Expression of a bacterial, phenylpropanoid-metabolizing enzyme in tobacco reveals essential roles of phenolic precursors in normal leaf development and growth

Tobacco plants (Nicotiana tabacum cv XHFD 8) were genetically modified to express a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) enzyme which is active with intermediates of the phenylpropanoid pathway. We have previously shown that HCHL expression in tobacco stem resulted in various pleiotropic effects, indicative of a reduction in the carbon flux through the phenylpropanoid pathway, accompanied by an abnormal phenotype. Here, we report that in addition to the reduction in lignin and phenolic biosynthesis, HCHL expression also resulted in several gross morphological changes in poorly lignified tissue, such as abnormal mesophyll and palisade. The effect of HCHL expression was also noted in lignin-free single cells, with suspension cultures displaying an altered shape and different growth patterns. Poorly/non-lignified cell walls also exhibited a greater ease of alkaline extractability of simple phenolics and increased levels of incorporation of vanillin and vanillic acid. However, HCHL expression had no significant effect on the cell wall carbohydrate chemistry of these tissues. Evidence from this study suggests that changes in the transgenic lines result from a reduction in phenolic intermediates which have an essential role in maintaining structural integrity of low-lignin or lignin-deprived cell walls. These results emphasize the importance of the intermediates and products of phenylpropanoid pathway in modulating aspects of normal growth and development of tobacco. Analysis of these transgenic plants also shows the plasticity of the lignification process and reveals the potential to bioengineer plants with reduced phenolics (without deleterious effects) which could enhance the bioconversion of lignocellulose for industrial applications.     

Metabolic diversion of the phenylpropanoid pathway causes cell wall and morphological changes in transgenic tobacco stems

Studies involving transgenic plants with modifications in the lignin pathway reported to date, have received a relatively preliminary characterisation in relation to the impact on vascular integrity, biomechanical properties of tissues and carbon allocation to phenolic pools. Therefore, in this study transgenic tobacco plants (Nicotiana tabacum cv XHFD 8) expressing various levels of a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) gene have been characterised for cell wall and related morphological changes. The HCHL enzyme converts p-coumaroyl-CoA to 4-hydroxybenzaldehyde thereby rerouting the phenylpropanoid pathway. Plants expressing high levels of HCHL activity exhibited reduced lignin deposition, impaired monolignol biosynthesis and vascular integrity. The plants also exhibited reduction in stem toughness concomitant with a massive reduction in both the cell wall esterified and soluble phenolics. A notable result of redirecting the carbon flux was the wall-bound accretion of vanillin and vanillic acid, probably due to the shunt pathway. Intracellular accumulation of novel metabolites such as hydroxybenzoic and vanillic acid derivatives also occurred in the transgenic plants. A line with intermediate levels of HCHL expression conferred correspondingly reduced lignin deposition, toughness and phenolics. This line displayed a normal morphology but distorted vasculature. Coloration of the xylem has been previously attributed to incorporation of alternative phenolics, whereas results from this study indicate that the coloration is likely to be due to the association of low molecular weight phenolics. There was no evidence of increased growth or enhanced cellulose biosynthesis as a result of HCHL expression. Hence, rerouting the phenylpropanoid biosynthetic pathway quantitatively and qualitatively modifies cell wall-bound phenolics and vascular structure.     

金鱼藻的化学成分

从金鱼藻( Ceratophyllum demersum L .) 全草的乙醇提取物中分离鉴定了其中7 个, 分别为: 苜蓿素-7- O-β- D-葡萄糖苷( 1) , naringenin-7- O-β- D-葡萄糖苷(2 ) , 七叶内酯(3) , β-谷甾醇(4 ) , 7α-羟基-β-谷甾醇(5) , 7α-甲氧基-β-谷甾醇(6) , 十六碳脂肪酸(7) 。化合物(3) 为首次从金鱼藻中分离得到。     

Effect of VULM 1457, an ACAT inhibitor, on serum lipid levels and on real time red blood cell flow in diabetic and non-diabetic hamsters fed high cholesterol-lipid diet

