Chapter 4. Salicylic acid accumulation in transgenic Brassica rapa transformed with a gene encoding bacterial isochorismate synthase

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1 Chapter 4 Salicylic acid accumulation in transgenic Brassica rapa transformed with a gene encoding bacterial isochorismate synthase Sanimah Simoh 1,2, Natali R Mustafa 1, Huub JM Linthorst 3, Robert Verpoorte 1 1 Section Metabolomics, Institute of Biology, Leiden University, Leiden, The Netherlands 2 Biotechnology Research Centre, Malaysian Agricultural Research & Development Institute (MARDI), Kuala Lumpur, Malaysia 3 Section Plant Cell Physiology, Institute of Biology, Leiden University, Leiden, The Netherlands Abstract The accumulation of salicylic acid (SA) in Brassica rapa ssp. oleifera transformed with a bacterial isochorismate synthase (ICS) gene was examined in the old and young leaves of the primary transformants through high performance liquid chromatography analysis. The level of SA and SA glucoside (SAG) in the transformed plants varied between the individual plants but the SA contents of the leaves in all transgenic plants were increased significantly in comparison to the control plants. Increased SA levels were accompanied by increased accumulation of the SAG. This finding suggests that the ICS gene was expressed and functional in inducing SA biosynthesis in B. rapa. The primary transformed plants showed a normal phenotype, but the flowering plants produced less seeds. Keywords: salicylic acid, isochorismate synthase, Brassica rapa, transgenic

2 4.1 Introduction Salicylic acid (SA, 2-hydroxybenzoic acid) was first discovered as one of the main components from bark extracts of the willow tree (Salix). Since ancient times SA is well known to possess therapeutic properties such as relieving headache and fever. Aspirin, the acetylated form of SA is the first synthetic drug in the world, produced by the Bayer Company as anti-inflammatory drug in 1897 (Weissman, 1991). Physiological and biochemical effects of SA in plants, such as to induce flowering or to inhibit biosynthesis of ethylene, and potassium/phosphate uptake have long been known (Lee et al., 1995). Nowadays, the central role of SA in inducing the plant defense response, both in local and systemic acquired resistance (SAR), is broadly accepted (Malamy et al., 1990; Delaney, 1997). Upon pathogen attack, SA mediates the oxidative burst that causes rapid cell death at the point of infection leading to the hypersensitive response (HR). The HR prevents the systemic spread of the pathogen and this is usually accompanied by the induction of SAR. Once SAR is established, the plant s resistance to subsequent infections by a broad spectrum of viral, bacterial and fungal pathogens is enhanced. The induction of SAR is strongly associated with the expression of a set of genes that leads to the accumulation of pathogenesis-related (PR) proteins (Yalpani et al., 1991; Linthorst, 1991). In addition to these proteins, salicylic acid glucoside (SA 2-O-β-glucoside, SAG) is a major glucose conjugate of SA that is also found during SAR development (Ryals et al., 1996). Once the plant is infected with the pathogen, the SAG which serves as a storage form is rapidly converted to free SA at the site of infection (Chen et al., 1995). This conversion is catalyzed by SA β-glucosidase. The importance of SA in the plant defense response has stimulated interest to study SA and its relation to SAR. SA is proven to enhance the defense gene expression in many plants. Transgenic tobacco plants expressing the Pseudomonas putida gene encoding salicylate hydroxylase (NahG) which converts SA to catechol, accumulated little or no SA and the plants lost the ability to develop SAR (Gaffney et al., 1993). Arabidopsis challenged with Pseudomonas syringae pv. syringae developed SAR and accumulated SA in both local and systemic tissues. This is accompanied by the increase of PR proteins, chitinase and peroxidase activities (Summermatter et al., 1995). Several mutants of Arabidopsis defective in SA

