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. 2016 May 10;6:25352.
doi: 10.1038/srep25352.

Characterization of a Citrus R2R3-MYB Transcription Factor That Regulates the Flavonol and Hydroxycinnamic Acid Biosynthesis

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Free PMC article

Characterization of a Citrus R2R3-MYB Transcription Factor That Regulates the Flavonol and Hydroxycinnamic Acid Biosynthesis

Chaoyang Liu et al. Sci Rep. .
Free PMC article

Abstract

Flavonols and hydroxycinnamic acids are important phenylpropanoid metabolites in plants. In this study, we isolated and characterized a citrus R2R3-MYB transcription factor CsMYBF1, encoding a protein belonging to the flavonol-specific MYB subgroup. Ectopic expression of CsMYBF1 in tomato led to an up-regulation of a series of genes involved in primary metabolism and the phenylpropanoid pathway, and induced a strong accumulation of hydroxycinnamic acid compounds but not the flavonols. The RNAi suppression of CsMYBF1 in citrus callus caused a down-regulation of many phenylpropanoid pathway genes and reduced the contents of hydroxycinnamic acids and flavonols. Transactivation assays indicated that CsMYBF1 activated several promoters of phenylpropanoid pathway genes in tomato and citrus. Interestingly, CsMYBF1 could activate the CHS gene promoter in citrus, but not in tomato. Further examinations revealed that the MYBPLANT cis-elements were essential for CsMYBF1 in activating phenylpropanoid pathway genes. In summary, our data indicated that CsMYBF1 possessed the function in controlling the flavonol and hydroxycinnamic acid biosynthesis, and the regulatory differences in the target metabolite accumulation between two species may be due to the differential activation of CHS promoters by CsMYBF1. Therefore, CsMYBF1 constitutes an important gene source for the engineering of specific phenylpropanoid components.

