The Onion (Allium cepa L.) R2R3-MYB Gene MYB1 Regulates Anthocyanin Biosynthesis

Abstract

Bulb color is an important consumer trait for onion (Allium cepa L., Allioideae, Asparagales). The bulbs accumulate a range of flavonoid compounds, including anthocyanins (red), flavonols (pale yellow), and chalcones (bright yellow). Flavonoid regulation is poorly characterized in onion and in other plants belonging to the Asparagales, despite being a major plant order containing many important crop and ornamental species. R2R3-MYB transcription factors associated with the regulation of distinct branches of the flavonoid pathway were isolated from onion. These belonged to sub-groups (SGs) that commonly activate anthocyanin (SG6, MYB1) or flavonol (SG7, MYB29) production, or repress phenylpropanoid/flavonoid synthesis (SG4, MYB4, MYB5). MYB1 was demonstrated to be a positive regulator of anthocyanin biosynthesis by the induction of anthocyanin production in onion tissue when transiently overexpressed and by reduction of pigmentation when transiently repressed via RNAi. Furthermore, ectopic red pigmentation was observed in garlic (Allium sativum L.) plants stably transformed with a construct for co-overexpression of MYB1 and a bHLH partner. MYB1 also was able to complement the acyanic petal phenotype of a defined R2R3-MYB anthocyanin mutant in Antirrhinum majus of the asterid clade of eudicots. The availability of sequence information for flavonoid-related MYBs from onion enabled phylogenetic groupings to be determined across monocotyledonous and dicotyledonous species, including the identification of characteristic amino acid motifs. This analysis suggests that divergent evolution of the R2R3-MYB family has occurred between Poaceae/Orchidaceae and Allioideae species. The DNA sequences identified will be valuable for future analysis of classical flavonoid genetic loci in Allium crops and will assist the breeding of these important crop species.

Keywords: Asparagales; R2R3-MYB; anthocyanin; garlic; onion; regulation; transgenic.

FIGURE 1

Graphic representation of the MYB1 gene of onion (Allium cepa). Exons, introns, 5′-UTR, the three potential ATGs, and the putative TATA box are marked. Positions of notable sequence motifs for transcriptional regulation identified using the PLACE website analysis are shown by the purple bars. Locations of DNA primers used for genome walking for promoter isolation are indicated in green. The upstream genomic sequence annotated for the specific DNA-binding motifs is given in Supplementary Figure 1.

FIGURE 2

Phylogenetic relationships of onion (Allium cepa) flavonoid-related transcription factors to those of other species. Full-length deduced amino acid sequences, Ac-MYB1, Ac-MYB4, Ac-MYB5, and Ac-MYB29 were used to form phylogenetic trees with selected flavonoid-related transcription factors from other plant species. The regulators of the different flavonoid types form separate sub-groups (SGs), as indicated, with a further division of anthocyanin regulators into two subgroups (SG5 and SG6 as indicated by TT2 and PAP1 of A. thaliana, respectively). GenBank accession numbers of the sequences used are listed in “Materials and Methods.” Bootstrap values >60% are shown (1000 replicates). Asparagales, Liliales, or Poaceae species are shown in red, orange, and green, respectively.

FIGURE 3

Alignment of part of the C-terminal region of R2R3-MYB sequences showing the SG5 and SG6 motifs. Amino acids matching either the SG6 motif or the SG5 (C1-TT2) clade motif are indicated in red. Species names are as given in the “Materials and Methods,” with Asparagales, Liliales, Poaceae, Gymnospermae, and dicot species shown in red, orange, green, blue, and black, respectively. The starting amino acid position of the sequences is given in the second column.

FIGURE 4

Increased MYB1 transcript abundance is associated with anthocyanin production. Quantitative RT-PCR (qPCR) analysis of red onion (Allium cepa) seedling leaf sheath tissue for expression of MYB1 and two anthocyanin biosynthetic genes, chalcone synthase-A (CHS) and dihydroflavonol 4-reductase-A (DFR). Transcript abundance was normalized to the geometric mean of the transcript quantities of UBQ and GAPDH. The sample consists of pooled tissue from three seedlings. Means ± SEM, n = 3 technical replicates are shown. A replicate qPCR experiment gave results consistent with the data presented. Top panels illustrate the phenotype and the type of tissue analyzed rather than an actual sample.

FIGURE 5

Transient overexpression of MYB1 induces anthocyanin pigmentation in onion (Allium cepa) and garlic (Allium sativum). Tissue was bombarded with constructs for 35S:MYB1+35S:Zm-Lc+35S:GFP, 35S:MYB1+35S:GFP, or 35S:Zm-Lc+35S:GFP. Tissue from acyanic red onion seedling leaf sheaths and garlic cloves was used. Images are shown under white light (Top panels) for anthocyanin pigmentation or blue light (Lower panels) for GFP fluorescence. A close up of one region is shown for easier comparison of the co-localisation of GFP signal and red pigmentation. Representative images are shown.

FIGURE 6

Transient RNAi inhibition of MYB1 expression inhibits anthocyanin pigment formation in onion (Allium cepa) seedlings. Acyanic sheath tissue was removed from inner layers of red onion seedlings and biolistically transformed with gene constructs for 35S:GFP and 35S:MYB1-RNAi (two different example regions are presented in C–H) or 35S:GFP alone (example region in A,B). They were then incubated for around 72 h on moistened filter paper in a Petri dish and exposed to a 16:8 h light:dark cycle. White regions, lacking anthocyanin pigmentation, were observed only when 35S:MYB1-RNAi was used. (B,D,G) Are blue light images for GFP visualization. (F,G) Show a close up of one white region (boxed in E), and (H) shows a further close up in which a partially transparent version of the white light image for anthocyanins (F) is overlaid on the GFP image (H). Representative images are shown.

FIGURE 7

Phenotypes of garlic (Allium sativum) plants stably transformed with a construct for 35S:MYB1 and 35S:ZmLc. (A) shows examples of regenerating shoots of independent transgenic events in culture. (B–D) show tissue from, respectively, the leaf, stem base, and bulb, with the MYB1+Lc transgenic (line 1111-8A) on the right and a control plant on the left. The control was a transgenic carrying a construct unrelated to pigmentation (an antiviral construct).

FIGURE 8

Complementation of the white petal phenotype of the Antirrhinum majus R2R3-MYB mutant rosea^dorsea by MYB1. Representative image shown of cell autonomous anthocyanin production after bombarding 35S:MYB1+35S:GFP into the inner epidermis of petal tissue. Anthocyanin production co-localizes with GFP expression. Images were photographed under white light for pigmentation and blue light for GFP fluorescence.