Induction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis)

(+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O2-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl2, camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn2+-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases.     

Relationship of Camphor Biosynthesis to Leaf Development in Sage (Salvia officinalis)

The camphor content of sage (Salvia officinalis L.) leaves increases as the leaves expand, and the increase is roughly proportional to the number of filled peltate oil glands which appear on the leaf surface during the expansion process. ¹⁴CO₂ is more rapidly incorporated into camphor and its direct progenitors in expanding leaves than in mature leaves, and direct in vitro measurement of the key enzymes involved in the conversion of geranyl pyrophosphate to camphor indicates that these enzymes, including the probable rate-limiting cyclization step, are at the highest levels during the period of maximum leaf expansion. These results clearly demonstrate that immature sage leaves synthesize and accumulate camphor most rapidly.     

An improved procedure for the isolation of 3-phosphoglycerate kinase from yeast

1. (+)-gamma-Carboxymethyl-gamma-methyl-Delta(alpha)-butenolide was isolated from resting-cell cultures of Pseudomonas desmolyticum incubated in the presence of 5-methylsalicylic acid. 2. The structure of this metabolite was deduced from physical and chemical evidence. 3. The isolated compound must be formed in an enzymic reaction since it shows optical activity. 4. The degradative pathway of 4-methylcatechol by Ps. desmolyticum is discussed.     

Biosynthesis of monoterpenes: demonstration of a geranyl pyrophosphate:(-)-bornyl pyrophosphate cyclase in soluble enzyme preparations from tansy (Tanacetum vulgare)

Tansy (Tanacetum vulgare L.) produces an essential oil containing the optically pure monoterpene ketone, (-)-camphor, as a major constituent. A soluble enzyme preparation from immature leaves of this plant converts the acyclic precursor [1-3H]geranyl pyrophosphate to the bicyclic monoterpene alcohol borneol in the presence of MgCl2, and oxidizes a portion of the borneol to camphor in the presence of a pyridine nucleotide. The identity of the major biosynthetic product as borneol was confirmed by chemical oxidation to camphor and crystallization of the derived oxime to constant specific radioactivity. The stereochemistry of the borneol was verified as the (-)-(1S,4S) isomer by oxidation to camphor, conversion to the corresponding ketal with D-(-)-2,3-butanediol, and separation of diastereoisomers by radio-gas-liquid chromatography. When enzyme reaction mixtures were treated with a mixture of acid phosphatase and apyrase, following an initial ether extraction of labeled borneol, additional quantities of borneol were generated, indicating the presence of a phosphorylated derivative of borneol. This water-soluble metabolite was prepared by large-scale enzyme incubations with [1-3H]geranyl pyrophosphate (plus phosphatase inhibitor), and the identity of the initial cyclization product was established as (-)-bornyl pyrophosphate by direct ion-exchange chromatographic analysis and enzymatic hydrolysis. The pathway for the formation of (-)-(1S,4S)-camphor was therefore identical to that previously demonstrated for the (+)-(1R,4R) isomer, involving cyclization of geranyl pyrophosphate to bornyl pyrophosphate, hydrolysis of this intermediate to borneol, and oxidation of the alcohol to the ketone. The labeling pattern of the product derived from [1-3H2, U-14C]geranyl pyrophosphate was determined by oxidation of the biosynthetic borneol to camphor and selective removal of tritium by exchange of the alpha hydrogens at C3 of the ketone. This labeling pattern was identical to that observed previously for the (+) isomer, suggesting the same mechanism of cyclization, but of opposite enantiospecificity. Some properties of the antipodal (+)- and (-)-bornyl pyrophosphate cyclases were compared.     

Metabolism of Monoterpenes : Metabolic Fate of (+)-Camphor in Sage (Salvia officinalis)

The bicyclic monoterpene ketone (+)-camphor undergoes lactonization to 1,2-campholide in mature sage (Salvia officinalis L.) leaves followed by conversion to the β-d-glucoside-6-O-glucose ester of the corresponding hydroxy acid (1-carboxymethyl-3-hydroxy-2,2,3-trimethyl cyclopentane). Analysis of the disposition of (+)-[G-³H]camphor applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis of the bis-glucose derivative and its transport from leaf to root to be determined, and gave strong indication that the transport derivative was subsequently metabolized in the root. Root extracts were shown to possess β-glucosidase and acyl glucose esterase activities, and studies with (+)-1,2[U-¹⁴C]campholide as substrate, using excised root segments, revealed that the terpenoid was converted to lipid materials. Localization studies confirmed the radiolabeled lipids to reside in the membranous fractions of root extracts, and analysis of this material indicated the presence of labeled phytosterols and labeled fatty acids (C₁₄ to C₂₀) of acyl lipids. Although it was not possible to detail the metabolic steps between 1,2-campholide and the acyl lipids and phytosterols derived therefrom because of the lack of readily detectable intermediates, it seemed likely that the monoterpene lactone was degraded to acetyl CoA which was reincorporated into root membrane components via standard acyl lipid and isoprenoid biosynthetic pathways. Monoterpene catabolism thus appears to represent a salvage mechanism for recycling mobile carbon from senescing oil glands on the leaves to the roots.     

