A MYB Triad Controls Primary and Phenylpropanoid Metabolites for Pollen Coat Patterning

Abstract

The pollen wall is a complex, durable structure essential for plant reproduction. A substantial portion of phenylpropanoids (e.g. flavonols) produced by pollen grain tapetal cells are deposited in the pollen wall. Transcriptional regulation of pollen wall formation has been studied extensively, and a specific regulatory mechanism for Arabidopsis (Arabidopsis thaliana) pollen flavonol biosynthesis has been postulated. Here, metabolome and transcriptome analyses of anthers from mutant and overexpression genotypes revealed that Arabidopsis MYB99, a putative ortholog of the petunia (Petunia hybrida) floral scent regulator ODORANT1 (ODO1), controls the exclusive production of tapetum diglycosylated flavonols and hydroxycinnamic acid amides. We discovered that MYB99 acts in a regulatory triad with MYB21 and MYB24, orthologs of emission of benzenoids I and II, which together with ODO1 coregulate petunia scent biosynthesis genes. Furthermore, promoter-activation assays showed that MYB99 directs precursor supply from the Calvin cycle and oxidative pentose-phosphate pathway in primary metabolism to phenylpropanoid biosynthesis by controlling TRANSKETOLASE2 expression. We provide a model depicting the relationship between the Arabidopsis MYB triad and structural genes from primary and phenylpropanoid metabolism and compare this mechanism with petunia scent control. The discovery of orthologous protein triads producing related secondary metabolites suggests that analogous regulatory modules exist in other plants and act to regulate various branches of the intricate phenylpropanoid pathway.

Figure 1.

Schematic representation of the evolutionary relationship of the 126 Arabidopsis R2R3-MYB family proteins and the petunia ODO1, EOBI, and EOBII proteins. Arabidopsis proteins relevant to this research are marked in blue, while petunia proteins are marked red. Protein sequences were aligned using the MUSCLE algorithm, and then the unconserved regions in N and C termini were removed in an unbiased manner. The tree was inferred using the maximum-likelihood method and 1,000 bootstraps using MEGA 7. The MYB99 subclade, MYB21/MYB24, and EOBI/EOBII are highlighted. Bootstrap values are presented as percentages, and the scale bar indicates 0.2 amino acid substitutions per position.

Figure 2.

Phenotypic characterization of plants with altered MYB99 expression. A, Open wild-type flower after removal of sepals and petals. B, myb99 knockout mutant with shorter stamen filaments. C, Stamen filaments of a MYB99-overexpressing plant with dwarf phenotype are shorter than the gynoecium, disrupting pollen adhesion to the stigma. D, Normal-looking anthers from MYB99-ox1. E, Plants overexpressing MYB99 (right) exhibit stunted growth when compared with wild-type plants (left). F, Sterile MYB99-overexpressing plant with undeveloped siliques (right) compared with a wild-type plant (left) of the same age.

Figure 3.

Malformation of pollen grains derived from myb99 mutants. A, Pollen grains of wild-type plants after Alexander staining. Wild-type pollen was stained red, indicative of viable pollen with an intact exine wall. B, Arrows point to aborted pollen grains isolated from myb99 mutants after Alexander staining. C, SEM of pollen grains adhered to the anther locule derived from wild-type plants displaying normal morphology. D, Pollen grains of myb99-1 mutants that adhered to the anther locule appeared malformed and collapsed. Arrows point to pollen surface malformations.

Figure 4.

myb99 knockout mutation leads to down-regulation in expression of genes in primary and phenylpropanoid metabolic pathways. RT-qPCR of anthers collected from preanthesis flowers of the wild type (WT) and myb99-1 mutants is shown. Down-regulation of TK2, PAL1, PAL4, 4CL2, LAP5, LAP6, CSE, UGT72B1, and UGT73B2 metabolic genes was detected in myb99-1. Significant decrease in expression was also measured for the transcripts of MYB21 and MYB24. Shown are means and se of four biological replicates. Asterisks denote statistically significant differences from the wild type calculated using Student’s t test: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Figure 5.

Production of pollen-specific flavonols by the consecutive reactions of UGT73B2 and UGT79B6. A, The glycosylation of quercetin on the 3-hydroxyl group by the catalytic activity of UGT73B2 was confirmed using a quercetin 3-glucoside standard. B, UGT79B6, as expected, did not show any activity toward quercetin. C, The combined activity of UGT73B2 and UGT79B6 resulted in the production of quercetin 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosides. A.U, Arbitrary units; m/z, mass-to-charge ratio.

Figure 6.

myb21-5, myb24-5, and myb21-24 knockout mutants are down-regulated in the expression of genes from primary and phenylpropanoid metabolic pathways. RT-qPCR of anthers collected from preanthesis flowers is shown. Down-regulation of TK2, PAL4, UGT73B2, and UGT79B6 was detected in the anthers of the mutant lines as well as a significant increase in expression of MYB99 transcripts. Shown are means and se of four biological replicates. Asterisks denote statistically significant differences from the wild type (WT) calculated using Student’s t test: *, P < 0.05 and **, P < 0.01.

Figure 7.

MYB99 activates promoters of structural genes and regulators of the phenylpropanoid pathway. A, Putative promoters of genes from primary and phenylpropanoid metabolic pathways coinfiltrated with a vector containing MYB99 under the regulation of the ubiquitin promoter. B, Coinfiltration of MYB21, MYB24, and MYB99 with their putative promoters. Background promoter activity was assayed by coinfiltration with an empty vector of the same type. Shown are means and se of six biological replicates. Asterisks denote statistically significant differences from the wild type calculated using Student’s t test: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Figure 8.

Scheme depicting the regulation of phenylpropanoid metabolism in Arabidopsis anthers and petunia flowers. Solid arrows represent one enzymatic reaction, and dashed arrows represent multiple reactions. Down-regulated genes in myb99 anthers are marked in red, and gene promoters that were tested positive in activation assays are marked with superscript 1, 2, or 3 for MYB99, MYB21, or MYB24, respectively. Underlined superscripts 1, 2, or 3 represent petunia orthologs that were shown to be regulated by ODO1, EOBI, or EOBII, respectively. IGS, Isoeugenol synthase; SHT, spermidine hydroxycinnamoyl transferase.