Loss of FERULATE 5-HYDROXYLASE Leads to Mediator-Dependent Inhibition of Soluble Phenylpropanoid Biosynthesis in Arabidopsis

Abstract

Phenylpropanoids are phenylalanine-derived specialized metabolites and include important structural components of plant cell walls, such as lignin and hydroxycinnamic acids, as well as ultraviolet and visible light-absorbing pigments, such as hydroxycinnamate esters (HCEs) and anthocyanins. Previous work has revealed a remarkable degree of plasticity in HCE biosynthesis, such that most Arabidopsis (Arabidopsis thaliana) mutants with blockages in the pathway simply redirect carbon flux to atypical HCEs. In contrast, the ferulic acid hydroxylase1 (fah1) mutant accumulates greatly reduced levels of HCEs, suggesting that phenylpropanoid biosynthesis may be repressed in response to the loss of FERULATE 5-HYDROXYLASE (F5H) activity. Here, we show that in fah1 mutant plants, the activity of HCE biosynthetic enzymes is not limiting for HCE accumulation, nor is phenylpropanoid flux diverted to the synthesis of cell wall components or flavonol glycosides. We further show that anthocyanin accumulation is also repressed in fah1 mutants and that this repression is specific to tissues in which F5H is normally expressed. Finally, we show that repression of both HCE and anthocyanin biosynthesis in fah1 mutants is dependent on the MED5a/5b subunits of the transcriptional coregulatory complex Mediator, which are similarly required for the repression of lignin biosynthesis and the stunted growth of the phenylpropanoid pathway mutant reduced epidermal fluorescence8. Taken together, these observations show that the synthesis of HCEs and anthocyanins is actively repressed in a MEDIATOR-dependent manner in Arabidopsis fah1 mutants and support an emerging model in which MED5a/5b act as central players in the homeostatic repression of phenylpropanoid metabolism.

Figure 1.

Disruption of phenylpropanoid biosynthetic genes results in the accumulation of normally low-abundance HCEs. Shown are quantifications by HPLC of total HCEs (A) and total flavonol glycosides (B) extracted from 3-week-old whole rosettes of wild-type plants and plants with the indicated mutations. Sinapoylmalate in A is represented in dark gray, and the sum of all other hydroxycinnamoyl esters is represented in light gray. Graphs show means ± sd of three biological replicates.

Figure 2.

Loss of F5H does not lead to decreased levels of cell wall-bound HCAs. Shown are quantifications by HPLC of p-coumarate and ferulate released by saponification from whole rosettes (A) and inflorescence stems (B) of wild-type plants and plants with the indicated mutations. Whole rosettes were harvested 3 weeks after planting and inflorescence stems were harvested 10 weeks after planting. Multiple plants were pooled for each biological replicate. Graphs show means ± sd of three biological replicates.

Figure 3.

Overexpression of neither UGT84A1 nor UGT84A3 rescues the HCE deficiency of the fah1 mutant. Shown are steady-state transcript levels of UGT84A1 and UGT84A3 (A), ferulate:UDP-Glc glucosyltransferase activity (B), and hydroxycinnamoylmalate content (C) of 3-week-old whole rosettes of wild-type plants, fah1 plants, and T1 progeny of fah1 plants transformed with either a UGT84A1 or UGT84A3 overexpression construct. Transcript abundance in A is shown relative to that of the control gene At1g13320. Graphs in A and C show means ± sd of three biological replicates of individual plants, whereas those in B show means ± sd of three technical replicates of pooled samples.

Figure 4.

Disruption of MED5a and MED5b substantially alleviates the HCE deficiency of the fah1 mutant. Shown is the quantification by HPLC of total methanol-soluble HCEs extracted from rosette leaves of fah1, med5a/5b, and med5a/5b fah1 mutants 3 weeks after planting. The graph shows means ± sd of three biological replicates.

Figure 5.

Inhibition of anthocyanin biosynthesis in fah1 mutants is dependent on the Mediator subunits MED5a and MED5b. A, Soil-grown wild-type and fah1 plants 3 weeks after planting. B, Representative HPLC chromatograms at 525 nm of seedlings after the induction of anthocyanin biosynthesis with Suc stress. For clarity, chromatograms from wild-type and med5a/5b samples are overlaid at top and those from fah1 and med5a/5b fah1 samples are overlaid at bottom. C, Quantification by HPLC of anthocyanins in Suc-stressed seedlings. Each bar shows the mean of three biological replicates for each genotype, with error bars indicating sd.

Figure 6.

Transcriptional induction of anthocyanin biosynthetic genes in fah1 mutants is indistinguishable from that of wild-type plants. A and B, Transcript levels of the anthocyanin biosynthetic genes PAL1, CHS, CHI, DFR, F3ʹH, LDOX, and UGT79B1 in wild-type seedlings and seedlings with the indicated mutations grown in either 0.5% Suc (A) or 3% Suc (B). C, Transcript levels of the same genes in rosette leaf blades of soil-grown wild-type, fah1, pap1-D, and fah1 pap1-D plants 3 weeks after planting. Transcript abundances shown are relative to those of the control gene At1g13320 for all experiments. Graphs show means ± sd of three biological replicates. CHI, Chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol reductase; F3′H, flavonoid 3′-hydroxylase; LDOX, leucoanthocyanidin dioxygenase; PAL1, phenylalanine ammonia lyase1.

Figure 7.

Overexpression of the anthocyanin-specific transcription factor PAP1 fails to overcome the anthocyanin repression of fah1. A, Soil-grown pap1-D and fah1 pap1-D plants 3 weeks after planting. B, Representative chromatograms of dissected leaf blades (top) and midribs (bottom) of pap1-D and fah1 pap1-D plants. C, Quantification of total anthocyanin levels in the leaf sections shown in B together with wild-type and fah1 controls. Each bar shows the mean of three samples, with error bars indicating sd.

Figure 8.

Anthocyanin accumulation is inhibited in the cortex of fah1 pap1-D inflorescence stems but is unaffected in vascular bundles. A, Representative inflorescence stems of pap1-D and fah1 pap1-D. B, Semithin sections of pap1-D and fah1 pap1-D inflorescence stems taken from the bottom of the second internode as viewed without staining using a light microscope. C, Representative chromatograms showing anthocyanins in pap1-D and fah1 pap1-D inflorescence stems.

Figure 9.

ref8 is epistatic to fah1 with respect to anthocyanin inhibition. Shown is the quantification by HPLC of anthocyanins in whole rosettes of wild-type plants and plants with the indicated mutations 3 weeks after planting. Each bar shows the mean of three biological replicates, with error bars indicating sd.