a single gene corresponded to multiple genes, for example, StBBX10 corresponded to StBBX17 and StBBX16.

3.6. Gene Expression Patterns of StBBXs in Different Potato Tissues

The gene expression levels of StBBXs in different potato tissues were evaluated by 17 RNA-seq data points in the potato genome database (cultivar, RH89-039-16). A heatmap of all StBBX genes with TPM (Transcripts Per Million) values was constructed by TBtools v2.102 [47]. It was found that 22 StBBX genes were expressed in different tissues of potato, but the remaining 8 StBBX genes were rarely expressed in potato tissues (Figure 4; Supplementary Table S7). StBBX14 was highly expressed in leaf. StBBX09 and StBBX17 were highly expressed in flower. High expression levels of StBBX20 were found in root. StBBX03, StBBX04, StBBX05, StBBX24, StBBX06, StBBX11, StBBX19 and StBBX31 were highly expressed in stem, leaf, stolon, petiole, stamen and flower, indicating that these genes may play an important role in potato tuberization. StBBX17 was more highly expressed in tuber sprout, young tuber and stem.

3.7. Expression Analysis of StBBXs Under Cold Stress

Base on the expression pattern of StBBX genes under cold stress in our previous study (Supplementary Table S8) [29], six differentially expressed StBBX genes were selected to validate by qRT-PCR (Figure 5). The results revealed that the expression levels of StBBX01, StBBX17 and StBBX23 were significantly increased after 1 h and 6 h of treatment. The expression level of StBBX07 was highly expressed at 1 h and 8 h. The expression level of StBBX28 was relatively high at 1 h but then decreased at subsequent cold treatment time points. The expression level of StBBX05 was significantly increased after 1 h, 6 h and 8 h of treatment.

3.8. Subcellular Localization of StBBX17

The coding sequence (CDS) of StBBX17 contained 393 bp and encoded a protein of 130 amino acids. The CDS of StBBX17 was cloned in PBWA(V)-GFP constructs and transformed in Agrobacterium tumefaciens GV3101. The GV3101 cells contained 35S::PBWA(V)-StBBX17-GFP, and 35S::GFP constructs were injected into Nicotiana benthamiana. StBBX17 did not have an RFP signal on the endoplasmic reticulum. The green GFP signals were observed and overlapped with the nuclear 4′,6-diamidino-2-phenylindole (DAPI) signals. The result indicates that the StBBX17 protein was localized in the nucleus of N. benthamiana (Figure 6).

3.9. Overexpression of StBBX17 Enhanced Cold Resistance in Potato

To investigate the function of the StBBX17 response to cold stress in potato, the overexpression construct of StBBX17 was transformed into S. tuberosum cv. Desiree. A total of 18 transgenic lines of StBBX17 were obtained by hygromycin resistance. After performing 35S primer validation and measuring gene expression levels, seven positive transgenic lines were obtained. After gene expression analysis of StBBX17, two transgenic lines, StBBX17-OE-3 and StBBX17-OE-23 plants with the highest gene expression levels of StBBX17, were selected for further analysis (Figure 7A). The electrolyte leakage of the two transgenic lines was significantly lower than that of wild-type (WT) plants at −1 °C (Figure 7B). Compared with wild-type plants, the two transgenic lines exhibited no morphological changes under normal growth conditions (Figure 7C). The two overexpressing lines and WT plants were subjected to cold treatment (−1 °C for 14 h and recovery for 3 days). The StBBX17 overexpressing lines showed dramatically improved cold tolerance compared with WT. After cold treatment, the overexpressing lines (StBBX17-OE) showed a less injured area of leaves compared with the WT plants (Figure 7D). These results suggest that StBBX17 acts as a positive regulator of cold tolerance in potato.

