A higher plant FAD synthetase is fused to an inactivated FAD pyrophosphatase.

The riboflavin derivatives FMN and flavin adenine dinucleotide (FAD) are critical cofactors for wide-ranging biological processes across all kingdoms of life. Although it is well established that these flavins can be readily interconverted, in plants, the responsible catalysts and regulatory mechanisms remain poorly understood. Here, we report the cloning and biochemical characterization of an FAD synthetase encoded by the gene At5g03430, which we have designated AtFADS1 (A. thaliana FADS1). The catalytic properties of the FAD synthetase activity are similar to those reported for other FAD synthetases, except that we observed maximum activity with Zn2+ as the associated divalent metal cation. Like human FAD synthetase, AtFADS1 exists as an apparent fusion with an ancestral FAD pyrophosphatase, a feature that is conserved across plants. However, we detected no pyrophosphatase activity with AtFADS1, consistent with an observed loss of a key catalytic residue in higher plant evolutionary history. In contrast, we determined that algal FADS1 retains both FAD synthetase and pyrophosphatase activity. We discuss the implications, including the potential for yet-unstudied biologically relevant noncatalytic functions, and possible evolutionary pressures that have led to the loss of FAD pyrophosphatase activity, yet universal retention of an apparently nonfunctional domain in FADS of land plants.

Revisiting the dual pathway hypothesis of Chorismate production in plants.

The shikimate pathway, the seven enzymatic steps that synthesize chorismate from phosphoenolpyruvate and erythrose 4-phosphate, produces the last common precursor of the three aromatic amino acids. It is firmly established that all seven enzymes are present in plastids, and it is generally accepted that this organelle is likely the sole location for production of chorismate in plants. However, recently a growing body of evidence has provided support for a previous proposal that at least portions of the pathway are duplicated in the cytosol, referred to as the Dual Pathway Hypothesis. Here I revisit this obscure hypothesis by reviewing the findings that provided the original basis for its formulation as well as more recent results that provide fresh support for a possible extra-plastidial shikimate pathway duplication. Similarities between this possible intercompartmental metabolic redundancy and that of terpenoid metabolism are used to discuss potential advantages of pathway duplication, and the translational implications of the Dual Pathway Hypothesis for metabolic engineering are noted.

Adaptive mechanisms of plant specialized metabolism connecting chemistry to function.

As sessile organisms, plants evolved elaborate metabolic systems that produce a plethora of specialized metabolites as a means to survive challenging terrestrial environments. Decades of research have revealed the genetic and biochemical basis for a multitude of plant specialized metabolic pathways. Nevertheless, knowledge is still limited concerning the selective advantages provided by individual and collective specialized metabolites to the reproductive success of diverse host plants. Here we review the biological functions conferred by various classes of plant specialized metabolites in the context of the interaction of plants with their surrounding environment. To achieve optimal multifunctionality of diverse specialized metabolic processes, plants use various adaptive mechanisms at subcellular, cellular, tissue, organ and interspecies levels. Understanding these mechanisms and the evolutionary trajectories underlying their occurrence in nature will ultimately enable efficient bioengineering of desirable metabolic traits in chassis organisms.

Overexpression of arogenate dehydratase reveals an upstream point of metabolic control in phenylalanine biosynthesis.

Out of the three aromatic amino acids, the highest flux in plants is directed towards phenylalanine, which is utilized to synthesize proteins and thousands of phenolic metabolites contributing to plant fitness. Phenylalanine is produced predominantly in plastids via the shikimate pathway and subsequent arogenate pathway, both of which are subject to complex transcriptional and post-transcriptional regulation. Previously, it was shown that allosteric feedback inhibition of arogenate dehydratase (ADT), which catalyzes the final step of the arogenate pathway, restricts flux through phenylalanine biosynthesis. Here, we show that in petunia (Petunia hybrida) flowers, which typically produce high phenylalanine levels, ADT regulation is relaxed, but not eliminated. Moderate expression of a feedback-insensitive ADT increased flux towards phenylalanine, while high overexpression paradoxically reduced phenylalanine formation. This reduction could be partially, but not fully, recovered by bypassing other known metabolic flux control points in the aromatic amino acid network. Using comparative transcriptomics, reverse genetics, and metabolic flux analysis, we discovered that transcriptional regulation of the d-ribulose-5-phosphate 3-epimerase gene in the pentose phosphate pathway controls flux into the shikimate pathway. Taken together, our findings reveal that regulation within and upstream of the shikimate pathway shares control over phenylalanine biosynthesis in the plant cell.

