These authors contributed equally to this paper.
Expression of a bacterial bi-functional chorismate mutase/prephenate dehydratase modulates primary and secondary metabolism associated with aromatic amino acids in Arabidopsis
Article first published online: 3 JUL 2009
© 2009 The Authors. Journal compilation © 2009 Blackwell Publishing Ltd
The Plant Journal
Volume 60, Issue 1, pages 156–167, October 2009
How to Cite
Tzin, V., Malitsky, S., Aharoni, A. and Galili, G. (2009), Expression of a bacterial bi-functional chorismate mutase/prephenate dehydratase modulates primary and secondary metabolism associated with aromatic amino acids in Arabidopsis. The Plant Journal, 60: 156–167. doi: 10.1111/j.1365-313X.2009.03945.x
- Issue published online: 24 SEP 2009
- Article first published online: 3 JUL 2009
- Received 27 March 2009; revised 20 May 2009; accepted 22 May 2009; published online 3 July 2009.
- chorismate mutase;
- prephenate dehydratase;
- Top of page
- Experimental procedures
- Supporting Information
Plants can synthesize the aromatic amino acid Phe via arogenate, but it is still not known whether they also use an alternative route for Phe biosynthesis via phenylpyruvate, like many micro-organisms. To examine this possibility, we expressed a bacterial bi-functional PheA (chorismate mutase/prephenate dehydratase) gene in Arabidopsis thaliana that converts chorismate via prephenate into phenylpyruvate. The PheA-expressing plants showed a large increase in the level of Phe, implying that they can convert phenylpyruvate into Phe. In addition, PheA expression rendered the plants more sensitive than wild-type plants to the Trp biosynthesis inhibitor 5-methyl-Trp, implying that Phe biosynthesis competes with Trp biosynthesis from their common precursor chorismate. Surprisingly, GC-MS, LC-MS and microarray analyses showed that this increase in Phe accumulation only had a very minor effect on the levels of other primary metabolites as well as on the transcriptome profile, implying little regulatory cross-interaction between the aromatic amino acid biosynthesis network and the bulk of the Arabidopsis transcriptome and primary metabolism. However, the levels of a number of secondary metabolites derived from all three aromatic amino acids (Phe, Trp and Tyr) were altered in the PheA plants, implying regulatory cross-interactions between the flux of aromatic amino acid biosynthesis from chorismate and their further metabolism into various secondary metabolites. Taken together, our results provide insights into the regulatory mechanisms of aromatic amino acid biosynthesis and their interaction with central primary metabolism, as well as the regulatory interface between primary and secondary metabolism.
- Top of page
- Experimental procedures
- Supporting Information
Micro-organisms use at least two metabolic routes for synthesis of the aromatic amino acid Phe from chorismate. The first enzyme, common to both routes, is chorismate mutase (CM), which converts chorismate to prephenate (Figure 1). Prephenate is then converted to Phe via either phenylpyruvate (PPY) or arogenate as intermediates (Patel et al., 1977; Haslam, 1993). The PPY route involves conversion of prephenate into PPY by the enzyme prephenate dehydratase (PDT), and the subsequent conversion of PPY into Phe by the enzyme aromatic amino acid aminotransferase (Kuramitsu et al., 1985). This route exists in Escherichia coli (E. coli) and Bacillus subtilis (Zhang et al., 1998). The arogenate route comprises the conversion of prephenate into arogenate by the enzyme prephenate aminotransferase (PAT), and the subsequent conversion of arogenate to Phe by the enzyme arogenate dehydratase (ADT) (Herrmann, 1995; Cho et al., 2007; Yamada et al., 2008). Some cyanobacteria, coryneform bacteria and spore-forming actinomycetes use the arogenate route (Keller et al., 1983).
