Figure 1.
Protein sequence alignment of the R2R3 DNA-binding domains of LcMYB1 and the other known anthocyanin MYB regulators in other species.
The R2 and R3 domains are underlined. The bHLH-binding motif is boxed in the R3 domain. Motif 6 was previously identified in the C-terminal domain of anthocyanin-related MYBs in Arabidopsis [39]. The accession number of these proteins (or translated products) are as follows in the GenBank database: MrMYB1, GQ340767; MdMYB1, ABK58136; MdMYB10, DQ267896; PyMYB10, ADN26574; GmMYB10, ACM62751.1; PhAN2, AAF66727; LeANT1, AAQ55181; CsRuby, AFB73909; MrMYB1, GQ340767; VlMYBA1-1, BAC07537; VvMYBA1, BAD18977; VvMYBA2, BAD18978; GhMYB10, CAD87010; AtPAP1, AAG42001; AtPAP2, AAG42002; AtMYB113, NP_176811; AtMYB114, NP_176812; AmROSEA1, ABB83826; AmROSEA2, DQ275530; AmVENOSA, DQ275531; and FaMYB1, AF401220.
Figure 2.
Phylogenetic relationships between Arabidopsis MYB transcription factors and anthocyanin-related MYBs in other species.
LcMYB1 clusters next to CsRudy, within the anthocyanin subgroup 6 MYBs. The subgroup numbers were previously described [12]. The tree was constructed using MEGA 5, neighboring-joining phylogeny testing, and 1,000 bootstrap replicates. The accession numbers for the genes in the other species are provided in Figure 1.
Figure 3.
Anthocyanin contents and expressions of biosynthetic genes.
(A) Anthocyanin contents and expression analysis of LcMYB1 and anthocyanin biosynthetic structural genes in different tissues of ‘NMC’ litchi including red pericarp, mature and young leaves, young stem and aril. (B) Anthocyanin contents and expression analysis of LcMYB1 and anthocyanin biosynthetic structural genes in the pericarp of ‘NMC’ litchi during fruit development. The Lcactin gene was used to normalize the expression levels of the genes under identical conditions. The vertical bars represent the standard error of triplicate experiments.
Figure 4.
LcMYB1 expression in the pericarp of different cultivars and manipulation treatments.
A, LcMYB1 expression of in the pericarp of twelve cultivars. B, The correlationship between LcMYB1 expression and anthocyanin content among the twelve cultivars. Anthocyanin contents were obtained from Wei et al. [7], Effects of ABA and CPPU on the expression of LcMYB1. D, Effects of bagging and bag removal on LcMYB1 expression.
Figure 5.
Color development in Nicotiana tabacum leaves following transient transformation.
Microscopic images showing anthocyanin accumulation in tobacco leaf infiltrated with: A) an empty vector (1× magnification) or B–C) 35S:LcMYB1 (8× magnification). D) Anthocyanin HPLC profiles of 35S:LcMYB1 extracts from tobacco leaf (top line) and empty vector (bottom line). Peaks identified at 520 nm: cy-glu, cyanidin-3-glucoside; cy-rut, cyanidin-3-rutinoside.
Figure 6.
Images of the transgenic tobacco lines and the anthocyanin content, and the expression of anthocyanin biosynthetic pathway structural and regulatory genes in transgenic tobacco lines.
A, Images of tobacco lines containing the empty vector (wild type) or the LcMYB1 allele with the CaMV 35S promoter (Line 1–2) (Scale bars for images = 1cm. B, Anthocyanin content in different organs of transgenic tobacco lines. C, Regulatory gene expression in the anthocyanin biosynthetic pathway in transgenic tobacco. D, Structural gene expression in the anthocyanin biosynthetic pathway in transgenic tobacco lines. The Lcactin gene was used to normalize gene expression of the genes under identical conditions. The vertical bars represent the standard error of triplicate experiments.
Table 1.
Putative cis-elements identified in the LcMYB1 promoter.
Table 2.
LcMYB1 3′ RACE, 5′ RACE, ORF and RT-PCR primers used in this study.
Table 3.
Primers used to isolated the LcMYB1 promotor region.