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.

Circulating microRNA Signatures in Rodent Models of Pain.

MicroRNAs (miRNAs) remain stable in circulation and have been identified as potential biomarkers for a variety of conditions. We report miRNA changes in blood from multiple rodent models of pain, including spinal nerve ligation and spared nerve injury models of neuropathic pain; a complete Freund's adjuvant (CFA) model of inflammatory pain; and a chemotherapy-induced model of pain using the histone deacetylase inhibitor JNJ-26481585. The effect of celecoxib, a cyclooxygenase-2-selective nonsteroidal anti-inflammatory drug, was investigated in the CFA model as proof of principle for assessing the utility of circulating miRNAs as biomarkers in determining treatment response.

Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits.

Geranyl diphosphate (GPP), the precursor of most monoterpenes, is synthesized in plastids from dimethylallyl diphosphate and isopentenyl diphosphate by GPP synthases (GPPSs). In heterodimeric GPPSs, a non-catalytic small subunit (GPPS-SSU) interacts with a catalytic large subunit, such as geranylgeranyl diphosphate synthase, and determines its product specificity. Here, snapdragon (Antirrhinum majus) GPPS-SSU was over-expressed in tomato fruits under the control of the fruit ripening-specific polygalacturonase promoter to divert the metabolic flux from carotenoid formation towards GPP and monoterpene biosynthesis.

Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.

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.

MicroRNA modulation in complex regional pain syndrome.

Background: Aberrant expression of small noncoding RNAs called microRNAs (miRNAs) is a common feature of several human diseases. The objective of the study was to identify miRNA modulation in patients with complex regional pain syndrome (CRPS) a chronic pain condition resulting from dysfunction in the central and/or peripheral nervous systems. Due to a multitude of inciting pathologies, symptoms and treatment conditions, the CRPS patient population is very heterogeneous.

Analysis of Amisulpride in Human Plasma by SPE and LC with Fluorescence Detection.

A rapid, precise, accurate, and selective high-performance liquid chromatographic method with fluorescence detection has been validated and used for analysis of amisulpride in human plasma after a simple solid-phase extraction procedure. Compounds were separated on a CN column with 0.03 M potassium dihydrogen phosphate (pH 6.

RNAi suppression of Arogenate Dehydratase1 reveals that phenylalanine is synthesized predominantly via the arogenate pathway in petunia petals.

l-Phe, a protein building block and precursor of numerous phenolic compounds, is synthesized from prephenate via an arogenate and/or phenylpyruvate route in which arogenate dehydratase (ADT) or prephenate dehydratase, respectively, plays a key role. Here, we used Petunia hybrida flowers, which are rich in Phe-derived volatiles, to determine the biosynthetic routes involved in Phe formation in planta. Of the three identified petunia ADTs, expression of ADT1 was the highest in petunia petals and positively correlated with endogenous Phe levels throughout flower development.

The small subunit of snapdragon geranyl diphosphate synthase modifies the chain length specificity of tobacco geranylgeranyl diphosphate synthase in planta.

Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein.

The lack of floral synthesis and emission of isoeugenol in Petunia axillaris subsp. parodii is due to a mutation in the isoeugenol synthase gene.

Floral scent has been extensively investigated in plants of the South American genus Petunia. Flowers of Petunia integrifolia emit mostly benzaldehyde, while flowers of Petunia axillaris subsp. axillaris emit a mixture of volatile benzenoid and phenylpropanoid compounds that include isoeugenol and eugenol.

The multiple phenylpropene synthases in both Clarkia breweri and Petunia hybrida represent two distinct protein lineages.

Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, isoeugenol synthase 1 (PhIGS1) that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, eugenol synthase 1 (ObEGS1), that produces eugenol.

Regulation of actomyosin contractility by PI3K in sensory axons.

Phosphatidylinositol 3-kinase (PI3K) activity is known to be required for the extension of embryonic sensory axons. Inhibition of PI3K has also been shown to mediate axon retraction and growth cone collapse in response to semaphorin 3A. However, the effects of inhibiting PI3K on the neuronal cytoskeleton are not well characterized.

Reduction of benzenoid synthesis in petunia flowers reveals multiple pathways to benzoic acid and enhancement in auxin transport.

In plants, benzoic acid (BA) is believed to be synthesized from Phe through shortening of the propyl side chain by two carbons. It is hypothesized that this chain shortening occurs via either a beta-oxidative or non-beta-oxidative pathway. Previous in vivo isotope labeling and metabolic flux analysis of the benzenoid network in petunia (Petunia hybrida) flowers revealed that both pathways yield benzenoid compounds and that benzylbenzoate is an intermediate between L-Phe and BA.

Eugenol and isoeugenol, characteristic aromatic constituents of spices, are biosynthesized via reduction of a coniferyl alcohol ester.

Phenylpropenes such as chavicol, t-anol, eugenol, and isoeugenol are produced by plants as defense compounds against animals and microorganisms and as floral attractants of pollinators. Moreover, humans have used phenylpropenes since antiquity for food preservation and flavoring and as medicinal agents. Previous research suggested that the phenylpropenes are synthesized in plants from substituted phenylpropenols, although the identity of the enzymes and the nature of the reaction mechanism involved in this transformation have remained obscure.

Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation.

We have isolated and characterized Petunia hybrida cv. Mitchell phenylacetaldehyde synthase (PAAS), which catalyzes the formation of phenylacetaldehyde, a constituent of floral scent. PAAS is a cytosolic homotetrameric enzyme that belongs to group II pyridoxal 5'-phosphate-dependent amino-acid decarboxylases and shares extensive amino acid identity (approximately 65%) with plant L-tyrosine/3,4-dihydroxy-L-phenylalanine and L-tryptophan decarboxylases.

The nonmevalonate pathway supports both monoterpene and sesquiterpene formation in snapdragon flowers.

Terpenoids, the largest class of plant secondary metabolites, play essential roles in both plant and human life. In higher plants, the five-carbon building blocks of all terpenoids, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate, are derived from two independent pathways localized in different cellular compartments. The methylerythritol phosphate (MEP or nonmevalonate) pathway, localized in the plastids, is thought to provide IPP and dimethylallyl diphosphate for hemiterpene, monoterpene, and diterpene biosynthesis, whereas the cytosol-localized mevalonate pathway provides C5 units for sesquiterpene biosynthesis.

Understanding in vivo benzenoid metabolism in petunia petal tissue.

In vivo stable isotope labeling and computer-assisted metabolic flux analysis were used to investigate the metabolic pathways in petunia (Petunia hybrida) cv Mitchell leading from Phe to benzenoid compounds, a process that requires the shortening of the side chain by a C(2) unit. Deuterium-labeled Phe ((2)H(5)-Phe) was supplied to excised petunia petals. The intracellular pools of benzenoid/phenylpropanoid-related compounds (intermediates and end products) as well as volatile end products within the floral bouquet were analyzed for pool sizes and labeling kinetics by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry.

Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases.

The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5 compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A.

Transformation of tobacco with a gene for the thermophilic acyl-lipid desaturase enhances the chilling tolerance of plants.

The desC gene for the acyl-lipid Delta9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus was introduced into Nicotiana tabacum under control of the 35S promoter. Expression of the desaturase was confirmed by Western blotting. Lipid analysis revealed that lipid content and the extent of fatty acid unsaturation significantly increased in leaves of transgenic plants.