Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling - PubMed (original) (raw)

Ralf Stracke et al. Plant J. 2007 May.

Abstract

The genes MYB11, MYB12 and MYB111 share significant structural similarity and form subgroup 7 of the Arabidopsis thaliana R2R3-MYB gene family. To determine the regulatory potential of these three transcription factors, we used a combination of genetic, functional genomics and metabolite analysis approaches. MYB11, MYB12 and MYB111 show a high degree of functional similarity and display very similar target gene specificity for several genes of flavonoid biosynthesis, including CHALCONE SYNTHASE, CHALCONE ISOMERASE, FLAVANONE 3-HYDROXYLASE and FLAVONOL SYNTHASE1. Seedlings of the triple mutant myb11 myb12 myb111, which genetically lack a complete subgroup of R2R3-MYB genes, do not form flavonols while the accumulation of anthocyanins is not affected. In developing seedlings, MYB11, MYB12 and MYB111 act in an additive manner due to their differential spatial activity; MYB12 controls flavonol biosynthesis mainly in the root, while MYB111 controls flavonol biosynthesis primarily in cotyledons. We identified and confirmed additional target genes of the R2R3-MYB subgroup 7 factors, including the UDP-glycosyltransferases UGT91A1 and UGT84A1, and we demonstrate that the accumulation of distinct and structurally identified flavonol glycosides in seedlings correlates with the expression domains of the different R2R3-MYB factors. Therefore, we refer to these genes as PFG1-3 for 'PRODUCTION OF FLAVONOL GLYCOSIDES'.

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Figures

Figure 1

Figure 1. Simplified, schematic representation of the biosynthesis of flavonols and anthocyanins in the A. thaliana Col-0 seedling

To make this figure more comprehensive, the results from this paper have been included. Enzymes are indicated by bold, capital letters. Regulatory proteins are given in gray boxes beside their target genes. Abbreviations are as follows: GPP, general phenylpropanoid pathway; PAL, phenylalanine ammonium lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumaroyl-CoA synthase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; F3′H, flavonoid 3′-hydroxylase; DFR, dihydroflavonol 4-reductase; F3GT, flavonol 3-_O_-glycosyltransferase; F7RT, flavonol 7-_O_-rhamnosyltransferase; FGT, flavonol glycosyltransferase; K, kaempferol; Q, quercetin; K 3-_O_-[glc-rha]-7-_O_-rha, kaempferol 3-_O_-[(rhamnosyl) glucoside]-7-_O_-rhamnoside; Q 3-_O_-[glc-rha]-7-_O_-rha, quercetin 3-_O_-[(rhamnosyl) glucoside]-7-_O_-rhamnoside; PFG, PRODUCTION OF FLAVONOL GLYCOSIDES. Note that the action of FLS is required for the biosynthesis of flavonols (light gray box) while DFR is necessary for the formation of anthocyanidins. Adapted from Routaboul et al. (2006). The pathway leading to the formation of isorhamnetin is not illustrated.

Figure 2

Figure 2. Co-transfection analysis of target gene specificities in At7 protoplasts

Results from co-transfection experiments in A. thaliana protoplasts. Promoter fragments of the CHS, CHI, F3H, FLS, F3′H and DFR genes (reporters) were assayed for their responsiveness to the effectors (a) MYB11 (b) MYB111 and (c) MYB12, respectively. The reporter constructs are as described in Mehrtens et al. (2005). The figure shows GUS′ activity resulting from the influence of tested effector proteins on different reporters. The values above or beside the error bars indicate fold induction values. The mean value of luciferase activity was 6094 relative luminescence units (RLU) μg protein−1 sec−1 using MYB11 as effector, 1825 RLU μg protein−1 sec−1 with MYB111 and 6800 RLU μg protein−1 sec−1 with MYB12.

Figure 3

Figure 3. Wild-type and mutant alleles of R2R3-MYB SG7 genes

(a) SALK077068 line with T-DNA insertion in At3g62610/MYB11. The mutant allele contains a premature Stop codon at codon position 79 of the MYB11 open reading frame that encodes for 343 amino acids in the wild type. (b) GABI-Kat291D01 line with a T-DNA insertion in the third exon of At5g49330/MYB111 at amino acid position 204 of 342. (c) _myb12_-1f knock-out line (Mehrtens et al., 2005). In each panel of (a)–(c) the exon/intron structure is depicted, where black boxes indicate coding regions and thin black lines symbolize non-coding regions. Positions of T-DNA insertions are given as vertical black lines; primers used in PCR genotyping are indicated by arrows and named. See Experimental procedures for primer sequences. (d) Results from RT-PCR showing the expression of ACT2, MYB11 and MYB111 in 5-dag A. thaliana wild type and SG7 triple mutant seedlings, indicating that myb11 and myb111 are null alleles. (e) Multiple alignment of SG7 R2R3-MYB proteins. Amino acids identical in all three proteins are marked black, amino acids found in two proteins are marked in gray. The R2 and R3 MYB domains as the SG7 motif are marked according to Stracke et al. (2001).