3 accumulation and failure to develop SAR and PR proteins have been identified (Dong, 2001; Nawrath et al., 1999). Previous work suggests that in the plant, SA is synthesized via the phenylalanine pathway (Mauch-Mani et al., 1996; Yalpani et al., 1993a). However, in some bacteria, SA is synthesized via isochorismate involving two enzymes: isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL) (Figure 4.1). Recent studies have revealed that SA in plants also can be synthesized from chorismate through isochorismate, similar to bacteria (Mustafa, 2007; van Tegelen et al., 1999; Wildermuth et al., 2001). By using the Arabidopsis defense related mutant sid2, harboring a mutant gene for ICS, Wildermuth et al. (2001) showed that isochorismate synthase is required for SA synthesis and induction of SAR. The sid2 mutant plants, after infection with pathogen showed an SA accumulation of only 5-10% of that of the wild type and the PR-1 protein was expressed at a very low level. Previous research has shown that the introduction of ICS and IPL genes into plants enhances SA and SAG accumulation as well as disease resistance. Transgenic tobacco overexpressing two bacterial genes encoding ICS and IPL accumulated up to 100-fold higher levels of SA and SAG and showed constitutive PR-1 gene expression (Verberne et al., 2000). Mauch et al. (2001) observed a 20-fold increase of SA and SAG levels compared to wild type in transgenic Arabidopsis plants expressing a fusion product of ICS and IPL from Pseudomonas aeruginosa. They also observed that the transgenic plants showed an increased resistance to Peronospora parasitica, a fungus causing mildew disease. In both cases, the product of ICS and IPL were targeted to the chloroplast. In the present study, our aim is to investigate the SA and SAG accumulation in transgenic Brassica rapa ssp. oleifera transformed with a gene encoding bacterial ICS. Our previous work on tobacco had shown that ICS overexpression alone also results in increased constitutive SA production. Therefore we aimed at overexpression of ICS only, to avoid that overexpression of IPL would result in channeling isochorismate away from essential metabolic pathways, such as phylloquinone (vitamin K 1 ).

4 Shikimate Chorismate Prephenate Arogenate H O O NH 2 Isochorismate synthase (ICS) Phenylalanine ammonia lyase (PAL) Isochorismate H Phenylalanine O NH 2 Isochorismate pyruvate lyase (IPL) COO- trans-cinnamic acid SALICYLIC ACID Benzoic acid Figure 4.1 Pathways of SA biosynthesis in plants via the phenylpropanoid pathway and in microorganisms via the chorismate/isochorismate pathway (adapted from Verberne et al., 2000). 4.2 Materials and methods Growth of plant materials Nine and eighteen months old primary transformants of B. rapa transformed with a gene encoding bacterial ICS ( ss entc plants) and untransformed control plants grown in in the greenhouse at 2 C with a 16:8 light/dark photoperiod per day were used for the experiments. Old (lowest green leaf) and young (upper three/four) leaves of transgenic and control plants were harvested at the same time and ground in liquid nitrogen prior to extraction. Control plants used for the experiments were tissue culture generated plants