4. Discussion

The plant gene BBX belongs to the zinc finger gene family and is involved in biological processes such as photoperiod, flowering, cold stress, and anthocyanin [48]. Whole-genome identification of BBX has been widely reported in crops such as Arabidopsis [14], rice [49], and tomato [26]. In this study, a systematic whole genome identification and characterization of the BBX gene family were conducted in potato. In the current study, 30 StBBX genes were identified in potato; the gene number was consistent with previous studies [28]. Many studies have reported that BBX was mainly classified into five categories [50,51]. In this study, StBBX proteins were grouped into five subfamilies by sequence similarity to Arabidopsis and tomato BBX proteins. The segmental and tandem duplication played an important role in the expansion of the gene family [48,49]. The 15 pairs of StBBX genes were identified to duplicated genomic regions, and no tandem duplicated gene was determined, similarly to previous studies [28]. Syntenic analysis among different plant genomes can acquire the conserved features in closely related species [52]. The 52 pairs of orthologous BBXs between tomato and potato and 47 pairs of orthologous BBXs between Arabidopsis and potato were obtained, respectively (Figure 3B). These results provide important information for understanding the functions of StBBX genes in potato.
Many studies have revealed that B-box genes play important roles in light-mediated developmental processes and response to cold stress in plants [20,24]. Overexpression of PpBBX18 promotes anthocyanin accumulation under light treatments [18]. HY5 is a central regulator in light signaling that interacts with BBX21, BBX22, BBX24, BBX25 and BBX28 [53]. The promoters of all StBBX genes with light-responsive elements were identified in this study. Compared with wild-type, overexpression of StCO restrains potato tuberization under a weakly inductive photoperiod [54]. In this study, the StCO gene was StBBX03, which was highly expressed in leaf and stolon according to transcriptome data. Compared with WT, silencing of StBBX24 induced an early flowering phenotype [55]. The StBBX24 identified in this study was the StBBX24 identified in previous study. StBBX09 and StBBX17 were highly expressed in flower, and StBBX04, StBBX05, StBBX06, StBBX24, and StBBX31 were higher expressed in stem, leaf tuber, stolon, and petiole flower. These BBX genes provide data support for further exploration of their functions in potato tuberization. In this study, nine StBBX genes contained low-temperature responsive and drought responsive cis-elements, respectively, indicating that they might participate in the response to low-temperature and drought stress.
Tetraploid potato is not tolerant of low temperature and frost, which seriously affects the growth of the above-ground parts of potato and eventually leads to a decrease in yield. Many studies have reported that cold-resistant genes isolated from wild materials were transferred into potato tetraploids, enhancing their cold resistance [34,56]. The genes for cold resistance traits in potato have been reported, but their molecular mechanisms underlying cold stress are not yet fully understood. Previous studies found that the CRY2-COP1-HY5-BBX7/8 module regulated blue light-dependent cold acclimation in Arabidopsis [20]. StBBX07 was found to homologous to AtBBX7 and AtBBX8, which was differentially expressed under cold stress by qRT-PCR in this study [20]. In apple, MdBBX37 binds to the MdCBF1 and MdCBF4 promoters regulating cold stress tolerance [19]. In this study, no potato StBBX gene homologous to apple MdBBX37 was found, demonstrating that the BBX gene has a certain specificity among different species. According to the results from the evolutionary tree, StBBX07, StBBX09, StBBX20 and StBBX31 in potato were homologous to SlBBX7, SlBBX9, SlBBX20 and SlBBX31, which regulated cold tolerance in tomato [24]. The StBBX29 in potato was homologous to Arabidopsis negative regulator AtBBX29 [25]. qRT-PCR results found that expression of four StBBX genes was significantly increased after 1 h; StBBX01, StBBX07, StBBX17 and StBBX23 genes maintained relatively high expression levels at 1 h and 6 h, respectively. SlBBX17 interacts with SlHY5 and positively regulates cold tolerance in tomato [24]. StBBX17 was homologous to SlBBX17, and StBBX17 was differential expressed under cold stress in our previous studies [45]. Compared to baseline at 0 h, StBBX17 was upregulated in S. cardiophyllum under 4 °C treatment for 14 d and exposure to −2 °C for 6 h after cold acclimation for 14 d [45]. In this study, the gene expression levels of StBBX17 were significantly increased after 2 °C treatment for 1 h and 6 h as compared with 0 h by qRT-PCR results. These results demonstrated that the StBBX17 gene was upregulated under both diploid and tetraploid cold treatments. Overexpression of StBBX17 in ‘Desiree’ corresponded with less leaf damage and relatively low electrolyte leakage as compared with the WT, indicating the StBBX17 gene positively regulates cold stress in potato. Therefore, the function of the StBBX gene under cold stress in potato and whether it depends on the CBF pathway require further study.

