Joshua R. Widhalm

Contribution of CoA Ligases to Benzenoid Biosynthesis in Petunia Flowers

Biochemical and genetic characterization of two petunia CoA ligases reveals that subcellular compartmentalization of enzymes determines their involvement in the benzenoid metabolic network. Additional evidence shows that formation of cinnamoyl-CoA in peroxisomes is the committed step in the synthesis of benzoyl-CoA via the β-oxidative pathway. Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway.

MutS HOMOLOG1 Is a Nucleoid Protein That Alters Mitochondrial and Plastid Properties and Plant Response to High Light

This work provides evidence, using genetic perturbation of the MSH1 nuclear gene in five plant species, that MSH1 functions within the mitochondrion and plastid to influence organellar genome behavior and plant growth patterns. Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but measurable demonstration of this intimate interaction is difficult. At the level of cellular metabolism, several biosynthetic pathways involve both mitochondrial- and plastid-localized steps. However, at an environmental response level, it is not clear how the two organelles intersect in programmed cellular responses. Here, we provide evidence, using genetic perturbation of the MutS Homolog1 (MSH1) nuclear gene in five plant species, that MSH1 functions within the mitochondrion and plastid to influence organellar genome behavior and plant growth patterns. The mitochondrial form of the protein participates in DNA recombination surveillance, with disruption of the gene resulting in enhanced mitochondrial genome recombination at numerous repeated sequences. The plastid-localized form of the protein interacts with the plastid genome and influences genome stability and plastid development, with its disruption leading to variegation of the plant. These developmental changes include altered patterns of nuclear gene expression. Consistency of plastid and mitochondrial response across both monocot and dicot species indicate that the dual-functioning nature of MSH1 is well conserved. Variegated tissues show changes in redox status together with enhanced plant survival and reproduction under photooxidative light conditions, evidence that the plastid changes triggered in this study comprise an adaptive response to naturally occurring light stress.

An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase

Phenylalanine is a vital component of proteins in all living organisms, and in plants is a precursor for thousands of additional metabolites. Animals are incapable of synthesizing phenylalanine and must primarily obtain it directly or indirectly from plants. Although plants can synthesize phenylalanine in plastids through arogenate, the contribution of an alternative pathway via phenylpyruvate, as occurs in most microbes, has not been demonstrated. Here we show that plants also utilize a microbial-like phenylpyruvate pathway to produce phenylalanine, and flux through this route is increased when the entry point to the arogenate pathway is limiting. Unexpectedly, we find the plant phenylpyruvate pathway utilizes a cytosolic aminotransferase that links the coordinated catabolism of tyrosine to serve as the amino donor, thus interconnecting the extra-plastidial metabolism of these amino acids. This discovery uncovers another level of complexity in the plant aromatic amino acid regulatory network, unveiling new targets for metabolic engineering.

Completion of the core β-oxidative pathway of benzoic acid biosynthesis in plants

Despite the importance of benzoic acid (BA) as a precursor for a wide array of primary and secondary metabolites, its biosynthesis in plants has not been fully elucidated. BA formation from phenylalanine requires shortening of the C3 side chain by two carbon units, which can occur by a non–β-oxidative route and/or a β-oxidative pathway analogous to the catabolism of fatty acids. Enzymes responsible for the first and last reactions of the core BA β-oxidative pathway (cinnamic acid → cinnamoyl-CoA → 3-hydroxy-3-phenylpropanoyl-CoA → 3-oxo-3-phenylpropanoyl-CoA → BA-CoA) have previously been characterized in petunia, a plant with flowers rich in phenylpropanoid/benzenoid volatile compounds. Using a functional genomics approach, we have identified a petunia gene encoding cinnamoyl-CoA hydratase-dehydrogenase (PhCHD), a bifunctional peroxisomal enzyme responsible for two consecutively occurring unexplored intermediate steps in the core BA β-oxidative pathway. PhCHD spatially, developmentally, and temporally coexpresses with known genes in the BA β-oxidative pathway, and correlates with emission of benzenoid volatiles. Kinetic analysis of recombinant PhCHD revealed it most efficiently converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA, thus forming the substrate for the final step in the pathway. Down-regulation of PhCHD expression in petunia flowers resulted in reduced CHD enzyme activity, as well as decreased formation of BA-CoA, BA and their derived volatiles. Moreover, transgenic lines accumulated the PhCHD substrate cinnamoyl-CoA and the upstream pathway intermediate cinnamic acid. Discovery of PhCHD completes the elucidation of the core BA β-oxidative route in plants, and together with the previously characterized CoA-ligase and thiolase enzymes, provides evidence that the whole pathway occurs in peroxisomes.

A Familiar Ring to It: Biosynthesis of Plant Benzoic Acids

Plant benzoic acids (BAs) are building blocks or important structural elements for numerous primary and specialized metabolites, including plant hormones, cofactors, defense compounds, and attractants for pollinators and seed dispersers. Many natural products derived from plant BAs or containing benzoyl/benzyl moieties are also of medicinal or nutritional value to humans. Biosynthesis of BAs in plants is a network involving parallel and intersecting pathways spread across multiple subcellular compartments. In this review, a current overview on the metabolism of plant BAs is presented with a focus on the recent progress made on isolation and functional characterization of genes encoding biosynthetic enzymes and intracellular transporters. In addition, approaches for deciphering the complex interactions between pathways of the BAs network are discussed.

