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Article

Transcript-Wide Identification and Characterization of the BBX Gene Family in Trichosanthes kirilowii and Its Potential Roles in Development and Abiotic Stress

by
Weiwen Li
1,2,*,
Rui Xiong
1,2,
Zhuannan Chu
1,2,
Xingxing Peng
1,2,
Guangsheng Cui
1,2 and
Ling Dong
1,2,*
1
Key Laboratory of Horticultural Crop Germplasm Innovation and Utilization (Co-Construction by Ministry and Province), Institute of Horticulture, Anhui Academy of Agricultural Sciences, Hefei 230001, China
2
Anhui Provincial Key Laboratory for Germplasm Resources Creation and High-Efficiency Cultivation of Horticultural Crops, Hefei 230001, China
*
Authors to whom correspondence should be addressed.
Plants 2025, 14(6), 975; https://doi.org/10.3390/plants14060975
Submission received: 5 February 2025 / Revised: 10 March 2025 / Accepted: 19 March 2025 / Published: 20 March 2025
(This article belongs to the Special Issue Abiotic and Biotic Stress of the Crops and Horticultural Plants)
Browse Figures
Versions Notes

Abstract

The B-box (BBX) protein has an impact on flowering physiology, photomorphogenesis, shade effects, and responses to both biotic and abiotic stresses. Although recent research described the BBX gene family in numerous plants, knowledge of the BBX gene in Trichosanthes kirilowii was sparse. In this study, we identified a total of 25 TkBBX genes, and phylogenetic analysis showed that these genes were divided into five subfamilies. Analyses of gene structure and motifs for each group found relative conservation. Ka/Ks values showed that most TkBBX genes have undergone negative selection. qRT-PCR analyses revealed that TkBBX1, TkBB4, TkBBX5, TkBBX7, TkBBX15, TkBBX16, TkBBX17, TkBBX19, and TkBBX21 genes respond to salt and drought treatment. Furthermore, we cloned TkBBX7 and TkBBX17 genes and performed a subcellular localization experiment, which revealed that these two genes were both located in the nucleus. Transgenic yeast experiments demonstrated that TkBBX7 and TkBBX17 enhanced yeast tolerance to both salt and drought stresses. These findings provide a theoretical foundation for further investigation on the functions of TkBBX genes in Trichosanthes kirilowii.