In contrast to micro-organisms, the metabolic route from chorismate to Phe in plants is still not entirely known. Plants, like micro-organisms, use CM to convert chorismate into prephenate en route to Phe biosynthesis (Eberhard et al., 1996; Mobley et al., 1999). However, the subsequent enzymatic steps for synthesis of Phe from prephenate in plants are still not clear. Several lines of evidence suggest that plants can synthesize Phe via the arogenate route. PAT enzymatic activity, converting prephenate into arogenate, has been reported in plants (Siehl et al., 1986; De-Eknamkul and Ellis, 1988). However, no plant gene encoding such activity has so far been reported (Boatright et al., 2004). The conversion of arogenate into Phe by ADT has also been demonstrated in tobacco (Nicotiana sylvestris) and spinach (Spinacia oleracea) chloroplasts and etiolated sorghum (Sorghum bicolor) seedlings (Siehl and Conn, 1988). Six genes showing homology to bacterial PDT genes of Phe biosynthesis have recently been characterized in Arabidopsis (Cho et al., 2007). Biochemical characterization of the recombinant enzymes encoded by these six genes implied that three of them only use arogenate as a substrate, while the other three utilize both arogenate and prephenate, but exhibit a preference for arogenate. This study classified all six of these enzymes as ADTs (Cho et al., 2007). A recent report demonstrated that over-accumulation of Phe, Trp and several phenylpropanoids in the Mtr1 rice (Oryza sativa) mutant (5-methyl-Trp-resistant 1) was the result of a point mutation in a gene encoding an enzyme possessing both ADT and PDT activities, rendering these activities insensitive to feedback inhibition by Phe (Yamada et al., 2008). However, this enzyme possessed activity with a preference for arogenate, suggesting that it functions primarily as an ADT. Plants, like many bacterial species, also utilize arogenate for synthesis of Tyr by arogenate dehydrogenase (ADS; Rippert and Matringe, 2002). ADS activity has been demonstrated in tobacco (Nicotiana sylvestris) (Gaines et al., 1982), maize (Zea mays) (Byng et al., 1981), sorghum (Sorghum bicolor) (Connelly and Conn, 1986) and Arabidopsis (Rippert and Matringe, 2002).
In contrast to the extensive experimental evidence supporting the use of arogenate in plant Phe biosynthesis, it is still not clear whether PPY also serves as an intermediate in Phe biosynthesis. Nevertheless, a number of plants species contain PPY, which also serves as a precursor for a number of secondary metabolites such as phenylacetaldehyde, 2-phenylethanol and 2-phenylethyl-β-d-glucopyranoside (Watanabe et al., 2002; Kaminaga et al., 2006). The biosynthesis of PPY from chorismate in E. coli is catalyzed by a single bi-functional CM/PDT enzyme, which contains both CM and PDT activities and is encoded by a single gene termed PheA (Romero et al., 1995). The catalytic activities of the CM and PDT domains in the bi-functional enzyme are located at amino acids 1-285 (Zhang et al., 1998), while the C-terminal domain is responsible for the allosteric feedback inhibition by Phe (Zhang et al., 1998). A truncated CM/PDT protein containing only amino acid residues 1-285 (or 1–300) retained the CM and PDT activities. However, it did not exhibit feedback inhibition by Phe, and its expression in E. coli resulted in Phe over-production (Zhang et al., 1998).
In this study, we have expressed a truncated bacterial PheA gene (termed PheA*) in Arabidopsis, encoding a feedback-insensitive CM/PDT enzyme that lacks the C-terminal allosteric domain. This was performed in order to: (i) test whether PPY serves as a precursor for plant Phe biosynthesis, and (ii) elucidate the regulatory relationship between Phe metabolism and the metabolism of the two other aromatic amino acids (Trp and Tyr), all three of which competing for the common precursor chorismate (Figure 1). Our results imply that Arabidopsis plants possess a functional metabolic route from prephenate via PPY into Phe, and also demonstrate some regulatory differences between the arogenate and PPY metabolic routes of Phe biosynthesis. Moreover, our results provide novel information regarding cross-regulation of the biosynthesis of the three aromatic amino acids, as well as the regulation between their primary and secondary metabolism.
