Next Article in Journal
Transcriptome Analysis Provides Insights into Copulation, Fertilization, and Gestation in Sebastes schlegelii
Previous Article in Journal
Basement-Membrane-Related Gene Signature Predicts Prognosis in WHO Grade II/III Gliomas
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 13
Issue 10
10.3390/genes13101811
genes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

PyunBBX18 Is Involved in the Regulation of Anthocyanins Biosynthesis under UV-B Stress

by
Qin Zhang
1,2,†,
Dongxiao Ma
1,2,†,
Zhixu Hu
1,2,
Dan Zong
1,2,3,* and
Chengzhong He
1,2,3
1
College of Life Sciences, Southwest Forestry University, Kunming 650224, China
2
Key Laboratory for Forest Genetic and Tree Improvement and Propagation in Universities of Yunnan Province, Southwest Forestry University, Kunming 650224, China
3
Key Laboratory of Biodiversity Conservation in Southwest China, State Forestry Administration, Southwest Forestry University, Kunming 650224, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2022, 13(10), 1811; https://doi.org/10.3390/genes13101811
Submission received: 15 September 2022 / Revised: 2 October 2022 / Accepted: 5 October 2022 / Published: 7 October 2022
(This article belongs to the Section Plant Genetics and Genomics)
Browse Figures
Versions Notes

Abstract

:
(1) Background: Populus yunnanensis Dode (P. yunnanensis) grows in the low-latitude and high-altitude areas of southwest China. In low-latitude and high-altitude areas, plants suffer from the high intensity of UV-B (ultraviolet-b) radiation, and they have a complete regulation system to adapt to the environment of the high UV-B radiation. As natural antioxidants, anthocyanins play an important role in scavenging free radicals. BBX (B-box) genes are involved in anthocyanins biosynthesis. (2) Methods: By exploring the gene structure and motifs of PyunBBX genes (genes of P. yunnanensis BBX family) and the evolutionary relationship between PyunBBX genes and other species BBX genes, six PyunBBX genes that responded to UV-B and participated in anthocyanins biosynthesis were screened. BBX, with the potential to regulate anthocyanins biosynthesis, was further investigated by anthocyanins content determination and RT-qPCR (real-time quantitative polymerase chain reaction); (3) Results: After 7 days of UV-B treatment, anthocyanins were significantly accumulated, and the expression of PyunBBX18 was up-regulated for 7 days. The expression of PyunBBX12 was inhibited by UV-B treatment. By analyzing the RNA-seq data of leaves and bark of P. yunnanensis, we found that PyunBBX18 was highly expressed in leaves and young bark; (4) Conclusions: These results showed that PyunBBX18 and PyunBBX12 may be involved in the response process of UV-B stress, in which PyunBBX18 may regulate the anthocyanins biosynthesis to resist UV damage.

1. Introduction

Transcription factors (TFs) play an important role in plant growth and development and stress defense. BBX is a kind of TF containing a zinc finger structure that mediates and regulates many biological processes in plants, including photomorphogenesis, flowering regulation, and response to biological and abiotic stresses [1]. The N-terminal of the BBX protein contains one B-box-conserved domain or two B-box-conserved domains, namely B-box1 and B-box2. Some BBX have a CCT (CONSTANS, CO-like, and TOC1) domain at the C-terminal. The B-box conserved domain is involved in protein interaction and transcriptional regulation, while the CCT domain is mainly involved in nuclear transport and transcriptional regulation [2,3].
Over the years, research has shown that the majority of BBX gene family members can interact with HY5 (ELONGATED HYPOCOTYL5, an important TF of light signal) or regulate transcription of HY5, thereby regulating photomorphogenesis and anthocyanins biosynthesis in plants [4,5,6,7,8,9,10]. For instance, AtBBX21 binds to T/G-box in the AtHY5 promoter through its second B-box domain, thereby regulating the expression of AtHY5 and HY5-regulated genes to promote photomorphogenesis in plants [5]. Under UV-B treatment, the module SIBBX20/21-SIHY5 could activate the transcription of SIHY5, and the excessive accumulation of SIHY5 protein inhibited its transcription, forming a negative feedback loop to maintain the level of SIHY5 in plants, thus regulating the photomorphogenesis of tomato [11]. PtrBBX23 showed a high expression response in Populus trichocarpa (P. trichocarpa) after different treatments, including high light, blue light, and UV-B radiation. The interaction experiment proved that the transcription of MYB115/119 and structural genes (CHS, F3H), which are associated with anthocyanins biosynthesis, was activated by PtrBBX23 bound to their promoters directly, and the interaction between PtrBBX23 and HY5 enhanced the activation activity of PtrBBX23, thus promoting the accumulation of proanthocyanidins and anthocyanins [12]. MdBBX22 can enhance the binding of HY5 with MdMYB10 and MdCHS by interacting with HY5, thereby promoting anthocyanins biosynthesis of apple fruit response to UV-B signal [13], and a similar mechanism exists in PpBBX16 [10] and MdBBX20 [14].
Anthocyanins are plant pigment with natural activity, and it is a kind of secondary metabolite produced in plants. It is distributed in flowers, leaves, fruits, and other organs. Biosynthesis and accumulation of anthocyanins play an important role in plant adaptation and resistance to harsh environment and can enhance plant resistance to biological and abiotic stresses [15,16,17,18,19,20,21,22,23,24]. Anthocyanin accumulation is not only affected by plants’ own factors but also by external environmental factors, among which light signal is one of the most important environmental factors affecting anthocyanins biosynthesis [25].
P. yunnanensis is an endemic Tacamahaca in China and a representative species of Populus in low-latitude and high-altitude areas of southwest China. It is mainly distributed in high-altitude mountains from 1600 to 3200 m, which have suitable characteristics of strong adaptability, rapid growth, and easy rooting, and it plays an important role in forestry production, afforestation, and environmental protection [26]. Previous studies have shown that the UV-B radiation intensity will increase by 10–16% in every 1000 m rise in altitude [27]. So, in high-altitude areas, P. yunnanensis suffers from the high intensity of UV-B radiation, and it has a complete regulation system to adapt to the environment of the UV-B radiation. As natural antioxidants, anthocyanins play an important role in scavenging free radicals. It can protect plant tissues from excessive UV-B radiation, and enhanced UV-B can promote and induce the biosynthesis of anthocyanins [28,29,30]. However, the fundamental research on molecules of P. yunnanensis in response to UV-B stress is limited. Therefore, further molecular studies are needed to explain the underlying mechanism of adaptation to UV-B radiation in P. yunnanensis. In this study, we identified 43 PyunBBX genes and investigated their structural domains and the effects of UV-B irradiation on the BBX gene expression and anthocyanins content in P. yunnanensis. This work will be helpful for future studies of BBX gene functions under UV-B radiation stress in P. yunnanensis.

2. Materials and Methods

2.1. Identification of BBX Gene Family Members in P. yunnanensis

The genome at the chromosome level of P. yunnanensis was obtained from our laboratory group (Southwest Forestry University, Kunming 6500224, China). In order to identify the BBX in P. yunnanensis genome, firstly, the 32 members of BBX proteins sequences in Arabidopsis thaliana (A. thaliana) genome were downloaded from the TAIR 10 (https://www.arabidopsis.org/, accessed on 1 December 2021) database [3]. The 32 proteins sequences were used to search the BBX family members in P. yunnanensis by Blastp (E-value < 1E-05) [31]. The profile of the hidden Markov model (HMM) for the B-box domain (Pfam00643) and CCT domain (Pfam06203) was obtained from Pfam (https://pfam.xfam.org/, accessed on 2 December 2021) [32]. The hmmsearch program of the HMMER 3.0 software was used to search for the putative BBX gene family members in the P. yunnanensis genome, and the E-value was set to 1E-05 [33]. If a gene had multiple transcripts, the longest transcript was selected as the representative protein sequence for further analysis. After removing the duplicate for candidate BBX gene family members, the reserved protein sequences of BBX gene family members were submitted to the SMART database (http://smart.embl-heidelberg.de/, accessed on 3 December 2021) [34], CDD (Conserved Domain Database, https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, accessed on 3 December 2021) [35], and InterPro database (http://www.ebi.ac.uk/interpro/, accessed on 3 December 2021) [36] in order to analyze whether it contained unique B-box conservative domain structure. The B-box conservative domain structure of candidate genes was identified as the BBX gene family members of P. yunnanensis.

