Identification of BpR2R3-MYBs
On the basis of the sequencing data of the full-length transcriptome of B. platyphylla, the genes with R2R3-MYB conserved structure domains were screened using the HMMer database, and the screened BpR2R3-MYB family genes were further verified using Pfam and CDD databases. Forty-four BpR2R3-MYB family genes were identified and numbered according to the order in which they were found (Table 1). The 44 BpR2R3-MYB proteins contained 186 (BpMYB24) to 484 (BpMYB41) amino acids, with molecular weights of 20.97 KDa (BpMYB24) and 54.60 KDa (BpMYB41) and an isoelectric point ranging from 5.12 (BpMYB38) to 9.57 (BpMYB10). The subcellular location predicted that most of the proteins were nuclear proteins. Only BpMYB2, BpMYB27, and BpMYB27 were distributed in the chloroplast. BpMYB16 and BpMYB23 were distributed in the cytoplasm and mitochondria, respectively.
Chromosome distribution of BpR2R3-MYBs
The 44 BpR2R3-MYB family genes were unevenly distributed in 11 chromosomes of B. platyphylla, and no distribution of BpR2R3-MYB was observed in chromosomes 1, 7, and 10 (Fig. 1). The maximum number of BpR2R3-MYB genes in one chromosome was six and located in chromosomes 3, 6, and 11. Two chromosomes (8 and 14) carried five BpR2R3-MYB genes, two chromosomes (5 and 12) had four BpR2R3-MYB genes, and four chromosomes (2, 4, 9, and 13) harbored two BpR2R3-MYB genes.
Analysis of the gene structure and conserved BpR2R3-MYB domains
The gene structure and domains of the BpR2R3-MYB proteins were analyzed using the online software MEME and TBtools. As shown in Fig. 2A, motifs with similar structures and domains were clustered into one clade, indicating that they had an analogous function. A total of 10 conserved amino acid motifs were identified in the BpR2R3-MYB proteins (Fig. 2B). Among them, Motifs 5, 6, 7, 8, 9, and 10 had no more than 5 occurrences in BpR2R3-MYB proteins, 20 BpR2R3-MYB proteins had Motif 4, 28 BpR2R3-MYB proteins had Motif 3, and all 44 BpR2R3-MYB proteins had the highly conserved R2-R3 structural domain of Motifs 1 and 2. Motif 1 was the R2-MYB structural domain (-W-(X19)-W-(X19)-W-), and Motif 2 was the R3-MYB structural domain (-F-(X19)-W-(X19)-W-) (Fig. 3). In the R3-MYB structural domain, the first W residue (position 9) was frequently substituted by phenylalanine (F) and less frequently by isoleucine (I), leucine (L), methionine (M), or tyrosine (Y).
To understand the structural diversity of BpR2R3-MYB, an exon–intron analysis was performed on the 44 BpR2R3-MYBs (Fig. 2C). The results showed that 90.90% (40/44) of the BpR2R3-MYBs had introns varying from 1 (BpR2R3-MYB1, 2, 5, 7, 12, 21, 27, 36, 42) to 12 (BpR2R3-MYB 41), and the four intron-less genes were BpR2R3-MYB 3, 14, 16, and 17. In addition, 75% (33/44) of the BpR2R3-MYBs had untranslated regions (UTRs) varying from 1 (BpR2R3-MYB1, 2, 5, 7, 12, 21, 27, 36, 42) to 3 (BpR2R3-MYB 41), and the 11 genes (BpR2R3-MYB1, 3, 14, 16, 17, 21, 22, 26, 27, 31, 36) had no UTR.
Evolutionary analysis of BpR2R3-MYBs
The Ka/Ks ratio was calculated to explore the evolutionary constraints on the BpR2R3-MYB gene family. The results showed that 20% (9/44) of the BpR2R3-MYB genes exhibited fragment duplication, and they were scattered in chromosomes 2, 3, 4, 6, 11, 12, and 14 (Fig. 4). Five gene pairs with segment duplication, namely, BpMYB11&BpMYB19, BpMYB43&BpMYB22, BpMYB6&BpMYB26, BpMYB3&BpMYB17, and BpMYB3&BpMYB14 (Table 2), were found in the chromosomes. The Ka/Ks ratios of the five gene pairs were less than 1. In addition, the syntenic relationships of the R2R3-MYB genes showed that 30 orthologs existed between B. platyphylla and A. thaliana, and 68 orthologs existed between B. platyphylla and Populus trichocarpa (Fig. 5).
Phylogenetic analyses of BpR2R3-MYBs
In accordance with the classification of Arabidopsis R2R3-MYB proteins, we divided the BpR2R3-MYB proteins into 13 subgroups (Fig. 6). The average size of the subgroups was 3.38, and the size range was 1–7. Four R2R3-MYB proteins (Fragaria x ananassa FaMYB9/FaMYB11, Prunus avium PacMYBA, and Triticum aestivum TaMyb1D) related to flavonoid synthesis were also used to cluster with the 44 BpR2R3-MYB proteins. BpR2R3-MYB15 and BpR2R3-MYB21, BpR2R3-MYBB36, and BpR2R3-MYB12 and BpR2R3-MYB37 were clustered with FaMYB9/FaMYB11, PacMYBA, and TaMyb1D, respectively. We deduced that the five genes were related to flavonoid synthesis.
Correlation analysis of the flavonoid content and gene expression of BpMYB15 and BpMYB21
The transcriptome sequencing data of nitrosoglutathione reductase (GSNOR) gene-silenced B. platyphylla plants (BpGSNOR-RNAi) and wild-type plants (WT) in our laboratory were used to analyze the correlation coefficients of the gene expression of the five BpR2R3-MYBs (BpR2R3-MYB12, 15, 21, 36, and 37) and the key enzyme genes of flavonoid synthesis (S Fig. 1 and S Table 2). The results showed that the correlation coefficients of BpR2R3-MYB15 and BpR2R3-MYB21 were higher than those of the three other genes. Hence, we cloned BpR2R3-MYB15 and BpR2R3-MYB21 via PCR (S Figs. 2 and 3).
The flavonoid content and gene expression of BpR2R3-MYB15 and BpR2R3-MYB21 were further analyzed under one daily cycle and 90 μmol·L-1 Cd treatment (Fig. 7). In one daily variation, the flavonoid content peaked at 18:00, and the time with high flavonoid content was from 15:00 to 0:00. The gene expression of BpR2R3-pMYB15 and BpR2R3-MYB21 peaked at 21:00 and 12:00, respectively. The flavonoid content and gene expression of BpR2R3-pMYB15 and BpR2R3-MYB21 in the leaves of the B. platyphylla plants reached the highest one day after Cd treatment, but the gene expression of BpR2R3-pMYB15 and BpR2R3-MYB21 in the stem and root of B. platyphylla mostly decreased after Cd treatment. The correlation coefficient of the gene expression of BpR2R3-pMYB15 and flavonoid content was higher than that of of BpR2R3-pMYB21 and flavonoid content (Table 3).
Overexpression of BpR2R3-MYB15 enhanced flavonoid production
After three days of Agrobacterium-mediated transient transformation, the overexpression of BpR2R3-MYB15 in B. platyphylla calli (5.72 times than that of untransformed calli) significantly enhanced the flavonoid and procyanidin contents and increased the gene expression of BpCHI1, BpF3H, and BpDFR, which are key enzyme genes for flavonoid synthesis. The silencing of BpR2R3-MYB15 in B. platyphylla calli (0.68 times than that of untransformed calli) decreased the flavonoid and procyanidin contents and reduced the gene expression of BpCHI1, BpF3H, and BpDFR (Fig. 8).