- Top of page
- Experimental procedures
- Supporting Information
Generation of transgenic Arabidopsis plants expressing a bacterial feedback-insensitive chorismate mutase/prephenate dehydratase gene (PheA*)
To study the significance of PPY as a precursor for the production of Phe, we expressed a truncated bacterial PheA* gene under the control of the 35S CaMV promoter, fused in-frame at the 3′ end of the coding sequence to DNA encoding a hemagglutinin (HA) epitope tag. PheA* encodes a bi-functional, Phe-insensitive CM/PDT enzyme that catalyzes the conversion of chorismate via prephenate into PPY (see Introduction). Two chimeric constructs were produced. In one, DNA encoding a Rubisco small subunit-3A plastid transit peptide (Shaul and Galili, 1993) was fused in-frame to the 5′ end of the PheA* open reading frame (Figure 2a) to direct the bacterial enzyme in to the plastid where aromatic amino acid biosynthesis is localized. The second construct lacked the Rubisco small subunit-3A plastid transit peptide in order to test whether aromatic amino acid metabolism is strictly localized tp the plastid or whether at least some parts of it operate in the cytosol. The two constructs were transformed into Arabidopsis plants, and homozygous T2 plants were generated. To examine the polypeptides produced by the chimeric PheA* transgene, proteins from independently transformed plants were subjected to immunoblot analysis using anti-HA antibodies. Immunoblot analysis with anti-HA antibodies showed that transgenic plants containing the PheA* construct lacking the plastid transit peptide produced a polypeptide band corresponding in size to the full-length bacterial PheA* polypeptide (data not shown). However, GC-MS analysis revealed that these plants have metabolic patterns that are highly comparable to those of the control plants, indicating that there is no major operation of aromatic amino acid biosynthesis in the cytosol. Immunoblot analysis with anti-HA antibodies showed that transgenic plants expressing the plastidic PheA* construct (termed pPheA*) produced two PheA*-derived polypeptide bands, one corresponded in size to the mature PheA* polypeptide, while the second, which migrates slightly more slowly, corresponds in size to unprocessed PheA* containing the plastid transit peptide (Figure 2b). This indicated that a high proportion of the PheA* polypeptide produced by this transgene was translocated into the plastids.
Transgenic pPheA* plants accumulate Phe and are hypersensitive to its external application
To test whether pPheA* expression alters the biosynthesis of Phe, we analyzed Phe levels in rosette leaves of ten independently transformed plants, compared to control plants. As shown in Figure 3, the individual pPheA*-expressing transgenic genotypes exhibited various degrees of increase in the levels of Phe (up to 100-fold compared to control plants). We next selected three independent pPheA* genotypes (PheA5, PheA9 and PheA17), exhibits 3:1 segregation Kanamycin resistance, indicating a single T-DNA insertion, as well as on the immunoblot analysis and increased Phe accumulation (Figures 2 and 3). These three independently transformed genotypes exhibited a normal phenotype, apart from the PheA17 genotype, which in some cases showed minor alterations in leaf structure when grown in soil for long periods (Figure 4a).
Amino acids are generally toxic to plants when added externally at relatively high concentrations. To further confirm that plants expressing the pPheA* gene over-produce Phe, we tested their sensitivity to external application of Phe. As shown in Figure 4(b), although the control and pPheA*-expressing genotypes germinated to the same extent on medium lacking Phe, germination of the pPheA*-expressing genotypes was markedly inhibited compared to the controls when grown on medium containing 4 mm Phe.
Effect of PheA* expression on primary metabolism as well as on secondary metabolites derived from aromatic amino acids
The aromatic amino acid biosynthesis network is located in a central position linking primary and secondary metabolism. To test whether the alteration of aromatic amino acid biosynthesis in the pPheA* plants influences the levels of primary metabolism as well as the interaction between primary and secondary metabolism, we performed non-targeted metabolite analyses using both GC-MS and high-resolution LC-MS metabolomics technologies. The GC-MS derivatization method enables analysis of mostly primary metabolites (e.g. sugars, organic acids and amino acids), LC-MS profiling allows the study of secondary metabolites (e.g. phenylpropanoids and glucosinolates). To identify differential metabolites between the three pPheA* transgenic genotypes and wild-type plants, the data from both GC-MS and LC-MS were converted into a mass-intensity matrix. Processing of the GC-MS dataset revealed 2487 mass fragments (a total of 55 identified metabolites; Table S1). In addition, 12 136 mass signals were detected in an LC-MS assay performed in the negative mode (a total of 47 identified metabolites; Table S2). Of these mass signals, 202 GC-MS mass signals and 1384 LC-MS mass signals were altered significantly in the pPheA* genotypes, i.e. common to all three transgenic genotypes compared to the wild-type. Principal component analysis (PCA) based on either the GC-MS or LC-MS mass signals (Figure 5) demonstrated that the metabolic profiles of the three pPheA* transgenic genotypes were clearly separable from that of the control.