Metabolism of monoterpenes: demonstration that (+)-cis-isopulegone, not piperitenone, is the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone in peppermint (Mentha piperita)

Piperitenone is commonly considered to be the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone in peppermint; however, [3H]piperitenone gave rise only to the inert metabolite (+)-piperitone when incubated with peppermint leaf discs. Under identical conditions, (-)-[3H]isopiperitenone was efficiently incorporated into (+)-pulegone, (-)-menthone, and (+)-isomenthone in leaf discs, and yielded an additional metabolite identified as (+)-cis-isopulegone; piperitenone was poorly labeled. Moreover, (+)-cis-[3H]isopulegone was rapidly converted to (+)-pulegone, (-)-menthone, and (+)-isomenthone in leaf discs, and the reduction of (+)-[3H]pulegone to (-)-menthone and (+)-isomenthone was similarly documented. Each step of the pathway was demonstrated in a crude soluble preparation from peppermint leaf epidermis and each of the relevant enzymes was partially purified in order to compare relative rates of catalysis. The results of these studies indicate that the endocyclic double bond of (-)-isopiperitenone is reduced to yield (+)-cis-isopulegone, which is isomerized to (+)-pulegone as the immediate precursor of (-)-menthone and (+)-isomenthone, and they rule out piperitenone as an intermediate of the pathway.     

Metabolism of Monoterpenes in Cell Cultures of Common Sage (Salvia officinalis) : Biochemical Rationale for the Lack of Monoterpene Accumulation

Leaves of common sage (Salvia officinalis) accumulate monoterpenes in glandular trichomes at levels exceeding 15 milligrams per gram fresh weight at maturity, whereas sage cells in suspension culture did not accumulate detectable levels of monoterpenes (<0.3 nanograms per gram fresh weight) at any stage of the growth cycle, even in the presence of a polystyrene resin trap. Monoterpene biosynthesis from [U-¹⁴C]sucrose was also virtually undetectable in this cell culture system. In vitro assay of each of the enzymes required for the sequential conversion of the ubiquitous isoprenoid precursor geranyl pyrophosphate to (+)-camphor (a major monoterpene product of sage) in soluble extracts of the cells revealed the presence of activity sufficient to produce (+)-camphor at a readily detectable level (>0.3 micrograms per gram fresh weight) at the late log phase of growth. Other monoterpene synthetic enzymes were present as well. In vivo measurement of the ability to catabolize (+)-camphor in these cells indicated that degradative capability exceeded biosynthetic capacity by at least 1000-fold. Therefore, the lack of monoterpene accumulation in undifferentiated sage cultures could be attributed to a low level of biosynthetic activity (relative to the intact plant) coupled to a pronounced capacity for monoterpene catabolism.     

Effect of salinity and abscisic acid on accumulation of glycinebetaine and betaine aldehyde dehydrogenase mRNA in Sorghum leaves (Sorghum bicolor)

The effects of salt stress and abscisic acid (ABA) on the expression of betaine aldehyde dehydrogenase (BADH) were determined in sorghum (Sorghum bicolor L.) plants. BADH mRNA expression was induced by salinity, and the timing coincided with the observed glycinebetaine (betaine) accumulation. The leaf water potential in the leaves of the sorghum plants was significantly affected by salinity. In response to salinity, betaine, ABA, Na and Cl accumulations increased 6-, 16-, 90-, and 3-fold, respectively. In the leaf disks from unsalinized plants incubated on NaCl, or ABA solution, the BADH mRNA level was lower than in the ABA-treated disks. Exogenous application of the ABA biosynthetic inhibitor fluridone to the NaCl-treated disks reduced the ABA accumulation and BADH mRNA levels compared with NaCl-treated leaves. The results indicate that the salt-induced accumulation of betaine and BADH mRNA coincides with the presence of ABA.     