Acyl-coenzyme A: cholesterol O-acyltransferase (ACAT) catalyzes the formation of cholesterol/fatty acyl-coenzyme A esters. Accumulation of cholesterol esters leads to pathological changes connected with atherosclerosis. We have evaluated effects of a newly synthesized ACAT inhibitor, 1-(2,6-diisopropyl-phenyl)-3-[4-(4'-nitrophenylthio)phenyl] urea (VULM 1457), on serum lipid (cholesterol and triglycerides) levels and velocity of red blood cells (RBC) in non-diabetic and diabetic hamsters fed on high cholesterol-lipid (HCHL) diet during 3 months. The VULM 1457 effects on the paw microcirculation were assessed using capillary microscopy by measuring (RBC) velocity in vivo. Hamsters fed on HCHL diet became hypercholesterolemic with a dramatic increase in serum lipids accompanied with significantly decreased RBC velocity. Diabetic hamsters fed on HCHL diet had further increased serum lipids with reduction of RBC velocity. The VULM 1457 inhibitor lowered cholesterol levels in both non-diabetic and diabetic hamsters fed on HCHL diet. The greater VULM 1457 effect was shown in diabetic hamsters fed on HCHL diet where VULM 1457 expressed hypotriglycerides effects, too. An improved RBC velocity-pronounced effect was observed in diabetic hamsters fed on HCHL diet treated with VULM 1457. These results suggest that the ACAT inhibitor, VULM 1457, is a prospective hypolipidemic and anti-atherogenic drug which treats diabetes.     

High yield mixotrophic cultures of the marine microalga Tetraselmis suecica (Kylin) Butcher (Prasinophyceae)

The effects of three organic compounds were tested on one of the most used marine micro-algae in the aquaculture of molluscs and crustaceans, Tetraselmis suecica. Studies were made in axenic conditions with yeast extract, peptone and glucose added to the culture medium, each alone, in combinations of two or all together. Medium without any organic compound was used for the control. Cultures containing yeast extract grew best, reaching maximum cell density of 3.79 × 10⁶ and 3.84 × 10⁶ cells ml⁻¹. The organic carbon source affected the biochemical composition. The components most affected were the carbohydrates, with values between 6.5 pg cell⁻¹ in control cultures and 48.5 pg cell⁻¹ in glucose cultures. Protein content ranged between 27.5 pg cell⁻¹ in control cultures and 88.6 pg cell⁻¹ in yeast + glucose + peptone cultures. The lipid content changed little. Maximum protein yields were reached in cultures with yeast + glucose and with yeast - glucose - peptone, with values of 24.6 and 28.2 mg 1⁻¹ d⁻¹, respectively. These values are 22 and 25 times those in control cultures. A maximum carbohydrate yield of 7.9 mg carbohydrate per litre per day was obtained in yeast + glucose + peptone cultures, 27 times that in the control cultures. The maximum lipid yield was obtained with yeast + glucose + peptone and yeast + glucose. Maximum energy values were 308 kcal 1⁻ in yeast extract - glucose - peptone cultures and 279 kcal 1⁻¹ in yeast extract + glucose cultures. Gross energy values in control cultures were 24.5 kcal 1⁻¹, but peptone cultures presented the minimum energy value, 22 kcal 1⁻¹. The yeast extract: glucose ratio in the culture medium was optimized. A ratio 2:1 produced the best yields in cells, protein, carbohydrate and gross energy.     

A ternary complex of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism

HCHL (hydroxycinnamoyl-CoA hydratase-lyase) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and is exploited in whole-cell systems for the bioconversion of ferulic acid into natural equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active-site residues identified using the crystal structure of HCHL revealed that while Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the K(M) for the substrate ferulic acid, fulfilling its anticipated role as a factor in substrate binding. Structures of WT (wild-type) HCHL and of the S123A mutant, each of which had been co-crystallized with feruloyl-CoA, reveal a subtle helix movement upon ligand binding, the consequence of which is to bring the phenolic hydroxyl of Tyr-239 into close proximity to Tyr-75 from a neighbouring subunit in order to bind the phenolic hydroxyl of the product vanillin, for which electron density was observed. The active-site residues of ligand-bound HCHL display a remarkable three-dimensional overlap with those of a structurally unrelated enzyme, vanillyl alcohol oxidase, that also recognizes p-hydroxylated aromatic substrates related to vanillin. The data both explain the observed substrate specificity of HCHL for p-hydroxylated cinnamate derivatives and illustrate a remarkable convergence of the molecular determinants of ligand recognition between the two otherwise unrelated enzymes.     

