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Article

An R2R3-MYB Transcription Factor RoMYB10 Regulates Anthocyanin Biosynthesis in Black Raspberry

by
Tiemei Li
,
Wenjiao Xin
,
Yutao Li
,
Aoxue Wang
and
Guohui Yang
*
Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of Northeast Region, Ministry of Agriculture and Rural Affairs, College of Horticulture & Landscape Architecture, Northeast Agricultural University, Harbin 150030, China
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(7), 1823; https://doi.org/10.3390/agronomy13071823
Submission received: 30 May 2023 / Revised: 29 June 2023 / Accepted: 6 July 2023 / Published: 9 July 2023
(This article belongs to the Special Issue Decrypting the Mechanism of Protein Transcription in Plant Stress Resistance)
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Abstract

:
At present, there are many excellent cultivars of raspberry worldwide with different fruit colors, such as yellow, red, purple and black. Anthocyanin accumulation is responsible for flower and fruit coloration in plants. Anthocyanins belong to flavonoids which are natural pigments widely distributed in plants. The MYB transcription factor (TF) plays a vital regulatory role in the metabolic pathway of anthocyanins. Here, RoMYB10, an R2R3-MYB in black raspberry (Rubus occidentalis L.), was isolated. The basic physical and chemical properties, conserved motif distribution, evolutionary relationship and subcellular localization were analyzed for the RoMYB10 encoded amino acid sequence. The contents of anthocyanin were determined in four different colored raspberry fruits by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS). The expression levels of MYB10 genes in various tissues and fruits at different developmental stages of the four different colored raspberries were detected by real-time fluorescence quantitative PCR (qRT-PCR). The results showed that the contents of anthocyanin and the expression levels of the MYB10 gene were the highest in black raspberry. Moreover, the expression of MYB10 in the fruit was significantly higher than in other tissues, especially in ripe and mottled fruits. When ectopic overexpression of RoMYB10 occurred in tobacco, the expression levels of the RoMYB10 gene and total anthocyanin contents in transgenic tobacco plants were significantly higher than those in wild-type (WT) plants. In addition, overexpression of RoMYB10 up-regulated the key genes in the anthocyanin biosynthesis, such as NtCHS, NtCHI, NtF3H, NtANS and NtUFGT. Our study suggests that RoMYB10 is involved in the regulating of anthocyanin biosynthesis in raspberry and has potential as a molecular tool for manipulating biosynthesis of anthocyanins in fruits using metabolic engineering.

1. Introduction

As a shrubby small berry fruit tree, raspberry is widely distributed in Europe, North America and parts of Asia and also widely cultivated in northeast China and southwest China. Raspberry has high-quality fruits with good development prospects and economic value. Raspberry fruits have rich colors, such as yellow, red, purple and black. The types and contents of anthocyanins in different colored raspberry fruits are various. Due to their unique structural characteristics, anthocyanins have been sought after for their antioxidant function, disease prevention, vision protection, anti-cancer, anti-mutation and other physiological effects [1,2,3,4,5].
Anthocyanins are essential pigments in fruit coloration and profoundly affect the quality and nutritional value of fruits [6,7]. There are many enzymes involved in the process of anthocyanin metabolism, such as cinnamate 4-hydroxylase (C4H), chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid-3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) [8,9,10]. Genes encoding these enzymes are structural genes in anthocyanin metabolism and relatively conserved among plant species. These structural genes are generally regulated by a conserved MBW complex that consists of R2R3-MYB, bHLH and WD40 TFs [11]. Notably, MYB TFs commonly play central roles in distinguishing the target genes in the MBW complex [12,13].
MYB is a large transcription factor family with a conserved DNA-binding domain in the N-terminus of many members [14]. The domain generally includes up to four sequence repeats (R) which consist of three α-helices of approximately 52 amino acids [15]. Based on the number of adjacent R structures, MYB TFs are classified into several subfamilies [16]. Among these, R2R3-MYB and R3-MYB are mainly MYB transcription factors that regulate anthocyanin synthesis, among which R2R3-MYB plays a significant role [17].
The earliest identified transcription factor regulating anthocyanin synthesis was the maize C1 gene, an R2R3-type MYB TF regulating the anthocyanin synthesis in the endosperm aleurone layer by up-regulating the expression of CHS and DFR [18]. In plants, many MYB TFs are involved in regulating proanthocyanin, flavonoids and anthocyanin biosynthesis. In octoploid and diploid strawberry fruits, the R2R3-type FvMYB10/FaMYB10 could activate the downstream structural genes of anthocyanin biosynthesis [19,20]. VvMYBA1 and VvMYBA2 in grape (Vitis vinifera L.), AtMYB75/113/114 in Arabidopsis and PpMYB10.1 in peach (Prunus persica) were reported to up-regulate the biosynthesis of flavonoids, anthocyanin and proanthocyanin [6,9,21,22]. In apples, the expression of MdMYB10 was closely linked to anthocyanin contents during fruit development [23]. Overexpression of MdMYB110 (a paralogue of MdMYB10) in tobacco up-regulated anthocyanin biosynthesis and caused the red-fleshed cortex phenotype [24]. Similarly, ectopic overexpression of the McMYB10 gene from Malus crabapple in tobacco exhibited more substantial anthocyanin accumulation and deeper red petal color [1]. Moreover, PyMYB10 could bind to the G-box motifs of PyHY5 and promote anthocyanin synthesis in pear [25]. In strawberries, inhibition of the expression of FvMYB10 resulted in a color loss in strawberry fruit, but overexpression of FvMYB10 significantly increased the contents of anthocyanin [26,27]. These studies confirmed that MYB10 proteins played essential roles in regulating anthocyanin biosynthesis.
Conversely, some MYB10 genes have been reported to regulate anthocyanin synthesis negatively. Overexpression of PtoMYB10 from Populus trichocarpa in Populus resulted in a drastic increase in secondary cell wall thickening in xylem fiber cells and an inhibition of gene expression levels in the anthocyanin biosynthesis pathway [28]. The introduction of PpMYB10.2/3 from peach into tobacco did not change the anthocyanin level and flower color of transgenic plants [29]. In Chinese narcissus, NtMYB2 acted as a transcriptional repressor by down-regulating the transcripts of key enzyme genes in the anthocyanin biosynthesis pathway [30].
Although the roles of MYB10 genes in anthocyanin synthesis and color alteration have been reported in some plants, such as Arabidopsis, tomato, tobacco, apple, grape, pear, peach, strawberry, kiwi fruit and so on, the MYB10 gene in raspberry has not been studied. The black raspberry was first used to study the relationship between the transcription levels of RoMYB10 and anthocyanin accumulation in raspberry fruits. The present study aimed to explore the biological functions of the MYB10 gene in raspberries, lay a theoretical basis for further elucidation of the molecular regulation mechanism of anthocyanin formation in raspberries and also provide gene resources for the cultivation of high-anthocyanin raspberry varieties by transgenic technology.