Overcoming Bottlenecks for Metabolic Engineering of Sesquiterpene Production in Tomato Fruits.

Terpenoids are a large and diverse class of plant metabolites that also includes volatile mono- and sesquiterpenes which are involved in biotic interactions of plants. Due to the limited natural availability of these terpenes and the tight regulation of their biosynthesis, there is strong interest to introduce or enhance their production in crop plants by metabolic engineering for agricultural, pharmaceutical and industrial applications. While engineering of monoterpenes has been quite successful, expression of sesquiterpene synthases in engineered plants frequently resulted in production of only minor amounts of sesquiterpenes. To identify bottlenecks for sesquiterpene engineering in plants, we have used two nearly identical terpene synthases, snapdragon (Antirrhinum majus) nerolidol/linalool synthase-1 and -2 (AmNES/LIS-1/-2), that are localized in the cytosol and plastids, respectively. Since these two bifunctional terpene synthases have very similar catalytic properties with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), their expression in target tissues allows indirect determination of the availability of these substrates in both subcellular compartments. Both terpene synthases were expressed under control of the ripening specific PG promoter in tomato fruits, which are characterized by a highly active terpenoid metabolism providing precursors for carotenoid biosynthesis. As AmNES/LIS-2 fruits produced the monoterpene linalool, AmNES/LIS-1 fruits were found to exclusively produce the sesquiterpene nerolidol. While nerolidol emission in AmNES/LIS-1 fruits was 60- to 584-fold lower compared to linalool emission in AmNES/LIS-2 fruits, accumulation of nerolidol-glucosides in AmNES/LIS-1 fruits was 4- to 14-fold lower than that of linalool-glucosides in AmNES/LIS-2 fruits. These results suggest that only a relatively small pool of FPP is available for sesquiterpene formation in the cytosol. To potentially overcome limitations in sesquiterpene production, we transiently co-expressed the key pathway-enzymes hydroxymethylglutaryl-CoA reductase (HMGR) and 1-deoxy-D-xylulose 5-phosphate synthase (DXS), as well as the regulator isopentenyl phosphate kinase (IPK). While HMGR and IPK expression increased metabolic flux toward nerolidol formation 5.7- and 2.9-fold, respectively, DXS expression only resulted in a 2.5-fold increase.

Silent constraints: the hidden challenges faced in plant metabolic engineering.

Metabolic engineering is embraced as a method to sustainably enhance production of valuable phytochemicals with beneficial properties. However, successful production of these compounds in plants is not always predictable even when the pathways are fully known, frequently due to the lack of comprehensive understanding of plant metabolism as a whole, and interconnections between different primary, secondary, and hormone metabolic networks. Here, we highlight critical hidden constraints, including substrate availability, silent metabolism, and metabolic crosstalk, that impair engineering strategies. We explore how these constraints have historically been manifested in engineering attempts and propose how modern advancements will enable future strategies to overcome these impediments.

Cuticle thickness affects dynamics of volatile emission from petunia flowers.

The plant cuticle is the final barrier for volatile organic compounds (VOCs) to cross for release to the atmosphere, yet its role in the emission process is poorly understood. Here, using a combination of reverse-genetic and chemical approaches, we demonstrate that the cuticle imposes substantial resistance to VOC mass transfer, acting as a sink/concentrator for VOCs and hence protecting cells from the potentially toxic internal accumulation of these hydrophobic compounds. Reduction in cuticle thickness has differential effects on individual VOCs depending on their volatility, and leads to their internal cellular redistribution, a shift in mass transfer resistance sources and altered VOC synthesis. These results reveal that the cuticle is not simply a passive diffusion barrier for VOCs to cross, but plays the aforementioned complex roles in the emission process as an integral member of the overall VOC network.

Aromatic Amino Acids: A Complex Network Ripe for Future Exploration.

