Cytosolic geraniol and citronellol biosynthesis require a Nudix hydrolase in rose-scented geranium (Pelargonium graveolens).

Geraniol, citronellol and their esters are high-value acyclic monoterpenes used in food technology, perfumery and cosmetics. A major source of these compounds is the essential oil of rose-scented geraniums of the genus Pelargonium. We provide evidence that their biosynthesis mainly takes place in the cytosol of glandular trichomes via geranyl monophosphate (GP) through the action of a Nudix hydrolase. Protein preparations could convert geranyl diphosphate (GDP) to geraniol in in vitro assays, a process which could be blocked by inorganic phosphatase inhibitors, suggesting a two-step conversion of GDP to geraniol. Pelargonium graveolens chemotypes enriched in either geraniol or (-)-citronellol accumulate GP or citronellyl monophosphate (CP), respectively, the presumed precursors to their monoterpenoid end products. Geranyl monophosphate was highly enriched in isolated glandular trichomes of lines producing high amounts of geraniol. In contrast, (-)-isomenthone-rich lines are depleted in these prenyl monophosphates and monoterpene alcohols and instead feature high levels of GDP, the precursor to plastidic p-menthane biosynthesis. A Nudix hydrolase cDNA from Pelargonium glandular trichomes, dubbed PgNdx1, encoded a cytosolic protein capable of hydrolyzing GDP to GP with a KM of about 750 nm but is only weakly active towards farnesyl diphosphate. In citronellol-rich lines, GDP, GP and CP were detected in nearly equimolar amounts, while citronellyl diphosphate was absent, suggesting that citronellol biosynthesis may proceed by reduction of GP to CP in this species. These findings highlight the cytosol as a compartment that supports monoterpene biosynthesis and expands the roles of Nudix hydrolases in the biosynthesis of plant volatiles.

Characterization of 12-Oxophytodienoic Acid Reductases from Rose-scented Geranium (Pelargonium graveolens).

Pelargonium graveolens L'Hér, also referred to as rose geranium, is a popular herbal plant with typical rosy fragrance largely based on the blend of monoterpenoid constituents. Among them, citronellol, which is biosynthesized from geraniol via double bond reduction, is the most abundant scent compound. In this study, three 12-oxophytodienoic acid reductases (PgOPRl-3) hive been cloned from P. graveolens, as -possible candidates for the double-bond reductase involved in citronellol biosynthesis. The bacterially expressed recombinant PgOPRs did not reduce geraniol to citronellol, but stereoselectively converted citral into (S)-citronellal in the presence of NADPH. Thus, the a,-unsaturated carbonyl moiety in the substrate is essential for the catalytic activity of PgOPRs; as reported for OPRs from other plants and structurally related yeast old yellow enzymes. PgOPRs promiscuously accepted linear and cyclic α,ß- uisaturated carbonyl substrates, including methacrolein, a typical reactive carbonyl compound. The possible biotechnological applications for PgOPRs in plant metabolic'engineering, based on their catalytic properties, are discussed herein.

Carbonate extraction process for the metabolic, isozymic and proteomic profiling of rose-scented geranium (Pelargonium sp.), a hyper-acidic plant.

Rose-scented geranium (Pelargonium sp.) is a valuable monoterpene-yielding plant. It has been well characterised phytochemically through the isolation of >270 secondary metabolites, however, there is hardly any biochemical or metabolic information concerning this plant. Initial attempts to investigate its metabolism failed to produce any enzyme activity in the tissue extracts prepared in routine extraction buffers owing to the intrinsic properties of the tissue matrix. It was recognised that cellular hyper-acidity (cell sap pH approximately 3.0) gave rise to very low protein levels in the extracts, thus prohibiting detection of activities of even primary metabolic enzymes that are usually abundantly present in plants. Tissue extraction in Tris solution without pH adjustment (as used for studies involving citrus and banana) led to little or no improvement. Therefore, a novel approach using sodium carbonate solution as an efficient extraction system for enzymes and proteins from the plant was studied. Functionality of the carbonate extraction has been demonstrated through its effectiveness, a several-fold superior performance, in yielding protein, monitoring primary metabolism and secondary metabolic enzymes, and isozymic and polypeptide profiling. The process may also be helpful in the reliable analysis of other acidic plant tissues.

Purification and characterization of an anionic isoperoxidase from scented-geranium callus.

Secretory anionic isoperoxidase (EC 1.11.1.7), named PA1, was 68-fold purified from scented-geranium (Pelargonium graveolense) callus by using ion exchange chromatography and gel filtration. Isoperoxidase PA1 was a glycoprotein with an isoelectric point (pI) of 4.0. The molecular weight of PA1 was approximately 42.5 and 44 kDa, estimated by SDS-PAGE and Sephadex G-150 gel filtration, respectively. The optimum pH of the enzyme was 5.0 for guaiacol and H2O2, and the Km values for guaiacol and H2O2 were 1.96 and 8.5mM, respectively. Substrate studies in terms of optimum pHs and Km values with various synthetic and naturally occurring phenolic compounds were performed. In comparison with cationic isoperoxidase, PC3, which has been already characterized, anionic isoperoxidase PA1 had much lower Km values for synthetic phenolic compounds and much higher Km values for naturally occurring phenolic compounds than PC3. Moreover, anionic isoperoxidase PA1 could utilize ferulic acid as a substrate very well, while cationic isoperoxidase PC3 could not utilize ferulic acid as a substrate.