2.2. Analysis of Basic Physicochemical and Subcellular Localization of PyunBBX Genes

The molecular weight (kDa) and isoelectric point (PI) of each BBX protein were predicted by ExPASy (http://www.expasy.org/tools/, accessed on 4 December 2021) [37]. The online software Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 4 December 2021) was used to predict the subcellular localization of PyunBBX genes [38].

2.3. Gene Structure and Motif Analysis of PyunBBX Genes

The length and location of exons and introns of PyunBBX genes were obtained from the P. yunnanensis genome for further analysis. Each sequence of PyunBBX genes was submitted to MEME (https://meme-suite.org/meme/tools/meme, accessed on 18 December 2021) [39]. In the conservative base sequence analysis, the maximum was set to 10, the mode was selected as zoops, and the default values of other parameters were used. Finally, the results were visualized by TBtools [40] and WebLogo (http://weblogo.berkeley.edu/logo.cgi, accessed on 20 December 2021).

2.4. Cis-Acting Elements Analysis

The promoter-upstream region (~2000 bp) sequences were extracted from the genomic DNA sequence of P. yunnanensis, and the sequences were submitted to the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 2 January 2022) [41] for cis-acting element prediction. Excel was used to sort out and simplify the analysis results, and the cis-acting elements with the same biological functions were labeled with the same label notes.

2.5. Chromosomal Location, Gene Duplication, and Synteny Analysis

The chromosome location image of the PyunBBX genes was drawn using the TBtools software according to the physical positions of PyunBBX genes on the P. yunnanensis chromosomes. All the BBX amino acid sequences of A. thaliana, P. trichocarpa, and P. yunnanensis were included in a local database using DIAMOND [42]. The P. trichocarpa genome (version = 3.0) was derived from the Phytozome database (https://phytozome-next.jgi.doe.gov/info/Ptrichocarpa_v3_0, accessed on 15 March 2022). The BBX protein sequences of P. yunnanensis were used as queries to search the above-mentioned database with an E-value of 1E-05. The blast results were analyzed by the MCScanX (Multiple Collinear Scan Kit) [43] and TBtools to determine and analyze the duplication and synteny of BBX genes. The Ks (synonymous substitution rate) and Ka (nonsynonymous substitution rate) values of collinearity pairs in PyunBBX genes were estimated by the KaKs_calculator [44] procedure in TBtools. The divergence time was calculated with the formula T = Ks/2r, with Ks being the synonymous substitutions per site and r being the rate of divergence for nuclear genes from plants. The r was taken to be 1.5 × 10−8 synonymous substitutions per site per year for dicotyledonous plants [45].

2.6. Evolutionary Analysis of BBX Gene Family Members

The molecular evolutionary genetic analysis tool MEGA (Molecular Evolutionary Genetics Analysis) version 7.0.20 [46] was used for multiple sequence alignment and evolutionary analysis. The ClustalW program of MEGA was firstly used for multi-sequence alignment of the BBX amino acid sequences. Then, the gaps were manually sheared, and conservative region sequences were reserved. The maximum likelihood (ML) method was selected to construct the phylogenetic tree for PyunBBX genes, and the neighbor-joining (NJ) method constructed a phylogenetic tree for BBX genes of multispecies. Bootstrap was set to 1000.

2.7. Plant Materials, Growth Conditions, and UV Treatment

The plant material was from the branches of P. yunnanensis that had grown for one year in the greenhouse of Southwest Forestry University. They were pruned into cuttings of about 15 cm, and cuttings were made in March 2022. The cuttings were induced when they had reached 5–6 leaves. The cuttings of the same growth were placed in the constant temperature incubator (temperature: 25℃, humidity: 70%, light intensity: 90 µmol·m−2·s−1, photoperiod: 12 h light/12 h dark) for cultivation, a UV lamp (Telipu, Beijing Zhongyi Boteng Technology Company, power: 8 W, radiation intensity: 68 µW/cm2, wavelength: 280–320 nm) was placed 35 ± 1 cm above the cuttings, and UV irradiation was carried out for 12 h every day under normal light treatment. The cottage seedlings were sampled after UV treatment for 12 h, and the sampling time was 0, 1, 2, 3, 5, and 7 days. The control group was cottage seedlings that grew under normal light. The sampling sites were the second and third fully extended leaves. The samples were frozen with liquid nitrogen and immediately stored in the −80 °C refrigerator.

2.8. Total RNA Isolation and cDNA Synthesis

Total RNA was isolated from the leaf of P. yunnanensis using the E.Z.N.A.@ Plant RNA kit (Omega Bio-tek Inc., Norcross, USA) according to its manual. The quality of RNA was evaluated with K5800C (KAIAO, Beijing, China). A total of 500 ng RNA of each sample was used for 1st strand cDNA synthesis using Hifair® Ⅲ Reverse Transcriptase (YEASEN, Shanghai, China) according to the manufacturer’s protocols. cDNA was diluted 8-fold for RT-qPCR analysis.

2.9. Quantitative Real-Time PCR

Primers were designed based on CDS sequences for real-time PCR by using NCBI primer-blast (Table S1). The internal reference gene was HIS (histone) [47]. Real-time PCR application was carried out in a LightCycler® 96 Real-Time PCR Detection System (Roche, Hercules, Switzerland) with Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (YEASEN, Shanghai, China). A total of 20 μL reaction system contained 10 μL Blue qPCR SYBR Green Master Mix, 1 μL cDNA samples, 0.4 μL of each primer (1 μM), and 8.2 μL ddH2O. The PCR thermal cycle conditions were as follows: denaturation at 95 °C for 2 min, 45 cycles of 95 °C for 10 s, and 56 °C for 30 s. Fluorescence intensities were measured for RT-qPCR at the end of each cycle. A melting curve (1 cycle of 95 °C for 10 s, 65 °C for 10 s, and 97 °C for 1 s) was performed directly to check for specific amplification. The relative gene expression was calculated by using the 2−△△Ct method [48], and the experiments were performed in triplicate technological repeats.

2.10. Extraction and Determination of Anthocyanins

Determination of anthocyanins content used ethanol/hydrochloric acid assay [49]. Fresh leaves were cut into pieces and weighed with 0.1 g, placed in a 10 mL centrifuge tube, and 10 mL hydrochloric acid ethanol (0.1 mol/L) solution was added to the tube. It was warmed at 60 °C for 30 min in a water bath. Then, the supernatant was transferred to a 25 mL volumetric flask after cooling. Then, 5 mL hydrochloric acid ethanol solution was added to the tube and continued water bath for 15 min. This step was repeated three times, and the supernatant was combined. Finally, the volume was fixed to 25 mL, and the absorbance value (Abs) was measured at 530 nm. Substitute the measured results into the following formula for calculation:
C = 958 − 1 × A × V × m − 1 × 10 3
C: anthocyanins content (mg/100 g), A: Abs (nm), V: metered volume (mL), m: mass (g), 958: empirical coefficient of Abs conversion into mass volume ratio, 103: unit conversion coefficient.

2.11. Expression Patterns of BBX Family Members in P. yunnanensis by Rna-seq Data

Transcriptome data of leaves (PRJNA505895) and bark (PRJNA542544) at different mature sites (PRJNA506110) were downloaded from NCBI (National Center for Biotechnology Information). For raw data, fastQC (Version = 0.11.9) was used for quality inspection. Fastp (Version = 0.19.4) further filtered low-quality reads. The obtained high-quality clean reads were compared to the genome of P. yunnanensis using Hisat2 (version = 7.5.0). FeaturCounts (Version = 2.0.1) normalized FPKM (fragments per kilobase of exon model per million mapped fragments) values were obtained in the R language for expression pattern analysis. Heatmap (Version = 2.8.0) was used to draw the heat map of gene expression.