1. Introduction

The zinc finger protein (ZEP) transcription factor family is one of the largest plant transcription factor families and plays an important role in plant growth, development, and response to abiotic stress [1,2,3]. The B-box (BBX) protein family belongs to a subfamily of ZEP and contains one or two B-box domains, and some family factors also have CONSTANS, CO-like, and TOC1 (CCT) domains [4]. The B-box motif contains about 40 amino acid residues, which can be divided into two types, B-box 1 and B-box 2, based on the consistency and difference of amino acids and zinc ion binding sites. Both of them are highly conserved and mainly play roles in protein interaction and transcriptional regulation. The CCT domain, consisting of 42 to 43 amino acid residues, is highly conserved and mainly involved in protein intranuclear transport and transcriptional regulation [1].
BBX genes play an important role in plant growth and development [5], including flowering physiology, photomorphogenesis, and shade effects. CONSTANS (CO) in Arabidopsis thaliana was the first BBX protein discovered in plants [6]. AtBBX6 and AtBBX24 can promote early flowering [7,8], while AtBBX4, AtBBX7, and AtBBX32 are negative regulators of flower formation [9,10]. In chrysanthemums, overexpression of CmBBX8 promoted flowering under both long- and short-day conditions. CmFTL1 induced blooming under long-day circumstances, while CmBBX8 was proved to promote flowering by binding to the CmFTL1 promoter’s transient acting element (CCACA) [11]. OsCOL4, OsBBX14, and OsCOL9 in rice delay plant heading through the Ehd1 pathway [12,13]. BBX can regulate some biological processes such as the growth of the embryo axis, the growth of lateral roots, and the spreading of seed leaves [14]. In Arabidopsis thaliana, AtBBX4, AtBBX20, AtBBX21, and AtBBX22 can promote photomorphogenesis [15,16,17,18,19], while AtBBX18, AtBBX19, AtBBX24, AtBBX25, and AtBBX32 can inhibit it [20,21,22,23,24]. Proteins in apples with the same structural domain as AtBBX1, MdBBX22 [25], and MdBBX20 [26] were shown to positively regulate light-induced anthocyanin accumulation through the light input pathway. When the plant is in a shaded environment, the BBX protein can mediate cell elongation, thereby increasing the height of the plant [27]. In shaded environments, AtBBX19, AtBBX21, and AtBBX22 were proved to inhibit cell elongation, while AtBBX18, AtBBX24, and AtBBX25 can promote cell elongation, thereby causing hypocotyl elongation [20,28]. MdBBX37 can inhibit MdMYB1 and MdMYB9 binds to target genes, thereby inhibiting anthocyanin biosynthesis and hypocotyl elongation [29]. MdPIF7 interacted with MdBBX23, reducing its transcriptional control on MdHY5, which regulated anthocyanin production and hypocotyl development [30].
The BBX proteins are also involved in the hormone signal pathway and abiotic stress response [31]. In some plants, BBX was reported to be involved in the anthocyanin synthesis pathway. HY5 promoted the expression of CHS, CHI, F3H, F3`H, and DFR genes during proanthocyanidin production, AtBBX21 can promote its expression through interaction with HY5 [28], nevertheless AtBBX24 and AtBBX25 inhibit the expression of HY5 [20]. In apple, a BBX protein, MdCOL11, interacted with MdHY5 to increase anthocyanin accumulation and pigmentation in the peel [32]. PpBBX18 and PpBBX21 antagonistically regulate anthocyanin biosynthesis in pear fruit through competitive binding with PpHY5 [33]. BBX plays certain roles in response to salt, drought, and heat stress [1]. For example, the overexpression of AtBBX24 increased the salt tolerance of plants [34]. The overexpression of MdBBX10 in Arabidopsis thaliana significantly enhanced tolerance to salt and drought stress, and the germination rate and root length were higher than those of wild type plants [35]. Overexpression of Ginkgo biloba L. BBX25 in poplar trees increased salt tolerance [36], while overexpression of Chimonanthus praecox L. CpBBX19 in Arabidopsis also increased plant tolerance to salt stress [37]. The CmBBX22 and AtBBX22 genes were homologous, and CmBBX22 in transgenic Arabidopsis provided a drought resistant phenotype [38]. The Arabidopsis bbx18 mutant has higher heat resistance, whereas the AtBBX18 overexpression strain has lower heat resistance [39].
BBXs have been identified in Arabidopsis, grape, rice, apple, tomato, and pear, but not in Trichosanthes kirilowii Maxim. This plant belongs to the Cucurbitaceae family, and its root, peel, and seeds are all used as medicine. Due to its growing application in the therapeutic management of cardiovascular disease and cancer, T. kirilowii was widely cultivated in Anhui, Hebei, Shandong, Henan, and Jiangsu provinces. In the context of global climate change and land degradation, highly resistant varieties are urgently needed in daily production. Furthermore, since T. kirilowii is a dioecous plant, the flowering times of female and male flowers do not coincide. Studying genes that can regulate the flowering period, making the flowering time of female and male flowers consistent, can increase the yield. T. kirilowii, as an octaploid plant, does not have a published genome, and gene mining can only rely on transcriptome data. In this study, we analyzed the evolution of TkBBX, the expression levels of TkBBX during flowering and salt and drought treatments and screened genes for preliminary functional validation in yeast. These results can provide a reference for regulating the flowering period and cultivating new stress-resistant varieties of T. kirilowii.

2. Results

2.1. Identification of TkBBX Genes

According to the AtBBX Pfam number (PF00643), a total of 25 TkBBX genes named TkBBX1 to TkBBX25 were identified from the transcriptome data. Table 1 includes basic information on T. kirilowii BBX genes such as open reading frame (ORF) length, protein length, isoelectric point (PI), molecular weight (MW), and subcellular localization. The TkBBX length range varies from 228 bp (TkBBX22) to 1323 bp (TkBBX25) and MW from 8068.35 Da (TkBBX22) to 49,928 Da (TkBBX25). These genes encode proteins with an average size of 282 aa and a size range from 75 to 440 aa. The PI values of TkBBX genes range from 4.2 (TkBBX17) to 10.42 (TkBBX14). In terms of subcellular localization, all TkBBX genes were predicted to be located in the nucleus.