Rethinking how volatiles are released from plant cells

For plant volatile organic compounds (VOCs) to be emitted, they must cross membrane(s), the aqueous cell wall, and sometimes the cuticle, before moving into the gas phase. It is presumed that VOC movement through each barrier occurs via passive diffusion. However, VOCs, which are primarily nonpolar compounds, will preferentially partition into membranes, making diffusion into aqueous compartments slow. Using Ficks first law, we calculated that to achieve observed VOC emission rates by diffusion alone would necessitate toxic VOC levels in membranes. Here, we propose that biological mechanisms, such as those involved in trafficking other hydrophobic compounds, must contribute to VOC emission. Such parallel biological pathways would lower barrier resistances and, thus, steady-state emission rates could be maintained with significantly reduced intramembrane VOC concentrations.

Phylloquinone (vitamin K 1 ) biosynthesis in plants: two peroxisomal thioesterases of lactobacillales origin hydrolyze 1,4-dihydroxy-2-naphthoyl-coa

It is not known how plants cleave the thioester bond of 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA), a necessary step to form the naphthoquinone ring of phylloquinone (vitamin K(1) ). In fact, only recently has the hydrolysis of DHNA-CoA been demonstrated to be enzyme driven in vivo, and the cognate thioesterase characterized in the cyanobacterium Synechocystis. With a few exceptions in certain prokaryotic (Sorangium and Opitutus) and eukaryotic (Cyanidium, Cyanidioschyzon and Paulinella) organisms, orthologs of DHNA-CoA thioesterase are missing outside of the cyanobacterial lineage. In this study, genomic approaches and functional complementation experiments identified two Arabidopsis genes encoding functional DHNA-CoA thioesterases. The deduced plant proteins display low percentages of identity with cyanobacterial DHNA-CoA thioesterases, and do not even share the same catalytic motif. GFP-fusion experiments demonstrated that the Arabidopsis proteins are targeted to peroxisomes, and subcellular fractionations of Arabidopsis leaves confirmed that DHNA-CoA thioesterase activity occurs in this organelle. In vitro assays with various aromatic and aliphatic acyl-CoA thioester substrates showed that the recombinant Arabidopsis enzymes preferentially hydrolyze DHNA-CoA. Cognate T-DNA knock-down lines display reduced DHNA-CoA thioesterase activity and phylloquinone content, establishing in vivo evidence that the Arabidopsis enzymes are involved in phylloquinone biosynthesis. Extraordinarily, structure-based phylogenies coupled to comparative genomics demonstrate that plant DHNA-CoA thioesterases originate from a horizontal gene transfer with a bacterial species of the Lactobacillales order.

A dedicated thioesterase of the Hotdog-fold family is required for the biosynthesis of the naphthoquinone ring of vitamin K1

Phylloquinone (vitamin K1) is a bipartite molecule that consists of a naphthoquinone ring attached to a phytyl side chain. The coupling of these 2 moieties depends on the hydrolysis of the CoA thioester of 1,4-dihydroxy-2-naphthoate (DHNA), which forms the naphthalenoid backbone. It is not known whether such a hydrolysis is enzymatic or chemical. In this study, comparative genomic analyses identified orthologous genes of unknown function that in most species of cyanobacteria cluster with predicted phylloquinone biosynthetic genes. The encoded approximately 16-kDa proteins display homology with some Hotdog domain-containing CoA thioesterases that are involved in the catabolism of 4-hydroxybenzoyl-CoA and gentisyl-CoA (2,5-dihydroxybenzoyl-CoA) in certain soil-dwelling bacteria. The Synechocystis ortholog, encoded by gene slr0204, was expressed as a recombinant protein and was found to form DHNA as reaction product. Unlike its homologs in the Hotdog domain family, Slr0204 showed strict substrate specificity. The Synechocystis slr0204 knockout was devoid of DHNA-CoA thioesterease activity and accumulated DHNA-CoA. As a result, knockout cells contained 13-fold less phylloquinone than their wild-type counterparts and displayed the typical photosensitivity to high light associated to phylloquinone deficiency in cyanobacteria.

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

This work shows that plants derive the ring of ubiquinone from tyrosine and phenylalanine via two distinct pathways. A trans-cinnamate 4-hydroxylase, a peroxisomal p-coumarate-CoA ligase, and the peroxisomal transporter PXA1 were identified in the phenylalanine branch. 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 β-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch.

A bimodular oxidoreductase mediates the specific reduction of phylloquinone (vitamin K1) in chloroplasts

Plants and certain species of cyanobacteria are the only organisms capable of synthesizing phylloquinone (vitamin K₁ for vertebrates), which they use as an electron carrier during photosynthesis. Recent studies, however, have identified a plastidial pool of non-photoactive phylloquinone that could be involved in additional cellular functions. Here, we characterized an Arabidopsis bimodular enzyme--the At4g35760 gene product--comprising an integral domain homologous to the catalytic subunit of mammalian vitamin K₁ epoxide reductase (VKORC1, EC 1.1.4.1) that is fused to a soluble thioredoxin-like moiety. GFP-fusion experiments in tobacco mesophyll cells established that the plant protein is targeted to plastids, and analyses of transcript and protein levels showed that expression is maximal in leaf tissues. The fused and individual VKORC1 domains were separately expressed in yeast, removing their chloroplast targeting pre-sequence and adding a C-terminal consensus signal for retention in the endoplasmic reticulum. The corresponding microsomal preparations were equally effective at mediating the dithiotreitol-dependent reduction of phylloquinone and menaquinone into their respective quinol forms. Strikingly, unlike mammalian VKORC1, the Arabidopsis enzyme did not reduce phylloquinone epoxide, and was resistant to inhibition by warfarin. The isoprenoid benzoquinone conjugates plastoquinone and ubiquinone were not substrates, establishing that the plant enzyme evolved strict specificity for the quinone form of naphthalenoid conjugates. In vitro reconstitution experiments established that the soluble thioredoxin-like domain can function as an electron donor for its integral VKORC1 partner.