Enhanced production of resveratrol derivatives in tobacco plants by improving the metabolic flux of intermediates in the phenylpropanoid pathway

The biosynthesis of flavonoids such as anthocyanin and stilbenes has attracted increasing attention because of their potential health benefits. Anthocyanins and stilbenes share common phenylpropanoid precursor pathways. We previously reported that the overexpression of sweetpotato IbMYB1a induced anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum) plants. In the present study, transgenic tobacco (Nicotiana tabacum SR1) plants (STS-OX and ROST-OX) expressing the RpSTS gene encoding stilbene synthase from rhubarb (Rheum palmatum L. cv. Jangyeop) and the RpSTS and VrROMT genes encoding resveratrol O-methyltransferase from frost grape (Vitis riparia) were generated under the control of 35S promoter. Phenotypic alterations in floral organs, such as a reduction in floral pigments and male sterility, were observed in STS-OX transgenic tobacco plants. However, we failed to obtain STS-OX and ROST-OX plants with high levels of resveratrol compounds. Therefore, to improve the production of resveratrol derivatives in plants, we cross-pollinated flowers of STS-OX or ROST-OX and IbMYB1a-OX transgenic lines (SM and RSM). Phenotypic changes in vegetative and reproductive development of SM and RSM plants were observed. Furthermore, by HPLC and LC-MS analyses, we found enhanced production of resveratrol derivatives such as piceid, piceid methyl ether, resveratrol methyl ether O-hexoside, and 5-methyl resveratrol-3,4′-O-β-d-diglucopyranoside in SM and RSM cross-pollinated lines. Here, total contents of trans- and cis-piceids ranged from approximately 104–240 µg/g fresh weight in SM (F₂). Collectively, we suggest that coexpression of RpSTS and IbMYB1a via cross-pollination can induce enhanced production of resveratrol compounds in plants by increasing metabolic flux into stilbenoid biosynthesis.     

Vitamin K1 accumulation in tobacco plants overexpressing bacterial genes involved in the biosynthesis of salicylic acid

Phylloquinone (Vitamin K(1)) is an essential component of the photosynthetic electron transfer. As isochorismate is required for the biosynthesis of Vitamin K(1), isochorismate synthase (ICS) activity is expected to be present in all green plants. In bacteria salicylic acid (SA) is synthesized via a two step pathway involving ICS and isochorismate pyruvate lyase (IPL). The effect of the introduction in tobacco plants of the bacterial ICS and IPL genes on the endogenous isochorismate pathway was investigated. Transgenic tobacco plants in which IPL was targeted to the chloroplast suffered severe growth retardation and had low Vitamin K(1) content. Probably because isochorismate was channeled towards SA production, the plants were no longer able to produce normal levels of Vitamin K(1). Transgenic tobacco plants in which the bacterial ICS was present in the chloroplast showed higher Vitamin K(1) contents than wild type plants.     

Pathogens pulling the strings: Effectors manipulating salicylic acid and phenylpropanoid biosynthesis in plants

During evolution, plants have developed sophisticated ways to cope with different biotic and abiotic stresses. Phytohormones and secondary metabolites are known to play pivotal roles in defence responses against invading pathogens. One of the key hormones involved in plant immunity is salicylic acid (SA), of which the role in plant defence is well established and documented. Plants produce an array of secondary metabolites categorized in different classes, with the phenylpropanoids as major players in plant immunity. Both SA and phenylpropanoids are needed for an effective immune response by the plant. To successfully infect the host, pathogens secrete proteins, called effectors, into the plant tissue to lower defence. Secreted effectors can interfere with several metabolic or signalling pathways in the host to facilitate infection. In this review, we will focus on the different strategies pathogens have developed to affect the levels of SA and phenylpropanoids to increase plant susceptibility.     

Enzymes of the phenylpropanoid pathway and the necrotic reaction of hypersensitive tobacco to tobacco mosaic virus

Pronounced increases in activity of phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (CAH) and caffeic acid-O-methyltransferase (OMT)     

Prolonged root hypoxia effects on enzymes involved in nitrogen assimilation pathway in tomato plants

In order to investigate the effects of root hypoxia (1–2% oxygen) on the nitrogen (N) metabolism of tomato plants (Solanum lycopersicum L. cv. Micro-Tom), a range of N compounds and N-assimilating enzymes were performed on roots and leaves of plants submitted to root hypoxia at the second leaf stage for three weeks. Obtained results showed that root hypoxia led to a significant decrease in dry weight (DW) production and nitrate content in roots and leaves. Conversely, shoot to root DW ratio and nitrite content were significantly increased. Contrary to that in leaves, glutamine synthetase activity was significantly enhanced in roots. The activities of nitrate and nitrite reductase were enhanced in roots as well as leaves. The higher increase in the NH₄⁺ content and in the protease activities in roots and leaves of hypoxically treated plants coincide with a greater decrease in soluble protein contents. Taken together, these results suggest that root hypoxia leaded to higher protein degradation. The hypoxia-induced increase in the aminating glutamate dehydrogenase activity may be considered as an alternative N assimilation pathway involved in detoxifying the NH₄⁺, accumulated under hypoxic conditions. With respect to hypoxic stress, the distinct sensitivity of the enzymes involved in N assimilation is discussed.Key words: tomato, hypoxia, nitrogen, glutamine synthetase, protease, glutamate dehydrogenase     

Changes and subcellular localizations of the enzymes involved in phenylpropanoid metabolism during grape berry development

The phenylpropanoid pathway yields a variety of phenolics that are closely associated with fruit qualities in addition to structural and defense-related functions. However, very little has been reported concerning its metabolism in fruit. This experiment was designed to assess changes of eleven phenolic acids in grape berry (Vitis vinifera L. cv. Cabernet Sauvignon) and explore both the activities and amounts of three key enzymes--phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate:coenzyme A ligase (4CL)--catalyzing the biosynthesis of these compounds during berry development. Finally, the subcellular localizations of the enzymes within berry tissues were also investigated using immuno-gold electron microscopic technique. The results indicated that the contents of gallic, protocatechuic, gentisic and caffeic acid all changed drastically during berry development, while other compounds containing p-hydroxybenzoic, vanillic, syringic, chlorogenic, p-coumaric, ferulic and sinapic acid varied only slightly. Activities of PAL, C4H and 4CL showed similar pattern changes with two accumulated peaks throughout berry development. In addition, their activities all showed a highly positive correlation with the total contents of phenolic acids, whereas the immunoblotting analysis showed that changes in enzyme activities were independent of the enzyme amounts. Results from the subcellular-localization study revealed that PAL was mainly present in the cell walls, secondarily thickened walls, and the parenchyma cells of the berry mesocarp cells, C4H was found primarily in the chloroplast (plastid) and nucleus and 4CL predominantly in the secondarily thickened walls and the parenchyma cells of mesocarp vascular tissue.     

Modification of non-protein thiols contents in transgenic tobacco plants producing bacterial enzymes of cysteine biosynthesis pathway

Conditions of achieving the maximal accumulation of sulfhydryl metabolites in the leaves of tobacco were explored. Simultaneous production of bacterial O-acetylserine (thiol)-lyase and serine acetyltransferase resulted in the increased thiols contents as compared to single transformants and controls. However, leaf discs feeding experiments differently affected thiols concentration in different plant groups and suggested that the most promising strategy to obtain plants with a high level of non-protein thiol-containing compounds might be sulfate feeding to plants overproducing serine acetyltransferase.     

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.     

PeaT1-induced systemic acquired resistance in tobacco follows salicylic acid-dependent pathway

Systemic acquired resistance (SAR) is an inducible defense mechanism which plays a central role in protecting plants from pathogen attack. A new elicitor, PeaT1 from Alternaria tenuissima, was expressed in Escherichia coil and characterized with systemic acquired resistance to tobacco mosaic virus (TMV). PeaT1-treated plants exhibited enhanced systemic resistance with a significant reduction in number and size of TMV lesions on wild tobacco leaves as compared with control. The quantitative analysis of TMV CP gene expression with real-time quantitative PCR showed there was reduction in TMV virus concentration after PeaT1 treatment. Similarly, peroxidase (POD) activity and lignin increased significantly after PeaT1 treatment. The real-time quantitative PCR revealed that PeaT1 also induced the systemic accumulation of pathogenesis-related gene, PR-1a and PR-1b which are the markers of systemic acquired resistance (SAR), NPR1 gene for salicylic acid (SA) signal transduction pathway and PAL gene for SA synthesis. The accumulation of SA and the failure in development of similar level of resistance as in wild type tobacco plants in PeaT1 treated nahG transgenic tobacco plants indicated that PeaT1-induced resistance depended on SA accumulation. The present work suggested that the molecular mechanism of PeaT1 inducing disease resistance in tobacco was likely through the systemic acquired resistance pathway mediated by salicylic acid and the NPR1 gene.     

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAP^s) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lys^s) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.     

Activities of hydrolytic enzymes in callus cultures of tobacco during organogenesis

Starch, total sugars, reducing sugars and protein contents and the specific activities of hydrolytic enzymes such as amylase, Phosphorylase, soluble acid invertase, wall-bound acid invertase, sucrose synthetase, acid and alkaline phosphatases and ribonuclease were determined in root forming, shoot forming and non-organ-forming callus cultures of tobacco. Organ-forming cultures not only showed higher amounts of the above metabolites but also higher enzyme activities compared to non-organ-forming cultures. The activities of these enzymes in relation to organogenesis is discussed.     

Activities of enzymes involved in amino-acid metabolism in developing rat placenta

Aspartate transaminase, alanine transaminase, glutamate dehydrogenase, arginase, serine dehydratase, tyrosine transaminase, glutamine synthetase, glutaminase and adenylate deaminase activities were measured in crude homogenates of 12, 19 and 21-day rat placentae. There is a considerable quantitative importance in enzymes able to produce free ammonia, such as adenylate deaminase and glutamate dehydrogenase, activity that progressively decrease with the age of placenta. The glutamine synthetase and tyrosine transaminase activities increase with age, while serine dehydratase decreases considerably and aspartate and alanine transaminase do not change practically. Arginase shows a maximum at 19, with lower 12 and 21-day activities. No measurable glutaminase activity has been found. The possible implications of the enzymes studied upon the ammonia-producing activity of rat placenta are discussed together with the relative decreasing role of placenta for the overall metabolic activity of the foetus, especially during the last phases of its development.     

Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:coenzyme A (CoA) ligase (4CL) from Arabidopsis thaliana, and chalcone synthase (CHS) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans-cinnamic acid and p-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter(-1) of naringenin and 0.8 mg liter(-1) of pinocembrin. Several by-products, such as 2',4',6'-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor l-tyrosine.     

Activities of enzymes involved in lignification during the postharvest storage of etiolated asparagus spears

The content of lignin and the activities of 5 enzymes involved in lignification were monitored along the length of etiolated spears of asparagus ( Asparagus officinalis L., INRA Fl male hybrid n°156) stored for 22 h with their base in air (control), water or water containing the ethylene antagonist, silver thiosulfate (STS). At the time of harvest the lignin content increased basipetally, as did the activity of all the enzymes studied, viz., phenylalanine ammonia lyase (PAL; EC 4.3.1.5), hydroxycinnamate: CoA ligase (HCoAL; EC 6.2.1.12), cinnamoyl-CoA reductase (CCR: EC 1.2.1.44), cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) and syringaldazine oxidase (SyrOx. a peroxidase [POD; EC 1.11.1.7] with syringaldazine as substrate). Neither the lignin content nor the activity of any enzyme changed in the spear apex during storage, regardless of treatment. In the spear base. all enzyme activities decrased during the first 2 to 4 h in every storage treatment. Subsequently. PAL and HCoAL activities remained constant. whereas the activities of CAD and SyrOx gradually increased. Lignification in the spear base was not affected by storage in air. However, storage in water increased lignin formation and SyrOx activity, whereas treatment with STS prevented both of these increases. The results indicate that postharvest lignification in etiolated asparagus spears is caused primarily by enhanced SyrOx activity, and that ethylene is involved in the control of this activity.     

Biosynthesis of salicylic acid in plants

Salicylic acid (SA) is an important signal molecule in plants. Two pathways of SA biosynthesis have been proposed in plants. Biochemical studies using isotope feeding have suggested that plants synthesize SA from cinnamate produced by the activity of phenylalanine ammonia lyase (PAL). Silencing of PAL genes in tobacco or chemical inhibition of PAL activity in Arabidopsis, cucumber and potato reduces pathogen-induced SA accumulation. Genetic studies, on the other hand, indicate that the bulk of SA is produced from isochorismate. In bacteria, SA is synthesized from chorismate through two reactions catalyzed by isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL). Arabidopsis contains two ICS genes but has no gene encoding proteins similar to the bacterial IPL. Thus, how SA is synthesized in plants is not fully elucidated. Two recently identified Arabidopsis genes, PBS3 and EPS1, are important for pathogen-induced SA accumulation. PBS3 encodes a member of the acyl-adenylate/thioester-forming enzyme family and EPS1 encodes a member of the BAHD acyltransferase superfamily. PBS3 and EPS1 may be directly involved in the synthesis of an important precursor or regulatory molecule for SA biosynthesis. The pathways and regulation of SA biosynthesis in plants may be more complicated than previously thought.Key words: salicylic acid biosynthesis, isochorismate synthase, phenylalanine ammonia lyase