Our combined GC-MS and LC-MS analyses further revealed that all metabolites whose levels were affected by pPheA* expression were secondary metabolites. In addition to the increase in Phe, the levels of 11 Phe-derived secondary metabolites were increased in the pPheA* plants compared to the wild-type: sinapyl alcohol, coniferin, caffeoyl glucose, vanillate glucoside, 2-phenylethyl glucosinolate, 2-phenethyl isothiocyanate, benzyl glucosinolate, phenylacetonitrile, acetovanillone, coumarate hexose and ferulate hexose (Figure 6b–l). However, the levels of two other Phe-derived secondary metabolites, namely 3-carboxy-2-hydroxyphenylalanine and sinapoyl malate, were decreased in the pPheA* plants (Figure 6m,n). This indicates that the higher levels of some of the Phe-derived secondary metabolites were not necessarily due to the increased rate of Phe metabolism, but also due to pPheA*-induced flux changes between branches of the phenylpropanoid network. Given this observation, we also wished to test the effect of pPheA* expression on the levels of anthocyanins, which are located far downstream in the phenylpropanoid network. This analysis was performed on 18-day-old Arabidopsis plants, at which time anthocyanin levels are generally very low (Kubasek et al., 1992). To address this issue, we crossed pPheA* plants with the production of anthocyanin pigment 1-Dominant (pap1-D) mutant. This dominant mutant over-produces anthocyanins as a result of over-expression of the PAP1 (MYB75) transcription factor that activates structural genes for anthocyanin biosynthesis (Borevitz et al., 2000). Interestingly, homozygous PheA5::pap1-D plants contained slightly lower anthocyanin levels compared to their homozygous pap1-D parent (Figure S1), indicating that the levels of some of the Phe-derived metabolites (particularly metabolites associated with lignin biosynthesis; Figure 6) were increased at the expense of decreased production of anthocyanins. Thus, our results imply that flux changes in Phe biosynthesis generate flux changes in various branches of Phe-derived secondary metabolites, indicating a novel regulatory cross-interaction between primary and secondary metabolism.
Notably, the levels of six secondary metabolites derived from Trp were also significantly decreased in the pPheA* plants compared to the wild-type (Figure 6o–t and Table S2). The reduced levels of Trp-derived secondary metabolites could be explained by the fact that pPheA* expression increases the ability of the Phe biosynthesis branch compared with the Trp biosynthesis branch to compete for their common precursor chorismate. The Trp level was not significantly changed in the pPheA* plants, but as Trp is a minor amino acid, we decided to analyze the extent of its biosynthesis by an indirect approach that involved testing the sensitivity of the pPheA* plants to growth on 5-methyl-Trp (5MT). 5MT is a Trp analog that slows down Trp biosynthesis through feedback inhibition of the first Trp biosynthetic enzyme anthranilate synthase (AS) (Widholm, 1972; Kisaka et al., 1996). 5MT-resistant plants are also generally associated with increased levels of Trp (Li and Last, 1996). As shown in Figure 7, growth of the PheA*-expressing plants was much more sensitive to 5MT compared to that of the controls, indicating that pPheA* expression down-regulates Trp biosynthesis and as a result also down-regulates the production of the Trp-derived secondary metabolites.
Another interesting observation was the increase in the level of the Tyr-derived secondary metabolite homogentisate in the pPheA* plants compared to the control (Figure 6u). Homogentisate is produced from 4-hydroxyphenylpyruvate by the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD), which is strongly inhibited by several herbicide families, such as isoxazoles, triketones and pyroxazoles, causing bleaching symptoms (Schulz et al., 1993; Secor, 1994; Pallett et al., 1998). We therefore tested the response of the PheA*-expressing plants to growth on medium containing isoxaflutole. As shown in Figure 8(a), PheA*-expressing plants were more resistant to this herbicide than the control plants, in agreement with the increased homogentisate level in them. As homogentisate is also a precursor for the tocochromanols (tocopherols and tocotrienols, commonly referred as vitamin E; DellaPenna and Pogson, 2006), we also tested the levels of these metabolites using HPLC. As shown in Figure 8(b), among the three tocopherol isoforms (α, γ and δ) and three tocotrienol isoforms (α, γ and δ), only the levels of γ-tocopherol and γ-tocotrienol were significantly higher in plants expressing the pPheA* gene. α-tocopherols were detected in control and pPheA* plants, and their levels were comparable in the two genotypes, while the levels of δ-tocopherol, α-tocotrienol and δ-tocotrienol were below the detection limit in both the control and pPheA* genotypes (data not shown).
