Pinoresinol rescues developmental phenotypes of Arabidopsis phenylpropanoid mutants overexpressing FERULATE 5-HYDROXYLASE.

Most phenylpropanoid pathway flux is directed toward the production of monolignols, but this pathway also generates multiple bioactive metabolites. The monolignols coniferyl and sinapyl alcohol polymerize to form guaiacyl (G) and syringyl (S) units in lignin, components that are characteristic of plant secondary cell walls. Lignin negatively impacts the saccharification potential of lignocellulosic biomass. Although manipulation of its content and composition through genetic engineering has reduced biomass recalcitrance, in some cases, these genetic manipulations lead to impaired growth. The reduced-growth phenotype is often attributed to poor water transport due to xylem collapse in low-lignin mutants, but alternative models suggest that it could be caused by the hyper- or hypoaccumulation of phenylpropanoid intermediates. In Arabidopsis thaliana, overexpression of FERULATE 5-HYDROXYLASE (F5H) shifts the normal G/S lignin ratio to nearly pure S lignin and does not result in substantial changes to plant growth. In contrast, when we overexpressed F5H in the low-lignin mutants cinnamyl dehydrogenase c and d (cadc cadd), cinnamoyl-CoA reductase 1, and reduced epidermal fluorescence 3, plant growth was severely compromised. In addition, cadc cadd plants overexpressing F5H exhibited defects in lateral root development. Exogenous coniferyl alcohol (CA) and its dimeric coupling product, pinoresinol, rescue these phenotypes. These data suggest that mutations in the phenylpropanoid pathway limit the biosynthesis of pinoresinol, and this effect is exacerbated by overexpression of F5H, which further draws down cellular pools of its precursor, CA. Overall, these genetic manipulations appear to restrict the synthesis of pinoresinol or a downstream metabolite that is necessary for plant growth.

Genome-wide association identifies a BAHD acyltransferase activity that assembles an ester of glucuronosylglycerol and phenylacetic acid.

Genome-wide association studies (GWAS) are an effective approach to identify new specialized metabolites and the genes involved in their biosynthesis and regulation. In this study, GWAS of Arabidopsis thaliana soluble leaf and stem metabolites identified alleles of an uncharacterized BAHD-family acyltransferase (AT5G57840) associated with natural variation in three structurally related metabolites. These metabolites were esters of glucuronosylglycerol, with one metabolite containing phenylacetic acid as the acyl component of the ester. Knockout and overexpression of AT5G57840 in Arabidopsis and heterologous overexpression in Nicotiana benthamiana and Escherichia coli demonstrated that it is capable of utilizing phenylacetyl-CoA as an acyl donor and glucuronosylglycerol as an acyl acceptor. We, thus, named the protein Glucuronosylglycerol Ester Synthase (GGES). Additionally, phenylacetyl glucuronosylglycerol increased in Arabidopsis CYP79A2 mutants that overproduce phenylacetic acid and was lost in knockout mutants of UDP-sulfoquinovosyl: diacylglycerol sulfoquinovosyl transferase, an enzyme required for glucuronosylglycerol biosynthesis and associated with glycerolipid metabolism under phosphate-starvation stress. GGES is a member of a well-supported clade of BAHD family acyltransferases that arose by duplication and neofunctionalized during the evolution of the Brassicales within a larger clade that includes HCT as well as enzymes that synthesize other plant-specialized metabolites. Together, this work extends our understanding of the catalytic diversity of BAHD acyltransferases and uncovers a pathway that involves contributions from both phenylalanine and lipid metabolism.

UGT76F1 glycosylates an isomer of the C7-necic acid component of pyrrolizidine alkaloids in Arabidopsis thaliana.

