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. 2011 Apr;23(4):1512-22.
doi: 10.1105/tpc.111.084525. Epub 2011 Apr 12.

Negative Regulation of Anthocyanin Biosynthesis in Arabidopsis by a miR156-targeted SPL Transcription Factor

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Negative Regulation of Anthocyanin Biosynthesis in Arabidopsis by a miR156-targeted SPL Transcription Factor

Jin-Ying Gou et al. Plant Cell. .
Free PMC article

Abstract

Flavonoids are synthesized through an important metabolic pathway that leads to the production of diverse secondary metabolites, including anthocyanins, flavonols, flavones, and proanthocyanidins. Anthocyanins and flavonols are derived from Phe and share common precursors, dihydroflavonols, which are substrates for both flavonol synthase and dihydroflavonol 4-reductase. In the stems of Arabidopsis thaliana, anthocyanins accumulate in an acropetal manner, with the highest level at the junction between rosette and stem. We show here that this accumulation pattern is under the regulation of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are deeply conserved and known to have important roles in regulating phase change and flowering. Increased miR156 activity promotes accumulation of anthocyanins, whereas reduced miR156 activity results in high levels of flavonols. We further provide evidence that at least one of the miR156 targets, SPL9, negatively regulates anthocyanin accumulation by directly preventing expression of anthocyanin biosynthetic genes through destabilization of a MYB-bHLH-WD40 transcriptional activation complex. Our results reveal a direct link between the transition to flowering and secondary metabolism and provide a potential target for manipulation of anthocyanin and flavonol content in plants.

