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. 2018 Jul 4:9:941.
doi: 10.3389/fpls.2018.00941. eCollection 2018.

Isolation and Characterization of the Flavonol Regulator CcMYB12 From the Globe Artichoke [ Cynara cardunculus var. scolymus (L.) Fiori]

Emanuela Blanco  1 , Wilma Sabetta  1 , Donatella Danzi  1 , Donatella Negro  1 , Valentina Passeri  2 , Antonino De Lisi  1 , Francesco Paolocci  2 , Gabriella Sonnante  1
Affiliations

Isolation and Characterization of the Flavonol Regulator CcMYB12 From the Globe Artichoke [ Cynara cardunculus var. scolymus (L.) Fiori] (VSports最新版本)

Emanuela Blanco et al. Front Plant Sci. .

Abstract

Flavonoids are a well-studied group of secondary metabolites, belonging to the phenylpropanoid pathway. Flavonoids are known to exhibit health promoting effects such as antioxidant capacities, anti-cancer and anti-inflammatory activity. Globe artichoke is an important source of bioactive phenolic compounds, including flavonoids. To study the regulation of their biosynthesis, a R2R3-MYB transcription factor, CcMYB12, was isolated from artichoke leaves. Phylogenetic analysis showed that this protein belongs to the MYB subgroup 7 (flavonol-specific MYB), which includes Arabidopsis AtMYB12, grapevine VvMYBF1, and tomato SlMYB12. CcMYB12 transcripts were detected specifically in artichoke immature inflorescence and young leaves and overlapped with the profiles of flavonol biosynthetic genes VSports手机版. Electrophoretic mobility shift assays (EMSAs) revealed that recombinant CcMYB12 protein is able to bind to ACII element, a DNA binding site ubiquitously present in the promoters of genes encoding flavonol biosynthetic enzymes. In transgenic Arabidopsis plants, the overexpression of CcMYB12 activated the expression of endogenous flavonol biosynthesis genes, leading to an increase of flavonol accumulation and a decrease of anthocyanins in leaves. Likewise, in transgenic tobacco petals and leaves, the overexpression of CcMYB12 decreased anthocyanin levels and increased flavonols. .

Keywords: R2R3-MYB; artichoke; flavonoid biosynthesis; flavonol; flower color; healthy compounds; qRT-PCR; transcription factor. V体育安卓版.