Identification of unknown metabolites and their biosynthetic genes is an active research area in plant specialized metabolism. By following a gene-metabolite association from a genome-wide association study of Arabidopsis stem metabolites, we report a previously unknown metabolite, 2-hydroxy-2-(1-hydroxyethyl)pentanoic acid glucoside, and demonstrated that UGT76F1 is responsible for its production in Arabidopsis. The chemical structure of the glucoside was determined by a series of analyses, including tandem MS, acid and base hydrolysis, and NMR spectrometry. T-DNA knockout mutants of UGT76F1 are devoid of the glucoside but accumulate increased levels of the aglycone. 2-hydroxy-2-(1-hydroxyethyl)pentanoic acid is structurally related to the C7-necic acid component of lycopsamine-type pyrrolizidine alkaloids such as trachelantic acid and viridifloric acid. Feeding norvaline greatly enhances the accumulation of 2-hydroxy-2-(1-hydroxyethyl)pentanoic acid glucoside in wild-type but not the UGT76F1 knockout mutant plants, providing evidence for an orthologous C7-necic acid biosynthetic pathway in Arabidopsis despite the apparent lack of pyrrolizidine alkaloids.

Metabolic source isotopic pair labeling and genome-wide association are complementary tools for the identification of metabolite-gene associations in plants.

The optimal extraction of information from untargeted metabolomics analyses is a continuing challenge. Here, we describe an approach that combines stable isotope labeling, liquid chromatography- mass spectrometry (LC-MS), and a computational pipeline to automatically identify metabolites produced from a selected metabolic precursor. We identified the subset of the soluble metabolome generated from phenylalanine (Phe) in Arabidopsis thaliana, which we refer to as the Phe-derived metabolome (FDM) In addition to identifying Phe-derived metabolites present in a single wild-type reference accession, the FDM was established in nine enzymatic and regulatory mutants in the phenylpropanoid pathway. To identify genes associated with variation in Phe-derived metabolites in Arabidopsis, MS features collected by untargeted metabolite profiling of an Arabidopsis diversity panel were retrospectively annotated to the FDM and natural genetic variants responsible for differences in accumulation of FDM features were identified by genome-wide association. Large differences in Phe-derived metabolite accumulation and presence/absence variation of abundant metabolites were observed in the nine mutants as well as between accessions from the diversity panel. Many Phe-derived metabolites that accumulated in mutants also accumulated in non-Col-0 accessions and was associated to genes with known or suspected functions in the phenylpropanoid pathway as well as genes with no known functions. Overall, we show that cataloguing a biochemical pathway's products through isotopic labeling across genetic variants can substantially contribute to the identification of metabolites and genes associated with their biosynthesis.

H-lignin can be deposited independently of CINNAMYL ALCOHOL DEHYDROGENASE C and D in Arabidopsis.

Lignin contributes substantially to the recalcitrance of biomass toward saccharification. To circumvent this problem, researchers have genetically altered lignin, although, in a number of cases, these efforts have resulted in an undesirable yield penalty. Recent findings have shown that by knocking out two subunits (MED5A and MED5B) of the transcriptional regulatory complex Mediator, the stunted growth phenotype of mutants in p-coumaroyl shikimate 3'-hydroxylase, reduced epidermal fluorescence 8-1 (ref8-1), can be alleviated. Furthermore, these plants synthesize a lignin polymer almost entirely derived from p-coumaryl alcohol. Plants deficient in cinnamyl alcohol dehydrogenase (CAD) are notable in that they primarily incorporate coniferaldehyde and sinapaldehyde into their lignin. We tested the hypothesis that by stacking mutations in the genes encoding for the CAD paralogs C and D on an Arabidopsis (Arabidopsis thaliana) med5a/5b ref8-1 genetic background, the biosynthesis of p-coumaryl alcohol would be blocked, making p-coumaraldehyde available for polymerization into a novel kind of lignin. The med5a/5b ref8-1 cadc cadd plants are viable, but lignin analysis demonstrated that they continue to synthesize p-hydroxyphenyl lignin despite being mutated for the CADs typically considered to be required for monolignol biosynthesis. In addition, enzyme activity tests showed that even in the absence of CADC and CADD, there is high CAD activity in stems. We tested the potential involvement of other CADs in p-coumaraldehyde biosynthesis in the quintuple mutant by mutating them using the CRISPR/Cas9 system. Lignin analysis demonstrated that the resulting hextuple mutant plants continue to deposit p-coumaryl alcohol-derived lignin, demonstrating a route for the synthesis of p-hydroxyphenyl lignin in Arabidopsis independent of four CAD isoforms.