2. Materials and Methods

2.1. Materials

Assays for MYB10 expression were conducted on different tissues of the four color varieties of raspberries. They were yellow raspberry ‘Jeltii gigant’ (Rubus idaeus L.), red raspberry ‘Caroline’ (Rubus idaeus L.), purple raspberry ‘American 22’ (Rubus idaeus L.) and black raspberry (Rubus occidentalis L.). The four raspberry varieties were picked from Xiangyang Farm of Northeast Agricultural University, and the samples of different tissues were frozen in liquid nitrogen and immediately stored at −80 °C for the subsequent experiments. The tobacco used for genetic transformation was wild-type tobacco (Nicotiana tabacum), and for subcellular localization wild-type Nicotiana benthamiana retained in our laboratory was used. Seeds of Nicotiana benthamiana were planted into a large pot, covered with a layer of punctured plastic wrap, then placed in a light incubator at about 22–24 °C for culture with a 16 h/8 h light/dark cycle every day, and the light intensity was approximately 100 μmol·m−2·s−1.

2.2. Isolation of RoMYB10 Gene

Total RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Beijing, China). The synthesis of first-strand cDNA was carried out from total RNA according to the manufacturer’s instructions of AMV Ver.3.0 (Takara, Dalian, China). The RoMYB10 gene was isolated from black raspberry fruit cDNA using specific primers which were designed based on the red raspberry RiMYB10 (ABX79950.1) from NCBI. The forward primer with a BamHI restriction site was 5′-GGATCCATGGAGGTGAGAAAAGGTGCATG-3′, and the reverse primer with a SalI restriction site was 5′-GTCGACTCTCGCTTCTTCTTGAAAAAAATGCC-3′. The PCR was conducted in the following steps: 3 min at 94 °C, 35 cycles of 30 s at 95 °C, 30 s at 64 °C and 1 min at 72 °C, with a final extension of 72 °C for 5 min. Then, 1.0% agarose gels were used to analyze the PCR products, and a DNA purification kit (Bioteke, Beijing, China) was used to purify the single fragments. The purification products were linked to the pEASY-T5 Zero vector using the pEASY-T5 Zero Cloning Kit and transported to Trans1-T1 Phage Resistant Chemically Competent Cell (TransGen, Beijing, China). After overnight culture on the medium, monoclones were selected and sent to BGI for sequencing.

