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
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Summary
Higher plants, besides providing basic nutrients such as proteins, fats and carbohydrates also synthesize a myriad of low molecular weight compounds, so called secondary metabolites. These metabolites are essential elements for a plant to interact with and survive in its environment. In the past years, a lot of efforts have been made to exploit the potential of plant secondary metabolites. One of the ways is by manipulating the biosynthetic pathways of plants by means of metabolic engineering. The genes from other sources such as plants from different species or bacteria or the combination of those can be introduced and overexpressed to the target plant to induce or increase the production of the desired compounds. On the other hand, certain pathways can be blocked from accumulating unwanted metabolites.
This thesis focused on investigating the effect of introducing the bacterial isochorismate synthase (ICS) gene into Brassica rapa on its secondary metabolite production. Previous efforts from our group have been successful in introducing the bacterial salicylic acid (SA) pathway in tobacco plants (Verberne et al., 2000). SA is one of the signal compounds that induce systemic acquired resistant (SAR) in plants. The entC gene from Escherichia coli encoding ICS and the pmsB gene from Pseudomonas fluorescence encoding isochorismate pyruvate lyase (IPL) were overexpressed in transgenic tobacco 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. Brassica rapa is one of the economically most important crops in the world as a high nutritional quality vegetable, as fodder and for the production of seed oil. Further improvement of the plant performance, like increasing pathogen resistance, either by conventional breeding or by genetic engineering is required. Thus, it is of great interest to improve resistance by introduction of the bacterial SA pathways in this non-model plant. A single ICS gene is used in this study as the overexpession of IPL might result in channeling isochorismate away from essential metabolic pathways, such as for production of phylloquinone.
The success of plant genetic engineering relies on the efficiency of transformation and gene delivery system by which the gene of interest can be inserted, integrated and subsequently expressed in the plant genome. Agrobacterium tumefaciens-mediated gene transfer is the currently most used method. We review in Chapter 2 the mechanism of the bacterium infection in the host plants as well as the plant factors involved in this interaction. There are at least 5 major steps involved at molecular level beginning from the attachment of Agrobacterium to the host plants until the T-DNA is integrated into the plant genome. This includes the activation of virulence (vir) genes, generation of a T- DNA complex and the transfer of T-DNA into the plant cell. The efficiency of plant transformation is depending on many factors, some of them are highlighted at the end of the chapter.
The establishment of a transformation system is a prerequisite in order to obtain the transgenic plants. Thus, in Chapter 3 we report the method development of B. rapa transformation including the tissue culture system, regeneration and establishment of the transformed ICS plants. Prior to this, several important parameters were investigated to optimize the transformation efficiency, i.e. different strains of Agrobacterium, cocultivation period, pH and photoperiod conditions, concentration of the phenolic compound acetosyringone, dilution factor of Agrobacterium cultures and preconditioning. The presence of the transgenes in the genome of the greenhouse-grown transgenic plants of ssp. oleifera was confirmed by PCR whereas the expression of the entC gene encoding ICS in mRNA was confirmed by RT-PCR. We also observed that all the primary transformed plants showed a normal phenotype, some of them entered the flowering stage but produced less seeds. Most of the siliques were not able to develop into maturity.
The study was continued by analyzing SA and its glucoside (SAG) accumulation in the primary transgenic plants (Chapter 4). The SA and SAG contents in the transgenic plants taken at two different times (9 months and 18 months old) were increased significantly in comparison with the control plants. However, the level of SA and SAG in the transformed plants varied between the individual plants. These results suggest that the entC gene was expressed and functional in inducing SA biosynthesis in B. rapa.
The introduction of the entC gene encoding ICS into B. rapa might have an effect on the accumulation of certain metabolites originating from chorismate. For instance, phylloquinone (vitamin K1) is derived from chorismate via ICS, whereas the aromatic amino acids phenylalanine and tyrosine which lead among others to the synthesis of phenylpropanoids, flavonoids, SA and glucosinolates are derived from chorismate through chorismate mutase. Tryptophan is also derived from chorismate via anthranilate synthase. In this study, the phylloquinone and glucosinolate levels were investigated in the transgenic plants (Chapter 5). It was found that there is no significant different of phylloquinone level between the transgenic plants and the controls whereas the glucosinolate profile was altered particularly for indole and aliphatic glucosinolates. This result indicates that the introduction of the entC gene encoding ICS in B. rapa, which was expected to have an influence on the isochorismate production, did not have an important influence on phylloquinone accumulation. The altered profile of glucosinolates might be due to the increased level of SA produced via isochorismate that leads to the activation of plant defense.
Manipulating a biosynthetic pathway does not only increase the fluxes toward desirable metabolites, but often it also leads to the change of fluxes through other related metabolic pathways which may result in an altered profile of the primary and secondary metabolites in the target plant. The introduction of ICS may alter directly or indirectly the metabolites present in B. rapa. Therefore, it is crucial to perform an unbiased analysis through metabolomic approaches on the transgenic plants (Chapter 6). Analysis by 1H- NMR coupled with multivariate data analysis suggested that glucosinolates (neoglucobrassicin) and phenylpropanoids (sinapoyl malate, feruloyl malate, caffeoyl malate) and primary metabolites, i.e. some organic acid and sugars were higher in transgenic ICS plants. In contrast, only primary metabolites such as alanine, valine and threonine were higher in control plants.
In the past years, the many attempts to obtain transgenic B. rapa plants that can confer specific desired traits were not successful due to the lack of efficiency in the transgene transformation and the subsequent regeneration of transformed plant cells. It was shown that the plant gene expression system related to plant defense was altered as a result of A. tumefaciens infection (Ditt et al., 2001; Ditt et al., 2005). The influence of
bacterial infection on the transcriptome has extensively been studied, but not at the metabolome level. In Chapter 7 we report the metabolome analysis of B. rapa upon infection with disarmed and tumor-inducing (nopaline and octopine) A. tumefaciens strains. The results show that certain flavonoids and phenylpropanoids were suppressed upon infection of B. rapa leaves with a disarmed (LBA4404) strain. A similar phenomenon was also observed for the leaves and stems taken from tumor-bearing plants infected with octopine and nopaline strains.
The manipulation of plant secondary metabolism to achieve particular goals by means of metabolic engineering has received a lot of attention. This approach can be applied not only to introduce value adding traits to existing agronomic crops but also as a way to exploit the potential of biodiversity resources especially in developing countries. We describe the future perspective of this field in Chapter 8.
In conclusion, it has been shown by this thesis that it is possible to introduce the entC gene encoding ICS in B. rapa by means of metabolic engineering and subsequently obtain transgenic plants. However, as mentioned above, the establishment of a plant transformation and regeneration platform for B. rapa with a high efficiency must first be addressed. This is of utmost importance and a challenging task.