Figures

Figure 1.
Figure 1.
Anthocyanin Accumulation. (A) Schematic diagram of flavonoid biosynthetic pathways. Anthocyanin biosynthetic genes are labeled in red. 4CL, 4-coumarate:CoA ligase; AAT, anthocyanin acyltransferase; C4H, cinnamate-4-hydroxylase; OMT, O-methyltransferase; PAL, phenylalanine ammonia lyase; UGT, UDP-dependent glycosyltransferase. (B) and (C) Inflorescences of wild-type (Wt, ecotype Col-0) and Pro35S:MIR156 plants. Arrow indicates the purple pigment in the stem of Pro35S:MIR156 plants. Bar = 1 cm. (D) and (E) Stem-rosette junction (arrowheads). Compared with the wild type, Pro35S:MIM156 plants accumulated less purple pigment (arrowheads). Bar = 1 cm. (F) Quantification of anthocyanin in stems. Errors bars indicate ±sd (n = 3). *Student’s test, P < 0.01.
Figure 2.
Figure 2.
LC-MS Analyses of Flavonol Level. (A) to (C) LC-MS profiles of soluble phenolic compounds from the stems of wild-type (A), Pro35S:MIR156 (B), and Pro35S:MIM156 plants (C). Insets show the mass spectrum and structures of the peaks 1 and 2, which correspond to kaempferol 3-O-rhamnopyranoside 7-O-rhamnopyranoside (K-3-R-7-R; shown in [A]) and K-7-R (shown in [C]), respectively. mAU, milliabsorbance unit. (D) Quantification of flavonols in the stems of wild-type, Pro35S:MIR156, and Pro35S:MIM156 plants. Data represent means of three trials. Errors bars indicate ± sd (n = 3). *Student’s test, P < 0.01. Wt, wild type.
Figure 3.
Figure 3.
Expression of Flavonoid Biosynthetic and Regulatory Genes. (A) Expression of flavonoid biosynthetic genes. Anthocyanin biosynthetic genes are labeled in red. Expression level in the wild type (Wt) was set to 1. Errors bars indicate ± se (n = 3). Two biological replicates were analyzed, with similar results. (B) Expression of flavonoid regulatory genes. Two-centimeter-long stems above the rosette were harvested and subjected to qRT-PCR analyses. Errors bars indicate ± se (n = 3). Two biological replicates were analyzed, with similar results.
Figure 4.
Figure 4.
Spatial Expression of SPLs and DFR. (A) Transcript levels of SPLs and DFR. The apical, middle, and basal parts of stems were harvested and subjected to qRT-PCR analyses. Expression level in the basal part of stems was set to 1. Errors bars indicate ± se (n = 3). Two biological replicates were analyzed, with similar results. (B) GUS reporter for DFR promoter activity. Twenty-day-old plants expressing GUS under the control of the DFR promoter were stained for 1 h. Bar = 1 cm. Wt, wild type.
Figure 5.
Figure 5.
DFR Is a Direct Target of SPL9. (A) Seven-day-old, long day–grown DEX- (+) or mock-treated (−) seedlings. The accumulation of anthocyanin was greatly reduced in DEX-treated ProSPL9:rSPL9-GR plants (arrows). Wt, wild type. (B) Induced expression of DFR and FLS1 in ProSPL9:rSPL9-GR plants. Seven-day-old, long day–grown seedlings were treated with either DEX and CHX (DC) or CHX alone (C). Seedlings were harvested 6 h after treatment. Expression was normalized relative to that of β-TUBULIN-2. Errors bars indicate ± se (n = 3). Two biological replicates were analyzed, with similar results. (C) Diagram of DFR genomic region. Solid lines, black boxes, and arrow indicate promoter/intron, exons, and transcription start site, respectively. Three regions were chosen for qRT-PCR analyses. Black triangles stand for GTAC boxes. (D) ChIP analyses of 1-week-old wild-type and ProSPL9:GFP-rSPL9 seedlings. Crude chromatin extracts were immunopercipated with either anti-Myc or anti-GFP antibody. Purified ChIP and input DNAs were used for qRT-PCR analyses. Relative enrichment of each fragment was calculated by comparing the samples treated with anti-GFP or anti-Myc antibodies (2−(Ct@Myc–Ct@GFP)). Errors bars indicate ± se (n = 3). Two biological replicates were analyzed, with similar results. (E) GUS staining of ProDFR:GUS and ProDFR:GUS mu (GTAC boxes mutated) plants. Tissue of 20-d-old plants was stained for 1 h. Bar = 0.5 cm.
Figure 6.
Figure 6.
SPL9 Binds to PAP1. (A) SPL9 and TT8 binding to PAP1 in yeast. TT8 and SPL9 were in-frame fused to the GAL4 binding domain (BD) in pGBKT7, whereas PAP1 was fused to the GAL4 activation domain (AD) in pGADT7. Transformed yeast cells were grown on SD –Leu Trp (–LT) (top). The direct interactions between TT8-BD, SPL9-BD, and PAP1-AD were assayed on a SD –Leu Trp His (–LTH) plate, supplemented with 15 mM 3-amino-1,2,4,-triazole (3-AT) (bottom). The pGADT7 (AD) and pGBKT7 (BD) were used as controls. (B) Role of R2R3 domain of PAP1 in mediating binding of PAP1 to SPL9 in yeast. The R2R3 domain of PAP1 was fused to AD in pGADT7. (C) BiFC assay. The leaves of N. benthamiana were infiltrated with agrobacteria as indicated. Blue luminescence indicates a direct protein–protein interaction between rSPL9 and PAP1. Constructs were combined at a 1:1 ratio. (D) CoIP analysis. Proteins were transiently expressed in N. benthamiana. Protein was immunoprecipitated with anti-FLAG antibody, and the IP fraction was analyzed in a protein blot with anti-HA antibody. Input fraction was analyzed by immunoblotting using either anti-FLAG or anti-HA antibody. (E) Competition of SPL9 and TT8 for PAP1 binding. Constructs were combined at a 1:1:4 ratio for PAP1-LUC-N: LUC-C-TT8: LUC-N, LUC-C, or rSPL9. Blue luminescence indicates a direct interaction between TT8 and PAP1.
Figure 7.
Figure 7.
A Model for Negative Regulation of Anthocyanin Biosynthesis by miR156-Targeted SPLs. Anthocyanins accumulate in an acropetal manner, with the highest level at the junction between rosette and stem. This pattern is regulated by miR156-targeted SPL genes. High levels of SPLs in the inflorescences repress anthocyanin accumulation by directly preventing expression of anthocyanin biosynthetic genes, such as ANS, F3′H, DFR, and UGT75C1, through destabilization of the MYB-bHLH-WD40 transcriptional activation complex.

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