2.2. Phylogenetic and Classification Analysis of TkBBX Genes

To investigate the relationship and classification of BBX members in T. kirilowii, an unrooted neighbor-joining (NJ) phylogenetic tree of 25 TkBBX and 32 AtBBX genes was constructed (Figure 1). According to the classfication of A. thaliana, TkBBXs were divided into five subfamilies (I–V) with 4 (TkBBX6, 12, 14, 19), 7 (TkBBX1, 2, 4, 8, 10, 18, 21), 5 (TkBBX3, 5, 9, 11, 25), 7 (TkBBX13, 15, 16, 20, 22, 23, 24), and 2 (TkBBX7, 17) members, respectively.
The tree was constructed using the neighbor-joining (NJ) method with MEGA 11.0 based on BBX sequences from Trichosanthes kirilowii and Arabidopsis thaliana.

2.3. Conserved Motif and Selective Pressure Analysis of TkBBX Genes

Motif analysis of 25 TkBBX genes was performed using Multiple Expectation Maximization for Motif Elicitation (MEME) online tools to investigate the conserved domain characteristics (Figure 2). As shown in Figure 2, all TkBBX genes contain at least one B-box (motif 1/motif 2). The motifs of genes which belong to the same subfamily were almost identical. For instance, motifs 1, 2, 4, and 6 appear in all genes of subfamily II, while most genes in subfamily III contained motifs 1, 5, 7, and 10.
Homologous pairs of BBX genes between T. kirilowii and A. thaliana were identified and are listed in Table 2. Ten paralogous pairs in T. kirilowii and nine orthologous pairs between T. kirilowii and A. thaliana were identified. To further comprehend Darwinian evolutionary selection in the TkBBX gene family, we estimated non-synonymous substitution (Ka), the synonymous substitution rate (Ks), and Ka/Ks value combinations (Table 2). The results showed that all pairs had Ka/Ks values less than 1, which indicated that all TkBBX genes underwent strong purification selection.

2.4. Expression Analyses of TkBBX Genes During Flowering Stage

BBX transcript factors play roles in plant growth and have an impact on flowering physiology. To analyze BBX gene expression changes during flowering, the fragments per kilobase of exon model per million mapped reads (FPKM) of 25 TkBBX genes were counted and are shown in Figure 3. The expression changes of female and male flowers at different flowering stages were compared. For female flowers, seventeen genes were differentially expressed genes (DEGs). The expression levels of TkBBX5, TkBBX7, TkBBX9, TkBBX12, and TkBBX17 were down-regulated, and the expression levels of TkBBX1, TkBBX4, TkBBX8, TkBBX10, and TkBBX21 were highest at full bloom, while expression levels of TkBBX15 and TkBBX16 were highest at preliminary bloom (Figure 3A). For male flowers, seventeen genes were DEGs. TkBBX6, TkBBX7, TkBBX12, TkBBX13, TkBBX15, and TkBBX19 were down−regulated, and TkBBX16’s expression level was highest at preliminary bloom, while TkBBX1, TkBBX4, TkBBX8, TkBBX10, and TkBBX21 were highest at full bloom (Figure 3B).

2.5. TkBBX Expression Pattern Responds to Salt and Drought Stresses

Previous studies reported that BBX transcription factor families were involved in plant stress responses [1]. Based on the transcriptome results, nine TkBBX genes were selected to investigate their response to salt and drought stress. Under NaCl treatment (Figure 4A), the expression levels of TkBBX1, TkBBX4, TkBBX5, TkBBX15, and TkBBX21 were highest at 12 h, those of TkBBX7, TkBBX16, and TkBBX17 exhibited a continuous increase, while TkBBX19 showed a continuous decrease. Among them, the expression levels of TkBBX7 and TkBBX17 genes were strongly up-regulated at 12 h (more than 50- and 30-fold, respectively). Under PEG treatment (Figure 4B), expression levels of TkBBX1, TkBBX4, TkBBX15, and TkBBX21 were highest at 12 h, while that of TkBBX19 at 6 h. The expression levels of TkBBX7, TkBBX16, and TkBBX17 exhibited a continuous increase. The results were similar to salt treatment, in which the expression levels of TkBBX7 and TkBBX17 genes were strongly up-regulated at 12 h (more than 15-fold).