2.3. Expression Analysis of RoMYB10 and Anthocyanin Biosynthetic Genes

To detect the expression levels of RoMYB10 in different parts, RNA was isolated from the root, stem, leaf, green fruit, mottled fruit and ripe fruit of the four different colored raspberry fruits using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Beijing, China). First-strand cDNA was synthesized from 1 μg of total RNA in a 20μL reaction mixture using a PrimeScript RT reagent kit (Takara, Dalian, China). The synthesized cDNA was diluted 100-fold and 2.5 μL was mixed with 500 nm of each primer and LightCycler® 480 Sybr Green I master mix (Roche Applied Science) for qPCR analysis. Primers for these genes are listed in Supplementary Materials, Table S1. qRT-PCR was performed on the IQ5 real-time PCR system (Bio-Rad) with TransStart® Green qPCR SuperMix (TransGen Biotech, Beijing, China) using standard protocols. The thermal profile of the reaction was an initial denaturation at 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 10 s. This was followed by fluorescence acquisition after each cycle. Finally, a dissociation curve was generated by increasing temperature from 65 to 95 °C, to verify primer specificity. The data were normalized to Ri18s (KP125886.1) and NtActin (GQ339768). The relative expression was calculated according to the 2−ΔΔCT method, and the lowest expression level was set as 1 [31,32]. Data were from three technical replicates and the error bars represent means ± SE.

2.4. Sequence Analysis

The MYB amino acid sequences used for sequence analysis were some proteins reported to be related to anthocyanin synthesis and some were from BLAST on the NCBI website (http://www.ncbi.nlm.nih.gov/blast/, accessed on 1 March 2022) according to the similarity to RoMYB10 protein. Molecular Evolutionary Genetics Analysis (MEGA) version 5.1 was used for sequence alignment and homologous evolutionary analysis [33]. The theoretical isoelectric point and relative molecular mass of RoMYB10 protein were identified by the ExPASy database (https://web.expasy.org/protparam/, accessed on 12 March 2022).

2.5. Subcellular Localization of the RoMYB10 Protein

For the analysis of the subcellular localization of RoMYB10 protein, the coding sequence (CDS) of RoMYB10 was amplified by PCR and cut with BamHI/SalI and cloned into a pCAMBIA 2300 expression vector to generate a pCAMBIA 2300-35S::RoMYB10-GFP fusion expression vector. The fusion vector and the empty pCAMBIA 2300 vector were transformed into the Agrobacterium strain GV3101. Cells were grown overnight at 28 °C in a Luria–Bertani (LB) medium supplemented with 50 mg/L of kanamycin sulfate (Kana). After centrifugation, the collected cells were resuspended in the solution containing 10 mM MES pH 5.7, 10 mM MgCl2, 150 μM acetosyringone. Then, samples were incubated at room temperature for an additional one hour on a shaker. The bacteria were suspended in the fresh buffer and adjusted to a final density of OD600 = 0.8. Then, the suspensions were transformed into the abaxial side mesophyll cells of tobacco leaves using a needleless syringe. The treated plants were maintained in greenhouse conditions at 25 ± 1 °C with a 16 h/8 h light/dark cycle for 2–3 days before observation under a confocal microscope (Olympus FV1000) [34]. The staining of 4′, 6-diamidino-2-phenylindole (DAPI) was used as a nucleus marker.

2.6. Overexpression of RoMYB10 in Tobacco

The seeds of the tobacco (Nicotiana tabacum, wild-type) plants used were surface sterilized in 75% ethanol for 5 min and then 2% NaClO for 10 min, washed with sterile distilled water three times for 5 min and then plated on 1/2 MS solid medium supplemented with 3% (w/v) sucrose and 0.7% (w/v) agar (pH 5.8). The pCAMBIA 2300-RoMYB10 fusion expression vector was transformed into tobacco using the method of Agrobacterium-mediated transformation [35]. Transgenic plants were screened on 1/2 MS media with 50 μg/mL of Kana [34]. Kana-resistant seedlings were selected and transferred to the soil. RT-PCR and qRT-PCR were both used to identify transgenic lines and the T3 generation transgenic plants were used for further experiments.

2.7. Pigment Analysis

The samples (approximately 0.8–1.0 g fresh weight) were subjected to extraction overnight using 5 mL of methanol–1.5% HCl at 4 °C in the dark [36]. The supernatant was isolated by filtration through filter paper and a further filtration through a 0.22 μm MilliporeTM filter (Billerica, MA, USA). The extraction solution was passed through a preconditioned ACQUITY UPLC BEH-C18 (Waters, Milford, MA, USA, 2.1 × 100 mm, 1.7 µm). Mobile phase A was formic acid/water (0.1%, v/v), and mobile phase B was acetonitrile (100%). The HPLC column oven temperature was 30 °C. The gradient of mobile phase B was 0 to 3 min, 5–15%; 3 to 3.5 min, 15%; 3.5–6 min, 15–30%; 6–6.5 min, 30%; 6.5–12 min, 30–70%; 12–12.5 min, 70%; 12.5–18 min, 70–100%; 18–25 min, 100%; 25–25.5 min, 100–5%; 25.5–29 min, 5%. The MS analytical parameters were as follows: scan type, Q1 MS; polarity, positive; ion source, turbo spray (ESI); declustering potential (DP), 90 V; entrance potential (EP), 10 V; curtain gas, 30 psi; ion spray voltage (IS), 5500 V; temperature, 550 °C; ion source gas, 50 psi; interface heater, ON [37]. Three biological replicates of each sample were conducted.