Induction of phenylpropanoid and tyramine metabolism in pectinase- or pronase-elicited cell suspension cultures of tobacco (Nicotiana tabacum)

The induction of the phenylpropanoid pathway and of tyramine metabolism was monitored in cell suspension cultures of Nicotiana tabacum treated with cell wall-degrading enzymes, in an attempt to correlate the synthesis of hydroxycinnamic acid amides of tyramine with the formation of wall-bound phenolic polymers. Treatment with commercial pectinase (from Penicilium occitanis ) induced a rapid rise in phenylalanine ammonia-lyase (EC 4.3.1.5), 4-coumarate:CoA ligase (EC 6.2.1.12), tyramine hydroxycinnamoyltransferase (EC 2.3.1.110) and peroxidase (EC 1.11.1.7) activities, and a concomitant decline in cinnamyl alcohol dehydrogenase (EC 1.1.1.195) activity. The induction of the phenylpropanoid pathway and of the synthesis of cinnamoyl-tyramines preceded the death of a large proportion of the elicited cells. When the cultures were treated with pronase (from Streptomyces griseus ), most cells remained alive and the induction of enzymes of the phenylpropanoid pathway lasted for several days, resulting in an accumulation of cinnamoyltyramines in the cells and in the culture medium. Treatment with pronase induced an increase in the activity of moderately anionic isoperoxidases which were also induced in pectinase-treated cells. Cinnamyl alcohol dehydrogenase activity remained stable in pronase-elicited cells, which rapidly accumulated thioglycolic acid-extractable phenolic polymers in their cell walls. The accumulation of these polymers coincided with the induction of 4-coumarate:CoA ligase but preceded the rise in tyramine hydroxycinnamoyltransferase and peroxidase activities.     

In Situ Localization of Phenylpropanoid Biosynthetic mRNAs and Proteins in Parsley (Petroselinum crispum)

Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together consituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence.     

Phenylpropanoid compounds in primary leaf tissues of rye (Secale cereale). Light response of their metabolism and the possible role in UV-B protection

The present study was undertaken in order to investigate the suitability of certain markers for UV plant response. In addition, we attempted to link the internal tissue distribution of specific UV-absorbing compounds to profiles of radiation gradients within intact primary rye leaves ( Secale cereale L. cv. Kustro). Etiolated rye seedlings irradiated with low visible light (LL) and/or UV radiation were used to study enzyme activities of the two key enzymes, phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), together with the tissue-specific accumulation of soluble phenylpropanoid products. Plants grown under relatively high visible light (HL) with or without supplementary UV-B radiation were used for further characterization. Apparent quantum yield and fluorescence quenching parameters were monitored to assess potential physiological changes due to UV-B exposure in HL-grown seedlings. A quartz fibreoptic microprobe was used to characterize the internal UV-B gradient of the leaf. The response of the phenylpropanoid metabolism to UV radiation was similar in primary leaves of both etiolated and HL-treated green plants. The epidermis-specific flavonoids together with CHS activity turned out to be suitable markers for assessing the effect of UV on the phenolic metabolism. The functional role of phenylpropanoid compounds was strongly implicated in protecting rye from UV-B radiation.     

Structural complexity, differential response to infection, and tissue specificity of indolic and phenylpropanoid secondary metabolism in Arabidopsis roots

