The effect of bacterial isochorismate synthase on the Brassica rapa metabolome
Simoh, S.
Citation
Simoh, S. (2008, June 11). The effect of bacterial isochorismate synthase on the Brassica rapa metabolome. Retrieved from https://hdl.handle.net/1887/12944
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
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Chapter 1
General Introduction
Plants synthesize a diverse array of phytochemicals, products of primary and secondary metabolism. Primary metabolites are the compounds that are necessary for the processes required for cell maintenance and proliferation (Kliebenstein, 2004). Unlike primary metabolites, secondary metabolites are defined as the compounds that play a role in the interaction of the cell/organism with its environment to ensure the survival of the organism in its ecosystem (Verpoorte, 2000). More than 100,000 low molecular weight compounds have been discovered from higher plants. From the economical point of view, secondary metabolites are of major interest as they are valuable for human beings, e.g. as pharmaceuticals, antioxidants, dyes, flavors and fragrances. Environmental stressors as well as genetic factors are the common stimulators which regulate and control the biosynthesis of plant secondary metabolites (Laitinen et al., 2005). For instance, activation by biotic or abiotic stress induces changes in the plant cell leading to a cascade of reactions which results in accumulation of these compounds (Sudha and Ravishankar, 2002). Some groups of secondary metabolites act as direct plant defense compounds in case of herbivory and pathogen attack, e.g. glucosinolates, flavonoids, and phenylpropanoids. Others, like e.g. salicylic acid, act as signal compounds in plant defense mechanisms. In addition, some secondary metabolites function as attractants for pollination such as floral scent or play a protective role in the plant tissues against abiotic environmental stresses such as UV-B radiation and extreme temperatures (Lila, 2006).
The importance of these compounds as well as their potential has led research to focus on the manipulation of secondary metabolite pathways to increase the production of specific desired compounds or to produce novel compounds through metabolic
development of the plants, such manipulation of the biochemical pathways of specific secondary metabolites is thought not to be lethal to the plant (Kutchan et al., 2005).
Metabolic engineering is generally referred to the channeling of the flux towards particular desirable compounds through modification of endogenous pathways (Capell and Christou, 2004). Among the strategies adopted are the blocking of metabolic flux, redirecting the metabolic flux into another cell compartment or introducing new pathways into the tested organism. Over the years, a significant progress has been made in the plant metabolic engineering field. Examples of the metabolic engineering of secondary metabolite pathways in plants have been highlighted by Verpoorte et al.
(2000) and Dixon (2001). The major constraint is the lack of characterization of plant secondary metabolite pathways at the level of biosynthetic enzymes and intermediates and only few genes of certain pathways are known (Verpoorte and Memelink, 2002).
Nevertheless, the increasing knowledge on the biosynthesis of metabolites and the discovery of pathway genes, and the ability to manipulate the expression of certain genes in transgenic plants paved the way of this field. In recent years, the ‘omics’ technologies, including metabolomics and fluxomics, are increasingly being adopted to advance this field to a level where the elucidation and characterization of certain metabolic pathways can be done in a holistic manner (Capell and Christou, 2004).
Most of the plant secondary metabolites are derived from three different sources:
shikimate pathway, isoprenoid pathway and polyketide pathway. The shikimate pathway is found only in plants and microorganism and is one of the most important pathways, since this is the biosynthetic route for the aromatic amino acids; phenylalanine, tyrosine and tryptophan. It is estimated that under normal conditions, 20% of the fixed carbon in plants flows through this pathway (Haslam, 1993). Unlike the bacterial shikimate pathway which is mostly used for the biosynthesis of these amino acids, higher plants use the amino acids also as precursors for a broad range of secondary metabolites (Hermann, 1995). Chorismate, the end product of the shikimate pathway is the intermediate which serves as a starting point for the biosynthesis of the aromatic amino acids and secondary metabolites including salicylate, 2,3-dihydroxybenzoate and anthranilates. Due to the occurrence of this pathway in microorganisms and plants only, it is an attractive target for
agrochemicals and antibiotics as it is expected that the compounds affecting this pathway would not cause negative effects to the mammalian system.
Salicylic acid (SA, 2-hydroxybenzoic acid) is one of the signal compounds that plays a key role in plant defense responses including induction of systemic acquired resistance and regulation of pathogenesis-related (PR) proteins. Microorganisms and plants use different pathways for SA production. In plants, SA is thought to be formed via the phenylpropanoid pathway which is derived from chorismate through trans-cinnamate and benzoate, whereas in microorganisms, it proceeds through isochorismate which is directly derived from chorismate. However, the possibility that also plants use isochorismate to form SA cannot be excluded. By using the Arabidopsis SID mutant (defective in isochorismate synthase (ICS)), Wildermuth et al. (2001) observed that SA is synthesized from chorismate using ICS and this pathway is required to induce systemic acquired resistant (SAR) and local acquired resistance (LAR). Verberne et al. (2000) were successful in introducing bacterial genes encoding two enzymes involved in the bacterial biosynthetic pathway of SA in tobacco plants. The entC gene from Escherichia coli encoding ICS and pmsB gene from Pseudomonas fluorescence encoding isochorismate pyruvate lyase (IPL) were overexpressed in the transgenic plants and this resulted in constitutive accumulation of SA and enhanced resistance to viral and oomycete infection.
Tobacco plants transformed with a single ICS gene also produced SA, although the accumulation was lower than in the plants transformed with both ICS and IPL.
Although the bacterial SA synthesis pathway was successfully introduced in the model plants tobacco and Arabidopsis (Verberne et al., 2000; Mauch et al., 2001;
Wildermuth, 2001), however, nothing so far has yet been done on any non-model plants.
Brassica rapa is one of the important agricultural crops, contributing around 12% of the worldwide supply of edible vegetable oil, and comprising many of the vegetables of our daily diet (Paterson et al., 2001). This makes it an attractive crop for further improvement of performance, like increasing pathogen resistance, either by conventional breeding or by genetic engineering. Introduction of the bacterial SA synthesis pathway would be one way to achieve this.
Aim of the thesis
The aim of the present study was to investigate the effect of introducing the bacterial isochorismate synthase (ICS) gene into Brassica rapa on its secondary metabolite production.
Outline of the thesis
The thesis begins with a review of the mechanism of infection of Agrobacterium tumefaciens in plant cells including the plant/host factors involved in this infection. The factors influencing the Agrobacterium-mediated transformation are discussed (Chapter 2). Chapter 3 deals with the development of a method for Agrobacterium-mediated transformation of B. rapa and considers the various parameters tested to optimize the transformation, including the different varieties of the plants, physical conditions and chemical factors. The primary transformants of B. rapa ssp. oleifera were analyzed for accumulation of salicylic acid (SA) and its glucosidic form (SAG) as discussed in Chapter 4. Chapter 5 deals with the study of the phylloquinone (vitamin K1) and glucosinolate contents of the transgenic plants. The metabolomic profiles of primary transgenic plants of B. rapa ssp. oleifera were investigated as discussed in Chapter 6.
Metabolomic changes upon infection of B. rapa with disarmed and tumor-inducing Agrobacterium strains were investigated as reported in Chapter 7. Finally in Chapter 8 the future perspectives in engineering of plant secondary metabolism are discussed.
