Involvement of the R2R3-MYB transcription factor MYB21 and its homologs in regulating flavonol accumulation in Arabidopsis stamen

Abstract

Commonly found flavonols in plants are synthesized from dihydroflavonols by flavonol synthase (FLS). The genome of Arabidopsis thaliana contains six FLS genes, among which FLS1 encodes a functional enzyme. Previous work has demonstrated that the R2R3-MYB subgroup 7 transcription factors MYB11, MYB12, and MYB111 redundantly regulate flavonol biosynthesis. However, flavonol accumulation in pollen grains was unaffected in the myb11myb12myb111 triple mutant. Here we show that MYB21 and its homologs MYB24 and MYB57, which belong to subgroup 19, promote flavonol biosynthesis through regulation of FLS1 gene expression. We used a combination of genetic and metabolite analysis to identify the role of MYB21 in regulating flavonol biosynthesis through direct binding to the GARE cis-element in the FLS1 promoter. Treatment with kaempferol or overexpression of FLS1 rescued stamen defects in the myb21 mutant. We also observed that excess reactive oxygen species (ROS) accumulated in the myb21 stamen, and that treatment with the ROS inhibitor diphenyleneiodonium chloride partly rescued the reduced fertility of the myb21 mutant. Furthermore, drought increased ROS abundance and impaired fertility in myb21, myb21myb24myb57, and chs, but not in the wild type or myb11myb12myb111, suggesting that pollen-specific flavonol accumulation contributes to drought-induced male fertility by ROS scavenging in Arabidopsis.

Keywords: FLS1 promoter; Arabidopsis; MYB21; ROS scavenging; flavonol; transcription factors.

Fig. 1.

MYB mutations lead to reduced flavonol accumulation and flavonol synthase gene expression. (A) Flavonol glycoside accumulation patterns of the inflorescences of wild-type (Col-0) plants and myb21, myb24, myb57, myb21myb24myb57, myb11myb12myb111, and chs mutants. The flavonol accumulation of both myb11myb12myb111 and chs mutants is included as a reference. Methanolic extracts were separated by HPTLC followed by DPBA staining and UV illumination. (B) Quantification of flavonols in the inflorescences of wild-type plants and myb21, myb24, myb57, myb21myb24myb57, myb11myb12myb111, and chs mutants. The flavonol accumulation of both myb11myb12myb111 and chs mutants is included as a reference. nd, not detected. Error bars indicate the SD of three biological replicates. Different letters above the bars indicate significant differences in flavonol content between the wild type and other genotypes (P<0.05; ANOVA with Fisher’s LSD test). (C) Gene expression of flavonol biosynthesis genes in inflorescences of the wild type and the myb21, myb24, myb57, and myb21myb24myb57 mutants. The related gene expression of both myb11myb12myb111 and chs mutants is included as a reference. Transcripts were analyzed by qRT–PCR and β-TUBULIN2 was used as the internal standard. Error bars indicate the SD of three biological replicates.

Fig. 2.

MYB21 and its homologs mediate flavonol biosynthesis through the regulation of flavonol synthase gene expression. (A) In situ flavonol staining of wild-type (Col-0) and myb21 inflorescences. Flavonols in ethanol-bleached inflorescences were stained with DPBA to saturation and imaged by epifluorescence microscopy. Bars=1 mm (for inflorescences) or 500 μm (for buds, pistils, and stamens). (B) Gene expression of FLS1, MYB21, CHS, CHI, F3H, and F3′H in inflorescences of wild-type (Col-0) and Pro35S:MYB21-FLAG (MYB21OE1–3) plants. Transcripts were analyzed by qRT–PCR. Error bars indicate the SD of three biological replicates. (C) Flavonol glycoside accumulation patterns of the inflorescences of wild-type and Pro35S:MYB21-FLAG (MYB21OE1–3) plants. Methanolic extracts were separated by HPTLC followed by DPBA staining and UV illumination. (D) Quantification of flavonols in the inflorescences of wild-type and Pro35S:MYB21-FLAG (MYB21OE1–3) plants. Error bars indicate the SD of three biological replicates. Different letters above the bars indicate significant differences in flavonol content between the wild type and other genotypes (P<0.05; ANOVA with Fisher’s LSD test).

Fig. 3.

MYB21 directly regulates FLS1 gene expression and final products by increasing promoter activity. (A) Schematic of the structures of effector (35S:MYB21, 35S:MYB24, 35S:MYB57, 35S:MYB12, and 35S:MYB99) and reporter (FLS1Pro:LUC) constructions for the dual-luciferase transient expression assay. The 35S:REN gene (Renilla luciferase) in the pGreenII 0800-LUC vector was used as an internal control. (B) Relative LUC/REN ratio from the transient expression assays. The ratio of firefly luciferase (LUC) to REN represents the activity of the FLS1 promoter in the absence or presence of MYB21 and its homologs. MYB12 was included as a positive control. Error bars indicate the SD of three biological replicates. (C) Expression of MYB21 and FLS1 in ProAlcA:MYB21 inflorescences. The plants were sprayed with 1% ethanol, and the total RNA was isolated for analysis 1 h later. Error bars indicate the SD of three biological replicates. (D) Quantification of flavonols in the inflorescences of ProAlcA:MYB21. The plants were sprayed with 1% ethanol, and the flavonols were isolated after 12 h and 24 h of treatment. Error bars indicate the SD of three biological replicates. *P<0.05 (Student’s t-test). (E) GUS staining of inflorescences of ProFLS1:GUS in the wild-type (Col-0) and the myb21 background. ProFLS1:GUS T2-1/2/3 represents T₂ generations of three individual transgenic lines, whereas ProFLS1:GUS/myb21 plants were produced via crossing a single ProFLS1:GUS line with myb21. The florescence samples were incubated in GUS reaction mixture at 37 °C for 4 h. Bars=2 mm.