2.12. Data Analysis

Adobe Photoshop CS6 was used for image processing, and Adobe Illustrator 2020 was used for chart layout. The data obtained from the experiment were plotted and analyzed by Graphpad Prism 8.0.2 software.

3. Results

3.1. Analysis of The Basic Characteristics of PyunBBX Genes

Totally, 43 PyunBBX genes were identified from the P. yunnanensis genome. They were named PyunBBX1 to PyunBBX43 in the order of their position on chromosomes. They had different sequence lengths, resulting in different isoelectric points and molecular weights (Table 1). Amino acid sequences ranged in length from 184 aa (PyunBBX10) to 513 aa (PyunBBX1). The predicted molecular weight of the BBX gene was 12.11 kDa (PyunBBX5) to 59.46 kDa (PyunBBX9), and the theoretical isoelectric point was 4.12 (PyunBBX30) to 9.26 (PyunBBX29). Subcellular prediction showed that all of the PyunBBX genes were located in the nucleus.
On 19 chromosomes of the P. yunnanensis genome, there were no PyunBBX genes on chromosomes 12, 13, and 19, and the other chromosomes (chromosomes 1–11 and chromosomes 14–18) had PyunBBX genes (Figure 1).

3.2. Gene Structure and Characteristics of Conserved Sequences of PyunBBX Gene Family Members

The amino acid sequences of 43 members of the P. yunnanensis BBX gene family were analyzed, and the 3 most significant conserved sequences were extracted, namely B-box1, B-box2, and CCT (Figure 2, Figure 3, and Figure S1). B-box1 and B-box2 have a high similarity, with the structure of C2-X2-C-X7, 8-C-X7-C-X2-C-D-Xn-H. Besides PyunBBX5, each of them has five conserved cysteine (Cys, C) residues adjacent to aspartic acid (Asp, D). Moreover, the Cys on both sides are (Leu, L) linked. Such a structure can combine with Zn2+ and form a “finger” structure through self-folding. It can be further combined with RNA, DNA, and protein to carry out gene regulation at the level of transcription and translation. Some BBX members also have a CCT domain, which is R-X5-R-Y-X-E-X4-R-X3-K-X2-R-Y-X3-K-X5-R-X-R-X2-G, containing a large number of arginine (Arg, R), followed by conservative tyrosine (Tyr, Y) and lysine (Lys, K), participating in the nuclear localization of the protein [50].
The structure of PyunBBX genes is shown in Figure 3. The results showed that most PyunBBX genes contained at least one intron and up to five introns. PyunBBX genes could be divided into five groups. It was consistent with the classification of other species. The first group contains 12 PyunBBX genes with 2 B-box domains and 1 CCT domain. The second group contains six PyunBBX genes with two B-box domains and one CCT domain. The third group contains three PyunBBX genes with a B-box domain and a CCT structure. The fourth group contains 14 members of PyunBBX genes with 2 B-box domains. The fifth group contains eight PyunBBX genes with a B-box domain. In addition, some PyunBBX genes (PyunBBX12/17/18/21/37/38/41/42/43) also have a VP motif, and the VP peptide motif in the gene can bind to the WD40 domain of COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) and interact with it to participate in the light response [51].

3.3. Collinearity Analysis

The high sequence similarity between repetitive gene pairs indicated that they were likely involved in regulating similar biological processes. MCScanX was used to analyze the collinear blocks and gene duplication type. There were 14 gene pairs of PyunBBX genes, which were PyunBBX1/9, PyunBBX6/16, PyunBBX15/19, PyunBBX13/26, PyunBBX14/27, PyunBBX18/42, PyunBBX23/28, PyunBBX22/29, PyunBBX4/31, PyunBBX34/35, PyunBBX21/37, PyunBBX20/39, PyunBBX12/41, and PyunBBX17/43, and they belong to segmental in duplicated type (Figure 4, Table 2). The Ka/Ks values of 14 pairs of collinear blocks were all less than 1, indicating that these members may have been affected by purification selection in evolution. By calculating the divergence time of collinear blocks, the result showed that the average differentiation time of PyunBBX genes was 2.3 Mya (millions of years ago). Moreover, PyunBBX22 and PyunBBX29 differentiated at 3.92 Mya that they had the longest time of differentiation, and PyunBBX34 and PyunBBX35 differentiated at 0.24 Mya that they diverged more recently (Table 2).
We constructed comparative syntenic maps between P. yunnanensis and the other two species (P. trichocarpa and A. thaliana) to analyze the similarities of the BBX gene among species. Syntenic maps revealed that 86 pairs of homologous genes were found between P. trichocarpa and P. yunnanensis, and 46 pairs of homologous genes were found between P. yunnanensis and A. thaliana (Figure 5).

3.4. Cis-Acting Elements of PyunBBX Genes Analysis

The promoter-upstream region (~2000 bp) sequences were extracted from the genomic DNA sequence of PyunBBX genes. The cis-elements of the PyunBBX gene family promoters were analyzed by using the PlantCARE database (Figure 6, Table S2). Each promoter of PyunBBX genes contains abiotic and biotic stress response elements (such as ARE, GC-motif, MBS, LTR, TC-motif, DRE-core, STRE, WUN-motif, and WRE3), phytohormone correlation (such as as-1, TCA-element, W box, CGTCA-motif, TGACG-motif, ABRE, AAGAA-motif, ERE, p-box, GARE-motif, TATC-box, AuxRR-core, and TGA-element), and light responsiveness (such as G-Box, MRE, GT1-motif, Sp1, ACE, 3-AF1 binding site, and AAAC-motif). Some PyunBBX genes had the cis-acting elements of the MYB binding site involved in light responsiveness, flavonoid biosynthetic genes regulation, and drought-inducibility (Table S2). This showed the functional diversity of the PyunBBX genes.

3.5. Expression Patterns of PyunBBX Genes in Leaf and Bark of P. yunnanensis by Rna-seq Data

The expression patterns of PyunBBX genes in leaf and bark were analyzed using RNA-seq data of leaves and bark in NCBI. Compared to the bark, PyunBBX12/15/18/29/36/37/38 expression quantity is relatively high in the leaves. The expression level of PyunBBX13 was very low in bark, with almost no expression (average of FPKM= 0.014062), while it was expressed in leaves (average of FPKM = 4.59). Compared with the leaves, PyunBBX3/4/6/8/14/16/27/28/31/32/40 expressed in the bark of the organization’s quantity is relatively high (Figure 7). The expression patterns of PyunBBXs in bark tissues at different locations showed that the expression patterns of most PyunBBXs were basically consistent, indicating that they performed the same function in bark tissues at different locations (Figure S2) [52]. The expression level of PyunBBX16/36/42 in the younger bark position (EU, Figure S2) was lower than that of other bark tissues, and the expression level of PyunBBX18 in EU was higher than that of other bark tissues (Figure 8).

3.6. Expression Patterns of Six PyunBBXs under UV Treatment by RT-qPCR Analysis

In order to determine the PyunBBX genes in response to UV, we picked out the highly expressed PtrBBXs of P. trichocarpa under the UV-B treatment (PtrBBX1/2/5/8/23/24/25/29). Based on the relationship of homology and collinearity (Figure 9), PyunBBXs with high homology to these eight genes were selected from the BBX gene family of P. yunnanensis [12]. A total of 12 PyunBBX genes were obtained. In order to determine whether these 12 PyunBBX genes interact with MYB, PyunBBX genes with MYB binding sites were screened according to PlantCARE results, and finally, six PyunBX genes were determined (Figure S3, Table S2). Under UV treatment, the results of RT-qPCR showed that the six PyunBBX genes showed different expression patterns (Figure 10), and the relative expression level of PyunBBX3/4/42 increased compared with the control group. The expression of PyunBBX12 was inhibited, suggesting that PyunBBX12 may be involved in the negative feedback regulatory pathway. The expression level of PyunBBX18 was up-regulated on day 7, which was consistent with anthocyanins accumulation observed on day 7 (Figure 11), indicating that PyunBBX18 may be directly involved in regulating the anthocyanins biosynthesis pathway. Compared with the control group, the expression level of PyunBBX13 showed a high–low–high trend under UV treatment, indicating the complexity of PyunBBX13 response to UV.

