NtMYB3, an R2R3-MYB from Narcissus, Regulates Flavonoid Biosynthesis

Abstract

R2R3-MYB transcription factors play important roles in the regulation of plant flavonoid metabolites. In the current study, NtMYB3, a novel R2R3-MYB transcriptional factor isolated from Chinese narcissus (Narcissus tazetta L. var. chinensis), was functionally characterized. Phylogenetic analysis indicated that NtMYB3 belongs to the AtMYB4-like clade, which includes repressor MYBs involved in the regulation of flavonoid biosynthesis. Transient assays showed that NtMYB3 significantly reduced red pigmentation induced by the potato anthocyanin activator StMYB-AN1 in agro-infiltrated leaves of tobacco. Over-expression of NtMYB3 decreased the red color of transgenic tobacco flowers, with qRT-PCR analysis showing that NtMYB3 repressed the expression levels of genes involved in anthocyanin and flavonol biosynthesis. However, the proanthocyanin content in flowers of transgenic tobacco increased as compared to wild type. NtMYB3 showed expression in all examined narcissus tissues; the expression level in basal plates of the bulb was highest. A 968 bp promoter fragment of narcissus FLS (NtFLS) was cloned, and transient expression and dual luciferase assays showed NtMYB3 repressed the promoter activity. These results reveal that NtMYB3 is involved in the regulation of flavonoid biosynthesis in narcissus by repressing the biosynthesis of flavonols, and this leads to proanthocyanin accumulation in the basal plate of narcissus.

Keywords: Chinese narcissus; R2R3-MYB; anthocyanin; flavonoid repressor; flavonol.

Figure 1

Phylogenetic tree analyses of MYB repressors. Our candidate gene NtMYB3 are shown by red rectangle.

Figure 2

Multiple alignments of the protein sequences of NtMYB3 with other R2R3-MYB repressors belonging to subgroup 4. Blue shaded are indicated the 100 % homology suquences of amino acids.

Figure 3

Transient transformation of tobacco leaves (Nicotiana tabacum). R1, R2, and R3 represent three replicates. (A) Infiltration with StMYB-AN1; (B) Co-infiltration of pSAK277 with StMYB-AN1; (C) Infiltration with NtMYB3; (D) Co-infiltration of StMYB-AN1with NtMYB3; (E) Co-infiltration of StMYB-AN1, NtMYB3, and StbHLH. Photos were taken after five days of agro-infiltration injection. (Scale bar = 1 cm).

Figure 4

Floral phenotypes of transgenic tobacco plants carrying NtMYB3 gene.Wild type tobacco (WT) and transgenic lines of tobacco are (L-2, L-4 and L-5). Flower color comparison in wild type (WT) and transgenic lines (A); Comparison of pistil length in WT and transgenic tobacco (B). Blue circle indicated the top of the pistal length.

Figure 5

Estimation of total anthocyanin in wild type and transgenic lines (L-2, L-4, and L-5). The bars represent the standard error of thrice biological replication. Small letters showed a significant difference at the level of p < 0.05 by using LSD statistical analysis.

Figure 6

Expression analyses of flavonoid biosynthetic pathway key genes in transgenic flowers carrying NtMYB3 by qPCR. L-2, L-4, indicated two transgenic tobacco lines. PAL, Phenylalanine ammonia lyase; 4CL, 4-coumaroyl-CoA ligase, CHS, Chalcone synthase; CHI, Chalcone isomerase; F3H, Flavanone 3-hydroxylase; DFR, Dihydroflavonol 4-reductase; FLS, flavonol synthase LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; ANS, anthocyanin synthase; UFGT, UDP glucose-flavonoid 3-O glucosyltransferase. Small letter showed the significant difference from wild type at the level of p < 0.05 by using LSD test. The bar represents the standard error of thrice biological replications.

Figure 7

Quantification of proanthocyanin and flavonol in transgenic and wild type tobacco flowers. (A) PA contents and (B) flavonol contents. Small letters showed a significant difference at the level of p < 0.05 by using LSD statistical analysis.

Figure 8

Different tissues and organs of Chinese narcissus used in this experiment.

Figure 9

Expression analysis of NtMYB3 in various tissues of Narcissus tazetta. P, C, L, S, and BP indicate the petals, corona, leaves, bulb scales, and basal plates, respectively. Small letters showed the significant difference among different tissues at the level of p < 0.05 by using LSD test. Bars showed the standard error of three replicates.

Figure 10

Tobacco transient expression to investigate the regulation of the NtFLS promoter by NtMYB3. (A) Tobacco leaf injected with pNtFLS::GUS; (B) Tobacco leaf injected with pNtFLS::GUS +NtMYB3; (C) QPCR analysis of GUS expression levels in tobacco leaves injected with pNtFLS::GUS (1) and injected with pNtFLS::GUS +NtMYB3 (2). Small letters showed the significant difference at the level of p < 0.05 by using LSD test. (Scale bar = 1cm)

Figure 11

Dual luciferase assay confirms the repression of NtMYB3 to NtFLS promoter.