In plants, high carbon flux is committed to the biosynthesis of phenylalanine, tyrosine, and tryptophan, owing to their roles not only in the production of proteins, but also as precursors to thousands of primary and specialized metabolites. The core plastidial pathways that supply the majority of aromatic amino acids (AAAs) have previously been described in detail. More recently, the discovery of cytosolic enzymes contributing to overall AAA biosynthesis, as well as the identification of intracellular transporters and the continuing elucidation of transcriptional and post-transcriptional regulatory mechanisms, have revealed the complexity of this intercompartmental metabolic network. Here, we review the latest breakthroughs in AAA production and use the newest findings to highlight both longstanding and newly developed questions.

Modulation of auxin formation by the cytosolic phenylalanine biosynthetic pathway.

In plants, phenylalanine biosynthesis occurs via two compartmentally separated pathways. Overexpression of petunia chorismate mutase 2 (PhCM2), which catalyzes the committed step of the cytosolic pathway, increased flux in cytosolic phenylalanine biosynthesis, but paradoxically decreased the overall levels of phenylalanine and phenylalanine-derived volatiles. Concomitantly, the levels of auxins, including indole-3-acetic acid and its precursor indole-3-pyruvic acid, were elevated. Biochemical and genetic analyses revealed the existence of metabolic crosstalk between the cytosolic phenylalanine biosynthesis and tryptophan-dependent auxin biosynthesis mediated by an aminotransferase that uses a cytosolic phenylalanine biosynthetic pathway intermediate, phenylpyruvate, as an amino acceptor for auxin formation.

Retracing the molecular basis and evolutionary history of the loss of benzaldehyde emission in the genus Capsella.

The transition from pollinator-mediated outbreeding to selfing has occurred many times in angiosperms. This is generally accompanied by a reduction in traits attracting pollinators, including reduced emission of floral scent. In Capsella, emission of benzaldehyde as a main component of floral scent has been lost in selfing C. rubella by mutation of cinnamate-CoA ligase CNL1. However, the biochemical basis and evolutionary history of this loss remain unknown, as does the reason for the absence of benzaldehyde emission in the independently derived selfer Capsella orientalis. We used plant transformation, in vitro enzyme assays, population genetics and quantitative genetics to address these questions. CNL1 has been inactivated twice independently by point mutations in C. rubella, causing a loss of enzymatic activity. Both inactive haplotypes are found within and outside of Greece, the centre of origin of C. rubella, indicating that they arose before its geographical spread. By contrast, the loss of benzaldehyde emission in C. orientalis is not due to an inactivating mutation in CNL1. CNL1 represents a hotspot for mutations that eliminate benzaldehyde emission, potentially reflecting the limited pleiotropy and large effect of its inactivation. Nevertheless, even closely related species have followed different evolutionary routes in reducing floral scent.

Natural fumigation as a mechanism for volatile transport between flower organs.

Plants synthesize volatile organic compounds (VOCs) to attract pollinators and beneficial microorganisms, to defend themselves against herbivores and pathogens, and for plant-plant communication. In general, VOCs accumulate in and are emitted from the tissue of their biosynthesis. However, using biochemical and reverse genetic approaches, we demonstrate a new physiological phenomenon: inter-organ aerial transport of VOCs via natural fumigation. Before petunia flowers open, a tube-specific terpene synthase produces sesquiterpenes, which are released inside the buds and then accumulate in the stigma, potentially defending the developing stigma from pathogens. These VOCs also affect reproductive organ development and seed yield, which are previously unknown functions of terpenoid compounds.

Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants.

In addition to being a vital component of proteins, phenylalanine is also a precursor of numerous aromatic primary and secondary metabolites with broad physiological functions. In plants phenylalanine is synthesized predominantly via the arogenate pathway in plastids. Here, we describe the structure, molecular players and subcellular localization of a microbial-like phenylpyruvate pathway for phenylalanine biosynthesis in plants. Using a reverse genetic approach and metabolic flux analysis, we provide evidence that the cytosolic chorismate mutase is responsible for directing carbon flux towards cytosolic phenylalanine production via the phenylpyruvate pathway. We also show that an alternative transcription start site of a known plastidial enzyme produces a functional cytosolic prephenate dehydratase that catalyzes the conversion of prephenate to phenylpyruvate, the intermediate step between chorismate mutase and phenylpyruvate aminotransferase. Thus, our results complete elucidation of phenylalanine biosynthesis via phenylpyruvate in plants, showing that this pathway splits from the known plastidial arogenate pathway at chorismate, instead of prephenate as previously thought, and the complete pathway is localized in the cytosol.