4. Discussion

4.1. Identification and Analysis of PyunBBX Gene Family Members in P. yunnanensis

In this study, 43 BBX gene family members were identified from the genome of P. yunnanensis, which can be divided into five types (Figure 3). They all have typical B-box structures, but the B-box1 structure of PyunBBX5 lacks the amino acid sequence of C-X2-C (Figure S1). Therefore, PyunBBX5 is a type of BBX-like gene, and its functions need to be further verified.
The process of segmental and tandem duplication is crucial for the amplification of gene family members in the genome during evolution [53]. In this study, we analyzed tandem and segmental duplication events to study the evolutionary path of BBX genes in P. yunnanensis. We found only segmental duplication in BBX genes (Table 2), suggesting that fragment duplication is involved in the expansion of PyunBBX genes.
In the phylogenetic tree (Figure 7) of P. yunnanensis, P. trichocarpa, and A. thaliana, it was found that BBX family genes were not clustered strictly according to the combination of conserved domains, and the uneven distribution may be due to a large number of genes or slight differences in domain organization in plant species.
The promoter regions of 43 PyunBBX genes also contain cis-acting elements of various plant hormones, such as ABA (abscisic acid), JA (jasmonic acid), IAA (indoleacetic acid), GA (gibberellic acid), ethylene, etc. Their functions in P. yunnanensis deserve further study.
In order to identify the UV-responsive PyunBBX genes under UV stress, we selected the up-regulated BBX genes of P. trichocarpa under UV treatment [12]. According to homology, we obtained 12 candidate PyunBBX genes. Finally, six PyunBBX genes were identified by the cis-acting elements of the promoter. The results of RT-qPCR showed that only the expression of PyunBBX3/4/18/42 was increased under UV treatment, while the expression of PyunBBX12 was inhibited. Moreover, PyunBBX13 showed functional complexity. The above shows that homologous genes of near-origin species may have similarities and differences in performing functions.
There is a VP motif binding to COP1 on the C segment of PyunBBX18, indicating that it interacts with COP1. In addition, PyunBBX12/42 also has a VP motif, revealing that they are important factors involved in light signal transduction and regulatory pathways.

4.2. BBX Is Involved in Anthocyanins Biosynthesis

Structural genes encoding key enzymes in anthocyanins biosynthesis are regulated by transcription factors. Widely studied TFs include the MYB gene family, bHLH gene family, WD40 gene protein, etc., which regulate anthocyanins biosynthesis by combining with functional elements in the promoter region of structural genes. For example, overexpression of SIMYB75 in tomatoes can increase anthocyanin content in fruits [54]. MYB TFs Aft can negatively regulate anthocyanins biosynthesis by binding to the promoter of negative regulator SlMYBATV [55]. Two bHLH TFs, JAF13 and AN1, in petunias can directly or indirectly bind with MYB transcription factor AN2 to activate DFR expression and promote anthocyanins biosynthesis [56,57]. The expression level of the StWD40 gene in potatoes was significantly up-regulated in red and purple tubers, suggesting that the up-regulated expression level of WD40 could promote anthocyanins accumulation [58].
Studies have shown that UV-B light can induce anthocyanin accumulation in plants. In apples, both MdHY5 and MdBBX22 could independently increase anthocyanin accumulation in the callus of apples under UV-B induction, and further investigation revealed that MdBBX22 and MdHY5 had interaction to jointly promote anthocyanin accumulation. In addition, both MdBBX24 and MdBBX33 interact with MdHY5 to regulate anthocyanins biosynthesis [13], suggesting that BBX-HY5 interaction is a universal regulatory module [8,59]. Recent studies have revealed that the interaction between HY5 and the BBX TF family affects anthocyanin accumulation, and B-box proteins respond to light induction and assist HY5 proteins in participating in transcriptional regulation of anthocyanin biosynthesis in plants [60]. Therefore, whether PyunBBX18 interacts with HY5 to affect anthocyanin accumulation of P. yunnanensis under UV-B stress requires further experimental verification.
Flavonoids are usually accumulated in leaves, roots, and bark [61,62]. By analyzing RNA-seq data of leaves and bark of P. yunnanensis, PyunBBX18 was found to be highly expressed in leaves and young bark, suggesting that PyunBBX18 may be involved in the synthesis of flavonoids. According to PlantCARE prediction results, the promoter of PyunBBX18 has three MYB binding sites (Figure S3), so it may interact with MYB TFs to regulate the synthesis of flavonoids.

5. Conclusions

In this study, 43 PyunBBX genes were identified and analyzed at the genome-wide level. They were distributed unevenly on 15 chromosomes, and their evolutionary relationships, physical and chemical properties, gene structure, and conserved motifs were analyzed. Collinearity analysis revealed that there were 14 pairs of homologous genes in the P. yunnanensis BBX gene family, and these genes were purified and selected. In addition, RNA-seq showed that PyunBBX18 was highly expressed in leaves and younger bark. The results showed that PyunBBX18 may be a key gene in UV-induced anthocyanin biosynthesis through the determination of anthocyanins content and the expression of genes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes13101811/s1, Figure S1: Amino acid sequences of conserved domains (B-box1/B-box2, CCT, and VP) of BBX genes family in P. yunnanensis; Figure S2: Sampling positions on the cuttings of P. yunnanensis. Figure S3: Cis-acting elements of six PyunBBX genes; Table S1: Primers for RT-qPCR; Table S2: The results of PlantCARE for PyunBBX genes.

