Background

The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species.

Results

We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins.

Conclusions

This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

Red fruits are popular and widely accepted by consumers because of an enhanced appearance and enriched anthocyanins. The molecular mechanism of anthocyanin regulation in red-skinned pear (Pyrus) has been studied, and the genes encoding the biosynthetic steps and several transcription factors (TFs) have been characterized. In this study, a candidate R2R3 MYB TF, PyMYB114, was identified by linkage to the quantitative trait loci (QTL) for red skin color on linkage group 5 in a population of Chinese pear (Pyrus bretschneideri). The function of PyMYB114 was verified by transient transformation in tobacco (Nicotinana tabacum) leaves and strawberry (Fragaria) and pear fruits, resulting in the biosynthesis of anthocyanin. Suppression of PyMYB114 could inhibit anthocyanin biosynthesis in red-skinned pears. The ERF/AP2 TF PyERF3 was found to interact with PyMYB114 and its partner PybHLH3 to co-regulate anthocyanin biosynthesis, as shown by a dual luciferase reporter system and a yeast two-hybrid assay. In addition, the transcript abundance of PyMYB114 and PyMYB10 were correlated, and co-transformation of these two genes into tobacco and strawberry led to enhanced anthocyanin biosynthesis. This interaction network provides insight into the coloration of fruits and the interaction of different TFs to regulate anthocyanin biosynthesis.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/tpj.13666

Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis.

The MYB superfamily is large and functionally diverse in plants. To date, MYB family genes have not yet been identified in Chinese white pear (Pyrus bretschneideri), and their functions remain unclear. In this study, we identified 231 genes as candidate MYB genes and divided them into four subfamilies. The R2R3-MYB (PbrMYB) family shared an R2R3 domain with 104 amino acid residues, including five conserved tryptophan residues. The Pbr MYB family was divided into 37 functional subgroups including 33 subgroups which contained both MYB genes of Rosaceae plants and AtMYB genes, and four subgroups which included only Rosaceae MYB genes or AtMYB genes. PbrMYB genes with similar functions clustered into the same subgroup, indicating functional conservation. We also found that whole-genome duplication (WGD) and dispersed duplications played critical roles in the expansion of the MYB family. The 87 Pbr MYB duplicated gene pairs dated back to the two WGD events. Purifying selection was the primary force driving Pbr MYB gene evolution. The 15 gene pairs presented 1-7 codon sites under positive selection. A total of 147 expressed genes were identified from RNA-sequencing data of fruit, and six Pbr MYB members in subgroup C1 were identified as important candidate genes in the regulation of lignin synthesis by quantitative real-time PCR analysis. Further correlation analysis revealed that six PbrMYBs were significantly correlated with five structural gene families (F5H, HCT, CCR, POD and C3'H) in the lignin pathway. The phylogenetic, evolution and expression analyses of the MYB gene family in Chinese white pear establish a solid foundation for future comprehensive functional analysis of Pbr MYB genes.

Genome-Wide Identification, Evolution and Functional Divergence of MYB Transcription Factors in Chinese White Pear (Pyrus bretschneideri)

Anthocyanin concentration is the key determinant for red skin color in pear fruit. However, the molecular basis for development of red skin is complicated and has not been well-understood thus far. "Starkrimson" (Pyrus communis L.), an introduced red pear cultivated in the north of China and its green mutant provides a desirable red/green pair for identification of candidate genes involved in color variation. Here, we sequenced and annotated the transcriptome for the red/green color mutant at three stages of development using Illumina RNA-seq technology. The total number of mapped reads ranged from 26 to 46 million in six libraries. About 70.11-71.95% of clean reads could be mapped to the reference genome. Compared with green colored fruit, a total of 2230 differentially expressed genes (DEGs) were identified in red fruit. Gene Ontology (GO) terms were defined for 4886 differential transcripts involved in 15 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Three DEGs were identified as candidate genes in the flavonoid pathway, LAR, ANR, and C3H. Tellingly, higher expression was found for genes encoding ANR and LAR in the green color mutant, promoting the proanthocyanidin (PA) pathway and leading to lower anthocyanin. MYB-binding cis-motifs were identified in the promoter region of LAR and ANR. Based on these findings, we speculate that the regulation of PA biosynthesis might be a key factor for this red/green color mutant. Besides the known MYB and MADS transcription families, two new families, AP2 and WRKY, were identified as having high correlation with anthocyanin biosynthesis in red skinned pear. In addition, qRT-PCR was used to confirm the transcriptome results for 17 DEGs, high correlation of gene expression, further proved that AP2 and WARK regulated the anthocyanin biosynthesis in red skinned "Starkrimson," and ANR and LAR promote PA biosynthesis and contribute to the green skinned variant. This study can serve as a valuable new resource laying a solid foundation for functional gene identification in the anthocyanin pathway of red-skinned pear and provide a good reference for relevant research on molecular mechanisms of color variation in other pear species.

/pdf/transcriptome-profiling-reveals-differential-gene-expression-1v1bx4d956.pdf

Transcriptome profiling reveals differential gene expression in proanthocyanidin biosynthesis associated with red/green skin color mutant of pear (Pyrus communis L.)

The red color of fruit is an attractive plant trait for consumers. Plants with color-faded fruit have a lower commercial value, such as ‘Red Bartlett’ pears (Pyrus communis L.) that have dark-red fruit in the early stages of fruit development that subsequently fade to red-green at maturity. To identify the reason for color fading, we first analyzed the anthocyanin content of peel from ‘Red Bartlett’, which displays the color fading phenomenon, and ‘Starkrimson’, which has no color fading. Results showed that the anthocyanin content of ‘Red Bartlett’ peel decreased significantly late in fruit development, while in ‘Starkrimson’ there was no significant decrease. Next, RNA-Sequencing was used to identify 947 differentially expressed genes (DEGs) between ‘Red Bartlett’ and ‘Starkrimson’. Among them, 471 genes were upregulated and 476 genes were downregulated in ‘Red Bartlett’ at the late development stage relative to ‘Starkrimson’. During ‘Red Bartlett’ color fading, the structural gene LDOX and six GST family genes were downregulated, while FLS, LAC, POD, and five light-responding genes were significantly upregulated. Additionally, 45 genes encoding transcription factors MYB, bHLH, WRKY, NAC, ERF, and zinc finger were identified among 947 DEGs. Changes in the expression of these genes may be responsible for the decrease in anthocyanin accumulation in ‘Red Bartlett’ fruit. Taken together, this study demonstrated that color fading of ‘Red Bartlett’ was closely linked to reduced anthocyanin biosynthesis, increased anthocyanin degradation and suppression of anthocyanin transport. It also provided novel evidence for the involvement of light signals in the color fading of red-skinned pears.

/pdf/transcriptome-analysis-reveals-candidate-genes-related-to-43gnrbjpux.pdf

Transcriptome Analysis Reveals Candidate Genes Related to Color Fading of 'Red Bartlett' ( Pyrus communis L.).

Key message
This research reveals the different expression patterns of anthocyanin biosynthesis enzyme genes and transcription factors in six red-skinned pear cultivars with different genetic backgrounds.

Chao Wang

Papers

Expression differences of anthocyanin biosynthesis genes reveal regulation patterns for red pear coloration