Effect of PheA* expression on the Arabidopsis transcriptome
Given the minimal effect of pPheA* expression on the primary metabolism and its mostly local effect on secondary metabolism derived from the aromatic amino acids, we also decided to test the effect of this transgenic plant on the Arabidopsis transcriptome. Hence, 10-day-old seedlings of the control and PheA5 plants were subjected to microarray analysis using the Affymetrix AtH1 GeneChip. Hierarchical clustering showed that biological replicates of the PheA* plants were grouped together and were separated from the control datasets (Ward’s algorithm; Figure S2a). However, when differential gene expression was analyzed by anova with a false discovery rate (FDR) P < 0.05 threshold, only seven of the genome-wide genes exhibited more than two-fold change between the pPheA* and wild-type plants (Figure S2b and Table S3). Hence, we concluded that pPheA* expression, while apparently causing minor expression changes (less than two-fold) in a number of genome-wide genes (see separation of genotypes in Figure S2a), has only a limited influence on the Arabidopsis transcriptome.
- Top of page
- Experimental procedures
- Supporting Information
Impact of bacterial feedback-insensitive PheA* expression on the regulation of Phe biosynthesis in plants
Our observation that expression of the bi-functional feedback-insensitive E. coli CM/PDT enzyme in the plastid enhances Phe accumulation in Arabidopsis plants supports extensive published evidence showing that their activities of bacterial enzymes for amino acid metabolism are generally preserved in plant cells. This was previously shown for the aspartate kinase and dihydrodipicolinate synthase enzymes for lysine biosynthesis (Shaul and Galili, 1993). Moreover, as the E. coli CM/PDT enzyme converts chorismate via prephenate into PPY (Zhang et al., 1998), the increase in Phe level in the pPheA* plants also implies that Arabidopsis plants possess an endogenous activity that converts the PPY produced by the bacterial CM/PDT into Phe. In the absence of such an activity, we would have expected to observe a notable accumulation of PPY (the final product of the bi-functional bacterial PheA enzyme) rather than Phe, while the opposite was observed experimentally, i.e. we observed a significant accumulation of Phe while that of PPY remained below the detection level. In order to synthesize Phe via PPY, Arabidopsis plants must possess three distinct enzymatic activities, namely CM, PDT and aromatic amino acid aminotransferase (Figure 1). The presence of endogenous CM activity in plants is well documented (Eberhard et al., 1996;Mobley et al., 1999). In addition, some of the six putative Arabidopsis ADT/PDT isozymes were shown to possess PDT activity when expressed in E. coli, and also complemented a yeast PDT null mutant (Cho et al., 2007), implying that these isozymes can also convert prephenate into PPY in vivo. So far, no aminotransferase converting PPY into Phe has been described in plants. However, studies in bacteria have shown that the reciprocal conversion between PPY and Phe can be catalyzed by a number of aminotransferases (Gelfand and Rudo, 1977; see also http://www.genome.ad.jp/kegg/pathway.html), and it is thus very likely that analogous plant aminotransferases could also catalyze this reciprocal reaction. Moreover, plants naturally possess PPY, and it has also been shown that some plant species can convert Phe into PPY in vivo (Watanabe et al., 2002; Kaminaga et al., 2006).
It is also possible that part of the prephenate that was over-produced by the bacterial CM activity was converted via arogenate into Phe by endogenous plant enzymes. However, our results do not support a major increased flux through arogenate into Phe in the PheA*-expressing plants as the endogenous ADT activity in plants is naturally feedback-inhibited by Phe, and a mutation rendering this activity feedback-insensitive is required to over-produce Phe via the arogenate route (Yamada et al., 2008). Notably, even though arogenate has proven to be a precursor for both Phe and Tyr in plants, an enzyme converting prephenate into arogenate has not yet been identified. Thus, despite a number of missing links, the combination of the present report and previous reports supports the hypothesis that plants can synthesize Phe via both the arogenate and PPY routes.