PHYSIOLOGICAL STUDIES ON THE VERTICILLIUM WILT DISEASE OF TOMATO

The water loss per unit leaf area of tomato plants was decreased after inoculation with Verticillium albo-atrum. When diseased plants began to wilt water loss temporarily increased, but then rapidly decreased to become less than that of healthy plants grown under conditions of adequate or restricted water supply.
The transpiration of excised leaves from plants grown with a restricted water supply was reduced, but not so severely as that of comparable leaves from infected plants. Water loss from leaves on infected plants was reduced irrespective of any blocking of the petiolar xylem.
The rate of water loss from turgid leaf disks on mannitol solutions, and the rate of water uptake of leaf disks on water was similar for disks cut from wilting or turgid leaves of diseased plants or healthy plants grown with an adequate or restricted water supply.
Disease or poor water supply reduced leaf growth but had no effect on the rate of leaf initiation. Although the density of stomata was higher on leaves of diseased plants the stomatal area was less than on healthy plants.
The resistance to water flow in diseased stems was high and was correlated with vessel blockage. About half the blocked vessels contained hyphae. The severity and localization of symptoms in inoculated plants growing on susceptible or resistant rootstocks was directly related to the extent of invasion by the pathogen and to vessel blockage.
Experiments on the wilting activity of cell-free filtrates from cultures of the pathogen in vitro indicated that it produced a stable substance, not an enzyme, that caused wilting in cut shoots by blocking the end of the stem. It is suggested that an increasing internal water shortage causes major symptoms of disease.     

Lack of regio- and stereospecificity in oxidation of (+) camphor by Streptomyces griseus enriched in cytochrome P-450soy

Oxidation of (+) camphor by cytochrome P-450soy-enriched intact cells of Streptomyces griseus resulted in the formation of one major and several minor metabolites. The minor metabolites were identified as 3-endo-hydroxycamphor (2%), 5-endo-hydroxycamphor (7%), 5-exo-hydroxycamphor (9%), 2,5-diketobornane (2%), and camphorquinone (3%). The major metabolite was isolated and conclusively identified as 6-endo-hydroxycamphor (60%). When supplemented with NADPH, spinach ferredoxin:NADP oxidoreductase and spinach ferredoxin, homogeneous preparations of cytochrome P-450soy oxidized camphor to a mixture of 3-endo-, 5-endo-, 5-exo-and 6-endo-hydroxycamphor. The data presented indicates that cytochrome P-450soy resembles its mammalian counterparts in its lack of regio- and stereospecificity in camphor oxidation.     

ABA modulates the degradation of phosphoenolpyruvate carboxylase kinase in sorghum leaves

Salt stresses strongly enhance the phosphoenolpyruvate carboxylase kinase (PEPC-k) activity of sorghum leaves. This work shows that (1) abscisic acid (ABA) increased the rise in kinase activity in illuminated leaf disks of the non-stressed plant, (2) ABA decreased the disappearance of PEPC-k activity in the dark, (3) two PEPC-k genes expressed in sorghum leaves, PPCK1 and PPCK2, were not up-regulated by the phytohormone and, (4) ABA effects were mimicked by MG132, a powerful inhibitor of the ubiquitin-proteasome pathway. Collectively these data support a role for the ubiquitin-proteasome pathway in the rapid turnover of PEPC-k. The negative control by ABA on this pathway might account for the increase of kinase activity observed in salt-treated plants.     

Microbiological degradation of bile acids. Ring a cleavage and 7α,12α-dehydroxylation of cholic acid by Arthrobacter simplex

The metabolism of cholic acid by Arthrobacter simplex was investigated. This organism effected both ring a cleavage and elimination of the hydroxyl groups at C-7 and C-12 and gave a new metabolite, (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid, which was isolated and identified through its partial synthesis. A degradative pathway of cholic acid into this metabolite is tentatively proposed, and the possibility that the proposed pathway could be extended to the cholic acid degradation by other microorganisms besides A. simplex is discussed. The possibility that the observed reactions in vitro could occur during the metabolism of bile acids in vivo is considered.     