Transcript and metabolite network perturbations in lignin biosynthetic mutants of Arabidopsis.

Lignin, one of the most abundant polymers in plants, is derived from the phenylpropanoid pathway, which also gives rise to an array of metabolites that are essential for plant fitness. Genetic engineering of lignification can cause drastic changes in transcription and metabolite accumulation with or without an accompanying development phenotype. To understand the impact of lignin perturbation, we analyzed transcriptome and metabolite data from the rapidly lignifying stem tissue in 13 selected phenylpropanoid mutants and wild-type Arabidopsis (Arabidopsis thaliana). Our dataset contains 20,974 expressed genes, of which over 26% had altered transcript levels in at least one mutant, and 18 targeted metabolites, all of which displayed altered accumulation in at least one mutant. We found that lignin biosynthesis and phenylalanine supply via the shikimate pathway are tightly co-regulated at the transcriptional level. The hierarchical clustering analysis of differentially expressed genes (DEGs) grouped the 13 mutants into 5 subgroups with similar profiles of mis-regulated genes. Functional analysis of the DEGs in these mutants and correlation between gene expression and metabolite accumulation revealed system-wide effects on transcripts involved in multiple biological processes.

Manipulation of Lignin Monomer Composition Combined with the Introduction of Monolignol Conjugate Biosynthesis Leads to Synergistic Changes in Lignin Structure.

The complexity of lignin structure impedes efficient cell wall digestibility. Native lignin is composed of a mixture of three dominant monomers, coupled together through a variety of linkages. Work over the past few decades has demonstrated that lignin composition can be altered through a variety of mutational and transgenic approaches such that the polymer is derived almost entirely from a single monomer. In this study, we investigated changes to lignin structure and digestibility in Arabidopsis thaliana in near-single-monolignol transgenics and mutants and determined whether novel monolignol conjugates, produced by a FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) or a p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT), could be integrated into these novel polymers to further improve saccharification efficiency. Monolignol conjugates, including a new conjugate of interest, p-coumaryl p-coumarate, were successfully integrated into high-H, high-G and high-S lignins in A. thaliana. Regardless of lignin composition, FMT- and PMT-expressing plants produced monolignol ferulates and monolignol p-coumarates, respectively, and incorporated them into their lignin. Through the production and incorporation of monolignol conjugates into near-single-monolignol lignins, we demonstrated that substrate availability, rather than monolignol transferase substrate preference, is the most important determining factor in the production of monolignol conjugates, and lignin composition helps dictate cell wall digestibility.

Spatio-temporal control of phenylpropanoid biosynthesis by inducible complementation of a cinnamate 4-hydroxylase mutant.

Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyzes the second step of the general phenylpropanoid pathway. Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, which carry hypomorphic mutations in C4H, exhibit global alterations in phenylpropanoid biosynthesis and have developmental abnormalities including dwarfing. Here we report the characterization of a conditional Arabidopsis C4H line (ref3-2pOpC4H), in which wild-type C4H is expressed in the ref3-2 background. Expression of C4H in plants with well-developed primary inflorescence stems resulted in restoration of fertility and the production of substantial amounts of lignin, revealing that the developmental window for lignification is remarkably plastic. Following induction of C4H expression in ref3-2pOpC4H, we observed rapid and significant reductions in the levels of numerous metabolites, including several benzoyl and cinnamoyl esters and amino acid conjugates. These atypical conjugates were quickly replaced with their sinapoylated equivalents, suggesting that phenolic esters are subjected to substantial amounts of turnover in wild-type plants. Furthermore, using localized application of dexamethasone to ref3-2pOpC4H, we show that phenylpropanoids are not transported appreciably from their site of synthesis. Finally, we identified a defective Casparian strip diffusion barrier in the ref3-2 mutant root endodermis, which is restored by induction of C4H expression.

