R2R3-MYB Transcription Factor NtMYB330 Regulates Proanthocyanidin Biosynthesis and Seed Germination in Tobacco (Nicotiana tabacum L.)

Abstract

Proanthocyanidins (PAs) are important phenolic compounds and PA biosynthesis is regulated by a ternary MBW complex consisting of a R2R3-MYB regulator, a bHLH factor and a WDR protein. In this study, a tobacco R2R3-MYB factor NtMYB330 was characterized as the PA-specific regulator in which the PA biosynthesis was promoted in the flowers of NtMYB330-overexpressing lines while decreased in the flowers of ntmyb330 mutants. NtMYB330 can interact with flavonoid-related bHLH partner NtAn1b and WDR protein NtAn11-1, and the NtMYB330-NtAn1b complex is required to achieve strong transcriptional activation of the PA-related structural genes NtDFR1, NtANS1, NtLAR1 and NtANR1. Our data reveal that NtMYB330 regulates PA biosynthesis in seeds and affects seed germination, in which NtMYB330-overexpressing lines showed higher PA accumulations in seed coats and inhibited germination, while ntmyb330 mutants had reduced seed coat PAs and improved germination. NtMYB330 affects seed germination possibly through two mechanisms: modulating seed coat PAs to affect coat-imposed dormancy. In addition, NtMYB330 regulates the expressions of abscisic acid (ABA) and gibberellin acid (GA) signaling-related genes, affecting ABA-GA crosstalk and seed germination. This study reveals that NtMYB330 specifically regulates PA biosynthesis via formation of the MBW complex in tobacco flowers and affects germination through adjustment of PA concentrations and ABA/GA signaling in tobacco seeds.

Keywords: ABA/GA signaling; MYB-bHLH-WDR complex; R2R3-MYB transcription factor; proanthocyanidin; seed germination.

FIGURE 1

Multiple alignment and phylogenetic analysis of selected R2R3-MYB proteins. (A) The deduced amino acid sequence of NtMYB330 was aligned with R2R3-MYB homologous ZmC1 (AAA33482) from Zea mays, VvMYBPAR (BAP39802) and VvMYBPA1 (CAJ90831) from Vitis vinifera, AtTT2 (NP198405) from Arabidopsis thaliana, DkMYB4 (BAI49721) from Diospyros kaki. The R2 and R3 repeats are boxed in orange and green, respectively. The bHLH motif [D/E]Lx₂[R/K]x₃Lx₆Lx₃R, PA-clade 1 conserved motif K[I/V]x₂PKPx₁Rx₂S[I/L] and PA-clade 2 conserved motif VI[R/P]TKAx₁RC[S/T] are indicated in red, dark blue, and brown boxes, respectively. (B) The phylogenetic tree of NtMYB330 along with selected R2R3-MYBs was constructed using neighbor-joining method. Scale bar represents 0.05 substitutions per site and numbers next to nodes are bootstrap values from 1000 replicates. GenBank accession numbers are indicated next to the protein name.

FIGURE 2

Subcellular localization of NtMYB330 protein and NtMYB330 gene expression pattern. (A) Subcellular localization of GFP-NtMYB330 fusion protein and GFP control in N. benthamiana leaves transformed by Agrobacterium-infiltration. GFP, GFP fluorescence; DIC, differential interference contrast; MERGE, fluorescence and DIC merged images. Scale bar = 50 μm. (B) Relative expressions of NtMYB330 in different tobacco tissues. Data are the mean of three replicates with error bars indicating ± SD. Asterisks indicate statistically significant differences from Roots according to paired t-test (*P < 0.05; **P < 0.01).

FIGURE 3

Quantitative transcript analyses of NtMYB330 and LBGs in PA pathway in tobacco flowers. (A) Expressions of NtMYB330 in the flowers of different tobacco lines. (B) Alignment of reads with CRISPR/Cas9-induced mutagenesis in NtMYB330. The wild-type sequence was shown at the top, and the two sequences on the bottom were reads of ntmyb330 mutant lines (M1 and M2). The targeted sequence was shown in red, while mutations were shown in blue. gRNA sequences used for genome editing and PAM (protospacer adjacent motif) are indicated. The sizes of the InDels introduced are shown on the left. Analyses of expressions of (C) NtDFR1, (D) NtDFR2, (E) NtANS1, (F) NtANS2, (G) NtLAR1, (H) NtLAR2, (I) NtANR1 and (J) NtANR2 in the flower petals of wild-type (WT), vector control (VC), NtMYB330-OE lines (OE) and ntmyb330 mutants (M). Data are the mean of three replicates with error bars indicating ± SD. Asterisks indicate statistically significant differences from WT according to paired t-test (*P < 0.05; **P < 0.01).