5. Conclusions

In this study, 30 StBBX genes were identified in potato. The comprehensive analysis of phylogenetic relationships, conserved domains, chromosome locations, cis-acting regulatory elements and duplication events were performed in the StBBX gene family. The gene expression patterns of all StBBXs were assessed in different tissues using transcriptome data. qRT-PCR analysis indicated that six StBBX genes were differentially expressed under cold stress. Overexpression of StBBX17 enhanced the cold resistance of tetraploid potato. These results provide a theoretical reference for exploring the molecular mechanisms of StBBX genes involved in potato under cold stress and offer theoretical support for further genetic improvement of cold resistance traits in potato.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11101167/s1, Supplementary Figure S1. The motif types in all StBBX genes. The p-value was used to evaluate the significance of the motif. Supplementary Figure S2. The intraspecific collinearity of the StBBX gene family in the potato genome. Collinear blocks across the entire genome were depicted with a grey background, and red lines represented duplicate StBBX genes. Supplementary Tables S1–S8: Supplementary Table S1. Primer sequences used in this study. Supplementary Table S2. Information and chromosomal location of the StBBX genes. Supplementary Table S3. The infomation of BBXs in Arabidopsis and tomato. Supplementary Table S4. The cis-acting elements detected in the promoter StBBXs genes. Supplementary Table S5. The collinearity orthologous gene pairs among potato, tomato and Arabidopsis. Supplementary Table S6. Whole genome duplication events of StBBX. Supplementary Table S7. The gene expression data of StBBXs in different tissues in potato genome. Supplementary Table S8. The gene expression data of StBBXs in OE-StBBX14 and WT under cold stress (2 °C for 1 and 12 h).