2.6. Subcellular Localization of TkBBX Genes

Subcellular localization provides vital information about a protein’s function. TkBBX7 and TkBBX17 were selected for subcelluar localization experiments. GFP-TkBBX7 and GFP-TkBBX17 plasmids were made and transformed in tobacco. As shown in Figure 5, the green fluorescence of the empty vector (35S::GFP) was distributed on plasma membranes and the nucleus, while GFP-TkBBX7 and GFP-TkBBX17 were exclusively localized in the nucleus. This result was consistent with the website’s predictions and other species’ BBX proteins.

2.7. The Tolerance of TkBBXs to Salt and Drought Stresses in Yeast

Through the expression pattern of TkBBX genes under salt and drought treatment, TkBBX7 and TkBBX17, whose expression levels showed a continuous increase, were selected to investigate the biological functions. In order to detect the tolerance of TkBBX7 and TkBBX17 to abiotic stress, drought (1 M, 1.75 M mannitol) and salt (0.75 M, 1 M NaCl) were selected for the experiments (Figure 6). A bacterial solution containing only pYES2-NTB plasmids and SG/−Ura solid medium with no additional NaCl or mannitol added was the negative control. The results indicated that both the control (pYES2−NTB) and the two overexpressing yeast strains showed a consistent growth state on SG−Ura medium, indicating that the overexpression of TkBBXs in the INVSC1 strain had no effect on its growth under normal conditions. Meanwhile, in medium containing 1 M, 1.75 M mannitol (Figure 6A) or 0.75 M, 1 M NaCl (Figure 6B), TkBBX7 and TkBBX17 showed much greater growth than the control. This indicated their potential to enhance salt and drought tolerance in yeast strains.