5 developed by using similar media and growth conditions as transformed plants. In addition, wild type plants (WT) generated from seed were also used Reagents Salicylic acid as a standard was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Methanol (absolute AR and HPLC grade), ethylacetate and glacial acid acetic were purchased from Biosolve BV, Netherlands. Cyclohexane was from J.T. Baker, Netherlands, trichloroacetic acid (TCA) ACS Reagent was from Merck, Darmstadt, Germany and ammonia was from Fluka, Germany Extraction of free and conjugated SA in Brassica rapa leaf materials Extraction methods of free and conjugated salicylic acid (SAG) were mainly based on a modified method of Verberne et al. (2002). Prior to extraction, fresh leaf material was collected and pulverized in liquid nitrogen. Half a gram of ground plant materials was extracted with 1 ml 90% methanol followed by centrifugation at 13,000 g for 30 minutes. The pellets obtained were re-extracted with 0.5 ml 100% methanol and centrifuged for 15 minutes. The supernatants from both centrifuge steps were pooled in a 2 ml microcentrifuge tube into which 10 µl of 0.2 M Na was added. The mixtures were vortexed and evaporated in vacuo to remove the methanol. The residues obtained were resuspended in 5% TCA, vortexed, and twice subjected to liquid-liquid extraction with 800 ml ethyl acetate:cyclohexane (1:1). For determination of free SA, 60 µl of HPLC eluent was added to the upper aqueous layer and the mixtures were evaporated in vacuo. The residues were dissolved in 600 µl of HPLC eluent prior to injection. For conversion of SAG to free SA, the lower aqueous fraction containing SAG was acid hydrolyzed by adding 300 µl of 8 M HCl and heating at 80 C for 1 hour. The fraction was further subjected to two times liquid-liquid extraction with ethyl acetate:cyclohexane (1:1) and the steps described above for SA determination were followed. All samples were extracted in 3-4 replicates.

6 4.2.4 Determination of SA recovery These experiments were carried out in order to determine the recovery of SA obtained in the samples after the extraction. Three different sets of B. rapa ground leaf material (3 samples and 500 mg fresh weight each set) i) samples spiked with 1.0 µg SA, ii) samples spiked with 1.0 µg SA only, in the TCA water phase before acid hydrolysis, and iii) nonspiked samples, were extracted for free SA and conjugated SA (SAG) following the above method. Three SA standard solutions containing 1.0 µg SA were also made in 660 µl HPLC eluent. All the spiked and non-spiked samples were then injected into HPLC. The mean of the SA peak area differences between the spiked and non-spiked samples was corrected with the mean of the peak area of SA standard solution to obtain the recovery of SA and conjugated SA (SAG) HPLC analysis of SA contents Twenty microliters from the total SA extract were injected into a HPLC C18 column (Phenomenex, type LUNA 3µ C18 (2)) 150 x 4.60 mm 3 µm, with a Phenomenex Security Guard (Torrance, CA, USA) using a Gilson 234 auto-injector (Villiers Le Bel, France). The HPLC eluent was 0.2 M ammonium acetate buffer (ph 5.5) in 10% methanol and had a flow rate of 0.8 ml/min. SA and SAG (expressed as SA after acid hydrolysis) were detected with a Shimadzu RF-10Ax1 fluorescence detector (Tokyo, Japan) with an emission and excitation wavelength at 407 and 305 nm respectively. The peak of SA appeared around minutes Statistical analysis A one way analysis of variance (ANOVA, Minitab ) was used to analyze the statistical differences between the transgenic and control plants. Significant differences were indicated for P Results and discussion In order to determine whether the ss entc transgenic of B. rapa plants contained elevated levels of SA, HPLC analysis was carried out to determine free SA and glucosylated SA (SAG) in young and old leaves of the transformed plants. Analysis on six primary

7 transformants (L2, L4, L5, L9, L10, and L12) was carried out when the plants were nine and eighteen months old. Recovery experiments which were carried out in duplicate showed that 94% (SD ± 2.6) and 68% (SD ± 1.9) of free SA and SAG (expressed as SA after acid hydrolysis), respectively, were recovered from this extraction method. The results showed an increased amount of free SA in almost all the transgenic plants (L2-L12, Figure 4.2a, Figure 4.3a) in comparison with tissue culture-generated (CTRL) and wild type plants (WT) which served as controls. SAG levels were also significantly increased in parallel with the increases in SA levels in comparison to control plants (Figure 4.2b, Figure 4.3b). Previous work in tobacco by Verberne et al. (2000) showed that plants transformed with both ICS and IPL targeted at the chloroplast (CSA plants) accumulated high levels of SA and SAG, whereas the plants transformed with the single gene construct ICS or IPL showed only a slight increase of SA. In addition, Verberne et al. (2000) also observed elevated levels of SAG in the single gene transformed plants, similar as in our study here. They suggested that isochorismate, as a product of transgene ICS accumulated in the ss entc plants, is converted to SA, possibly via a spontaneous chemical conversion (Young et al., 1969). The result of our study also shows that the level of SA and SAG varied between the individual transgenic plants. The wide variation of SA and SAG accumulation observed in the transgenic plants is probably due to the segregation of the T-DNA loci (Nugroho et al., 2001). These results indicate that the bacterial isochorismate synthase (ICS) gene was expressed and functional in B. rapa. In this study, it was found that most of the transgenic and control plants accumulated higher levels of SAG than SA (the range is fold, Table 4.1). However, the ratio of SAG to SA varied between the tested individual transformed plants. In plant L5 the old leaves of 18 months old showed 300 fold higher levels of SAG as compared to SA. In contrast to this, the young leaves showed only a 6-fold increment, lower than in the control and wild type plants. This variation was also observed between transgenic lines of ss entc tobacco plants (Verberne et al., 2000). There the ratio observed was 1-8 fold. Glucosylation of SA is an important step in SA metabolism. Even though the mechanism is not clearly understood, it was suggested that SAG stored in vacuoles, may affect the signaling and defense response mediated by SA (Dean et al., 2003).

8 Old leaves Young leaves a Free SA (ng/g FW) * L2 L4 L5 L9 L10 CTRL 1 CTRL 2 WT1 WT2 SA (a fter hydrolysis) (ng/g FW ) Old leaves Young leaves b 0 L2 L4 L5 L9 L10 CTRL 1 CTRL 2 WT1 WT2 Figure 4.2 (a) Free SA and (b) SAG (SA after acid hydrolysis) accumulation in old and young leaves of 9 months old ss entc plants of Brassica rapa ssp. oleifera. All the individual plants showed significant difference at p 0.05 from the data set of control plants except indicated by asterisk (*). Vertical bars represent SDs obtained from three replicates. The data are not corrected for the extraction recovery of 94% and 68% for SA and SAG (SA after acid hydrolysis) respectively.

9 Old leaves Young leaves a Free SA (ng/g FW ) * L2 L4 L5 L10 L12 CTRL1 WT Old leaves Young leaves b SA (after hydrolysis) (ng/g FW ) L2 L4 L5 L10 L12 CTRL1 WT1 Figure 4.3 (a) Free SA and (b) SAG (SA after acid hydrolysis) accumulation in old and young leaves of 18 months old ss entc plants of Brassica rapa ssp. oleifera. All the individual plants showed significant difference at p 0.05 from the data set of control plants, except indicated by asterisk (*) Vertical bars represent SDs obtained from three replicates. The data are not corrected for the extraction recovery of 94% and 68% for SA and SAG (SA after acid hydrolysis) respectively.

