REF4 and RFR1, subunits of the transcriptional coregulatory complex mediator, are required for phenylpropanoid homeostasis in Arabidopsis

Abstract

The plant phenylpropanoid pathway produces an array of metabolites that impact human health and the utility of feed and fiber crops. We previously characterized several Arabidopsis thaliana mutants with dominant mutations in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) that cause dwarfing and decreased accumulation of phenylpropanoids. In contrast, ref4 null plants are of normal stature and have no apparent defect in phenylpropanoid biosynthesis. Here we show that disruption of both REF4 and its paralog, REF4-RELATED 1 (RFR1), results in enhanced expression of multiple phenylpropanoid biosynthetic genes, as well as increased accumulation of numerous downstream products. We also show that the dominant ref4-3 mutant protein interferes with the ability of the PAP1/MYB75 transcription factor to induce the expression of PAL1 and drive anthocyanin accumulation. Consistent with our experimental results, both REF4 and RFR1 have been shown to physically associate with the conserved transcriptional coregulatory complex, Mediator, which transduces information from cis-acting DNA elements to RNA polymerase II at the core promoter. Taken together, our data provide critical genetic support for a functional role of REF4 and RFR1 in the Mediator complex, and for Mediator in the maintenance of phenylpropanoid homeostasis. Finally, we show that wild-type RFR1 substantially mitigates the phenotype of the dominant ref4-3 mutant, suggesting that REF4 and RFR1 may compete with one another for common binding partners or for occupancy in Mediator. Determining the functions of diverse Mediator subunits is essential to understand eukaryotic gene regulation, and to facilitate rational manipulation of plant metabolic pathways to better suit human needs.

FIGURE 1.

Arabidopsis mutants containing an inactivating insertion in RFR1 or in both REF4 and RFR1 do not phenotypically resemble the previously reported ref4-3 mutant. A, comparison of 3-week-old rosettes from a wild-type plant and plants containing a single inactivating insertion in the REF4 gene (ref4), RFR1 gene (rfr1), or both (ref4 rfr1), together with plants homozygous for the previously described ref4-3 point mutation. B, mature plants and C, seeds of plants with the genotypes described in A.

FIGURE 2.

rfr1 and ref4 rfr1 mutant plants hyperaccumulate phenylpropanoids. A, quantification of sinapoylmalate in leaves of wild-type, ref4 null, rfr1 null, ref4 rfr1 null, and ref4-3 mutant plants. B, quantification of sinapoylmalate in inflorescence stems and C, sinapoylglucose in seeds of the plants described in A. Sinapate esters were quantified by HPLC by comparing their absorbance at 330 nm to the absorbance of a sinapic acid standard of known concentration. Values shown for leaves and inflorescence stems are the mean of six biological replicates, and those shown for seeds are the mean of three technical replicates on each of three biological replicates. Error bars represent S.D. in all cases.

FIGURE 3.

HPLC analysis reveals increased levels of many UV absorbing compounds in ref4 rfr1 plants relative to wild-type plants. HPLC profiles of methanol-soluble, UV absorbing metabolites from leaves of wild-type, ref4 null, rfr1 null, ref4 rfr1 null, and ref4-3 mutant plants.

FIGURE 4.

Early phenylpropanoid genes show increased expression in ref4 rfr1-deficient plants. Steady-state transcript levels of PAL1, PAL2, C4H, and 4CL1 genes relative to the reference gene At1g13220 at the indicated times after subjective dawn. Dark gray bars show transcript measurements from wild-type plants, and light gray bars show measurements from ref4 rfr1-deficient plants. Error bars represent the S.D. of three biological replicates.

FIGURE 5.

The ref4-3 mutant interferes with the ability of MYB75 to drive anthocyanin production and PAL1 expression. A, HPLC chromatograms of anthocyanins in 3-week-old rosette leaves of pap1-D and ref4-3 pap1-D plants grown in parallel. B, quantification of PAL1 transcript levels from 3-week-old rosette leaves of wild-type, pap1-D, ref4-3, and ref4-3 pap1-D plants, relative to the reference gene At1g13320.

FIGURE 6.

Phylogeny and rescue with modified Selaginella REF4. A, Bayesian phylogenetic tree of plant REF4/Med5p homologs. B, sinapoylmalate concentration in ref4 rfr1-deficient plants transformed with the indicated SmREF4 expression constructs.

FIGURE 7.

The presence of wild-type RFR1 alleles substantially mitigates the mutant phenotype resulting from the ref4-3 mutation. Representative plants with the phenotypes observed in the F2 progeny of a cross between ref4 rfr1 null and ref4-3 mutant plants. The genotype of each plant is indicated, and a close-up view of the most severely dwarfed plant on the far right is provided in the inset.