Levels of indolic and phenylpropanoid secondary metabolites in Arabidopsis (Arabidopsis thaliana) leaves undergo rapid and drastic changes during pathogen defense, yet little is known about this process in roots. Using Arabidopsis wild-type and mutant root cultures as an experimental system, and the root-pathogenic oomycete, Pythium sylvaticum, for infections, we analyzed the aromatic metabolite profiles in soluble extracts from uninfected and infected roots, as well as from the surrounding medium. A total of 16 indolic, one heterocyclic, and three phenylpropanoid compounds were structurally identified by mass spectrometry and nuclear magnetic resonance analyses. Most of the indolics increased strongly upon infection, whereas the three phenylpropanoids decreased. Concomitant increases in both indolic and phenylpropanoid biosynthetic mRNAs suggested that phenylpropanoids other than those examined here in "soluble extracts" were coinduced with the indolics. These and previous results indicate that roots differ greatly from leaves with regard to the nature and relative abundance of all major soluble phenylpropanoid constituents. For indolics, by contrast, our data reveal far-reaching similarities between roots and leaves and, beyond this comparative aspect, provide an insight into this highly diversified yet under-explored metabolic realm. The data point to metabolic interconnections among the compounds identified and suggest a partial revision of the previously proposed camalexin pathway.     

Growth dynamics of Agrobacterium tumefaciens in chemostat cultures limited by carbon source and mineral nutrients.

Agrobacterium tumefaciens was grown in a chemostat in a chemically-defined medium which hs alpha-methyl D-glucoside, magnesium, manganese, phosphate or urea as the growth-limiting nutrient. Steady-state biomass concentrations were dependent on the specific growth rate of the organism when alpha-methyl D-glucoside, manganese or phosphate were growth-limiting nutrients. During magnesium-limited growth, large undamped oscillations in biomass concentration occurred. In all chemostat cultures a variant organism was selected which had lost the ability to grow in the medium supplied, bur survived on products of carbon metabolism derived from the wild-type.     

Induction of Flavonoid Synthesizing Enzymes by Light in Etiolated Pea (Pisum sativum cv. Midfreezer) Seedlings

Etiolated pea (Pisum sativum cv. Midfreezer) seedlings respond to illumination with white light by changes in the activity of phenylpropanoid and flavonoid synthesizing enzymes. Unlike in cell cultures, changes in enzyme activity in pea seedlings are not concerted. Phenylalanine ammonia-lyase (EC 4.3.1.5) activity peaked approximately 18 hours after onset of illumination. The phenylacetate path did not interfere with the measurement of phenylalanine ammonia-lyase activity. Activity of cinnamic acid 4-hydroxylase (EC 1.14.13.11) showed an early peak after 8 hours illumination, declined thereafter sharply, then gradually increased during the remainder of the experiment. Activities of chalcone synthase and UDP glucose:flavonol 3-O-glucosyltransferase (EC 2.4.1.91) increased steadily and reached a plateau after approximately 70 hours illumination time. Activity of 4-hydroxycinnamate:coenzyme A ligase (EC 6.2.1.12) remained relatively unchanged, whereas that of chalcone isomerase (EC 5.5.1.6) declined steadily during the course of the experiment. The relative in vitro enzyme activities suggest that the rate-limiting step for the phenylpropanoid path is the cinnamic acid 4-hydroxylase, that of the flavonoid pathway is the chalcone synthase. Integration of enzyme activity curves, however, show that only the curve deriving from phenylanine ammonia-lyase activity matches closely the production of the flavonol glycosides.     

C(15)H(24) Volatile Compounds Unique to Aflatoxigenic Strains of Aspergillus flavus

Headspace volatiles from eight strains of Aspergillus flavus (four aflatoxigenic strains and four nonaflatoxigenic strains), grown for 1, 2, 3, 4, 8, and 10 days in submerged cultures, were collected in Tenax GC traps. The traps were desorbed onto a 50-m gas-liquid chromatography capillary column by heat and gas purge from an external direct injector device. The column was interfaced with a mass spectrometer data acquisition system. Peaks were identified by comparing retention times and mass spectra with those obtained from authentic compounds and by using a computer-assisted mass spectral data base. Aflatoxigenic strains of A. flavus produced several C(15)H(24) compounds (e.g., alpha-gurjunene, trans-caryophyllene, and cadinene) which peaked in 3-day cultures and were not present in earlier (1- and 2-day) or later (8- and 10-day) cultures. None of these volatiles were detected in nonaflatoxigenic strains of A. flavus. There was an apparent correlation between the release of C(15)H(24) volatile compounds and the initiation of aflatoxin biosynthesis, and a correlation between decline of aflatoxin synthesis and the disappearance of the C(15)H(24) compounds unique to aflatoxigenic A. flavus also existed.     