Author Contributions

Conceptualization, Q.Z.; Data curation, C.H.; Formal analysis, D.M.; Funding acquisition, C.H.; Investigation, Z.H.; Methodology, Q.Z., Z.H. and D.M.; Project administration, C.H.; Software, Q.Z.; Supervision, D.Z. and C.H.; Validation, Q.Z.; Visualization, Q.Z.; Writing—original draft, Q.Z. and D.M.; Writing—review and editing, Q.Z. and D.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31860219), Yunnan Provincial Expert Workstation (20200AF150020), and the Postgraduate Fund Project of Yunnan Education Department (2022Y590).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Genomic data of P. yunnanensis can be obtained by contacting the corresponding author. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Gangappa, S.N.; Botto, J.F. The BBX family of plant transcription factors. Trends Plant Sci. 2014, 7, 460–470. [Google Scholar] [CrossRef]
  2. Griffiths, S.; Dunford, R.P.; Coupland, G.; Laurie, D.A. The evolution of CONSTANS-like gene families in barley, rice, and Arabidopsis. Plant Physiol. 2003, 131, 1855–1867. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Khanna, R.; Kronmiller, B.; Maszle, D.R.; Coupland, G.; Holm, M.; Mizuno, T.; Wu, S. The Arabidopsis B-Box Zinc Finger Family. Plant Cell 2009, 21, 3416–3420. [Google Scholar] [CrossRef] [Green Version]
  4. Xu, D.; Jiang, Y.; Li, J.; Lin, F.; Holm, M.; Deng, X.W. BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation. Proc. Natl. Acad. Sci. USA 2016, 113, 7655–7660. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Xu, D.; Jiang, Y.; Li, J.; Holm, M.; Deng, X.W. The B-Box Domain Protein BBX21 Promotes Photomorphogenesis. Plant Physiol. 2018, 176, 2365–2375. [Google Scholar] [CrossRef] [PubMed]
  6. Job, N.; Yadukrishnan, P.; Bursch, K.; Datta, S.; Johansson, H. Two B-Box Proteins Regulate Photomorphogenesis by Oppositely Modulating HY5 through their Diverse C-Terminal Domains. Plant Physiol. 2018, 176, 2963–2976. [Google Scholar] [CrossRef]
  7. Heng, Y.; Lin, F.; Jiang, Y.; Ding, M.; Yan, T.; Lan, H.; Zhou, H.; Zhao, X.; Xu, D.; Deng, X.W. B-Box Containing Proteins BBX30 and BBX31, Acting Downstream of HY5, Negatively Regulate Photomorphogenesis in Arabidopsis. Plant Physiol. 2019, 180, 497–508. [Google Scholar] [CrossRef]
  8. Bai, S.; Tao, R.; Yin, L.; Ni, J.; Yang, Q.; Yan, X.; Yang, F.; Guo, X.; Li, H.; Teng, Y. Two B-box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit. Plant J. 2019, 100, 1208–1223. [Google Scholar] [CrossRef]
  9. Gangappa, S.N.; Crocco, C.D.; Johansson, H.; Datta, S.; Hettiarachchi, C.; Holm, M.; Botto, J.F. The Arabidopsis B-BOX protein BBX25 interacts with HY5, negatively regulating BBX22 expression to suppress seedling photomorphogenesis. Plant Cell 2013, 25, 1243–1257. [Google Scholar] [CrossRef] [Green Version]
  10. Bai, S.; Tao, R.; Tang, Y.; Yin, L.; Ma, Y.; Ni, J.; Yan, X.; Yang, Q.; Wu, Z.; Zeng, Y.; et al. BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear. Plant Biotechnol. J. 2019, 17, 1985–1997. [Google Scholar] [CrossRef]
  11. Yang, G.; Zhang, C.; Dong, H.; Liu, X.; Guo, H.; Tong, B.; Fang, F.; Zhao, Y.; Yu, Y.; Liu, Y.; et al. Activation and negative feedback regulation of SlHY5 transcription by the SlBBX20/21–SlHY5 transcription factor module in UV-B signaling. Plant Cell 2022, 34, 2038–2055. [Google Scholar] [CrossRef]
  12. Li, C.; Pei, J.; Yan, X.; Cui, X.; Tsuruta, M.; Liu, Y.; Lian, C. A poplar B-box protein PtrBBX23 modulates the accumulation of anthocyanins and proanthocyanidins in response to high light. Plant Cell Environ. 2021, 44, 3015–3033. [Google Scholar] [CrossRef] [PubMed]
  13. An, J.; Wang, X.; Zhang, X.; Bi, S.; You, C.; Hao, Y. MdBBX22 regulates UV-B-induced anthocyanin biosynthesis through regulating the function of MdHY5 and is targeted by MdBT2 for 26S proteasome-mediated degradation. Plant Biotechnol. J. 2019, 17, 2231–2233. [Google Scholar] [CrossRef] [PubMed]
  14. Fang, H.; Dong, Y.; Yue, X.; Hu, J.; Jiang, S.; Xu, H.; Wang, Y.; Su, M.; Zhang, J.; Zhang, Z.; et al. The B-box zinc finger protein MdBBX20 integrates anthocyanin accumulation in response to ultraviolet radiation and low temperature. Plant Cell Environ. 2019, 42, 2090–2104. [Google Scholar] [CrossRef]
  15. Zhuang, W.B.; Liu, T.Y.; Shu, X.C.; Shen-Chun, Q.U.; Zhai, H.H.; Wang, T.; Zhang, F.J.; Wang, Z. The molecular regulation mechanism of anthocyanin biosynthesis and coloration in plants. Plant Physiol. J. 2018, 11, 1630–1644. [Google Scholar] [CrossRef]
  16. Xie, Y.; Tan, H.; Ma, Z.; Huang, J. DELLA Proteins Promote Anthocyanin Biosynthesis via Sequestering MYBL2 and JAZ Suppressors of the MYB/bHLH/WD40 Complex in Arabidopsis thaliana. Mol. Plant 2016, 9, 711–721. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Mahmood, K.; Xu, Z.; El-Kereamy, A.; Casaretto, J.A.; Rothstein, S.J. The Arabidopsis Transcription Factor ANAC032 Represses Anthocyanin Biosynthesis in Response to High Sucrose and Oxidative and Abiotic Stresses. Front. Plant Sci. 2016, 7, 1548. [Google Scholar] [CrossRef] [Green Version]
  18. Pradeep, S.; Oleg, F.; Dalia, M.; Noam, A. Improved Cold Tolerance of Mango Fruit with Enhanced Anthocyanin and Flavonoid Contents. Molecules 2018, 23, 1832. [Google Scholar] [CrossRef] [Green Version]
  19. Cui, Z.; Bi, W.; Hao, X.; Li, P.; Duan, Y.; Walker, M.A.; Xu, Y.; Wang, Q. Drought Stress Enhances Up-Regulation of Anthocyanin Biosynthesis in Grapevine leafroll-associated virus 3-Infected in vitro Grapevine (Vitis vinifera) Leaves. Plant Dis. 2017, 101, 1605–1615. [Google Scholar] [CrossRef] [Green Version]
  20. Bi, H.; Guo, M.; Wang, J.; Qu, Y.; Du, W.; Zhang, K. Transcriptome analysis reveals anthocyanin acts as a protectant in Begonia semperflorens under low temperature. Acta Physiol. Plant. 2018, 40, 10. [Google Scholar] [CrossRef]
  21. Roberts, J.A.; Evan, D.; Mcmanus, M.T.; Rose, J.K.C. Annual Plant Reviews. Functions of Flavonoid and Betalain Pigments in Abiotic Stress Tolerance in Plants. Annu. Plant Rev. Online 2018, 1, 1–41. [Google Scholar] [CrossRef]
  22. Zhang, T.; Chow, W.S.; Liu, X.; Zhang, P.; Liu, N.; Peng, C. A magic red coat on the surface of young leaves: Anthocyanins distributed in trichome layer protect Castanopsis fissa leaves from photoinhibition. Tree Physiol. 2016, 36, 1296–1306. [Google Scholar] [CrossRef] [Green Version]
  23. Zhang, X.; Zheng, X.; Sun, B.; Peng, C.; Chow, W.S. Over-expression of the CHS gene enhances resistance of Arabidopsis leaves to high light. Environ. Exp. Bot. 2018, 154, 33–43. [Google Scholar] [CrossRef] [Green Version]
  24. Ahmed, N.U.; Park, J.; Jung, H.; Yang, T.; Hur, Y.; Nou, I. Characterization of dihydroflavonol 4-reductase (DFR) genes and their association with cold and freezing stress in Brassica rapa. Gene 2014, 550, 46–55. [Google Scholar] [CrossRef]
  25. Hong, Y.; Wu, Y.; Song, X.; Li, M.; Dai, S. Molecular mechanism of light-induced anthocyanin biosynthesis in horticultural crops. Acta Hortic. Sin. 2021, 48, 1983–2000. [Google Scholar] [CrossRef]
  26. Li, X.; Yang, Y.; Sun, X.; Lin, H.; Chen, J.; Ren, J.; Hu, X.; Yang, Y. Comparative Physiological and Proteomic Analyses of Poplar (Populus yunnanensis) Plantlets Exposed to High Temperature and Drought. PLoS ONE 2014, 9, e107605. [Google Scholar] [CrossRef] [PubMed]
  27. Schmucki, D.A. Ultraviolet radiation in the Alps: The altitude effect. Proc. Spie 2002, 41, 3090–3095. [Google Scholar] [CrossRef]
  28. Costa, D.; Galvão, A.M.; Di Paolo, R.E.; Freitas, A.A.; Lima, J.C.; Quina, F.H.; Maçanita, A.L. Photochemistry of the hemiketal form of anthocyanins and its potential role in plant protection from UV-B radiation. Tetrahedron 2015, 71, 3157–3162. [Google Scholar] [CrossRef]
  29. Tsurunaga, Y.; Takahashi, T.; Katsube, T.; Kudo, A.; Kuramitsu, O.; Ishiwata, M.; Matsumoto, S. Effects of UV-B irradiation on the levels of anthocyanin, rutin and radical scavenging activity of buckwheat sprouts. Food Chem. 2013, 141, 552–556. [Google Scholar] [CrossRef]
  30. Kalidhasan, N.; Bhagavan, N.B.; Dhiraviam, K. Ultraviolet B (280-320 nm) enhanced radiation induced changes in secondary metabolites and photosystem-II of medicinal plant Withania somnifera Dunal. J. Med. Plant Res. 2013, 7, 3112–3120. [Google Scholar] [CrossRef]
  31. Camacho, C.; Coulouris, G.; Avagyan, V.; Ma, N.; Papadopoulos, J.; Bealer, K.; Madden, T.L. BLAST+: Architecture and applications. BMC Bioinform. 2009, 10, 421. [Google Scholar] [CrossRef] [Green Version]
  32. Mistry, J.; Chuguransky, S.; Williams, L.; Qureshi, M.; Salazar, G.A.; Sonnhammer, E.L.L.; Tosatto, S.C.E.; Paladin, L.; Raj, S.; Richardson, L.J.; et al. Pfam: The protein families database in 2021. Nucleic Acids Res. 2021, 49, D412–D419. [Google Scholar] [CrossRef] [PubMed]
  33. Söding, J. Protein homology detection by HMM–HMM comparison. Bioinformatics 2005, 21, 951–960. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Letunic, I.; Khedkar, S.; Bork, P. SMART: Recent updates, new developments and status in 2020. Nucleic Acids Res. 2021, 49, D458–D460. [Google Scholar] [CrossRef] [PubMed]
  35. Marchler-Bauer, A.; Bryant, S.H. CD-Search: Protein domain annotations on the fly. Nucleic Acids Res. 2004, 32, W327–W331. [Google Scholar] [CrossRef] [PubMed]
  36. Blum, M.; Chang, H.; Chuguransky, S.; Grego, T.; Kandasaamy, S.; Mitchell, A.; Nuka, G.; Paysan-Lafosse, T.; Qureshi, M.; Raj, S.; et al. The InterPro protein families and domains database: 20 years on. Nucleic Acids Res. 2021, 49, D344–D354. [Google Scholar] [CrossRef]
  37. Wilkins, M.R.; Gasteiger, E.; Bairoch, A.; Sanchez, J.; Williams, K.L.; Appel, R.D.; Hochstrasser, D.F. Protein Identification and Analysis Tools in the ExPASy Server. In 2-D Proteome Analysis Protocols; Link, A.J., Ed.; Humana Press: Totowa, NJ, USA, 1999; Volume 112. [Google Scholar]
  38. Chou, K.C.; Shen, H.B. Plant-mPLoc: A Top-Down Strategy to Augment the Power for Predicting Plant Protein Subcellular Localization. PLoS ONE 2010, 5, e11335. [Google Scholar] [CrossRef] [Green Version]
  39. Bailey, T.L.; Mikael, B.; Buske, F.A.; Martin, F.; Grant, C.E.; Luca, C.; Jingyuan, R.; Li, W.W.; Noble, W.S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, W202–W208. [Google Scholar] [CrossRef]
  40. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  41. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef]
  42. Buchfink, B.; Xie, C.; Huson, D.H. Fast and sensitive protein alignment using DIAMOND. Nat. Methods 2015, 12, 59–60. [Google Scholar] [CrossRef]
  43. Wang, Y.; Tang, H.; Debarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.; Jin, H.; Marler, B.; Guo, H.; et al. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, e49. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Zhang, Z.; Li, J.; Zhao, X.; Wang, J.; Wong, G.K.; Yu, J. KaKs_Calculator: Calculating Ka and Ks through model selection and model averaging. Genom. Proteom. Bioinform. 2006, 4, 259–263. [Google Scholar] [CrossRef] [Green Version]
  45. Huang, Z.; Duan, W.; Song, X.; Tang, J.; Wu, P.; Zhang, B.; Hou, X. Retention, Molecular Evolution, and Expression Divergence of the Auxin/Indole Acetic Acid and Auxin Response Factor Gene Families in Brassica Rapa Shed Light on Their Evolution Patterns in Plants. Genome Biol. Evol. 2016, 8, 302–316. [Google Scholar] [CrossRef] [Green Version]
  46. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2015, 33, 1870–1874. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Shuai, P.; Liang, D.; Tang, S.; Zhang, Z.; Ye, C.; Su, Y.; Xia, X.; Yin, W. Genome-wide identification and functional prediction of novel and drought-responsive lincRNAs in Populus trichocarpa. J. Exp. Bot. 2014, 65, 4975–4983. [Google Scholar] [CrossRef] [PubMed]
  48. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29, e45. [Google Scholar] [CrossRef]
  49. Fan, G.; Han, Y.; Gu, Z.; Chen, D. Optimizing conditions for anthocyanins extraction from purple sweet potato using response surface methodology (RSM). LWT Food Sci. Technol. 2008, 41, 155–160. [Google Scholar] [CrossRef]
  50. Strayer, C. Cloning of the Arabidopsis Clock Gene TOC1, an Autoregulatory Response Regulator Homolog. Science 2000, 289, 768–771. [Google Scholar] [CrossRef] [Green Version]
  51. Lau, K.; Podolec, R.; Chappuis, R.; Ulm, R.; Hothorn, M. Plant photoreceptors and their signaling components compete for COP1 binding via VP peptide motifs. EMBO J. 2019, 38, e102140. [Google Scholar] [CrossRef]
  52. Zhou, A.; Gan, P.; Zong, D.; Fei, X.; Zhong, Y.; Li, S.; Yu, J.; He, C. Bark tissue transcriptome analyses of inverted Populus yunnanensis cuttings reveal the crucial role of plant hormones in response to inversion. PeerJ 2019, 7, e7740. [Google Scholar] [CrossRef] [Green Version]
  53. Cannon, S.B.; Mitra, A.; Baumgarten, A.; Young, N.D.; May, G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004, 4, 10. [Google Scholar] [CrossRef] [Green Version]
  54. Mathews, H.; Clendennen, S.K.; Caldwell, C.G.; Liu, X.L.; Wagner, D.R. Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell 2003, 15, 1689–1703. [Google Scholar] [CrossRef] [Green Version]
  55. Yan, S.; Chen, N.; Huang, Z.; Li, D.; Zhi, J.; Yu, B.; Liu, X.; Cao, B.; Qiu, Z. Anthocyanin Fruit encodes an R2R3-MYB transcription factor, SlAN2-like, activating the transcription of SlMYBATV to fine-tune anthocyanin content in tomato fruit. New Phytol. 2020, 225, 2048–2063. [Google Scholar] [CrossRef]
  56. Quattrocchio, F.; Wing, J.F.; van der Woude, K.; Mol, J.N.; Koes, R. Analysis of bHLH and MYB domain proteins: Species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes. Plant J. Cell Mol. Biol. 1998, 13, 475–488. [Google Scholar] [CrossRef] [PubMed]
  57. Spelt, C.; Quattrocchio, F.; Joseph, N.M.M.; Koes, R. anthocyanin1 of Petunia Encodes a Basic Helix-Loop-Helix Protein That Directly Activates Transcription of Structural Anthocyanin Genes. Plant Cell 2000, 12, 1619–1631. [Google Scholar] [CrossRef] [Green Version]
  58. Payyavula, R.S.; Singh, R.K.; Navarre, D.A. Transcription factors, sucrose, and sucrose metabolic genes interact to regulate potato phenylpropanoid metabolism. J. Exp. Bot. 2013, 64, 5115–5131. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Gangappa, S.N.; Botto, J.F. The Multifaceted Roles of HY5 in Plant Growth and Development. Mol. Plant 2016, 9, 1353–1365. [Google Scholar] [CrossRef] [Green Version]
  60. Yadav, A.; Ravindran, N.; Singh, D.; Rahul, P.V.; Datta, S. Role of Arabidopsis BBX proteins in light signaling. J. Plant Biochem. Biot. 2020, 29, 623–635. [Google Scholar] [CrossRef]
  61. Gourlay, G.; Ma, D.; Schmidt, A.; Constabel, C.P. MYB134-RNAi poplar plants show reduced tannin synthesis in leaves but not roots, and increased susceptibility to oxidative stress. J. Exp. Bot. 2020, 71, 6601–6611. [Google Scholar] [CrossRef]
  62. Yoshida, K.; Ma, D.; Constabel, C.P. The MYB182 protein down-regulates proanthocyanidin and anthocyanin biosynthesis in poplar by repressing both structural and regulatory flavonoid genes. Plant Physiol. 2015, 167, 693–710. [Google Scholar] [CrossRef]
Genes 13 01811 g001 550
Figure 1. Chromosomes location of PyunBBX genes. The scale on the left is in megabases (Mb). The yellow lines within each chromosome represent gene density.
Figure 1. Chromosomes location of PyunBBX genes. The scale on the left is in megabases (Mb). The yellow lines within each chromosome represent gene density.
Genes 13 01811 g001
Genes 13 01811 g002 550
Figure 2. B-box1/B-box2 and CCT conserved domains and corresponding hidden Markov models (HMM) of BBX protein sequences in P. yunnanensis. The X-axis represents the conserved sequences of the domain. Conservation of each residue across all proteins is indicated by the height of each letter. The Y-axis is a scale of the relative entropy, which reflects the conservation rate of each amino acid.
Figure 2. B-box1/B-box2 and CCT conserved domains and corresponding hidden Markov models (HMM) of BBX protein sequences in P. yunnanensis. The X-axis represents the conserved sequences of the domain. Conservation of each residue across all proteins is indicated by the height of each letter. The Y-axis is a scale of the relative entropy, which reflects the conservation rate of each amino acid.
Genes 13 01811 g002
Genes 13 01811 g003 550
Figure 3. The phylogenetic tree and gene structures of PyunBBX genes. ML method with 1000 bootstrap replications to construct a phylogenetic tree. Starting from the left, the first scale below represents the number of amino acids (aa), and the second scale represents the number of bases (b).
Figure 3. The phylogenetic tree and gene structures of PyunBBX genes. ML method with 1000 bootstrap replications to construct a phylogenetic tree. Starting from the left, the first scale below represents the number of amino acids (aa), and the second scale represents the number of bases (b).
Genes 13 01811 g003
Genes 13 01811 g004 550
Figure 4. Gene duplication and synteny analysis of PyunBBX genes. The purple circles represent chromosomes. The outermost circle represents gene density, and the scale represents the number of bases (Mb). The 14 putative segmental-duplicated pairs of PyunBBX genes are linked by red lines, and the gene names are shown in the same color. The gray blocks represent replication events that occur on the chromosome of P. yunnanensisg.
Figure 4. Gene duplication and synteny analysis of PyunBBX genes. The purple circles represent chromosomes. The outermost circle represents gene density, and the scale represents the number of bases (Mb). The 14 putative segmental-duplicated pairs of PyunBBX genes are linked by red lines, and the gene names are shown in the same color. The gray blocks represent replication events that occur on the chromosome of P. yunnanensisg.
Genes 13 01811 g004
Genes 13 01811 g005 550
Figure 5. Synteny analysis between P. yunnanensis and the other two species. The putative collinear genes between P. yunnanensis and the other two species are marked in gray, while the syntenic BBX gene pairs are marked in red.
Figure 5. Synteny analysis between P. yunnanensis and the other two species. The putative collinear genes between P. yunnanensis and the other two species are marked in gray, while the syntenic BBX gene pairs are marked in red.
Genes 13 01811 g005
Genes 13 01811 g006 550
Figure 6. Analysis of cis-acting elements of PyunBBX genes promoters in P. yunnanensis. The number in the box represents the number of cis-acting elements. The shade of the color block depends on the number of cis-acting elements, and the deeper color, the more.
Figure 6. Analysis of cis-acting elements of PyunBBX genes promoters in P. yunnanensis. The number in the box represents the number of cis-acting elements. The shade of the color block depends on the number of cis-acting elements, and the deeper color, the more.
Genes 13 01811 g006
Genes 13 01811 g007 550
Figure 7. Expression patterns of 43 PyunBBX genes in leaf and bark. The FPKMs of 43 PyunBBX genes are taken as the logarithm with base 10 for standardization; the color patches of different colors indicate the expression levels of PyunBBX genes in the leaf and bark of P. yunnanensis. LC1/2/3: three biological repeats of leaves, BU1/2/3: three biological repeats of bark. The connecting lines on the left represent cluster analysis.
Figure 7. Expression patterns of 43 PyunBBX genes in leaf and bark. The FPKMs of 43 PyunBBX genes are taken as the logarithm with base 10 for standardization; the color patches of different colors indicate the expression levels of PyunBBX genes in the leaf and bark of P. yunnanensis. LC1/2/3: three biological repeats of leaves, BU1/2/3: three biological repeats of bark. The connecting lines on the left represent cluster analysis.
Genes 13 01811 g007
Genes 13 01811 g008 550
Figure 8. Gene expression levels of 43 PyunBBX genes in different parts of bark. The FPKMs of 43 PyunBBX genes are taken as the logarithm with base 10 for standardization; the color patches of different colors indicate the expression levels of PyunBBX genes in different parts of bark of P. yunnanensis. BU/CU/DU/EU: four positions of bark tissue: BU/CU: above and below the shoot site, DU: at the bottom of the new growth, EU: at the top (Figure S2). The number 1/2/3 represents three biological duplications. The connecting lines on the left represent cluster analysis.
Figure 8. Gene expression levels of 43 PyunBBX genes in different parts of bark. The FPKMs of 43 PyunBBX genes are taken as the logarithm with base 10 for standardization; the color patches of different colors indicate the expression levels of PyunBBX genes in different parts of bark of P. yunnanensis. BU/CU/DU/EU: four positions of bark tissue: BU/CU: above and below the shoot site, DU: at the bottom of the new growth, EU: at the top (Figure S2). The number 1/2/3 represents three biological duplications. The connecting lines on the left represent cluster analysis.
Genes 13 01811 g008
Genes 13 01811 g009 550