Contribution of isopentenyl phosphate to plant terpenoid metabolism.

Plant genomes encode isopentenyl phosphate kinases (IPKs) that reactivate isopentenyl phosphate (IP) via ATP-dependent phosphorylation, forming the primary metabolite isopentenyl diphosphate (IPP) used generally for isoprenoid/terpenoid biosynthesis. Therefore, the existence of IPKs in plants raises unanswered questions concerning the origin and regulatory roles of IP in plant terpenoid metabolism. Here, we provide genetic and biochemical evidence showing that IP forms during specific dephosphorylation of IPP catalysed by a subset of Nudix superfamily hydrolases. Increasing metabolically available IP by overexpression of a bacterial phosphomevalonate decarboxylase (MPD) in Nicotiana tabacum resulted in significant enhancement in both monoterpene and sesquiterpene production. These results indicate that perturbing IP metabolism results in measurable changes in terpene products derived from both the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways. Moreover, the unpredicted peroxisomal localization of bacterial MPD led us to discover that the step catalysed by phosphomevalonate kinase (PMK) imposes a hidden constraint on flux through the classical MVA pathway. These complementary findings fundamentally alter conventional views of metabolic regulation of terpenoid metabolism in plants and provide new metabolic engineering targets for the production of high-value terpenes in plants.

Characterization of a non-nudix pyrophosphatase points to interplay between flavin and NAD(H) homeostasis in Saccharomyces cerevisiae.

The flavin cofactors FMN and FAD are required for a wide variety of biological processes, however, little is known about their metabolism. Here, we report the cloning and biochemical characterization of the Saccharomyces cerevisiae pyrophosphatase Fpy1p. Genetic and functional studies suggest that Fpy1p may play a key role in flavin metabolism and is the first-reported non-Nudix superfamily enzyme to display FAD pyrophosphatase activity. Characterization of mutant yeast strains found that deletion of fpy1 counteracts the adverse effects that are caused by deletion of flx1, a known mitochondrial FAD transporter. We show that Fpy1p is capable of hydrolyzing FAD, NAD(H), and ADP-ribose. The enzymatic activity of Fpy1p is dependent upon the presence of K+ and divalent metal cations, with similar kinetic parameters to those that have been reported for Nudix FAD pyrophosphatases. In addition, we report that the deletion of fpy1 intensifies the FMN-dependence of null mutants of the riboflavin kinase Fmn1p, demonstrate that fpy1 mutation abolishes the decreased fitness resulting from the deletion of the flx1 ORF, and offer a possible mechanism for the genetic interplay between fpy1, flx1 and fmn1.

A peroxisomal thioesterase plays auxiliary roles in plant ß-oxidative benzoic acid metabolism.

Peroxisomal ß-oxidative degradation of compounds is a common metabolic process in eukaryotes. Reported benzoyl-coenzyme A (BA-CoA) thioesterase activity in peroxisomes from petunia flowers suggests that, like mammals and fungi, plants contain auxiliary enzymes mediating ß-oxidation. Here we report the identification of Petunia hybrida thioesterase 1 (PhTE1), which catalyzes the hydrolysis of aromatic acyl-CoAs to their corresponding acids in peroxisomes. PhTE1 expression is spatially, developmentally and temporally regulated and exhibits a similar pattern to known benzenoid metabolic genes. PhTE1 activity is inhibited by free coenzyme A (CoA), indicating that PhTE1 is regulated by the peroxisomal CoA pool. PhTE1 downregulation in petunia flowers led to accumulation of BA-CoA with increased production of benzylbenzoate and phenylethylbenzoate, two compounds which rely on the presence of BA-CoA precursor in the cytoplasm, suggesting that acyl-CoAs can be exported from peroxisomes. Furthermore, PhTE1 downregulation resulted in increased pools of cytoplasmic phenylpropanoid pathway intermediates, volatile phenylpropenes, lignin and anthocyanins. These results indicate that PhTE1 influences (i) intraperoxisomal acyl-CoA/CoA levels needed to carry out ß-oxidation, (ii) efflux of ß-oxidative products, acyl-CoAs and free acids, from peroxisomes, and (iii) flux distribution within the benzenoid/phenylpropanoid metabolic network. Thus, this demonstrates that plant thioesterases play multiple auxiliary roles in peroxisomal ß-oxidative metabolism.