3. Discussion

The BBX gene is a key transcription factor that regulates plant growth, development, and stress response [1,2]. The BBX gene family is a kind of zinc finger transcription factor consisting of B-box and CCT domains. It was identified and functionally studied in many plants [4,5,6,7,8,9,10,11,12,13], but not in T. kirilowii. In this study, the evolutionary relationships of the TkBBX gene family were analyzed using transcriptome data, and qRT-PCR was used to analyze the gene response to stress treatment. Furthermore, we selected TkBBX7 and TkBBX17 for subcellular localization assays and functional analysis.
We identified 25 TkBBX members and divided these proteins into five subfamilies. It is well known that, in Arabidopsis, the BBX family is divided into five subgroups based on the presence and number of B-box domains and CCT protein domains [6]. Both the first and second branches contain two B-box domains and one CCT protein domain. The third branch consists of a B-box domain and a CCT domain, the fourth branch contains two B-box domains, and the fifth branch has only one B-box domain. In this study, a neighbor-joining (NJ) evolutionary tree was constructed and divided into five subgroups based on the A. thaliana BBX protein sequence. However, unlike A. thaliana, T. kirilowii BBX cannot be grouped into subfamilies according to its domain (Figure 2). For each subfamily, TkBBX differs from AtBBX in both the type and number of domains. For example, TkBBX12 and TkBBX19 in subfamily I both contained two B-box domains and one CCT domain, which is the same as pineapple [40] and quinoa [31]. But, the members in A. thaliana subfamily I contained only one B-box domain. Previous studies suggested that this may be because some B-box 2 may have been removed during evolution. Researchers discovered significant variations in the gene structure and molecular characteristics of the BBX gene in plants by examining the development and growth of the BBX gene family, indicating that the BBX family is highly diverse [41].
Ka/Ks ratios were calculated to analyze the evolutionary process of the T. kirilowii BBX gene family. In general, a Ka/Ks ratio greater than 1 means positive selection for evolutionary acceleration, a Ka/Ks ratio equal to 1 represents neutral selection, and a Ka/KS ratio less than 1 signifies negative selection for evolutionary acceleration. In T. kirilowii, Ka/Ks values for all homologous pairs were less than 1, suggesting that these genes have undergone negative purification selection during evolution, and this is consistent with quinoa [31].
BBX genes were identified to be involved in many processes of plant growth and development, such as seedling photomorphogenesis, flowering, plant hormone signal transduction, pigment accumulation, and abiotic and biological stress [42]. Nevertheless, the function of TkBBXs has not been studied. BBX genes regulate flowering in herbaceous plants [43,44,45], and PtCO2 was shown to be associated with poplar growth arrest and flower bud formation [46]. And in Platanus × acerifolia, some BBX genes, such as PaBBX1-1, PaBBX4, PaBBX5-1/2, PaBBX7, PaBBX8-1/2, and PaBBX11-1, showed high expressions during the blossoming transition period [47]. These results suggested that the BBX gene may be involved in flowering and dormancy. In our study, some TkBBX genes were highly expressed during bud and fade bloom, for example, the TkBBX19 gene in female flowers and the TkBBX7 gene in male flowers. In addition, numerous genes, such as TkBBX12, 13, 15, 16, and 17 in female flowers and TkBBX3, 5, 15, 16, 19, and 25 in male flowers, were highly expressed in preliminary bloom. This indicated that these TkBBX genes may be involved in the flowering transition.
Because the BBX gene has multiple functions, we were still concerned about the response to abiotic stress. Previous studies reported that, in alfalfa, most MsBBX genes have positive responses to drought or salt stress [48]. And in T. kirilowii, a similar pattern was found. Only the TkBBX19 gene was down-regulated under NaCl treatment, and the TkBBX3 gene was down-regulated under PEG treatment. A similar situation occurred in other plants. For example, GmBBX5b/15c/15d/21d/2 1 g/24d/27a/28e/28f showed an increased expression pattern under salt stress conditions, and only GmBBX21c’s expression level was decreased [49]. All of these results suggested that these genes may have potential roles of plants in drought or salt tolerance and were positive regulators of drought and salt stress signaling in T. kirilowii. It is well known that salt and drought stress can cause crop growth restriction, affecting yield significantly and causing it to decline [50]. Increasing evidence indicated that BBX genes can affect both plant growth and development and plant response to stress. For example, overexpression of AtSTO (AtBBX24) promoted root growth of A. thaliana under high-salinity conditions [34]. The survival rate of MdBBX1 transgenic plants was higher under salt stress [51]. In our study, TkBBX7 was highly expressed not only in male and female flower buds but also in salt and drought treatment. We hypothesize that TkBBX7 played a role in both growth and development and in response to stress.
After a thorough analysis of all the results, two genes were identified, namely TkBBX7 and TkBBX17. Their expression levels are continuously up-regulated under NaCl and PEG stress conditions. Transgenic yeasts also show some resistance to stress. These findings suggest that the two genes may play a major role in the response of T. kirilowii to abiotic stress, and further study of their molecular regulatory mechanisms may help improve the biological and abiotic stress resistance of T. kirilowii.

4. Materials and Methods

4.1. Plant Materials, Growth Conditions, and Stress Treatments

The experimental materials (T. kirilowii tissue culture seedlings) used in this study were kept at the Institute of Horticulture, Anhui Province, China. Six-week-old seedlings grown on MS medium were used for relative expression level comparisons of candidate genes under abiotic stress. These seedings were grown under conditions of 16 h light/8 h dark at 22 °C. The leaves for tissue culture were treated with 20% PEG 6000 (Polyethylene Glycol 6000, Sangon, Shanghai, China) and 300 mM NaCl (Shanghai, Sangon) solution, respectively, to simulate drought and salt stress. All the leaves were harvested for 1, 3, 6, 12, 24 h treatments, while 0 h was used as control. Analyses were conducted with three biological and three technical replicates. The samples were frozen in liquid nitrogen and stored at −80 °C for RNA extraction.

4.2. Identification of the BBX Gene Family in T. kirilowii

The transcriptome data used in this study were uploaded to the National Center for Biotechnology Information (NCBI) website (https://www.ncbi.nlm.nih.gov/ (accessed on 20 January 2025)) (project accession number PRJNA858494). The Arabidopsis Information Resource (http://www.arabidopsis.org) was used to download Arabidopsis thaliana B-box protein sequences. The Pfam database (http://pfam.xfam.org/ (accessed on 20 January 2025)) was used to obtain TkBBX candidate genes and the complete B-box domain was verified using the NCBI online tool CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 20 January 2025)). The genes’ amino acid number, open reading frame (ORF) length (bp), isoelectric point (pI), and molecular weight (MW) were calculated on the ExPASy website (https://web.expasy.org/compute_pi/pi_tool-doc.html (accessed on 20 January 2025)) [52]. The WOLF PSORT (http://www.genscript.com/psort.html (accessed on 20 January 2025)) website was used to predict the subcellular localization of the TkBBX proteins [53].