Glucosinolate and phenylpropanoid biosynthesis are linked by proteasome-dependent degradation of PAL.

Plants produce several hundreds of thousands of secondary metabolites that are important for adaptation to various environmental conditions. Although different groups of secondary metabolites are synthesized through unique biosynthetic pathways, plants must orchestrate their production simultaneously. Phenylpropanoids and glucosinolates are two classes of secondary metabolites that are synthesized through apparently independent biosynthetic pathways. Genetic evidence has revealed that the accumulation of glucosinolate intermediates limits phenylpropanoid production in a Mediator Subunit 5 (MED5)-dependent manner. To elucidate the molecular mechanism underlying this process, we analyzed the transcriptomes of a suite of Arabidopsis thaliana glucosinolate-deficient mutants using RNAseq and identified misregulated genes that are rescued by the disruption of MED5. The expression of a group of Kelch Domain F-Box genes (KFBs) that function in PAL degradation is affected in glucosinolate biosynthesis mutants and the disruption of these KFBs restores phenylpropanoid deficiency in the mutants. Our study suggests that glucosinolate/phenylpropanoid metabolic crosstalk involves the transcriptional regulation of KFB genes that initiate the degradation of the enzyme phenylalanine ammonia-lyase, which catalyzes the first step of the phenylpropanoid biosynthesis pathway. Nevertheless, KFB mutant plants remain partially sensitive to glucosinolate pathway mutations, suggesting that other mechanisms that link the two pathways also exist.

Mediator Complex Subunits MED2, MED5, MED16, and MED23 Genetically Interact in the Regulation of Phenylpropanoid Biosynthesis.

The phenylpropanoid pathway is a major global carbon sink and is important for plant fitness and the engineering of bioenergy feedstocks. In Arabidopsis thaliana, disruption of two subunits of the transcriptional regulatory Mediator complex, MED5a and MED5b, results in an increase in phenylpropanoid accumulation. By contrast, the semidominant MED5b mutation reduced epidermal fluorescence4-3 (ref4-3) results in dwarfism and constitutively repressed phenylpropanoid accumulation. Here, we report the results of a forward genetic screen for suppressors of ref4-3. We identified 13 independent lines that restore growth and/or phenylpropanoid accumulation in the ref4-3 background. Two of the suppressors restore growth without restoring soluble phenylpropanoid accumulation, indicating that the growth and metabolic phenotypes of the ref4-3 mutant can be genetically disentangled. Whole-genome sequencing revealed that all but one of the suppressors carry mutations in MED5b or other Mediator subunits. RNA-seq analysis showed that the ref4-3 mutation causes widespread changes in gene expression, including the upregulation of negative regulators of the phenylpropanoid pathway, and that the suppressors reverse many of these changes. Together, our data highlight the interdependence of individual Mediator subunits and provide greater insight into the transcriptional regulation of phenylpropanoid biosynthesis by the Mediator complex.

Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant.

Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

Mutation of Mediator subunit CDK8 counteracts the stunted growth and salicylic acid hyperaccumulation phenotypes of an Arabidopsis MED5 mutant.

The Mediator complex functions as a hub for transcriptional regulation. MED5, an Arabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid homeostasis. A semidominant mutation (ref4-3) that causes a single amino acid substitution in MED5b functions as a strong suppressor of the pathway, leading to decreased soluble phenylpropanoid accumulation, reduced lignin content and dwarfism. By contrast, loss of MED5 results in increased concentrations of phenylpropanoids. We used a reverse genetic approach to identify suppressors of ref4-3 and found that ref4-3 requires CDK8, a kinase module subunit of Mediator, to repress plant growth. The genetic interaction between MED5 and CDK8 was further characterized using mRNA-sequencing (RNA-seq) and metabolite analysis. Growth inhibition and suppression of phenylpropanoid metabolism can be genetically separated in ref4-3 by elimination of CDK8 kinase activity; however, the stunted growth of ref4-3 is not dependent on the phosphorylation event introduced by the G383S mutation. In addition, rather than perturbation of lignin biosynthesis, misregulation of DJC66, a gene encoding a DNAJ protein, is involved in the dwarfism of the med5 mutants. Together, our study reveals genetic interactions between Mediator tail and kinase module subunits and enhances our understanding of dwarfing in phenylpropanoid pathway mutants.