Figure 4

Figure 4. MYB11, MYB12 and MYB111 are differentially expressed in distinct parts of 5-dag A. thaliana Col-0 wild type seedlings

(a) Arabidopsis thaliana seedlings were grown on vertically oriented MS-agar plates and cut into three parts as indicated by the white lines. These three parts represent the different regions of main flavonol accumulation and are referred to as C (cotyledons), H/TZ (hypocotyl/transition zone) and R (root). The whole, uncut seedling is referred to as S. (b) Quantitative PCR results showing relative expression levels referred to the MYB11 transcript level in whole seedlings (set as one). Complementary DNA concentrations were normalized to the 18S rRNA transcript levels as standard. Expression was determined in triplicate measurements in two independent biological replicates. (c) Representative whole mount GUS stainings of Pro MYB11 :GUS, Pro MYB12 :GUS and Pro MYB111 :GUS transformants.

Figure 5

Figure 5. Confirmation of candidate SG7 R2R3-MYB target genes

(a) Confirmation of microarray results. Results from qPCR showing the relative expression of At5g62210, At2g22590 and At4g15480 in roots of A. thaliana Col-0 wild type and myb11 myb12 myb111 triple mutant 5-dag seedlings. Columns represent the relative expression levels of distinct target genes tested. Values were normalized to 18S rRNA and relative expression levels were referenced to the amount of mRNA in the wild-type sample. The values above the bars indicate the fold increase in the mutant. (b) Co-transfection analysis of activation potential in At7 protoplasts. Results from co-transfection experiments in A. thaliana protoplasts. Promoter fragments of At5g62210, At2g22590, At4g15480 and DFR, CHS (as controls) were tested on their responsiveness to MYB11, MYB12 and MYB111 as effector. The figure shows absolute GUS′ activity resulting from the influence of MYB protein on the different reporters. The mean value of luciferase activity was 2516 RLU μg protein−1 sec−1 for MYB11, 3366 RLU μg protein−1 sec−1 for MYB12 and 2350 RLU μg protein−1 sec−1 for MYB111.

Figure 6

Figure 6. Metabolic analyses of methanolic extracts of mutant seedlings

(a) Typical representative HPLC-PDA chromatograms (monitored by absorbance at 354 nm) obtained from 80% methanolic extracts of 5-dag A. thaliana seedlings of the indicated genotypes. Peaks are numbered in the chromatogram of Col-0 wild-type seedlings, where IS is the abbreviation for the internal standard used for quantification, the digitoflavone luteolin-7-glycoside. Peaks were classified by UV spectral analysis as corresponding to flavonol or sinapate derivatives. Minor peaks with UV spectra that do not fit to the known spectrum of flavonols are indicated by asterisks. (b) Representative result of the same methanolic extracts analyzed in (a) after separation by HPTLC on silica gel-60 plates followed by DPBA staining. Pictures were taken under UV illumination. Color key: green, kaempferol derivative; orange, quercetin derivative; faint blue, sinapate derivative; pink, overlapping quercetin and sinapate derivatives; dark red, chlorophyll. Numbers given at the left correspond to the peak numbers as mentioned in (a). Names of identified structures are indicated in Table 2. (c) The HPTLC analysis of methanolic extracts showing different flavonoid compositions in distinct parts of 5-dag A. thaliana Col-0 wild-type seedlings. Peak numbers are as in (a) and (b). Arabidopsis thaliana seedlings were grown and cut as described in the legend of Figure 4: C, cotyledons; H/TZ, hypocotyl/transition zone; R, root; S, whole uncut seedling. (d) Photometric determination of the anthocyanin content in acidic methanolic extracts of A. thaliana seedlings: _A_530, absorption at 530 nm; _A_657, absorption at 657 nm.

Figure 7

Figure 7. Flavonol staining in wild-type and mutant seedlings

(a) Flavonols in intact, norflurazon-bleached seedlings were stained with DPBA until saturation and imaged by epifluorescence microscopy. Photographs of representative seedlings are shown. (b) Close-ups to show accumulations of flavonol at the apical meristem region and at the tip of cotyledons, indicated by arrows. The greenish color at the apical meristem region in the myb11 myb12 mutant indicates accumulation of kaempferol derivatives, while orange indicates quercetin derivatives in the other double mutants.

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