4.3. Phylogenetic and Conserved Motif Analysis of TkBBX Gene Family

An unrooted phylogenetic tree was constructed by MEGA 11.0 software using the neighbor-joining method with 1000 replications [54]. The MEME online tool (https://meme-suite.org/meme/ (accessed on 20 January 2025)) was used to identify conserved motifs [55]. For paralogous pairs in T. kirilowii and orthologous pairs between T. kirilowii and A. thaliana, Ka, Ks, and Ka/Ks values for homologous pairs were determined and obtained based on previous research [56].

4.4. Expression Pattern Analysis of TkBBX Genes in Different Flower Periods

The fold change (FC) and significant q-values were calculated by TBtools 2.136 software [57]. Those genes that satisfied both FC > 1 and p < 0.05 were defined as differentially expressed genes (DEGs). The FPKM values of TkBBX were used to draw heat maps.

4.5. RNA Extraction and qRT-PCR Analysis

Total RNA of leaves was extracted using the Spin Column Plant Total RNA Purification Kit (Shanghai, Sangon) according to the manufacturer’s instructions. The integrity of RNA was detected by 1% agarose gel electrophoresis, and a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Wilmington, DE, USA) was used to detected the concentration of purified RNAs. Total RNA was reverse transcribed into cDNA utilizing UnionScript First-stand cDNA Synthesis Mix (Genesand Biotech Co., Ltd., Beijing, China) and all the samples were stored at −20 °C. The qRT-PCR was performed using GS AntiQ qPCR SYBR Master Mix (Taraka, Beijing, China). The reaction procedure was carried out in an ABI7500 thermal circulator (Applied Bio-systems, Foster City, CA, USA). The relative expression was calculated using the 2−∆∆CT method [58]. GAPDH was used as an internal control [59] and GraphPad 8 software was used for statistical analysis [60]. The primers used in this experiment are listed in Table S1.

4.6. Subcellular Localization Assay

The full-length CDSs of TkBBX7 and TkBBX17 genes were cloned from T. kirilowii, and the products were inserted into a pMD43 vector containing the CaMV35S promoter and GFP. The recombinant vector plasmid and empty vector plasmid were transformed into Agrobacterium competent GV3101, respectively. The cell suspensions were injected into tobacco leaves for instantaneous transformation. After 48 h, the green fluorescence signal was observed by a laser scanning confocal microscope (LSM 880, Zeiss, Wetzlar, Germany).

4.7. Functional Verification of TkBBX7 and TkBBX17 in Yeast

The CDS of TkBBX7 and TkBBX17 were inserted into pYES2-NTB vectors to create the fusion plasmids pYES2-TkBBX7 and pYES2-TkBBX17. The empty vector and two fusion vectors were transformed into the yeast strain INVSC1, respectively. The yeast cell suspensions were coated on SD-Ura medium and cultured at 29 °C for 72–96 h. SD/-Ura liquid medium was used to culture positive colonies until OD600 was just above 1.2, then SG/-Ura liquid medium was used to induce expression. The yeast cell suspensions were diluted to an OD600 of 0.5 and further diluted 10, 100, and 1000 times. The bacterial solutions grown on SG/Ura plates and cultured at 30 °C were the control group. For drought and salt treatment, the continuously diluted bacterial solution was plated on SG/-Ura medium containing mannitol (1.5 M, 1.75 M) and NaCl (0.75 M, 1 M), respectively.

5. Conclusions

A total of 25 BBX genes were identified and systematically analyzed in the T. kirilowii transcriptome. The evolutionary features of TkBBX genes were examined through phylogenetic comparison and homology. Based on the expression levels during flowering and under drought and salt treatment, TkBBX7 and TkBBX17 were screened for further experiments. Subcellular localization experiments indicated that the candidate genes have typical transcription factor characteristics, that is, they are all located in the nucleus. Transgenic yeast experiments demonstrated that TkBBX7 and TkBBX17 both enhanced yeast tolerance to salt and drought. In summary, these findings offer a valuable reference for comprehending the distinct biological function of TkBBX genes in T. kirilowii.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14060975/s1, Table S1: The primers used in this study.