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VSports最新版本 - Figures

FIGURE 1
FIGURE 1
Protein sequence alignment of CcMYB12 deduced amino acid sequence with plant R2R3-MYB transcription factors and phylogenetic analysis. (A) Protein sequence alignment of CcMYB12 with representative members of R2R3-MYB-type transcriptional regulators of flavonols, proanthocyanidins and anthocyanins from other plant species. The position of the R2 and R3-type MYB domains is indicated below the alignment by gray bars, the SG7 domain is highlighted by a black box. Key amino acid residues are indicated by asterisks. (B) Phylogenetic analysis of deduced amino acid sequences of R2R3-MYB transcription factors present in higher plants. The Neighbor-Joining phylogenetic tree was generated using MEGA software (Tamura et al., 2013). Numerals next to branch nodes indicate bootstrap values from 10000 replications. The bar indicates an evolutionary distance of 0.1%. Protein accession numbers are as follows: apple MdMYB22 (AAZ20438 [Malus domestica]); Arabidopsis: MYB11 (NP_191820), MYB12 (CAB09172), MYB111 (AAK97396), AtMYB123/TT2, (CAC40021), AtMYB75/PAP1 (AAG42001), AtMYB90/PAP2 (AAG42002), AtMYB33 (NP_850779); grape: MYBF1 (ACT88298), MYBPA1 (CAJ90831.1) and MYBA1 (BAD18977); gentian: GtMYBP3 (BAM71801) and GtMYBP4 (BAF96934); gerbera: GhMYB1 (CAD87007); lotus LjMYB12 (BAF74782 MYB-related protein [Lotus japonicus]); maize: ZmMYBP (P27898) and ZmC1 (AAA33482); rice OsMYB3 (EAY89678 (hypothetical protein [Oryza sativa])); sorghum: Y1 (AAX44239); tomato: SlMYB12 (ACB46530) and LeANT1 (AAQ55181). AtMYB33, a GAMYB-like protein, was used as outgroup.
FIGURE 2
FIGURE 2
CcMYB12 gene expression in different organs of artichoke. Quantitative Real Time PCR analysis was performed in the organs depicted in the photograph: stem (St), receptacle (Rec), internal bracts (InB), intermediate bracts (ImB), external bracts (ExB), adult leaves (AL), young leaves (YL), St is the calibrator. Elongation Factor EF1α was employed as reference gene. Values are means ± SD of three biological replicates. Bars with different letters are statistically different to each other according to one-way ANOVA Tukey test (p < 0.05).
FIGURE 3
FIGURE 3
Analyses of CcMYB12 protein binding to AC elements. EMSA output of the purified recombinant CcMYB12 binding to the AC element ACII (left) or to its mutated counterpart (mACII). The biotin-labeled free probe (ACII∗) without added protein is designed as control. Binding of recombinant CcMYB12 to biotin-labeled probe ACII∗ can be outcompeted by cold competitor ACII. Nucleotide sequences of probes are shown on the top of the figure. The AC or mAC element is underlined.
FIGURE 4
FIGURE 4
Expression of CcMYB12 transgene (A) and of endogenous phenylpropanoid biosynthetic genes (B) in leaves of transgenic Arabidopsis plants. qRT-PCR experiments were conducted on cDNAs from various transgenic Arabidopsis T3 plants grouped in 4 different pools (pool 1, 2, 3, and 4), each constituted by 4 plants of the same transgenic T3 line. Actin was employed as reference gene. (A) qRT-PCR of CcMYB12 in transgenic Arabidopsis plants. Pool 4 as calibrator. (B) Expression levels of PAL, CHI, FLS, CHS, F3H, F3′H, DFR, ANS, AtMYB12 and AtMYB111 in CcMYB12 transgenic Arabidopsis plants. WT as calibrator. Values are means ± SD of three biological replicates. Asterisks indicate a statistical difference (∗p-value < 0.05; ∗∗p-value < 0.01) between the means for WT and tested transgenic samples, according to Student’s t-test.
FIGURE 5
FIGURE 5
HPLC flavonol and anthocyanin quantification in Arabidopsis plants overexpressing CcMYB12 protein. Leaves were analyzed from wild type (WT) plants and from four different Arabidopsis pools (1–4), each constituted by four plants of the same transgenic T3 line. (A) total kaempferols, (B) total quercetins, (C) total anthocyanins. Error bars indicate the SD of the average of quercetin, kaempferol or cyanidin equivalents determined as triplicates in three independent biological replicates. Asterisks ∗ and ∗∗ indicate statistically significant differences (P < 0.05 and P < 0.01 respectively) between the means for WT and tested transgenic samples according to Student’s t-test. DW, dry weight.
FIGURE 6
FIGURE 6
Anthocyanin content and qRT-PCR analysis in Arabidopsis seedlings grown on control and anthocyanin inductive medium. (A) Left panel: 20-days-old Arabidopsis wild type (WT) and CcMYB12 seedlings (1, 2, 3, and 4) grown in MS medium supplemented with 1 or 6% sucrose under long day condition. Right panel: Anthocyanin content in 2-weeks-old WT and CcMYB12 seedlings grown in 1 and 6% sucrose. Values are reported as relative to WT 1% sucrose, set as 1. Data represent mean values (+SD, n = 3). Within each growth condition, asterisks ∗ and ∗∗ indicate statistically significant differences (P < 0.05 and P < 0.01 respectively), between the means for WT and tested transgenic samples according to Student’s t-test. (B–D) qRT-PCR analysis of early (B) and late (C) flavonoid biosynthetic genes, and AtPAP1 and AtMYB12 TFs (D) in seedlings grown in 1 and 6% sucrose. Upper panel in (C) represents late biosynthetic genes with a low expression level in 1% sucrose condition, with the appropriate axis scale. Actin was employed as reference gene. WT grown in 1% sucrose is the calibrator. Values are means ± SD of three biological replicates. Bars with different letters are statistically different to each other according to one-way ANOVA Tukey test (p < 0.05).
FIGURE 7
FIGURE 7
Expression of CcMYB12 transgene and of endogenous phenylpropanoid biosynthetic genes in CcMYB12-expressing tobacco plants. (A) Color of tobacco flowers in transgenic CcMYB12 lines. The pigmentation of corolla limbs of three different transgenic lines expressing CcMYB12 was compared with corolla from WT. (A) line 3, (B) line 21, and (C) line 13. (B) qRT-PCR of exogenous CcMYB12 in transgenic tobacco plants. Line 13 as calibrator. (C,D), Expression levels of the early biosynthetic genes, namely PAL, HQT, CHS, CHI, F3H, F3′H, FLS, and ANS, DFR, UFGT and AN2in transgenic tobacco leaves (C) and flowers (D). WT as calibrator. qRT-PCR experiments were conducted on cDNAs from various transgenic tobacco T3 plants. Three independent preparations of total RNA from each line were assayed in triplicate. Actin was employed as reference gene. Values are means ± SD of three biological replicates. Asterisks indicate a statistical difference (∗p-value < 0.05; ∗∗p-value < 0.01) between the means for WT and tested transgenic samples, according to Student’s t-test.
FIGURE 8
FIGURE 8
HPLC analyses of flavonoids and caffeoyl-quinic acids in leaves and flowers of tobacco plants overexpressing CcMYB12 protein. Leaves (A–D) and flowers (E–H) from WT and T3 transgenic tobacco lines 3, 13, and 21 were tested. Total kaempferols (A,E), total quercetins (B,F), chlorogenic acid (CGA; C), total caffeoyl-quinic acids (CQAs; G), total anthocyanins (D,H). Total anthocyanin content is relative (in percentage) to the mean levels of the WT line. Error bars indicate the SD of the average of kaempferol, quercetin, CGA or cyanidin equivalents, and CGA determined as triplicates in three independent biological replicates. Asterisks indicate a statistical difference (∗p-value < 0.05; ∗∗p-value < 0.01) between the means for WT and for tested transgenic samples, according to Student’s t-test. DW, dry weight.
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