Multifaceted plant responses to circumvent Phe hyperaccumulation by downregulation of flux through the shikimate pathway and by vacuolar Phe sequestration.

Detrimental effects of hyperaccumulation of the aromatic amino acid phenylalanine (Phe) in animals, known as phenylketonuria, are mitigated by excretion of Phe derivatives; however, how plants endure Phe accumulating conditions in the absence of an excretion system is currently unknown. To achieve Phe hyperaccumulation in a plant system, we simultaneously decreased in petunia flowers expression of all three Phe ammonia lyase (PAL) isoforms that catalyze the non-oxidative deamination of Phe to trans-cinnamic acid, the committed step for the major pathway of Phe metabolism. A total decrease in PAL activity by 81-94% led to an 18-fold expansion of the internal Phe pool. Phe accumulation had multifaceted intercompartmental effects on aromatic amino acid metabolism. It resulted in a decrease in the overall flux through the shikimate pathway, and a redirection of carbon flux toward the shikimate-derived aromatic amino acids tyrosine and tryptophan. Accumulation of Phe did not lead to an increase in flux toward phenylacetaldehyde, for which Phe is a direct precursor. Metabolic flux analysis revealed this to be due to the presence of a distinct metabolically inactive pool of Phe, likely localized in the vacuole. We have identified a vacuolar cationic amino acid transporter (PhCAT2) that contributes to sequestering excess of Phe in the vacuole. In vitro assays confirmed PhCAT2 can transport Phe, and decreased PhCAT2 expression in PAL-RNAi transgenic plants resulted in 1.6-fold increase in phenylacetaldehyde emission. These results demonstrate mechanisms by which plants maintain intercompartmental aromatic amino acid homeostasis, and provide critical insight for future phenylpropanoid metabolic engineering strategies.

Emission of volatile organic compounds from petunia flowers is facilitated by an ABC transporter.

Plants synthesize a diversity of volatile molecules that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. Despite progress in deciphering plant volatile biosynthesis, their release from the cell has been poorly understood. The default assumption has been that volatiles passively diffuse out of cells. By characterization of a Petunia hybrida adenosine triphosphate-binding cassette (ABC) transporter, PhABCG1, we demonstrate that passage of volatiles across the plasma membrane relies on active transport. PhABCG1 down-regulation by RNA interference results in decreased emission of volatiles, which accumulate to toxic levels in the plasma membrane. This study provides direct proof of a biologically mediated mechanism of volatile emission.

Identification of a plastidial phenylalanine exporter that influences flux distribution through the phenylalanine biosynthetic network.

In addition to proteins, L-phenylalanine is a versatile precursor for thousands of plant metabolites. Production of phenylalanine-derived compounds is a complex multi-compartmental process using phenylalanine synthesized predominantly in plastids as precursor. The transporter(s) exporting phenylalanine from plastids, however, remains unknown. Here, a gene encoding a Petunia hybrida plastidial cationic amino-acid transporter (PhpCAT) functioning in plastidial phenylalanine export is identified based on homology to an Escherichia coli phenylalanine transporter and co-expression with phenylalanine metabolic genes. Radiolabel transport assays show that PhpCAT exports all three aromatic amino acids. PhpCAT downregulation and overexpression result in decreased and increased levels, respectively, of phenylalanine-derived volatiles, as well as phenylalanine, tyrosine and their biosynthetic intermediates. Metabolic flux analysis reveals that flux through the plastidial phenylalanine biosynthetic pathway is reduced in PhpCAT RNAi lines, suggesting that the rate of phenylalanine export from plastids contributes to regulating flux through the aromatic amino-acid network.