Figure 9. A rooted phylogenetic tree representing the relationships in P. yunnanensis, A. thaliana, and P. trichocarpa. We used the neighbor-joining (NJ) method with 1000 bootstrap replications to construct a phylogenetic tree. The BBX genes of a species are shown by genes name in the same colors and the same shapes in same colors (genes name in green and stars in red: PyunBBX genes of P. yunnanensis; genes name in orange and circles in yellow: PtrBBX genes of P. trichocarpa; genes name in purple and triangles in blue: AtBBX genes of A. thaliana).
Figure 9. A rooted phylogenetic tree representing the relationships in P. yunnanensis, A. thaliana, and P. trichocarpa. We used the neighbor-joining (NJ) method with 1000 bootstrap replications to construct a phylogenetic tree. The BBX genes of a species are shown by genes name in the same colors and the same shapes in same colors (genes name in green and stars in red: PyunBBX genes of P. yunnanensis; genes name in orange and circles in yellow: PtrBBX genes of P. trichocarpa; genes name in purple and triangles in blue: AtBBX genes of A. thaliana).
Genes 13 01811 g009
Genes 13 01811 g010 550
Figure 10. Relative expression analysis of six PyunBBX genes. CK: control check; UV: UV treatment; *: significant correlation at the 0.05 probability level; **: significant correlation at the 0.01 probability level; ***: significant correlation at the 0.001 probability level. Error bars indicate the SD of three independent biological and technical replicates. Time (d): days after UV treatment for 12 h a day.
Figure 10. Relative expression analysis of six PyunBBX genes. CK: control check; UV: UV treatment; *: significant correlation at the 0.05 probability level; **: significant correlation at the 0.01 probability level; ***: significant correlation at the 0.001 probability level. Error bars indicate the SD of three independent biological and technical replicates. Time (d): days after UV treatment for 12 h a day.
Genes 13 01811 g010
Genes 13 01811 g011 550
Figure 11. Anthocyanins content in leaves of P. yunnanensis in the six time periods. CK: control check; UV: UV treatment; **: significant correlation at the 0.01 probability level; Error bars indicate the SD of three independent biological and technical replicates. Time (d): days after UV treatment for 12 h a day.
Figure 11. Anthocyanins content in leaves of P. yunnanensis in the six time periods. CK: control check; UV: UV treatment; **: significant correlation at the 0.01 probability level; Error bars indicate the SD of three independent biological and technical replicates. Time (d): days after UV treatment for 12 h a day.
Genes 13 01811 g011
Table
Table 1. Basic characteristic of BBX family members in P. yunnanensis.
Table 1. Basic characteristic of BBX family members in P. yunnanensis.
Gene IDChromosomal LocationAmino Acid Length (aa)Isoelectric Point (pI)Molecular Weight (kDa)Subcellular
Localization
PyunBBX1Chr01: 5,540,646–5,543,9615135.7556.51Nucleus
PyunBBX2Chr01: 36,111,780–36,114,2904 305.5448.42Nucleus
PyunBBX3Chr01: 43,995,595–43,999,8662975.6831.88Nucleus
PyunBBX4Chr01: 44,107,170–44,111,3902975.9931.88Nucleus
PyunBBX5Chr01: 47,801,948–47,803,4972714.3129.56Nucleus
PyunBBX6Chr02: 1,909,069–1,910,5163116.0534.36Nucleus
PyunBBX7Chr02: 19,567,193–195713193625.740.72Nucleus
PyunBBX8Chr02: 20,923,421–20,934,9004165.6845.58Nucleus
PyunBBX9Chr03: 20,261,596–20,265,1455416.1959.46Nucleus
PyunBBX10Chr04: 1,800,513–1,801,0981844.1520.32Nucleus
PyunBBX11Chr04: 1,805,089–1,811,2552364.6725.65Nucleus
PyunBBX12Chr04: 11,046,033–11,048,0353695.9240.86Nucleus
PyunBBX13Chr04: 20,203,185–20,203,8932065.7222.9Nucleus
PyunBBX14Chr04: 20,382,296–20,384,2722035.9822.75Nucleus
PyunBBX15Chr05: 9,739,681–9,741,4761937.5621.42Nucleus
PyunBBX16Chr05: 25,453,505–25,455,1633116.234.34Nucleus
PyunBBX17Chr06: 18,887,979–18,889,9033846.2342.36Nucleus
PyunBBX18Chr06: 28,508,368–28,511,0983466.0937.58Nucleus
PyunBBX19Chr07: 1,484,594–1,486,8751926.4921.18Nucleus
PyunBBX20Chr07: 16,742,283–16,744,0394335.2647.03Nucleus
PyunBBX21Chr07: 17,319,158–17,322,0422354.826.05Nucleus
PyunBBX22Chr08: 292,896–293,9272508.6627.54Nucleus
PyunBBX23Chr08: 8,052,050–8,054,7304445.5849.27Nucleus
PyunBBX24Chr08: 8,604,637–8,611,2665005.8156.13Nucleus
PyunBBX25Chr08: 8,669,884–8,676,2755005.7856.09Nucleus
PyunBBX26Chr09: 12,288,823–12,289,5652176.2424.06Nucleus
PyunBBX27Chr09: 12,435,516–12,440,0142387.0526.38Nucleus
PyunBBX28Chr10: 156,680,74–15,670,3424475.148.93Nucleus
PyunBBX29Chr10: 24,419,560–24,421,3222639.2628.89Nucleus
PyunBBX30Chr11: 4,714,212–4,714,7751874.1220.59Nucleus
PyunBBX31Chr11: 15,010,674–15,015,1432985.8532.2Nucleus
PyunBBX32Chr11: 17,403,770–17,405,0353364.3437.13Nucleus
PyunBBX33Chr14: 9,996,595–10,004,3053155.335.29Nucleus
PyunBBX34Chr14: 13,912,158–13,917,9394165.0945.2Nucleus
PyunBBX35Chr14: 14,454,914–14,460,6894165.0445.34Nucleus
PyunBBX36Chr15: 8,537,097–8,539,4444265.1248.43Nucleus
PyunBBX37Chr17: 2,781,131–2,783,9092384.7726.02Nucleus
PyunBBX38Chr17: 3,054,640–3,057,4292384.7725.99Nucleus
PyunBBX39Chr17: 4,089,911–4,091,4814215.8545.62Nucleus
PyunBBX40Chr17: 4,094,941–4,096,9531088.6112.11Nucleus
PyunBBX41Chr17: 14,748,917–14,751,0953805.2541.61Nucleus
PyunBBX42Chr18: 966,086–968,2603505.637.89Nucleus
PyunBBX43Chr18: 12,620,866–12,622,5573825.9641.94Nucleus
Table
Table 2. Analysis of evolutionary selection pressure on co-linear members of PyunBBX genes.
Table 2. Analysis of evolutionary selection pressure on co-linear members of PyunBBX genes.
Syntenic Gene PairsMethodKsKaKa/KsDuplicated
Type		Divergence Time
(Mya.)
PyunBBX22-PyunBBX29MA *0.1175740.3507050.335249Segmental3.92
PyunBBX15-PyunBBX19MA0.09449590.3357610.281438Segmental3.15
PyunBBX13-PyunBBX26MA0.09228840.2659140.347061Segmental3.08
PyunBBX1-PyunBBX9MA0.08283910.3017190.274557Segmental2.76
PyunBBX23-PyunBBX28MA0.07359780.2177240.338032Segmental2.45
PyunBBX4-PyunBBX31MA0.07337250.2260660.324562Segmental2.45
PyunBBX14-PyunBBX27MA0.07268480.3788820.19184Segmental2.42
PyunBBX20-PyunBBX39MA0.06770180.1920950.35244Segmental2.26
PyunBBX17-PyunBBX43MA0.06722060.3559350.188856Segmental2.24
PyunBBX12-PyunBBX41MA0.06535160.30690.212941Segmental2.18
PyunBBX6-PyunBBX16MA0.05425110.2851740.190239Segmental1.81
PyunBBX21-PyunBBX37MA0.05308820.2375770.223457Segmental1.77
PyunBBX18-PyunBBX42MA0.04267610.3587640.118953Segmental1.42
PyunBBX34-PyunBBX35MA0.007179760.03006010.238847Segmental0.24
* Model averaging.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Zhang, Q.; Ma, D.; Hu, Z.; Zong, D.; He, C. PyunBBX18 Is Involved in the Regulation of Anthocyanins Biosynthesis under UV-B Stress. Genes 2022, 13, 1811. https://doi.org/10.3390/genes13101811

AMA Style

Zhang Q, Ma D, Hu Z, Zong D, He C. PyunBBX18 Is Involved in the Regulation of Anthocyanins Biosynthesis under UV-B Stress. Genes. 2022; 13(10):1811. https://doi.org/10.3390/genes13101811

Chicago/Turabian Style

Zhang, Qin, Dongxiao Ma, Zhixu Hu, Dan Zong, and Chengzhong He. 2022. "PyunBBX18 Is Involved in the Regulation of Anthocyanins Biosynthesis under UV-B Stress" Genes 13, no. 10: 1811. https://doi.org/10.3390/genes13101811

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop