Marianne C. Verberne

Are Natural Deep Eutectic Solvents the Missing Link in Understanding Cellular Metabolism and Physiology

Over the past decade, metabolomics has developed into a major tool for studying the metabolism of organisms and cells, and through this approach much has been learned about metabolic networks and the reactions of organisms to various external conditions ([Lay et al., 2006][1]). Most of this work

Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance.

After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens. There is compelling evidence that SA plays a crucial role in triggering SAR. We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process. When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumulation of SA and SA glucoside compared to control plants. Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants. This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants.

Chapter 13 – Salicylic acid biosynthesis

Salicylic acid (SA) is probably one of the best-known natural products, because of its acetyl-derivative, the widely used drug, aspirin. The application of exogenous SA or its acetyl-derivative induces pathogenesis-related genes and causes partial resistance to plant diseases. Thus, the biosynthesis of SA became a hot topic because of its important signaling role in plant defence against pathogens. SA also has a number of other functions in plants, such as stimulation of flowering in Lemnaceae, inhibition of the biosynthesis of the plant hormone ethylene, regulation of stomata1 closure and root ion uptake. Moreover, endogenous SA regulates heat production in the inflorescences of Arum lilies. However, despite the growing interest in SA as a natural signal in systemic acquired resistance in plants, the complete biosynthetic pathway has still not yet been resolved. Besides the phenylpropanoid pathway, which includes trans-cinnamic acid (CA) as a putative intermediate, SA can also be formed along a completely different biosynthetic pathway, via isochorismate, which is directly derived from chorismate. This chapter reviews both pathways. The identified intermediates and the enzymes involved have been described. The biosynthesis of the closely related 2,3-dihydroxybenzoic acid (2,3-DHBA) is also discussed. This pathway shares the first enzyme with the SA-biosynthesis from chorismate, and seems to occur in both plants and microorganisms.

Activities of enzymes involved in the phenylpropanoid pathway in constitutively salicylic acid-producing tobacco plants

Chorismate mutase (CM, EC 5.4.99.5), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and chalcone synthase (CHS, EC 2.3.1.74) activities were studied in constitutive salicylic acid-producing (CSA) tobacco plants in relation to the accumulation of flavonoids and chlorogenic acid. The CM, PAL and CHS activities in CSA-tobacco (Nicotiana tabacum cv. Samsun NN) plants were lower than in non-transgenic tobacco plants. Flavonoid and chlorogenic acid accumulation was suppressed in CSA-tobacco plants compared to those of non-transgenic tobacco plants.

Salicylic acid produced by isochorismate synthase and isochorismate pyruvate lyase in various parts of constitutive salicylic acid producing tobacco plants

Salicylic acid production in roots, stems, young leaves, medium leaves and old leaves of constitutive salicylic acid producing (CSA) tobacco plants was tested. In protein extracts of CSA-tobacco plants, the formation of isochorismate and of salicylic acid (SA) were measured to determine isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL) activity. The highest production of isochorismate and SA in vitro were produced by protein extracts of the young leaves of CSA-tobacco plants. The isochorismate and SA productions in vitro by protein extracts and the accumulation of SA and SA-glucoside in CSA-tobacco plants varied among the tested lines and within one line. The accumulation of SA in vivo is determined by the activity of IPL.

Cloning of a cDNA encoding 1-deoxy-d-xylulose 5-phosphate synthase from Morinda citrifolia and analysis of its expression in relation to anthraquinone accumulation ☆

Abstract In higher plants, there are two pathways leading to the formation of isoprenoids: the mevalonate pathway and the recently discovered methyl- d -erythritol 4-phosphate (MEP) pathway. It was shown that the isoprenoid moiety, constituting ring C of anthraquinones (AQs) in the plant family Rubiaceae, is formed from the MEP pathway. 1-Deoxy- d -xylulose 5-phosphate synthase (DXS), catalyzing the first step of the MEP pathway, is suggested to be a regulatory enzyme. In order to evaluate the potential regulatory role of the DXS in AQ biosynthesis, we identified a full-length cDNA encoding DXS from Morinda citrifolia, a member of the Rubiaceae family, by a PCR strategy. The cDNA encodes a protein of 722 amino acids with a calculated molecular mass of 78 kDa. The deduced amino acid sequence shares high homology with amino acid sequences of DXSs from other higher plants. Southern blot indicated the existence of a small family of DXS genes in M. citrifolia. Northern blot analysis showed that the DXS transcript levels were correlated with AQ accumulation, suggesting that AQ biosynthesis is regulated at the transcriptional level of the DXS gene.

Is accumulation of sesquiterpenoid phytoalexins induced in tobacco plants constitutively producing salicylic acid

The accumulation of sesquiterpenoid phytoalexins was screened in constitutive salicylic acid producing (CSA)-tobacco leaves. Only after tobacco mosaic virus (TMV)-inoculation the sesquiterpenoid phytoalexin capsidiol was identified in CSA- and non-transgenic tobacco leaves. Capsidiol was not accumulated in CSA- and non-transgenic tobacco leaves after mechanical wounding.

Isochorismate synthase transgenic expression in Catharanthus roseus cell suspensions

Abstract Cell suspensions of Catharanthus roseus produce 2,3-dihydroxybenzoic acid (2,3-DHBA) after elicitation with fungal elicitor. Together with the 2,3-DHBA production there is a strong increase in isochorismate synthase (ICS; EC 5.4.99.6) activity, an enzyme which converts chorismate into isochorismate. Bacteria also produce 2,3-DHBA which is an intermediate for the biosynthesis of siderophores. The biosynthesis pathway of 2,3-DHBA in bacteria proceeds via ICS. To investigate whether C. roseus cells use the same pathway to produce 2,3-DHBA, cells were transformed with constructs containing a C. roseus cDNA clone of ics in sense or antisense orientation. Although the transformation with ics in sense orientation resulted in constitutive ICS activity, no accumulation of 2,3-DHBA was observed. Transformation of ics in antisense orientation was not sufficient to block the ICS production. The biosynthesis of 2,3-DHBA in C. roseus probably involves a multi-enzyme pathway, which cannot simply be modulated by over-expression of ics .

Nicotine and related alkaloids accumulation in constitutive salicylic acid producing tobacco plants

Large variations in nicotine and related alkaloids accumulations were found in the leaves of constitutive salicylic acid producing (CSA)-tobacco and non transgenic tobacco plants. Statistical analysis showed that there was no significant correlation between salicylic acid (SA) levels and alkaloid accumulation in CSA-tobacco plants. The accumulation of nicotine after wounding and tobacco mosaic virus (TMV)-inoculation of leaves were measured as well. The accumulation of nicotine in the leaves of CSA-tobacco was suppressed after TMV-inoculation, whereas wounding enhanced nicotine accumulation. There was no effect of TMV-inoculation on nicotine accumulation in non-transgenic tobacco but wounding also increased nicotine accumulation.

Differential Metabolic Profiling by HPLC–PDA–MS of Wild Type and Transgenic Tobacco Plants Constitutively Producing Salicylic Acid

Abstract The metabolite profiles of wild type tobacco plants and transgenic tobacco plants, constitutively producing salicylic acid (SA), were compared by means of liquid chromatography with online photo diode array and mass spectrometer detector (LC–PDA–MS). Statistical analysis of the peak area of 15 major peaks showed significant differences in quantities for three compounds, which are present in the extract before acid hydrolysis, containing mainly aglucones. By comparison of the retention time, UV spectra, and molecular weight with reference compounds, two of these peaks were identified as chlorogenic acid and rutin. The extracts obtained after acidic hydrolysis did not show any significant difference.