Author Contributions

X.L., investigation, validation, writing—original draft, writing—review and editing. L.W., funding acquisition, writing—review and editing. F.S., writing—original draft, data curation, formal analysis, Software. Y.M., writing—original draft, data curation, formal analysis. D.Z., writing—review and editing. F.L., funding acquisition, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Guizhou Province (QianKeheJiChu-MS [2025] 301), Guizhou Academy of Agricultural Sciences Youth Fund Project (QianNongKeQingNianJiJing (2023) 06), the Guizhou Provincial Science and Technology Plan Project (Qian Kehe Support [2022] key 030), the Innovation Capacity Construction of Breeding Scientific Research Platform in Guizhou Province (QianKeHeFuQi [2022] 014), the Construction of Biological Breeding Platform for Important Crops in Karst Mountain Areas of Guizhou Province (QianKeHeZhongYinDi; [2023] 033), and National Potato Industry Technology System Guiyang Comprehensive Experimental Station (2025).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. Phylogenetic tree of BBX gene families in potato, Arabidopsis and tomato. The red star represents StBBXs, the blue circle represents AtBBXs, and the green triangle represents SlBBXs.
Figure 1. Phylogenetic tree of BBX gene families in potato, Arabidopsis and tomato. The red star represents StBBXs, the blue circle represents AtBBXs, and the green triangle represents SlBBXs.
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Figure 2. Evolutionary relationship, conserved motifs and protein domain analysis of StBBX gene family.
Figure 2. Evolutionary relationship, conserved motifs and protein domain analysis of StBBX gene family.
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Figure 3. Chromosome location and collinearity analysis of StBBX gene family. (A) Chromosome location of StBBX protein. The numbers on chromosomes represent physical distances. (B) Orthologous collinearity of StBBX genes among tomato, potato and Arabidopsis. Collinear relationships of StBBX gene pairs are indicated with red lines.
Figure 3. Chromosome location and collinearity analysis of StBBX gene family. (A) Chromosome location of StBBX protein. The numbers on chromosomes represent physical distances. (B) Orthologous collinearity of StBBX genes among tomato, potato and Arabidopsis. Collinear relationships of StBBX gene pairs are indicated with red lines.
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Figure 4. The gene expression levels of StBBX genes in different tissues. The expression level of StBBX genes in different tissues. The heatmap was constructed based on the TPM values of StBBXs.
Figure 4. The gene expression levels of StBBX genes in different tissues. The expression level of StBBX genes in different tissues. The heatmap was constructed based on the TPM values of StBBXs.
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Figure 5. qRT-PCR expression analysis of the StBBX genes in potato Desiree after treatment at 2 °C for 1 h, 2 h, 4 h, 6 h, 8 h, 16 h, and 1 d. The fold change represents units on the Y axis. All data points shown are mean  ±  SE (n  =  3). Different lowercase letters indicate a significant difference (p  <  0.05, one-way ANOVA).
Figure 5. qRT-PCR expression analysis of the StBBX genes in potato Desiree after treatment at 2 °C for 1 h, 2 h, 4 h, 6 h, 8 h, 16 h, and 1 d. The fold change represents units on the Y axis. All data points shown are mean  ±  SE (n  =  3). Different lowercase letters indicate a significant difference (p  <  0.05, one-way ANOVA).
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Figure 6. Subcellular localization of StBBX17-GFP and GFP in epidermic cells of Nicotiana benthamiana leaves. Bar = 10 μm.
Figure 6. Subcellular localization of StBBX17-GFP and GFP in epidermic cells of Nicotiana benthamiana leaves. Bar = 10 μm.
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Figure 7. Overexpression of StBBX17 enhanced the cold resistance in potato. (A) The gene expression levels of StBBX17 in WT, OE-StBBX17-3 and OE-StBBX17-23 plants. (B) The electrolyte leakage of genotypes at the indicated temperatures. (C,D) The WT and StBBX17 overexpressing transgenic lines exposed to freezing treatment, and after being subjected to −1 °C for 14 h, followed by 3 days of recovery under normal conditions. The different letters represent significant differences at p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test).
Figure 7. Overexpression of StBBX17 enhanced the cold resistance in potato. (A) The gene expression levels of StBBX17 in WT, OE-StBBX17-3 and OE-StBBX17-23 plants. (B) The electrolyte leakage of genotypes at the indicated temperatures. (C,D) The WT and StBBX17 overexpressing transgenic lines exposed to freezing treatment, and after being subjected to −1 °C for 14 h, followed by 3 days of recovery under normal conditions. The different letters represent significant differences at p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test).
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Luo, X.; Wang, L.; Shen, F.; Mei, Y.; Zhao, D.; Li, F. Genome-Wide Identification of the BBX Gene Family: StBBX17 Positively Regulates Cold Tolerance in Potato. Horticulturae 2025, 11, 1167. https://doi.org/10.3390/horticulturae11101167

AMA Style

Luo X, Wang L, Shen F, Mei Y, Zhao D, Li F. Genome-Wide Identification of the BBX Gene Family: StBBX17 Positively Regulates Cold Tolerance in Potato. Horticulturae. 2025; 11(10):1167. https://doi.org/10.3390/horticulturae11101167

Chicago/Turabian Style

Luo, Xiaobo, Luo Wang, Feng Shen, Yi Mei, Degang Zhao, and Fei Li. 2025. "Genome-Wide Identification of the BBX Gene Family: StBBX17 Positively Regulates Cold Tolerance in Potato" Horticulturae 11, no. 10: 1167. https://doi.org/10.3390/horticulturae11101167

APA Style

Luo, X., Wang, L., Shen, F., Mei, Y., Zhao, D., & Li, F. (2025). Genome-Wide Identification of the BBX Gene Family: StBBX17 Positively Regulates Cold Tolerance in Potato. Horticulturae, 11(10), 1167. https://doi.org/10.3390/horticulturae11101167

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