3. Results

3.1. Isolation and Sequence Analysis of RoMYB10

The ripe fruits of black raspberry were chosen to isolate the RoMYB10 gene due to their high anthocyanin contents. A 654 bp product was obtained using a pair of special primers. It encoded a predicted protein of 217 amino acids that had 100.0% similarity to the red raspberry RiMYB10 (ABX79950.1). So, the black raspberry clone was named the RoMYB10 gene. The calculated molecular mass of RoMYB10 protein was 25,341 Da, and the isoelectric point was 9.66. Alignment of the RoMYB10 protein with other MYBs indicated a high degree of homology in the R2R3 domain, especially some MYBs related to anthocyanin biosynthesis (Figure 1A) such as FaMYB10 from strawberry and PyMYB10 from Pyrus pyrifolia. RoMYB10 had 50% similarity to both FaMYB10 and PyMYB10. Moreover, in the R3 domain, all these MYB proteins contained the signature bHLH motif that is specific to the interaction between MYB and bHLH [38,39].
The result of phylogenetic analysis showed that RoMYB10 was in the same subgroup as strawberry FaMYB10, apple MdMYB10, pear PyMYB10 and peach PpMYB10 which were confirmed to regulate the synthesis of anthocyanin and belonged to subgroup-10 (Figure 1B) [40]. These results suggested that RoMYB10 might have the same functions as the other MYB10s in the subgroup which are involved in anthocyanin synthesis and promote anthocyanin accumulation.

3.2. Anthocyanin Contents and Gene Expression Analysis

With the application of HPLC-MS, twenty-four anthocyanins were identified in the four different colored raspberries. The molecular ions and fragment ions of these anthocyanins are summarized in Table 1. There were 7, 8, 13 and 16 anthocyanins in yellow raspberry ‘Jeltii gigant’, red raspberry ‘Caroline’, purple raspberry ‘American 22’ and black raspberry, respectively. Corresponding differences occurred in anthocyanin contents among the four cultivars. With the deepening of fruit color, the concentration of the total anthocyanins increased, and the highest concentration of the total anthocyanins was in the ripe black raspberry fruits, approximately 55.79 mg/g fresh weight (FW). The anthocyanin content in ‘Caroline’ was 7.28 mg/g FW, about three times that in ‘Jeltii gigant’ (2.4 mg/g FW), and had no significant difference to ‘American 22’ (10.89 mg/g FW) (Figure 2A).
Transcription of MYB10 was also detected in root, stem, young leaves and fruits at different development stages (green, mottled and ripe stages) in the four varieties (Figure 2B). The mRNA of RoMYB10 was detected in the tissues where anthocyanins appeared to accumulate, especially in the fruit. The transcript levels of RoMYB10 in mottled fruits and ripe fruits were approximately six hundred- and seven hundred-fold higher than in other tissues, respectively.

3.3. RoMYB10 Was Localized to the Nucleus

To explore the subcellular location of RoMYB10, the fusion protein (35S::RoMYB10-GFP) and the control protein (35S::GFP) were transiently expressed in tobacco leaves. The results showed that the RoMYB10 fusion protein was exclusively localized in the nucleus with DAPI staining. However, the control protein was distributed throughout the cell (Figure 3).