Biosynthesis of curcuminoids and gingerols in turmeric (Curcuma longa) and ginger (Zingiber officinale): identification of curcuminoid synthase and hydroxycinnamoyl-CoA thioesterases

Members of the Zingiberaceae such as turmeric (Curcuma longa L.) and ginger (Zingiber officinale Rosc.) accumulate at high levels in their rhizomes important pharmacologically active metabolites that appear to be derived from the phenylpropanoid pathway. In ginger, these compounds are the gingerols; in turmeric these are the curcuminoids. Despite their importance, little is known about the biosynthesis of these compounds. This investigation describes the identification of enzymes in the biosynthetic pathway leading to the production of these bioactive natural products. Assays for enzymes in the phenylpropanoid pathway identified the corresponding enzyme activities in protein crude extracts from leaf, shoot and rhizome tissues from ginger and turmeric. These enzymes included phenylalanine ammonia lyase, polyketide synthases, p-coumaroyl shikimate transferase, p-coumaroyl quinate transferase, caffeic acid O-methyltransferase, and caffeoyl-CoA O-methyltransferase, which were evaluated because of their potential roles in controlling production of certain classes of gingerols and curcuminoids. All crude extracts possessed activity for all of these enzymes, with the exception of polyketide synthases. The results of polyketide synthase assays showed detectable curcuminoid synthase activity in the extracts from turmeric with the highest activity found in extracts from leaves. However, no gingerol synthase activity could be identified. This result was explained by the identification of thioesterase activities that cleaved phenylpropanoid pathway CoA esters, and which were found to be present at high levels in all tissues, especially in ginger tissues. These activities may shunt phenylpropanoid pathway intermediates away from the production of curcuminoids and gingerols, thereby potentially playing a regulatory role in the biosynthesis of these compounds.     

Induction of phenylpropanoid compounds by UV-B irradiation in roots of seedlings and cell cultures from dill (Anethum graveolens L.)

In both cell cultures and seedling roots from dill (Anethum graveolens L.) UV irradiation selectively induced a quercetin arabinoside and two other phenylpropanoid compounds with caffeic acid as the aglycone. Only wavelengths below 360 nm were effective in this induction. Maximal effectiveness was observed below 320 nm.Supported by the Deutsche Forschungsgemeinschaft (SFB 46).     

Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway

3H-l-Phenylalanine is incorporated into a range of phenylpropanoid compounds when fed to tobacco cell cultures. A significant proportion of (3)H-trans-cinnamic acid formed from (3)H-l-phenylalanine did not equilibrate with exogenous trans-cinnamic acid and therefore may be rapidly channeled through the cinnamate 4-hydroxylase (C4H) reaction to 4-coumaric acid. Such compartmentalization of trans-cinnamic acid was not observed after elicitation or in cell cultures constitutively expressing a bean phenylalanine ammonia-lyase (PAL) transgene. Channeling between PAL and C4H was confirmed in vitro in isolated microsomes from tobacco stems or cell suspension cultures. This channeling was strongly reduced in microsomes from stems or cell cultures of transgenic PAL-overexpressing plants or after elicitation of wild-type cell cultures. Protein gel blot analysis showed that tobacco PAL1 and bean PAL were localized in both soluble and microsomal fractions, whereas tobacco PAL2 was found only in the soluble fraction. We propose that metabolic channeling of trans-cinnamic acid requires the close association of specific forms of PAL with C4H on microsomal membranes.     

Enhancement of (R,R)-2,3-butanediol production from xylose by Paenibacillus polymyxa at elevated temperatures

Conversion of xylose to (R,R)-2,3-butanediol by Paenibacillus polymyxa in anaerobic batch and continuous cultures was increased by 39% and 52%, respectively, by increasing the growth temperatures from 30 to 39 °C. There was no effect of temperature when glucose was used as substrate. 39 mM (R,R)-2,3-butanediol, 65 mM ethanol, and 47 mM acetate were obtained from 100 mM xylose after 24 h batch culture at 39 °C. With 100 mM glucose and 100 mM xylose used together in a batch culture at 39 °C, all xylose was consumed after 24 h and 82 mM (R,R)-2,3-butanediol, 124 mM ethanol and 33 mM acetate were produced.