Cross-regulation of Phe and Trp biosynthesis
pPheA* expression rendered the plants more sensitive than the control plants to growth on medium containing the Trp analog 5MT (Figure 8). In addition, pPheA* expression caused a reduction in the levels of the Trp-derived secondary metabolites 6-hydroxyindole-3-carboxylate-6-O-β-d-glucopyranoside, 6-hydroxyindole-3-carboxylate-β-d-glucopyranosyl ester and tryptophan-N-formyl-methyl ester (Figures 6 and 9). These results indicate that pPheA* expression slows down the flux of Trp biosynthesis. Interestingly, expression of a feedback-insensitive AS of Trp biosynthesis in transgenic rice increased Trp biosynthesis, but had no effect on the levels of Phe and Tyr (Dubouzet et al., 2007). In addition, Trp accumulation in Arabidopsis plants expressing a feedback-insensitive AS showed reduced levels of a number of Phe-derived secondary metabolites (Ishihara et al., 2006). Taken together, these results, combined with present report, support the existence of natural competition of the Trp and Phe/Tyr branches for their common substrate chorismate (Figure 1).
Expression of the PheA* gene indicates novel interactions between primary and secondary metabolism of Phe
Our observation that pPheA* expression leads to changes in the levels of a number of secondary metabolites derived from all three aromatic amino acids (Phe, Trp and Tyr) strongly indicates that fluxes of aromatic amino acid biosynthesis (primary metabolism) influence the levels of secondary metabolites derived from them. Of particular interest are the results indicating that pPheA* caused changes in the levels of the various classes of Phe-derived phenylpropanoid secondary metabolites (Figure 9). pPheA* expression in Arabidopsis enhanced the production of a number of lignin-associated metabolites as well as of vanillate glucoside, both derived from the Phe catabolic product caffeate. In addition, pPheA* expression also enhanced synthesis of the Phe glucosinolate derivatives benzyl glucosinolate and 2-phenylethyl glucosinolate, as well as their two isothiocyanates phenyacetonitrile and 2-phenylethyl isothiocyanate (Barillari et al., 2001; Brader et al., 2006). These Phe-derived secondary metabolites are produced either from Phe (Wittstock and Halkier, 2000) or from its catabolic products cinnamate and benzoate (Graser et al., 2001; Reichelt et al., 2002). In contrast, pPheA* expression reduced the synthesis of the Phe-derived anthocyanins (as observed for the cross between the pPheA* and pap-1D plants), which are located quite far downstream in the phenylpropanoid pathway, suggesting a pPheA*-induced alteration of the competition between the upstream lignin branch and the downstream anthocyanin branch. We also observed a minor reduction in the level of sinapyl malate in the pPheA* plants, apparently due to its negative competition with the increased level of sinapyl alcohol within the lignin metabolism network (Figure 9). The reasons behind the effect of pPheA* expression on the phenylpropanoid patterns are still unknown. It may result from a regulatory effect of the increased flux of Phe biosynthesis or alternatively from the competitive decreased flux of Trp biosynthesis and its further conversion into the Trp-derived secondary metabolites. Finally, our results support previous studies in which alteration of fluxes through various branches in the phenylpropanoid network were observed in response to other metabolic perturbations of Phe metabolism (Li et al., 1993; Howles et al., 1996).
The mechanistic reason for the increased levels of the Tyr-derived secondary metabolites homogentisate, γ-tocopherols and γ-tocotrienols in the pPheA* plants is also still unknown. One possibility to explain this is that the accelerated conversion of chorismate to prephenate by the bacterial CM activity was further channeled into arogenate and Tyr by the endogenous plant enzymes (Figure 1). Alternatively, these Tyr-derived secondary metabolites may be produced either by a putative plant prephenate dehydrogenase activity, for which no candidate gene has yet been reported, or by a putative cytochrome P450 enzyme that is able to convert PPY to 4-hydroxyphenylpyruvate (Figure 9, green arrows).
Finally, our results indicate additional regulatory aspects of aromatic amino acid metabolism compared to those obtained for a mutation reducing the feedback sensitivity of the rice ADT to Phe (Yamada et al., 2008). Although Phe over-production due to an insensitive ADT activity renders the plants more resistant to the Trp analog 5MT (Yamada et al., 2008), Phe over-production via pPheA* expression renders the plants more sensitive to 5MT. Understanding the nature of this difference requires further studies.