Author Contributions

Conceptualization, W.L. and R.X.; methodology, W.L.; software, Z.C.; validation, W.L., R.X. and Z.C.; formal analysis, W.L.; investigation, X.P.; resources, L.D.; data curation, G.C.; writing—original draft preparation, W.L.; writing—review and editing, W.L.; visualization, R.X.; supervision, G.C.; project administration, X.P.; funding acquisition, W.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Modern Agro-industry Technology Research System, grant number CARS-21; the ability establishment of sustainable use for valuable Chinese medicine resources, grant number 2060302.

Data Availability Statement

The raw sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) database under accession number PRJNA858494.

Acknowledgments

The transcriptome profiling was carried out by Biomarker Technologies.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic tree of full-length BBX genes from Trichosanthes kirilowii and Arabidopsis thaliana.
Figure 1. Phylogenetic tree of full-length BBX genes from Trichosanthes kirilowii and Arabidopsis thaliana.
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Figure 2. Domain, motif compositions, and distribution of conserved motifs of TkBBX proteins. (A): Phylogenetic tree of the TkBBX family. Colors represent the different groups. (B): Conserved motif analysis of TkBBX within each group. Different colored boxes represent different motifs. (C): Domain analysis of TkBBX within each group. (D): Conserved amino acid sequences and the length of each motif.
Figure 2. Domain, motif compositions, and distribution of conserved motifs of TkBBX proteins. (A): Phylogenetic tree of the TkBBX family. Colors represent the different groups. (B): Conserved motif analysis of TkBBX within each group. Different colored boxes represent different motifs. (C): Domain analysis of TkBBX within each group. (D): Conserved amino acid sequences and the length of each motif.
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Figure 3. Heat map of TkBBX genes at different flowering stages based on transcriptome data. (A): Female flower; (B): Male flower; F1: buds; F2: preliminary bloom; F3: full bloom; F4: fade bloom.
Figure 3. Heat map of TkBBX genes at different flowering stages based on transcriptome data. (A): Female flower; (B): Male flower; F1: buds; F2: preliminary bloom; F3: full bloom; F4: fade bloom.
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Figure 4. Expression analysis of 9 TkBBX genes under salt and drought treatments by qRT-PCR. (A): Nine TkBBXs’ expression levels in seedlings under 300 mM NaCl treatments. (B): Nine TkBBXs’ expression levels in seedlings under 20% PEG 6000 treatments. Error bars indicate standard deviations among three independent biological replications. **: p-value < 0.01.
Figure 4. Expression analysis of 9 TkBBX genes under salt and drought treatments by qRT-PCR. (A): Nine TkBBXs’ expression levels in seedlings under 300 mM NaCl treatments. (B): Nine TkBBXs’ expression levels in seedlings under 20% PEG 6000 treatments. Error bars indicate standard deviations among three independent biological replications. **: p-value < 0.01.
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Figure 5. The subcellular localization analysis of TkBBX7 and TkBBX17 proteins.
Figure 5. The subcellular localization analysis of TkBBX7 and TkBBX17 proteins.
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Figure 6. The function analysis of TkBBX genes under salt and drought stresses in yeast strain INVSC1. (A): The function analysis of control and TkBBX7 and TkBBX17 genes under different drought stresses in yeast strain INVSC1. (B): The function analysis of control and TkBBX7 and TkBBX17 genes under different salt stresses in yeast strain INVSC1.