3.4. Overexpression of RoMYB10 in Tobacco

To find the function of RoMYB10, the CDS of RoMYB10 was overexpressed in tobacco (Nicotiana tabacum) (Figure 4A). Interestingly, most transgenic tobacco plants with strong pigmentation died, but those that did not show visible pigmentation actually grew well. The same phenomenon occurred in Feng’s study [6]. So, these transgenic plants that grew well were selected for the next test. The expression of RoMYB10 was detected by RT-PCR and qRT-PCR (Figure 4B,C). Transcript levels of RoMYB10 were detected in the transgenic tobacco lines, and the expression levels in transgenic lines were more than fifty-fold that of the expression levels in WT plants.
To analyze whether there was any anthocyanin accumulation in the overexpression lines, the anthocyanin contents in the tobacco lines overexpressing RoMYB10 were detected. It was found that the anthocyanin contents in leaves of transgenic plants were significantly higher than that in WT plants. The anthocyanin contents were approximately seven to nine times higher than in WT (Figure 4D).
To elucidate the molecular mechanism of RoMYB10 in anthocyanin metabolism, transcription profiles of eight genes, NtCHS, NtCHI, NtF3H, NtDFR, NtLAR, NtANR, NtANS and NtUFGT, in anthocyanin metabolism were investigated in transgenic lines by qRT-PCR. The results showed that the expression of NtCHS, NtCHI, NtF3H, NtANS and NtUFGT was higher in transgenic lines, especially NtANS and NtUFGT which operated in the late steps of anthocyanin biosynthesis (Figure 5). The transcript levels of the two genes were more than four-fold higher in transgenic tobacco lines. Interestingly, the expression of NtDFR was lower than that of WT, and the transcript levels of NtLAR and NtANR had no significant difference from that of WT (Figure 5). Presumably, up-regulation of RoMYB10 and other genes associated with anthocyanin biosynthesis during raspberry fruit development contributed to the higher efficiency of anthocyanin accumulation. Based on these results, it can be speculated that higher transcription levels of RoMYB10 and some anthocyanin synthesis genes may account for the fruit color formation in black raspberry at a molecular level.

4. Discussion

Many MYB TFs regulating anthocyanin biosynthesis in horticulture crops have been reported, such as in apple, pear grape, peach, strawberry and so on [6,20,21,29,41,42]. However, it remains unknown whether MYB TFs in raspberries have the same functions. A 654 bp gene named RoMYB10 was isolated from black raspberry in this study. The R2R3 domain distribution of RoMYB10 had a high degree of similarity to other R2R3 MYBs, and there was a signature bHLH motif in the R3 domain of RoMYB10 [38,39]. Analysis of the phylogenetic relationship indicated that RoMYB10 was close to MYB TFs of subgroup-10, including FaMYB10, MdMYB10, PyMYB10 and PpMYB10 reported to promote anthocyanin biosynthesis in previous studies [6,20,22,40]. Moreover, the RoMYB10 protein was localized in the nucleus, supporting its potential function as a transcription factor. All these results revealed that RoMYB10 presumably promotes anthocyanin synthesis.
Fruit pigmentation differs significantly among raspberry cultivars. There are four colors of raspberries, but the molecular basis of the appearance of different colors is not clear. Therefore, four different colored raspberry varieties were used as materials in this study. It was found that the anthocyanin contents of black raspberry were about fifty times that of yellow raspberry and nearly five times that of red raspberry and purple raspberry. The transcription levels of RoMYB10 in different tissues of four varieties were also investigated by qRT-PCR. The expression level of the RoMYB10 gene in fruits was significantly higher than in other tissues. Otherwise, the RoMYB10 gene expression level increased dramatically from the mottled stage and reached its maximum when the fruit ripened. MYB regulatory genes exist in different tissues and parts in many plant species, such as roots, stems, leaves, flowers, fruits and seeds. The expression of the IbMYB1 gene from sweet potato was mainly detected in tuberous roots, the MdMYB10 gene from apple was expressed in fruits and leaves and PyMYB10 had higher expression in flower buds in pear [6,23,43,44]. In this study, the expression of RoMYB10 was detected in roots, stems, young leaves and fruits where anthocyanins concentrated. This phenomenon was in accordance with other fruit trees. These results suggested that the anthocyanin contents in raspberries closely correlated with the expression of RoMYB10, and RoMYB10 potentially regulates anthocyanin biosynthesis, especially in ripe fruits.
Ectopic overexpression of MYB might cause ectopic pigmentation accumulation in plants. MrMYB6 induced ectopic pigmentation accumulation in tobacco; apple MdMYB1, MdMYB10 and pear PyMYB10 induced ectopic pigmentation accumulation in Arabidopsis [6,10,23]. Ectopic overexpression of the RoMYB10 gene from black raspberry in tobacco led to abundant pigmentation. However, the plants with significant pigmentation died, and a few plants with inconspicuous pigmentation survived. The pigmentations were detected in the leaves of these transgenic plants (Figure 4D). This phenomenon also appeared in a previous study [6]. This suggested that RoMYB10 functions as a regulatory gene in anthocyanin synthesis in raspberry, but ectopic pigmentation accumulation may affect the normal growth of plants. Therefore, the reason for this phenomenon needs to be explored further. Other regulatory genes, including bHLH, WD40 and some other members of the MYB family, still need to be explored in raspberry.
In green apples, genes in the late steps of anthocyanin synthesis were present but barely detected by RNA gel blot analysis [45]. Some R2R3-MYBs, like NtAn2, VvMYBC2L2 and McMYB12, could regulate the expression of DFR and ANS, sequentially influencing anthocyanin accumulation and flower or fruit coloration [46,47,48]. MrMYB6 in Chinese bayberry (Morella rubra) could significantly decrease the expression of NtLAR and NtANR2 [10]. Overexpression of MYB10/MYB110 in Actinidia chinensis increased the anthocyanin contents, but the expression levels of F3′H and F3′5′H genes were not strongly related to MYB10/MYB110 [49]. In nectarine (Prunus persica), PpMYBPA1 transactivates the promoters of proanthocyanidin (PA) pathway genes DFR and LAR but not UFGT [50]. The peach PpMYB7 activated transcription of PpLAR1 but not PpANR [51]. The molecular mechanism of MYB transcription factors to regulate flavonoid structural genes is complex. In this study, transcription levels of the genes in the anthocyanin synthesis pathway were higher in transgenic tobacco except NtDFR, NtLAR and NtANR. The relative transcription level of NtDFR in transgenic tobacco plants was lower than that in WT plans. The result was different from most studies. But, in peach, PpMYB17-20 showed a strong repressive effect on transcription of flavonoid pathway genes such as DFR [52]. Anthocyanin accumulation in plants was coordinately regulated by a set of R2R3-MYB genes. The MYB TFs were functionally divergent, activating or inhibiting the expression of different structural genes. The expression of these structural genes may be affected by the endogenous MYBs. Thus, the regulatory role of RoMYB10 needs further investigation in raspberry rather than model plants. Our results showed that RoMYB10 has a crucial part in regulating the anthocyanin biosynthetic genes. To some extent, it could determine the gradation of fruit color and content of anthocyanin in raspberries. However, the deeper molecular mechanisms of RoMYB10 need to be explored. Our study provides the primary molecular mechanism of anthocyanin accumulation in black raspberry and offers important information for fruit tree breeding.

5. Conclusions

It was found in this study that RoMYB10 played an essential part in positively regulating the synthesis of anthocyanins by up-regulating the critical genes in anthocyanin synthesis. The current results suggested that the expression of MYB10 was the highest in black raspberry, especially in ripe and mottled fruits. Our study provides a basis for further exploring the complex regulatory mechanisms of RoMYB10. In the future, we will devote ourselves to the downstream genes of RoMYB10, to finding the bound elements, to creating genetic transformation materials of the downstream target gene and to analyzing the molecular mechanisms of anthocyanin accumulation in depth.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13071823/s1, Table S1: List of qRT-PCR primers.

Author Contributions

G.Y. and A.W. conceived the research. T.L. wrote and edited the manuscript. T.L. analyzed the data, W.X., Y.L. and T.L. performed the experiments. 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 (Grant No. 32202417); the Natural Science Fund Joint Guidance Project of Heilongjiang Province (Grant No. LH2020C009); China Postdoctoral Science Foundation (Grant No. 2022MD713727) and Postdoctoral Science Foundation of Heilongjiang Province, China (Grant No. LBH-Z21119).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the data in this study are presented in the manuscript and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Comparison and phylogenetic relationship of RoMYB10 with anthocyanin-related MYB proteins of other species. (A) Comparison of protein sequences, the R2 and R3 domain are underlined and the blue box indicates the bHLH motif specific to the interaction between MYB and bHLH proteins. The sequences in dark blue, red, light blue and white indicate the similarity is, respectively, 100%, greater than 75%, greater than 50% and less than 50%. (B) Evolutionary tree analysis of RoMYB10 (indicated by a red circle) and other MYB proteins. The accession numbers of these proteins in the GenBank database are as follows: RoMYB10 (ABX79950.1), NtMYB114 (NP_001306786.1, Nicotiana tomentosiformis L.), PeAN2 (ABO21072.1, Petunia integrifolia L.), VvMYBA6 (ACL97979.1, Vitis vinifera L.), CsAN2 (AOY10781.1, Camellia sinensis L.), PhMYB (ADW94951.1, Petunia × hybrida L.), StMYB114 (ALA13583.1, Solanum tuberosum L.), MdMYB10(ACQ45201.1, Malus domestica L.), FtMYB10 (ALT31509.1, Fagopyrum tataricum L.), GhMYB10 (ABR01222.1, Gossypium hirsutum L.), EsMYB10 (AFH03062.1, Epimedium sagittatum L.), PpMYB10 (AKI23599.1, Prunus persica L.), FaMYB10 (ABX79947.1, Fragaria × ananassa L.), PyMYB10 (ADN26574.1, Pyrus pyrifolia var. culta L.),CsAN1 (AOM63231.1, Camellia sinensis L.), CtMYB (ANI87841.1, Citrus trifoliata L.), MrMYB1 (ADG21957.1, Morella rubra L.), NaMYB113 (OIS96677.1, Nicotiana attenuata L.), CmMYB (ANI87835.1, Citrus maxima L.), AtMYB75 (ABB03878.1, Arabidopsis thaliana), VvMYBA1(ABD72954.1, Vitis vinifera), AcMYB10 (QGA78460, Actinidia chinensis).
Figure 1. Comparison and phylogenetic relationship of RoMYB10 with anthocyanin-related MYB proteins of other species. (A) Comparison of protein sequences, the R2 and R3 domain are underlined and the blue box indicates the bHLH motif specific to the interaction between MYB and bHLH proteins. The sequences in dark blue, red, light blue and white indicate the similarity is, respectively, 100%, greater than 75%, greater than 50% and less than 50%. (B) Evolutionary tree analysis of RoMYB10 (indicated by a red circle) and other MYB proteins. The accession numbers of these proteins in the GenBank database are as follows: RoMYB10 (ABX79950.1), NtMYB114 (NP_001306786.1, Nicotiana tomentosiformis L.), PeAN2 (ABO21072.1, Petunia integrifolia L.), VvMYBA6 (ACL97979.1, Vitis vinifera L.), CsAN2 (AOY10781.1, Camellia sinensis L.), PhMYB (ADW94951.1, Petunia × hybrida L.), StMYB114 (ALA13583.1, Solanum tuberosum L.), MdMYB10(ACQ45201.1, Malus domestica L.), FtMYB10 (ALT31509.1, Fagopyrum tataricum L.), GhMYB10 (ABR01222.1, Gossypium hirsutum L.), EsMYB10 (AFH03062.1, Epimedium sagittatum L.), PpMYB10 (AKI23599.1, Prunus persica L.), FaMYB10 (ABX79947.1, Fragaria × ananassa L.), PyMYB10 (ADN26574.1, Pyrus pyrifolia var. culta L.),CsAN1 (AOM63231.1, Camellia sinensis L.), CtMYB (ANI87841.1, Citrus trifoliata L.), MrMYB1 (ADG21957.1, Morella rubra L.), NaMYB113 (OIS96677.1, Nicotiana attenuata L.), CmMYB (ANI87835.1, Citrus maxima L.), AtMYB75 (ABB03878.1, Arabidopsis thaliana), VvMYBA1(ABD72954.1, Vitis vinifera), AcMYB10 (QGA78460, Actinidia chinensis).
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Figure 2. Relative expression of the RoMYB10 genes in four colored raspberry varieties and in different tissues of these raspberries. (A) Relative expression of the MYB10 genes in four varieties. (B) Relative expression of the MYB10 genes in different tissues and in fruits at different developmental stages in four varieties. Error bars indicate the mean ± SE of three replicate reactions. The asterisks above columns indicate significant differences (* p ≤ 0.05, ** p ≤ 0.01).
Figure 2. Relative expression of the RoMYB10 genes in four colored raspberry varieties and in different tissues of these raspberries. (A) Relative expression of the MYB10 genes in four varieties. (B) Relative expression of the MYB10 genes in different tissues and in fruits at different developmental stages in four varieties. Error bars indicate the mean ± SE of three replicate reactions. The asterisks above columns indicate significant differences (* p ≤ 0.05, ** p ≤ 0.01).
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Figure 3. Subcellular localization of RoMYB10 in tobacco leaf epidermal cells. From left to right are photos of green fluorescent protein (GFP), bright field, DAPI staining and three channels of RoMYB10/empty vector, respectively. The scale bar represents 50 µm.
Figure 3. Subcellular localization of RoMYB10 in tobacco leaf epidermal cells. From left to right are photos of green fluorescent protein (GFP), bright field, DAPI staining and three channels of RoMYB10/empty vector, respectively. The scale bar represents 50 µm.
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Figure 4. Ectopic expression of RoMYB10 gene in tobacco. (A) Comparison of phenotypes of wild-type (WT) and two RoMYB10 transgenic tobacco lines (OERoMYB10-1 and OERoMYB10-2). (B) Detection of RoMYB10 transcript levels in WT and transgenic tobacco lines visualized by semi-quantitative RT-PCR. Lane M: marker (Trans DNA 2K); Lane 1: 35S::OERoMYB10 line 1; Lane 2: 35S::OERoMYB10 line 2; Lane 3: positive control, plasmid of 35S::OERoMYB10; Lane 4: products of RT-PCR without template DNA; Lane 5: WT. (C) Relative expression levels of RoMYB10 in transgenic lines and WT were analyzed by qRT-PCR. The data were normalized using the WT sample and the NtActin (GQ339768) gene as the internal controls and calculated using the 2−ΔΔCT method. (D) Anthocyanin content of WT and transgenic lines. The asterisks above columns indicate significant differences compared to WT (** p ≤ 0.01).
Figure 4. Ectopic expression of RoMYB10 gene in tobacco. (A) Comparison of phenotypes of wild-type (WT) and two RoMYB10 transgenic tobacco lines (OERoMYB10-1 and OERoMYB10-2). (B) Detection of RoMYB10 transcript levels in WT and transgenic tobacco lines visualized by semi-quantitative RT-PCR. Lane M: marker (Trans DNA 2K); Lane 1: 35S::OERoMYB10 line 1; Lane 2: 35S::OERoMYB10 line 2; Lane 3: positive control, plasmid of 35S::OERoMYB10; Lane 4: products of RT-PCR without template DNA; Lane 5: WT. (C) Relative expression levels of RoMYB10 in transgenic lines and WT were analyzed by qRT-PCR. The data were normalized using the WT sample and the NtActin (GQ339768) gene as the internal controls and calculated using the 2−ΔΔCT method. (D) Anthocyanin content of WT and transgenic lines. The asterisks above columns indicate significant differences compared to WT (** p ≤ 0.01).
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Figure 5. Relative expression of enzyme genes related to anthocyanin metabolism in transgenic tobacco lines and WT plants. The data were normalized using the WT sample and the NtActin (GQ339768) gene as the internal controls and calculated using the 2−ΔΔCT method. The asterisks above columns indicate significant differences compared to WT (* p ≤ 0.05, ** p ≤ 0.01).
Figure 5. Relative expression of enzyme genes related to anthocyanin metabolism in transgenic tobacco lines and WT plants. The data were normalized using the WT sample and the NtActin (GQ339768) gene as the internal controls and calculated using the 2−ΔΔCT method. The asterisks above columns indicate significant differences compared to WT (* p ≤ 0.05, ** p ≤ 0.01).
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Table
Table 1. Identification of anthocyanin in four colored raspberry fruits by HPLC-MS. Asterisk (*) denotes presence of anthocyanin in raspberry varieties.
Table 1. Identification of anthocyanin in four colored raspberry fruits by HPLC-MS. Asterisk (*) denotes presence of anthocyanin in raspberry varieties.
Peak Retention Time (min)[M]+ (m/z)Fragment Ions (m/z)Compound NameRaspberry Varieties
Jeltii GigantCarolineAmerican 22Black Raspberry
11.76757595,449,287Cyanidin-3-O-(6″-O-coumaroyl)-glucoside,5-O-glucoside *
22.17773611,287Cyanidin-3-O-caffeoylsophoroside **
32.51611287Cyanidin-3-O-di-hexoside **
42.57449287Cyanidin-3-O-glucoside****
52.80595449,287Cyanidin 3-O-rutinose * *
62.77757611,287Cyanidin-3-(2G-glucosylrutinoside) *
72.86581287Delphinidin-3-O-sambubioside chloride **
82.87727581,287Cyanidin-3-xylosylrutinoside *
92.99595287Cyanidin-3-O-coumaryl-glucoside* *
103.29625301Peonidin 3-O-di hexoside *
113.31433271Peonidin-3-O-arabinoside *
123.37463301Peonidin-3-O-galactoside **
133.43463301Peonidin-3-O-glucoside
143.49579433,271Pelargonidin 3-O-rutinoside *
153.54609463,301Peonidin-3-O-coumarylglucoside **
164.38449287Cyanidin-3-O-glucoside****
174.98493331Malvidin-3-glucoside****
185.10611303Delphinidin-3-O-β-D-(6-(E)-p-coumaroyl) galactopyranoside *
195.17465303Delphinidin-3-galactoside *
205.22435303Delphinidin-3-arabinoside*
215.38519271Pelargonidin-3-(6″malonylglucoside)* **
225.42433271Pelargonidin-3-glucoside **
235.99507303Delphinidin-3-(6″acetylglucoside)
246.53465303Delphinidin-3-glucoside****
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Li, T.; Xin, W.; Li, Y.; Wang, A.; Yang, G. An R2R3-MYB Transcription Factor RoMYB10 Regulates Anthocyanin Biosynthesis in Black Raspberry. Agronomy 2023, 13, 1823. https://doi.org/10.3390/agronomy13071823

AMA Style

Li T, Xin W, Li Y, Wang A, Yang G. An R2R3-MYB Transcription Factor RoMYB10 Regulates Anthocyanin Biosynthesis in Black Raspberry. Agronomy. 2023; 13(7):1823. https://doi.org/10.3390/agronomy13071823

Chicago/Turabian Style

Li, Tiemei, Wenjiao Xin, Yutao Li, Aoxue Wang, and Guohui Yang. 2023. "An R2R3-MYB Transcription Factor RoMYB10 Regulates Anthocyanin Biosynthesis in Black Raspberry" Agronomy 13, no. 7: 1823. https://doi.org/10.3390/agronomy13071823

APA Style

Li, T., Xin, W., Li, Y., Wang, A., & Yang, G. (2023). An R2R3-MYB Transcription Factor RoMYB10 Regulates Anthocyanin Biosynthesis in Black Raspberry. Agronomy, 13(7), 1823. https://doi.org/10.3390/agronomy13071823

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