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.
Highlights
Mediator is a conserved eukaryotic transcriptional coregulatory complex
We 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
Neither rfr1- nor ref4 rfr1-deficient Plants Morphologically Resemble Plants Carrying the ref4-3 Mutation—In contrast to the dominant ref4-3 mutant, which exhibits a severe dwarf phenotype, light-colored seeds, and dark, spatulate leaves, we previously reported that the morphological phenotype of plants containing a T-DNA insertion in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) was indistinguishable from that of wild-type plants [31]
Summary
Mediator is a conserved eukaryotic transcriptional coregulatory complex. Results: Disruption of two putative Mediator subunits leads to hyperaccumulation of phenylpropanoids, metabolites important to both the fitness of plants and their utility to humans. 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. Soluble phenylpropanoids are important in plant ecology, where they act variously to absorb damaging ultraviolet light, defend against pathogens, repel herbivores, or attract pollinators [5] In addition to their essential roles in ensuring plant fitness, phenylpropanoid metabolites exert a substantial influence on the utility of plants to suit human needs. Dominant ref mutants exhibit a reduced epidermal fluorescence phenotype due to a deficiency in the synthesis of sinapoylmalate, a UV-absorbent and fluorescent specialized metabolite accumulated in the epidermis of Arabidopsis leaves, as well as dwarfing, decreased lignin deposition, and a pale seed coat. We present evidence that REF4 and RFR1 are required for phenylpropanoid homeostasis in wild-type plants, acting directly or indirectly to repress the transcription of phenylpropanoid biosynthetic genes
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