Figures

Figure 1
Figure 1. Schematic representation of shikimate and phenylpropanoid biosynthesis pathways in plants.
Enzyme names are abbreviated as follows: DHAPS, DAHP synthase; DHQS, dehydroquinate synthase; DHD-SDH, 3-dehydroquinate dehydratase/shikimate 5-dehydrogenase; SK, shikimate kinase; EPSPS, EPSP synthase; CS, chorismate synthase; PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxy-cinnamoyl CoA ligase; C3H, ρ-Coumarate 3-hydrolase; COMT, caffeic acid 3-O-methyltransferase; CCR, cinnamoyl CoA reductase; CAD, cinnamyl alcohol dehydrogenase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3-hydroxylase; F3′H, flavonoid-3′-hydroxylase; F3′5′H, flavonoid-3′5′-hydroxylase; FLS, flavonol synthase; FNS, flavone synthase; HCT, cinnamoyl CoA shikimate/quinate transferase; HQT, hydroxycinnamoyl CoA quinate transferase.
Figure 2
Figure 2. Sequence comparison, expression pattern and subcellular localization of CsMYBF1.
(a) Phylogenetic analysis of selected plant MYB proteins. The scale bar represents 0.05 substitutions per site. GenBank accession numbers of the proteins are listed in Supplementary Table S1. (b) Expression pattern of CsMYBF1 in different tissues of citrus. Data are means ± SE of three replicate PCRs. (c) The subcellular localization of CsMYBF1 in citrus protoplast. OsGhd7-CFP and CsMYBF1-GFP were co-transformed into citrus mesophyll protoplasts. OsGhd7-CFP was used as a nuclear marker. (i) OsGhd7-CFP , (ii) CsMYBF1-GFP, (iii) bright field, (iv) merged picture.
Figure 3
Figure 3. Integrated metabolome and transcriptome analysis for the CsMYBF1-overexpressing tomato fruits.
Differences between metabolic profiles of WT/transgenic lines were adopted by PCA of LC-MS (a) and GC-MS data sets (b). (c) Hierarchical cluster analysis of secondary metabolites data in the WT and transgenic tomato fruits. (d) HPLC analysis of methanol extracts from WT and CsMYBF1-overexpressing tomato fruits. S1, 3-Caffeoylquinic acid; S2, 4-Caffeoylquinic acid; S3, Quercetin glucosyl-glucoside rhamnoside; S4, Kaempferol glucosyl-glucoside rhamnoside; S5, Rutin; S6, Phloretin 3′,5′-di-C-glucoside; S7, Kaempferol rutinoside; S8, Dicaffeoylquinic acid; S9, Dicaffeoylquinic acid; S10, Naringenin chalcone-glucoside; S11, Tricaffeoylquinic acid; S12, Naringenin chalcone (e) Hierarchical cluster analysis of primary metabolites data in the WT and transgenic tomato fruits. Data were processed with Z-score transformation and hierarchically clustered using Spearman distance. (f) Analysis of transcript levels of endogenous phenylpropanoid related genes in transgenic tomato fruits. Data are means ± SE of three replicate PCRs. Asterisks (P < 0.05, Student’s t-test) indicate significant differences compared with WT.
Figure 4
Figure 4. Effects of RNAi suppression of CsMYBF1 in citrus callus on the endogenous gene expression and metabolite accumulation.
Expression analyses of CsMYBF1 expression in WT and transgenic lines by quantitative RT-PCR (a) and semi-quantitative RT-PCR (b). (c) Contents of five representative phenylpropanoid compounds in WT and transgenic callus lines determined by LC-MS. Data are means of three replicates and error bars indicating SD. DW, dry weight. Asterisks indicate significant differences as determined by t-test analysis (P < 0.05). (d) Differences between metabolic profiles of WT and RNAi callus lines detected by PCA of LC-MS data sets. (e) Visualized flavonol accumulation in transgenic calli. Representative images for the calli of WT (i), RNAi lines (ii) and negative control (iii) were shown. Bars = 1mm. (f) HPLC analysis of methanol extracts from WT and RNAi callus lines. C1, Kaempferol derivative; C2, Rutin; C3, Kaempferol-rutinside; C4, Quercetin derivative; C5, Kaempferol derivative; C6, Quercetin derivative (g) Analysis of transcript levels of endogenous phenylpropanoid biosynthesis genes in transgenic lines by quantitative RT-PCR. The WT expression data were normalized to 1. Data are means ± SD of two independent biological replicates. Asterisks (P < 0.05, Student’s t-test) indicate significant differences compared with WT.
Figure 5
Figure 5
Effects of CsMYBF1 on promoter activities of phenylpropanoid pathway genes from tomato (a) and citrus (b) in transient expression assays. LUC, Firefly luciferase activity; REN, Renilla luciferase activity. The ratio of LUC/REN of the empty vector (EV) plus promoter was used as a calibrator (set as 1). Error bars indicate SE from six replicates. Asterisks indicate significant differences as determined by t-test analysis (P < 0.05).
Figure 6
Figure 6. CsMYBF1 specifically activates the three selected promoters via the MYBPLANT cis-element.
Diagrams showing various DNA fragments of the three selected promoters inked to the firefly luciferase reporter: (a) ProCs4CL, (b) ProSlFLS and (c) ProCsCHS. The detailed promoter lengths and the position of the cis-elements were shown in Figure S4. (d) Diagrams showing four copies of wild type/mutant MYBPLANT cis-elements linked to the minimal 35S promoter and the firefly luciferase reporter. The corresponding relative ratio of LUC/REN was shown on the right. The ratio of LUC/REN of the empty vector (EV) plus promoter was used as a calibrator (set as 1). Error bars indicate SE from six replicates. Asterisks indicate significant differences as determined by t-test analysis (P < 0.05).
Figure 7
Figure 7. Yeast one-hybrid assays.
(a) Schematic structures of the yeast one hybrid effector (pGADT7-CsMYBF1) and reporter vector. (b) Growth of yeast cells transformed with the effector plasmid and the reporter plasmid on SD-Trp-Leu-His supplemented with or without 50 mM 3-AT. P. Control, positive control (p53HIS2 plus pGAD-p53); N. Control, negative control (p53HIS2 plus pGAD-CsMYBF1)
Figure 8
Figure 8. CsMYBF1 binds to the promoters containing MYBPLANT cis-elements.
(a) Sequences for oligonucleotides used in the EMSA. The red bold letters highlight MYBPLANT sequences, mutated positions are underlined. (b) EMSA of the CsMYBF1 binding to the promoters of Cs4CL, SlFLS and CsCHS. The “+” and “−” indicate the presence and absence of the corresponding probe or protein, respectively. Arrows indicate the positions of protein-DNA probe complex and free probes, respectively.

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