10 Glucosylation may also act as a protection to the cell from toxic levels (<0.1mM, Lee et al., 1995) of SA. Enyedi et al. (1992) observed that most SA found in the leaf after TMV infection is present in a conjugated form. Tobacco leaves heavily infected with TMV accumulated 40-fold higher SAG levels than SA, whereas the non-inoculated plants accumulated very low levels of SAG. The results from our study show that the ratio of SAG to SA in some of the transgenic plants is lower than in the control plants, which might be because the level of SA as a result of the introduction of the single ICS gene construct is not sufficient to induce the conversion to SAG as it occurs in the CSA tobacco plants with much higher SA levels (Verberne et al., 2000). Considerably higher levels of SAG as compared to SA were also observed in wild type and non-transformed plants (Table 4.1). This may be due to the glucosylated form of SA being the major product of SA metabolism (Enyedi et al., 1992). SAG is the predominant form of stable metabolites of SA in various plants (Song, 2006) such as tobacco and soybean (Dean et al., 2003), Vicia faba (Schulz et al., 1993) and rice (Tanaka et al., 1990). The results also show that 9 months old plants have significantly a higher accumulation of free SA in young leaves than old leaves (Figure 4.2a) except in L4 (p=0.434). The highest accumulation of SA was observed in L2 (445 ng/g FW), 5-6 fold more than the control plants. In plants, leaf aging is induced by biotic or abiotic stress (Gan and Amasino, 1997). This causes oxidative stress, which in turn causes degradation of the chloroplast and leads to the decrease of the photosynthetic efficiency (Mýtinova et al., 2006). In our study, the entc gene encoded a ICS protein containing a signal sequence for targeting to the chloroplast where the substrate chorismate is available. Chloroplast degradation may have an effect on the level of the chorismate substrate, which leads to the decrease of SA. This may explain why old leaves in 9 months old plants accumulated lower levels of SA than young leaves. In contrast to this, in most 9 months old plants, SAG accumulation was higher in old leaves than young leaves (Figure 4.2b), except in L4 which also showed highest level of SAG (3300 ng/g FW) in young leaves. Although the accumulation of SAG in L4 was slightly higher in young leaves, this was not significantly different from old leaves (p=0.299). Nugroho et al. (2001) observed that both SA and SAG accumulation was higher in young leaves than old leaves. The age-

11 Table 4.1 The ratio of SAG to SA in the transgenic of ss entc and control plants of B. rapa ssp. oleifera. Individual plants tested Old leaves The ratio of SAG to SA Eighteen months old Young leaves L L L L L CTRL WT Nine months old L L L L L CTRL WT CTRL1: tissue culture generated plant; WT: wild type plant dependent variation of SA was also reported in Arabidopsis when challenged with Pseudomonas syringae (Zeier et al., 2005).In contrast to 9 months old plants, most of the 18 months old plants showed higher accumulation of free SA in old leaves than young leaves (Figure 4.3a) except in L5 and L10. The highest accumulation of SA was observed in L5 (335 ng/g FW). A similar pattern was observed for SAG accumulation in most of the tested plants (Figure 4.3b). This result is an agreement with Yalpani et al. (1993b) who showed that older leaves of tobacco plants at the beginning of flowering accumulated a five-fold higher level of SA. The change in the endogenous level of SA might be related with the transition to the flowering phase (Martinez et al., 2004). In our case, three of the transgenic plants of B. rapa (L5, L10, L12) started to flower when they were months old.

12 Different phenotypes of the transgenic plants were observed by previous researchers following introduction of single or double constructs of the bacterial genes ICS and IPL. Tobacco plants with both ICS and IPL targeted to the chloroplast showed a normal phenotype and viable seeds. Plants with a single IPL gene showed strong dwarfism, whereas plants with a single ICS construct developed normally (Verberne et al., 2000). Mauch et al. (2001) reported that Arabidopsis plants transformed with both ICS and IPL targeted to the chloroplast showed strong dwarfism. In our case, primary transformants of ssentc B. rapa plants showed a phenotype similar to control plants. Three of the transgenic plants entered the flowering stage although they produced only few seeds. It was observed that most of the siliques from these flowering plants did not develop to maturity. Our previous experience showed that this Finnish variety of B. rapa required cross pollination to set seeds. This may explain why our transgenic and control plants were not able to set viable seeds since the flowering time differed between the individual plants and the cross pollination could not be carried out. 4.4 Conclusion It was shown that by introducing the bacterial isochorismate synthase (ICS) gene into B. rapa ssp. oleifera, salicylic acid (SA) and its conjugated form (SAG) accumulated at a level that was significantly higher than in the control plants. This indicated that the ICS gene was expressed and functional in B. rapa. However, the level of SA and SAG in the transformed plants varied between the individual plants. 4.5 Acknowledgements We thank Malaysian Agricultural Research and Development Institute (MARDI), Malaysia for the Ph.D grant to Sanimah Simoh.