Mediator function in plant metabolism revealed by large-scale biology.

Mediator is a multisubunit transcriptional co-regulator that is involved in the regulation of an array of processes including plant metabolism. The pathways regulated by Mediator-dependent processes include those for the synthesis of phenylpropanoids (MED5), cellulose (MED16), lipids (MED15 and CDK8), and the regulation of iron homeostasis (MED16 and MED25). Traditional genetic and biochemical approaches laid the foundation for our understanding of Mediator function, but recent transcriptomic and metabolomic studies have provided deeper insights into how specific subunits cooperate in the regulation of plant metabolism. In this review, we highlight recent developments in the investigation of Mediator and plant metabolism, with particular emphasis on the large-scale biology studies of med mutants.

The genetics of lignin biosynthesis: connecting genotype to phenotype.

The processes underlying lignification, which for many years have been the near-exclusive purview of chemists and biochemists, have more recently been approached using both classical forward genetic screens and targeted reverse genetic approaches such as antisense suppression, RNAi, and characterization of insertional mutants. In this review, we provide an overview of the current understanding of lignin biosynthesis and structure, with emphasis on mutant and transgenic plants that have contributed to this knowledge. We also discuss ongoing work aimed at elucidating the relationship between lignin structure and function in vivo, as well as the phenotypic consequences arising from genetic manipulation of the lignin biosynthetic pathway.

Dynamic modeling of subcellular phenylpropanoid metabolism in Arabidopsis lignifying cells.

Lignin is a polymer that significantly inhibits saccharification of plant feedstocks. Adjusting the composition or reducing the total lignin content have both been demonstrated to result in an increase in sugar yield from biomass. However, because lignin is essential for plant growth, it cannot be manipulated with impunity. Thus, it is important to understand the control of carbon flux towards lignin biosynthesis such that optimal modifications to it can be made precisely. Phenylalanine (Phe) is the common precursor for all lignin subunits and it is commonly accepted that all biosynthetic steps, spanning multiple subcellular compartments, are known, yet an in vivo model of how flux towards lignin is controlled is lacking. To address this deficiency, we formulated and parameterized a kinetic model based on data from feeding Arabidopsis thaliana basal lignifying stems with ring labeled [13C6]-Phe. Several candidate models were compared by an information theoretic approach to select the one that best matched the experimental observations. Here we present a dynamic model of phenylpropanoid metabolism across several subcellular compartments that describes the allocation of carbon towards lignin biosynthesis in wild-type Arabidopsis stems. Flux control coefficients for the enzymes in the pathway starting from arogenate dehydratase through 4-coumarate: CoA ligase were calculated and show that the plastidial cationic amino-acid transporter has the highest impact on flux.

Indole Glucosinolate Biosynthesis Limits Phenylpropanoid Accumulation in Arabidopsis thaliana.

Plants produce an array of metabolites (including lignin monomers and soluble UV-protective metabolites) from phenylalanine through the phenylpropanoid biosynthetic pathway. A subset of plants, including many related to Arabidopsis thaliana, synthesizes glucosinolates, nitrogen- and sulfur-containing secondary metabolites that serve as components of a plant defense system that deters herbivores and pathogens. Here, we report that the Arabidopsis thaliana reduced epidermal fluorescence5 (ref5-1) mutant, identified in a screen for plants with defects in soluble phenylpropanoid accumulation, has a missense mutation in CYP83B1 and displays defects in glucosinolate biosynthesis and in phenylpropanoid accumulation. CYP79B2 and CYP79B3 are responsible for the production of the CYP83B1 substrate indole-3-acetaldoxime (IAOx), and we found that the phenylpropanoid content of cyp79b2 cyp79b3 and ref5-1 cyp79b2 cyp79b3 plants is increased compared with the wild type. These data suggest that levels of IAOx or a subsequent metabolite negatively influence phenylpropanoid accumulation in ref5 and more importantly that this crosstalk is relevant in the wild type. Additional biochemical and genetic evidence indicates that this inhibition impacts the early steps of the phenylpropanoid biosynthetic pathway and restoration of phenylpropanoid accumulation in a ref5-1 med5a/b triple mutant suggests that the function of the Mediator complex is required for the crosstalk.

Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure.

Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content.

Discovery of a novel amino acid racemase through exploration of natural variation in Arabidopsis thaliana.

Plants produce diverse low-molecular-weight compounds via specialized metabolism. Discovery of the pathways underlying production of these metabolites is an important challenge for harnessing the huge chemical diversity and catalytic potential in the plant kingdom for human uses, but this effort is often encumbered by the necessity to initially identify compounds of interest or purify a catalyst involved in their synthesis. As an alternative approach, we have performed untargeted metabolite profiling and genome-wide association analysis on 440 natural accessions of Arabidopsis thaliana. This approach allowed us to establish genetic linkages between metabolites and genes. Investigation of one of the metabolite-gene associations led to the identification of N-malonyl-D-allo-isoleucine, and the discovery of a novel amino acid racemase involved in its biosynthesis. This finding provides, to our knowledge, the first functional characterization of a eukaryotic member of a large and widely conserved phenazine biosynthesis protein PhzF-like protein family. Unlike most of known eukaryotic amino acid racemases, the newly discovered enzyme does not require pyridoxal 5'-phosphate for its activity. This study thus identifies a new d-amino acid racemase gene family and advances our knowledge of plant d-amino acid metabolism that is currently largely unexplored. It also demonstrates that exploitation of natural metabolic variation by integrating metabolomics with genome-wide association is a powerful approach for functional genomics study of specialized metabolism.

Conservation and Divergence of Mediator Structure and Function: Insights from Plants.

The Mediator complex is a large, multisubunit, transcription co-regulator that is conserved across eukaryotes. Studies of the Arabidopsis Mediator complex and its subunits have shown that it functions in nearly every aspect of plant development and fitness. In addition to revealing mechanisms of regulation of plant-specific pathways, studies of plant Mediator complexes have the potential to shed light on the conservation and divergence of Mediator structure and function across Kingdoms and plant lineages. The majority of insights into plant Mediator function have come from Arabidopsis because it is the only plant from which Mediator has been purified and from which an array of Mediator mutants have been isolated by forward and reverse genetics. So far, these studies indicate that, despite low sequence similarity between many orthologous subunits, the overall structure and function of Mediator is well conserved between Kingdoms. Several studies have also expanded our knowledge of Mediator to other plant species, opening avenues of investigation into the role of Mediator in plant adaptation and fitness.

The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in Arabidopsis.

It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. Here, we demonstrate that Arabidopsis thaliana uses for that purpose two separate biosynthetic branches stemming from phenylalanine and tyrosine. Gene network modeling and characterization of T-DNA mutants indicated that acyl-activating enzyme encoded by At4g19010 contributes to the biosynthesis of ubiquinone specifically from phenylalanine. CoA ligase assays verified that At4g19010 prefers para-coumarate, ferulate, and caffeate as substrates. Feeding experiments demonstrated that the at4g19010 knockout cannot use para-coumarate for ubiquinone biosynthesis and that the supply of 4-hydroxybenzoate, the side-chain shortened version of para-coumarate, can bypass this blockage. Furthermore, a trans-cinnamate 4-hydroxylase mutant, which is impaired in the conversion of trans-cinnamate into para-coumarate, displayed similar defects in ubiquinone biosynthesis to that of the at4g19010 knockout. Green fluorescent protein fusion experiments demonstrated that At4g19010 occurs in peroxisomes, resulting in an elaborate biosynthetic architecture where phenylpropanoid intermediates have to be transported from the cytosol to peroxisomes and then to mitochondria where ubiquinone is assembled. Collectively, these results demonstrate that At4g19010 activates the propyl side chain of para-coumarate for its subsequent ß-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch.