Abstract

Background

   Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don] (syn. Prunus
   pseudocerasus Lindl.) is an economically important fruiting cherry
   species with a diverse range of attractive colors, spanning from the
   lightest yellow to the darkest black purple. However, the MYB
   transcription factors involved in anthocyanin biosynthesis underlying
   fruit color variation in Chinese cherry remain unknown.

Results

   In this study, we characterized the R2R3-MYB gene family of Chinese
   cherry by genome-wide identification and compared it with those of 10
   Rosaceae relatives and Arabidopsis thaliana. A total of 1490 R2R3-MYBs
   were classified into 43 subfamilies, which included 29 subfamilies
   containing both Rosaceae MYBs and AtMYBs. One subfamily (S45) contained
   only Rosaceae MYBs, while three subfamilies (S12, S75, and S77)
   contained only AtMYBs. The variation in gene numbers within identical
   subfamilies among different species and the absence of certain
   subfamilies in some species indicated the species-specific expansion
   within MYB gene family in Chinese cherry and its relatives. Segmental
   and tandem duplication events primarily contributed to the expansion of
   Chinese cherry R2R3-CpMYBs. The duplicated gene pairs underwent
   purifying selection during evolution after duplication events.
   Phylogenetic relationships and transcript profiling revealed that
   CpMYB10 and CpMYB4 are involved in the regulation of anthocyanin
   biosynthesis in Chinese cherry fruits. Expression patterns, transient
   overexpression and VIGS results confirmed that CpMYB10 promotes
   anthocyanin accumulation in the fruit skin, while CpMYB4 acts as a
   repressor, inhibiting anthocyanin biosynthesis of Chinese cherry.

Conclusions

   This study provides a comprehensive and systematic analysis of R2R3-MYB
   gene family in Chinese cherry and Rosaceae relatives, and identifies
   two regulators, CpMYB10 and CpMYB4, involved in anthocyanin
   biosynthesis in Chinese cherry. These results help to develop and
   utilize the potential functions of anthocyanins in Chinese cherry.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12864-024-10675-7.

   Keywords: Chinese cherry, Fruit color, Genome-wide identification,
   R2R3-MYB transcription factor, Regulation of anthocyanin biosynthesis,
   CpMYB10, CpMYB4

Introduction

   Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don] (syn. Prunus
   pseudocerasus Lindl.), belonging to the Rosaceae family, is an
   economically important tetraploid fruiting cherry species
   [[54]1–[55]3]. Cherry cultivation has been rapidly developing in China
   and has increasingly contributed to poverty alleviation and rural
   revitalization. Chinese cherry exhibits a wide range of fruit colors,
   including yellow, vermilion on yellow ground, red, purple red, and
   black purple [[56]4]. The fruit coloration is attributed to the
   accumulation of anthocyanins, which are controlled by a distinct group
   of R2R3-MYB transcription factors [[57]5, [58]6]. In apple, MdMYB10,
   MdMYB1, and MdMYBA are the main determinants of fruit color variations
   among cultivars [[59]7–[60]9]. In sweet cherry, three PavMYB10.1
   alleles determine the fruit skin color: yellow (PavMYB10.1c), blush
   (homozygous for PavMYB10.1b) and red (at least one intact PavMYB10.1a)
   [[61]10]. Allelic variation of MYB10 is the major force controlling
   natural variations in both skin and flesh color in strawberry fruits
   [[62]11]. Therefore, R2R3-MYB TFs play key roles in regulating the
   anthocyanin biosynthesis in Rosaceae fruit crops.

   Numerous reports have demonstrated that the R2R3-MYB TFs typically form
   a well-conserved MYB-bHLH-WD40 (MBW) complex to regulate the
   anthocyanin biosynthesis [[63]12–[64]14]. In Arabidopsis,
   AtMYB11/12/111 independently participate in transcriptional activation
   of the early biosynthetic genes (EBGs), while AtMYB75/90/113/114 of
   subfamily S6 activate the late biosynthetic genes (LBGs) by the
   formation of MBW complex [[65]15]. These functions are conserved in
   other species, such as apple MdMYB10 [[66]16, [67]17], strawberry
   FvMYB10 [[68]11, [69]18] and FaMYB5 [[70]19], and sweet cherry PavMYB10
   and PacMYBA [[71]10, [72]20, [73]21]. In addition to MYB activators, it
   has been reported that R2R3-MYB TFs belonging to subfamily S4 can
   repress anthocyanin accumulation. Two types of MYB repressors have been
   identified, one of which is dependent on its own EAR (ERF-associated
   amphiphilic repression) inhibitory sequence, while the other is
   independent [[74]22–[75]24]. Furthermore, two ways to inhibit
   anthocyanin biosynthesis have been proposed: the AtMYB4-like type
   [[76]25] and the FaMYB1-like type [[77]26]. The AtMYB4-like type acts
   directly on the promoters of target structural genes [[78]27], such as
   MdMYB6, MdMYB16 in apple [[79]24, [80]28] and SlMYB7 in tomato
   [[81]29]. The FaMYB1-like type functions as a corepressor, which is
   incorporated into or binds MBW complexes to alter the complex activity
   and transform from activation to inhibition [[82]27], including grape
   VvMYBC2, VvMYBC2-L3, and apple MdMYB15L [[83]23, [84]30, [85]31]. These
   findings have highlighted the core roles of transcriptional regulations
   of R2R3-MYB in the control of anthocyanin biosynthesis.

   In Chinese cherry, cyanidin and its glycoside derivatives have been
   identified as the primary anthocyanins responsible for fruit coloration
   [[86]32–[87]34]. Compared to yellow fruits, (dark)-red fruits were
   found to accumulate significantly higher level of
   cyanidin-3-rutinoside, but lower levels of flavanol and
   proanthocyanidin [[88]33]. The up-regulation of structural genes in
   cyanidin biosynthesis, including EBGs (CpF3H, CpF3’H) and LBGs (CpDFR,
   CpANS, and CpUFGT), was observed, contributing to the formation of
   dark-red fruits [[89]33]. On the contrary, higher expression of CpLAR
   was observed in yellow fruits. In addition, eight regulatory genes,
   including MYB TFs, have been identified as candidate determinants of
   fruit color in Chinese cherry [[90]33]. However, the genome-wide
   characterization of MYB gene family and their regulatory roles in
   anthocyanin biosynthesis in Chinese cherry has not been previously
   reported.

   To gain further insights into the regulatory network underlying fruit
   color variation in Chinese cherry, we conducted a genome-wide
   characterization of the R2R3-MYB gene family in Chinese cherry and
   relative species in Rosaceae family. Based on transcriptomic profiling
   and function verification, we identified the key genes involved in
   anthocyanin biosynthesis in Chinese cherry fruits. Our objectives were
   (i) to characterize the R2R3-MYB gene family of Chinese cherry through
   genome-wide identification and compare it with that of 10 Rosaceae
   relatives and Arabidopsis thaliana; (ii) to identify key MYB TFs
   related to anthocyanin biosynthesis in Chinese cherry; and (iii) to
   preliminarily verify the functions of CpMYB10 and CpMYB4. This study
   provides a starting point for further analysis of MYB functions in
   Chinese cherry and establishes a solid foundation for utilizing
   candidate genes in breeding programs aimed at improving anthocyanin
   accumulation.

Materials and methods

Plant materials

   Three Chinese cherry accessions, namely ‘PZB’, ‘HF’ and ‘HP600’,
   representing yellow, red and black-purple fruit colors respectively,
   were used in this study. They were cultivated under field conditions at
   Cherry Germplasm Repository of Sichuan Province in Sichuan Agricultural
   University (Chengdu), China. The fruits from three accessions were
   harvested at full maturity stage. Tissue samples including roots,
   stems, leaves, flower buds (red), and open flowers (white) were
   specifically collected from ‘HF’ accession. All samples were
   immediately frozen in liquid nitrogen and stored at − 80 °C for
   subsequent analysis, with three biological replicates per sample.

Determination of fruit color and total anthocyanin content

   Fruit color parameters (lightness L*, redness a*, and yellowness b*)
   were measured using a KONICA MINOLTA CM-2600d spectrophotometer
   (Japan), and the color ratio (a*/b*) was calculated [[91]35]. Ten
   cherries for each replicate were used, with three biological replicates
   per sample point.

   Total anthocyanin content was determined using the pH differential
   method described by Lee et al. [[92]36]. Approximately 0.5 g of fruit
   tissue was extracted with 5 mL of extraction solution (acetone:
   methanol: water: acetic acid = 2:2:1:0.5) and heated in a water bath at
   40 °C. The mixture was centrifuged at 8,000 ×g for 25 min, and the
   supernatant was used for analysis. Two buffer systems were used with
   0.4 M potassium chloride (pH 1.0) and 0.4 M dibasic sodium (pH 4.5).
   The total anthocyanin content was calculated using the equation: A =
   [(A[510] − A[700]) pH[1.0] − (A[510] − A[700]) pH[4.5]] and converted
   into mg cyanidin 3-glucoside per 1,000 g fresh weight (mg⋅kg^− 1 FW).
   Three independent biological replicates per sample point were analyzed.

Genome-wide identification of R2R3-MYB gene family

   A total of 12 genomes from Chinese cherry (unpublished) and related
   species within Rosaceae family, and Arabidopsis thaliana were selected
   for analysis (Table [93]1).

Table 1.

   The information of reference genomes for Chinese cherry and other
   eleven species
   Species Release Source References