Fig. 4.

MYB21 recognizes and binds to the GARE elements of the FLS1 promoter. (A) Schematic diagrams of the FLS1 promoter constructs in yeast one-hybrid assays. Black triangles indicate GARE cis-elements (TTGTTA), and blue and orange triangles indicate the PY-BOX (TTTTTTCC) and TA-BOX (TATCCA), respectively. (B) Yeast one-hybrid assays of the interactions between MYB21 and FLS1 promoter fragments. The empty vectors of pGADT7-Rec were used as a negative control. (C) EMSA indicating that MYB21 binds to the GARE motifs. Recombinant GST-MYB21 was purified from E. coli. The promoter fragment containing the two GARE motifs of the FLS1 promoter was labeled with biotin. A competition assay for the protein–DNA binding was performed using 100× unlabeled wild-type (Competitor) and mutant probes. (D) ChIP enrichment of FLS1 promoter regions bound by MYB21-FLAG. Ten-day-old Pro35S:MYB21-FLAG and wild-type (Col-0) seedlings were used; DNA fragments were analyzed by quantitative PCR, with the Actin8 promoter as a reference. Enrichments in Pro35S:MYB21-FLAG were compared with wild-type seedlings. Error bars indicate the SD of three PCR repeats of four separate samples.

Fig. 5.

Treatment with kaempferol partially rescues stamen filament growth and fertility in the myb21 mutant. (A) Phenotype of wild-type (Col-0) and myb21 flowers with or without kaempferol (K) treatment. Bar=1 mm. (B) Ratio of filament length to pistil length in wild-type and myb21 plants before and after kaempferol treatment. Error bars indicate the SD of three biological replicates. Different letters above the bars indicate significant differences between groups (P<0.05; ANOVA with Fisher’s LSD test). (C) Main shoot bearing siliques of wild-type and myb21 plants with and without kaempferol treatment. (D) Percentage of siliques with seeds in wild-type and myb21 plants with and without kaempferol treatment. Error bars indicate the SD of three biological replicates. Different letters above the bars indicate significant differences between groups (P<0.05; ANOVA with Fisher’s LSD test).

Fig. 6.

Overexpression of Pro35S:FLS1 partially complements the phenotype of the myb21 mutant. (A) Phenotype of wild-type (Col-0), myb21, FLS1OE1myb21, and FLS1OE2myb21 flowers. Bar=1 mm. (B) Ratio of filament length to pistil length in wild-type, myb21, FLS1OE1myb21, and FLS1OE2myb21 plants. Error bars indicate the SD of three biological replicates. Different letters above the bars indicate significant differences between groups (P<0.05; ANOVA with Fisher’s LSD test). (C) Main shoot bearing siliques of wild-type, myb21, FLS1OE1myb21, and FLS1OE2myb21 plants. Bar=2 cm. (D) Percentage of siliques with seeds in wild-type, myb21, FLS1OE1myb21, and FLS1OE2myb21 plants. Error bars indicate the SD of three biological replicates. Different letters above the bars indicate significant differences between groups (P<0.05; ANOVA with Fisher’s LSD test).(E) Quantification of flavonols in the inflorescences of wild-type, myb21, FLS1OE1myb21, and FLS1OE2myb21 plants. Error bars indicate the SD of three biological replicates. Different letters above the bars indicate significant differences between groups (P<0.05; ANOVA with Fisher’s LSD test). (F) Images of pollen grains of wild-type, myb21, FLS1OE1myb21, and FLS1OE2myb21 plants, stained with CM-H₂DCFDA. Bar=50 μm. (G) Quantification of DCF fluorescence in pollen grains of wild-type plants and flavonoid biosynthesis mutants. Error bars indicate the SD of three biological replicates. Different letters above the bars indicate significant differences between groups (P<0.05; ANOVA with Fisher’s LSD test).

Fig. 7.

Treatment with DPI partially rescues stamen filament growth and fertility in the myb21 and myb21myb24myb57 mutants. (A) Main shoot bearing siliques of wild-type (Col-0), myb21, myb21myb24myb57, myb11myb12myb111, and chs plants with and without DPI treatment. (B) Percentage of siliques with seeds in wild-type, myb21, myb21myb24myb57, myb11myb12myb111, and chs plants with and without DPI treatment. Error bars indicate the SD of three biological replicates. *P<0.05 (Student’s t-test).

Fig. 8.

Pollen-specific flavonol accumulation contributes to male fertility by ROS scavenging in Arabidopsis. (A) Images of CM-H₂DCFDA-stained pollen grains of wild-type (Col-0), myb21, myb21myb24myb57, myb11myb12myb111, and chs with and without drought treatment. Bars=50 μm. (B) Quantification of DCF fluorescence in pollen grains of the wild type and the flavonoid biosynthesis mutants. Different letters above the bars indicate significant differences between groups (P<0.05; ANOVA with Fisher’s LSD test). (C) Main shoot bearing siliques of wild-type, myb21, myb21myb24myb57, myb11myb12myb111, and chs plants with and without drought treatment. (D) Percentage of siliques with seeds in wild-type, myb21, myb21myb24myb57, myb11myb12myb111, and chs plants with and without drought treatment. Error bars indicate the SD of three biological replicates. Different letters above the bars indicate significant differences between groups (P<0.05; ANOVA with Fisher’s LSD test).