FIGURE 4

NtMYB330 regulates PA biosynthesis in tobacco flowers. (A) Concentrations of catechins and epicatechins in the flower petals of WT, VC, NtMYB330-OE and ntmyb330 mutant plants. Data are the mean of three replicates with error bars indicating ± SD. Asterisks indicate statistically significant differences from WT according to paired t-test (*P < 0.05; **P < 0.01). (B) DMACA staining of PAs accumulated in the flower petals of WT, VC, NtMYB330-OE and ntmyb330 mutant plants. Scale bar = 0.5 cm.

FIGURE 5

NtMYB330 interacts with NtAn1b_MIR and NtAn11-1. (A) Y2H assays reveal the interactions between NtMYB330 and NtAn1b_MIR, and between NtMYB330 and NtAn11-1. The coding sequence of the MYB-interacting region (MIR) of NtAn1b and the full-length coding sequence of NtAn11-1 were cloned into pGBKT7 to make fusion constructs BD-NtAn1b_MIR and BD-An11-1, respectively, and were co-transformed with pAD-NtMYB330. The interactions were indicated by yeast growth and X-α-Gal staining. Yeasts grown on medium with double (SD/-Leu/-Trp), triple (SD/-Leu/-Trp/-His) or quadruple (SD/-Leu/-Trp/-His/-Ade) selection were indicated. (B) BiFC assay shows the localization of NtMYB330–NtAn1b_MIR or NtMYB330-NtAn11-1 complex in epidermal cells of N. benthamiana. The full-length cDNA of NtMYB330 was fused to N-terminal half of the yellow fluorescent protein (YFP), while cDNA of NtAn1b_MIR (aa 1–195) or the full-length cDNA of NtAn11-1 was fused to C-terminal half of YFP. NYFP-NtMYB330 plus YFPC, NYFP plus NtAn1b_MIR-YFPC, and NYFP plus NtAn11-1-YFPC were used as the controls. Yellow signals indicate the positive interactions. Scale bar = 50 μm. Experiment was repeated three times, representative data from one experimental replicate presented here.

FIGURE 6

Transactivation assays of NtMYB330 and NtAn1b in N. benthamiana leaves. The reporter plasmids contain a firefly luciferase gene driven by the promoters of (A) NtDFR1, (B) NtANS1, (C) NtLAR1, or (D) NtANR1. The effector plasmids contain the full-length cDNA of either NtMYB330 or NtAn1b, and were co-transformed, alone or in combination, with the reporter plasmid into the N. benthamiana leaf. A pGreenII 62-SK plasmid was used as the vector control (62SK). Results are presented as fold activation compared to the vector control. Error bars represent the mean ± SD of nine biological replicates. Asterisks indicate statistically significant differences from the vector control (*P < 0.05; **P < 0.01) using one-way ANOVA followed by Bonferroni test.

FIGURE 7

NtMYB330 regulates PA biosynthesis in tobacco seeds. (A) DMACA staining of PAs accumulated in the seed coats of WT, VC, NtMYB330-OE and ntmyb330 mutant plants. Scale bar = 500 μm. Relative expression levels of (B) NtDFR1, (C) NtANS1, (D) NtLAR1 and (E) NtANR1 in the seeds of WT, VC, NtMYB330-OE and ntmyb330 mutant plants. Data are the mean of three replicates with error bars indicating ± SD. Asterisks indicate statistically significant differences from WT according to paired t-test (*P < 0.05; **P < 0.01).

FIGURE 8

Seed germination phenotypes of WT, VC, NtMYB330-OE lines and ntmyb330 mutant plants. (A,C) Seed germination percentages of WT, VC, NtMYB330-OE lines (OE1, OE2) and ntmyb330 mutant plants (M1, M2) in nine consecutive days. Values are means of three independent replicates. Error bars denote standard deviations. Different letters indicate statistically significant differences at P < 0.05 according to Tukey’s HSD test. (B,D) Seedlings of WT, VC, NtMYB330-OE lines (OE1, OE2) and ntmyb330 mutants (M1 and M2) grown on 1/2 MS medium for 3 days. The entire experiment was repeated three times, representative data from one experimental replicate presented here.

FIGURE 9

Quantitative analyses of transcript levels of the genes involved in homeostasis and signal transduction of ABA/GA in tobacco seeds. Relative expression levels of (A) NtABI3 (ABA signaling-related gene), (B) NtNCED1 (gene involved in ABA biosynthesis), (C) NtCYP707A (gene involved in ABA catabolism), (D) NtGID2 (GA signaling-related gene), (E) NtGA20ox2 (gene involved in GA biosynthesis), and (F) NtGA2ox2 (gene involved in GA catabolism) in the seeds of WT, VC, NtMYB330-OE lines and ntmyb330 mutant plants. Data are the mean of three replicates with error bars indicating ± SD. Asterisks indicate statistically significant differences from WT according to paired t-test (*P < 0.05; **P < 0.01).