Theacrine (1,3,7,9-tetramethyluric acid) synthesis in leaves of a Chinese tea,kucha (Camellia assamica var. kucha)

Theacrine (1,3,7,9-tetramethyluric acid) and caffeine were the major purine alkaloids in the leaves of an unusual Chinese tea known as kucha (Camellia assamica var. kucha). Endogenous levels of theacrine and caffeine in expanding buds and young leaves were ca. 2.8 and 0.6-2.7% of the dry wt, respectively, but the concentrations were lower in the mature leaves. Radioactivity from S-adenosyl-L-[methyl-14C]methionine was incorporated into theacrine as well as theobromine and caffeine by leaf disks of kucha, indicating that S-adenosyl-L-methionine acts as the methyl donor not only for caffeine biosynthesis but also for theacrine production. [8-14C]Caffeine was converted to theacrine by kucha leaves with highest incorporation occurring in expanding buds. When [8-14C]adenosine, the most effective purine precursor for caffeine biosynthesis in tea (Camellia sinensis), was incubated with young kucha leaves for 24 h, up to 1% of total radioactivity was recovered in theacrine. However, pulse-chase experiments with [8-14C]adenosine demonstrated much more extensive incorporation of label into caffeine than theacrine, possibly because of dilution of [14C]caffeine produced by the large endogenous caffeine pool. These results indicate that in kucha leaves theacrine is synthesized from caffeine in what is probably a three-step pathway with 1,3,7-methyluric acid acting an intermediate. This is a first demonstration that theacrine is synthesized from adenosine via caffeine.     

Effect of Gibberellic Acid on Growth, Gibberellin Content, and Chlorophyll Content of Leaves of Potato (Solanum tuberosum)

Spraying potato plants with a solution of gibberellic acid (GA)when the 15th leaf was emerging increased the area of this leafand its total gibberellin content, assayed by dwarf French beanleaf disks. GA, assayed by lettuce hypocotyls, was not detectedin leaves from untreated plants. The GA content of leaves fromGA-treated plants decreased after 2 weeks and none was detectableafter 5 weeks; apparently it was converted to another gibberellin,possibly the same as the natural gibberellin. GA increased chlorophyllcontent per leaf but increased leaf area more so that the chlorophyllper unit area decreased, and the leaves were paler than untreatedleaves.     

Manganese and antibiotic biosynthesis. I. A specific manganese requirement for patulin production in Penicillium urticae

The effect of trace metal nutrition on the functioning of the patulin biosynthetic pathway in submerged cultures of Penicillium urticae (NRRL 2159A) was examined by both chromatographic and enzymological means. Comprehensive metal ion analysis showed generally low levels of contaminating metal ions in media components. Of eight metal ions examined, only manganese strongly influenced secondary metabolite production. In control cultures or cultures deficient in calcium, iron, cobalt, copper, zinc, or molybdenum, pathway metabolites appeared in the medium at about 25 h after inoculation. The first pathway-specific metabolite, 6-methylsalicylic acid, accumulated only transiently before being converted to patulin whose concentration steadily increased. In manganese-deficient cultures, however, 6-methylsalicylic acid continued to accumulate, with only minor amounts of patulin being produced. Additionally, a marker enzyme for the pathway showed only 0-20% of control activity. Clear dose responses (patulin versus manganese) were found in different media, with no effect on growth yield. Addition of manganese to depleted cultures at 18, 26, or 36 h resulted in increasing marker enzyme activity and patulin concentrations. It is concluded that manganese exerts a specific, positive effect on patulin biosynthesis and may in some way control the section of the patulin pathway occurring after 6-methylsalicylic acid.     

The Botrytis cinerea phytotoxin botcinic acid requires two polyketide synthases for production and has a redundant role in virulence with botrydial

The grey mould fungus Botrytis cinerea produces two major phytotoxins, the sesquiterpene botrydial, for which the biosynthesis gene cluster has been characterized previously, and the polyketide botcinic acid. We have identified two polyketide synthase (PKS) encoding genes, BcPKS6 and BcPKS9, that are up-regulated during tomato leaf infection. Gene inactivation and analysis of the secondary metabolite spectra of several independent mutants demonstrated that both BcPKS6 and BcPKS9 are key enzymes for botcinic acid biosynthesis. We showed that BcPKS6 and BcPKS9 genes, renamed BcBOA6 and BcBO9 (for B. cinerea botcinic acid biosynthesis), are located at different genomic loci, each being adjacent to other putative botcinic acid biosynthetic genes, named BcBOA1 to BcBOA17. Putative orthologues of BcBOA genes are present in the closely related fungus Sclerotinia sclerotiorum, but the cluster organization is not conserved between the two species. As for the botrydial biosynthesis genes, the expression of BcBOA genes is co-regulated by the Gα subunit BCG1 during both in vitro and in planta growth. The loss of botcinic acid production does not affect virulence on bean and tomato leaves. However, double mutants that do not produce botcinic acid or botrydial (bcpks6Δbcbot2Δ) exhibit markedly reduced virulence. Hence, a redundant role of botrydial and botcinic acid in the virulence of B. cinerea has been demonstrated.     

Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus. Identification of a gene encoding a senescence-specific metallothionein-like protein.

cDNA clones representing genes that are expressed during leaf senescence in Brassica napus were identified by differential screening of a cDNA library made from RNA isolated from leaves at different stages of senescence. The expression of these genes at different stages of leaf development was examined by northern blot analysis, and several different patterns of expression were observed. One of the clones, LSC54, represented a gene that is expressed at high levels during leaf senescence. Analysis of this gene indicated strong expression in flowers as well as in senescing leaves. DNA sequence analysis of the LSC54 cDNA indicated a similarity between the deduced amino acid sequence and several metallothionein-like proteins previously identified in plants.     

Influence of ionic interactions on essential oil and phenolic diterpene composition of Dalmatian sage (Salvia officinalis L.)

The potential of four essential cations (K⁺, Ca²⁺, Mg²⁺ and Fe²⁺) to alleviate salt toxicity was studied in sage (Salvia officinalis L.) plants grown in pots. Two concentrations of the following chloride salts: KCl, CaCl₂, MgCl₂ and FeCl_3, were used together with 100 mM NaCl to study the effects of these nutrients on plant growth, leaf essential oils (EOs) and phenolic diterpenes composition. The sage plants accumulated Na⁺ in their leaves (includers); this has affected secondary metabolites’ biosynthesis. Treatment with 100 mM NaCl slightly decreased borneol and viridiflorol, while increased manool concentrations. Addition of KCl, CaCl₂ and MgCl₂ increased considerably in a dose-dependent manner the oxygen-containing monoterpenes (1.8-cineole, camphor, β-thujone and borneol) in 100 mM NaCl-treated sage. Whereas, the contents of viridiflorol decreased further with the addition of KCl in 100 mM NaCl-treated sage. Our results suggest that the changes in EOs composition were more related to K⁺ and Ca²⁺ availability than to Na⁺ toxicity. Furthermore, treatment with NaCl decreased by 50% carnosic acid (CA), a potent antioxidant, content in the leaves. K⁺ and Ca²⁺ promoted the accumulation of CA and its methoxylated form (MCA) in the leaves. The concentration of CA was positively correlated with leaf K⁺ (r = 0.56, P = 0.01) and Ca²⁺ (r = 0.44, P = 0.05) contents. It appears that different salt applications in combination with NaCl treatments had a profound effect on EOs and phenolic diterpene composition in sage. Therefore, ionic interactions may be carefully considered in the cultivation of this species to get the desired concentrations of these secondary metabolites in leaf extracts.     

Metabolism of limonoids via a deoxylimonoid pathway in Citrus

Methyl deacetylnomilinate was metabolized in leaves of calamondin (Citrus microcarpa) to form a deoxy derivative. This reaction indicated the presence in citrus of an epoxidase, which is required for the first step of the deoxylimonoid pathway. The product of the second step, deoxylimonic acid, was isolated from grapefruit seeds. Deoxylimonate A-ring lactone hydrolase, which is involved in the third step of the deoxylimonoid pathway, was also found in grapefruit seeds. These results and the previously reported presence of the first metabolite of this pathway, deoxylimonin, show that limonoids are metabolized in citrus, not only via the 17-dehydrolimonoid pathway as previously established, but also via the deoxylimonoid pathway.     

Metabolic networks of Cucurbita maxima phloem

Metabolomic analysis aims at a comprehensive characterization of biological samples. Yet, biologically meaningful interpretations are often limited by the poor spatial and temporal resolution of the acquired data sets. One way to remedy this is to limit the complexity of the cell types being studied. Cucurbita maxima Duch. vascular exudates provide an excellent material for metabolomics in this regard. Using automated mass spectral deconvolution, over 400 components have been detected in these exudates, but only 90 of them were tentatively identified. Many amino compounds were found in vascular exudates from leaf petioles at concentrations several orders of magnitude higher than in tissue disks from the same leaves, whereas hexoses and sucrose were found in far lower amounts. In order to find the expected impact of assimilation rates on sugar levels, total phloem composition of eight leaves from four plants was followed over 4.5 days. Surprisingly, no diurnal rhythm was found for any of the phloem metabolites that was statistically valid for all eight leaves. Instead, each leaf had its own distinct vascular exudate profile similar to leaves from the same plant, but clearly different from leaves harvested from plants at the same developmental stage. Thirty to forty per cent of all metabolite levels of individual leaves were different from the average of all metabolite profiles. Using metabolic co-regulation analysis, similarities and differences between the exudate profiles were more accurately characterized through network computation, specifically with respect to nitrogen metabolism.