Figure 6. The function analysis of TkBBX genes under salt and drought stresses in yeast strain INVSC1. (A): The function analysis of control and TkBBX7 and TkBBX17 genes under different drought stresses in yeast strain INVSC1. (B): The function analysis of control and TkBBX7 and TkBBX17 genes under different salt stresses in yeast strain INVSC1.
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Table 1. Characterization of TkBBXs identified in Trichosanthes kirilowi.
Table 1. Characterization of TkBBXs identified in Trichosanthes kirilowi.
NameORF Length (bp)ProteinSubcellular Localization
Length (aa)PIMW (Da)
TkBBX112514165.3445,315.65nucleus
TkBBX25911965.42 21,528.00nucleus
TkBBX310713564.74 40,194.50nucleus
TkBBX412274085.48 44,870.00nucleus
TkBBX511973986.22 44,811.11nucleus
TkBBX69333105.30 34,189.24nucleus
TkBBX78342774.24 30,376.77nucleus
TkBBX812514165.27 45,256.58nucleus
TkBBX99543174.79 35,435.72nucleus
TkBBX1011013664.82 39,790.15nucleus
TkBBX116872284.86 25,319.24nucleus
TkBBX129543178.22 34,707.13nucleus
TkBBX134801596.86 17,469.76nucleus
TkBBX1431210310.42 11,615.83nucleus
TkBBX158882955.36 32,186.03nucleus
TkBBX167082355.02 25,915.49nucleus
TkBBX178462814.20 31,019.26nucleus
TkBBX186512166.37 23,578.75nucleus
TkBBX199513167.51 34,439.71nucleus
TkBBX204561515.70 16,623.66nucleus
TkBBX2112274085.31 44,798.88nucleus
TkBBX22228755.13 8068.35nucleus
TkBBX236692225.60 24,372.74nucleus
TkBBX244621536.74 17,176.36nucleus
TkBBX2513234406.12 49,928.00nucleus
Table 2. Ka, Ks, and Ka/Ks values for the BBX genes in Trichosanthes and Arabidopsis.
Table 2. Ka, Ks, and Ka/Ks values for the BBX genes in Trichosanthes and Arabidopsis.
Paralogous PairsKaKsKa/Ks
TkBBX1/TkBBX180.0628110.0849510.73938
TkBBX2/TkBBX40.0133960.0223760.598669
TkBBX3/TkBBX250.0275150.0558710.492471
TkBBX5/TkBBX250.3498043543.0313009590.115397434
TkBBX8/TkBBX180.0649636280.0774805060.838451254
TkBBX7/TkBBX170.0077620360.0109791530.706979455
TkBBX9/TkBBX110.0085416930.0097933930.872189379
TkBBX10/TkBBX210.004717460.0122243790.385905915
TkBBX13/TkBBX200.0172793820.061415370.281352732
TkBBX22/TkBBX240.0646952580.185556130.348656001
Orthologous pairsKaKsKa/Ks
TkBBX8/AtBBX70.2574203382.437984840.105587342
TkBBX11/AtBBX150.3645691
TkBBX12/AtBBX60.3737134243.0800753120.12133256
TkBBX18/AtBBX250.223789243
TkBBX18/AtBBX80.356460289
TkBBX20/AtBBX180.256192316
TkBBX23/AtBBX200.324254708
TkBBX24/AtBBX190.168224215
TkBBX25/AtBBX140.419126264
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Li, W.; Xiong, R.; Chu, Z.; Peng, X.; Cui, G.; Dong, L. Transcript-Wide Identification and Characterization of the BBX Gene Family in Trichosanthes kirilowii and Its Potential Roles in Development and Abiotic Stress. Plants 2025, 14, 975. https://doi.org/10.3390/plants14060975

AMA Style

Li W, Xiong R, Chu Z, Peng X, Cui G, Dong L. Transcript-Wide Identification and Characterization of the BBX Gene Family in Trichosanthes kirilowii and Its Potential Roles in Development and Abiotic Stress. Plants. 2025; 14(6):975. https://doi.org/10.3390/plants14060975

Chicago/Turabian Style

Li, Weiwen, Rui Xiong, Zhuannan Chu, Xingxing Peng, Guangsheng Cui, and Ling Dong. 2025. "Transcript-Wide Identification and Characterization of the BBX Gene Family in Trichosanthes kirilowii and Its Potential Roles in Development and Abiotic Stress" Plants 14, no. 6: 975. https://doi.org/10.3390/plants14060975

APA Style

Li, W., Xiong, R., Chu, Z., Peng, X., Cui, G., & Dong, L. (2025). Transcript-Wide Identification and Characterization of the BBX Gene Family in Trichosanthes kirilowii and Its Potential Roles in Development and Abiotic Stress. Plants, 14(6), 975. https://doi.org/10.3390/plants14060975

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