pPheA* expression has a minimal effect on primary metabolism and on the Arabidopsis transcriptome
In contrast to the effect of pPheA* expression on the interaction between primary and secondary metabolism of the aromatic amino acid network, this transgene had a minor effect on the primary metabolism (Table S1). This implies that, under normal (non-stress) growth conditions, the network of aromatic amino acid biosynthesis from chorismate (Figure 1) shows minor cross-interactions with the primary metabolism. In addition, pPheA* expression also had a minor effect, if at all, on the Arabidopsis transcriptome (Figure S2b and Table S3). This further indicates that, under normal (non-stress) growth conditions, flux changes in the biosynthesis pathway of the aromatic amino acids from chorismate are not recognized in Arabidopsis plants as signals that influence gene expression programs. Interestingly, similar minor effects on global gene expression were also observed in Trp-over-producing rice plants expressing a feedback-insensitive AS, an enzyme of Trp biosynthesis (Dubouzet et al., 2007). This indicating that under normal (non-stress) growth conditions, fluxes within the entire network of aromatic amino acid biosynthesis from chorismate have a minor influence on gene expression programs.
- Top of page
- Experimental procedures
- Supporting Information
Plant material and growth conditions
Seeds were imbibed for 48 h at 4°C, germinated on Nitsch complete medium (Duchefa; http://www.duchefa.com/) supplemented with 1% sucrose and 50 μg/ml kanamycin, and then resistant seedlings were transferred to soil. Plants were grown in a climate-controlled growth room at 22°C with a 16 h light/8 h dark regime. To test the response of plant growth to the various compounds, homozygous pPheA*-expressing plants were germinated on Nitsch medium as described above, which was supplemented with various compounds as described by the manufacturers: Phe (Sigma–Aldrich; http://www.sigmaaldrich.com/) (Voll et al., 2004); 5MT (Sigma–Aldrich; http://www.sigmaaldrich.com/) (Li and Last, 1996) and the HPPD inhibitor isoxaflutole (BALANCE™, Bayer CropScience; http://www.bayercropscience.com/) (Rippert et al., 2004). The pap1-D mutant (Borevitz et al., 2000) was obtained from the SALK collection (stock name CS3884). The amt1-1 dominant mutant resistant to 5MT (Kreps et al., 1996) was obtained from the European Arabidopsis Stock Center (NASC; http://arabidopsis.info/; stock number N6168).
Plasmid construction and Arabidopsis transformation
The truncated coding DNA sequence of the E. coli PheA gene, encoding the CM and PDT domains, was amplified by PCR using primers 5′-GCCAAGCTTATGGGCATGCCATCGGAAAACCCGTTACTGGC-3′, which introduces an SphI restriction site (underlined), and 5′-CCCCGGAATTCCAACGTCGTTTTCGCCGGAACCTG-3′, which introduces an EcoRI restriction site (underlined). The Rubisco small subunit-3A plastid transit peptide (Shaul and Galili, 1993) was fused in-frame to the 5′ end of the PheA* open reading frame. The PheA* 3′ end was fused to three copies of a HA epitope tag fused to an octopine synthase terminator, and the entire fragment was sub-cloned into the Ti plasmid pART27 (Gleave, 1992). The chimeric pPheA* gene was introduced into Agrobacterium tumefaciens strain EHA-105 and transformed into Arabidopsis plants as previously described (Clough and Bent, 1998).
Immunoblot analysis, chlorophyll analysis and anthocyanin analysis
Immunoblots were performed as previously described (Stepansky and Galili, 2003) using monoclonal anti-HA antibodies (Sigma-Aldrich). Chlorophyll analysis was performed as previously described (Lichtenthaler et al., 1986). Anthocyanin content was determined as previously described (Mita et al., 1997).
Targeted analysis of tocochromanols
Tocopherol and tocotrienol extraction was performed essentially as previously described (Fraser et al., 2000; Bino et al., 2005) with several modifications: aerial tissues of 10-day-old Arabidopsis seedlings (100 mg frozen powder) were extracted with 0.5 ml methanol containing 0.1% butylated hydroxytoluene. The samples were shaken for 5 min at 4°C, and then 0.5 ml of 50 mm Tris/HCl pH 7.5 was added, and the samples were shaken for 10 min at 4°C. Subsequently, 0.4 ml of cold chloroform (4°C) was added, samples were shaken for 10 min (4°C), centrifuged at 10 000 g (4°C) for 10 min, and the supernatant was collected in a new tube. The supernatant was re-extracted with 0.2 ml cold chloroform, and samples were shaken for 10 min (4°C) and centrifuged at 10 000 g (4°C) for 10 min. The chloroform fractions were combined, dried under a stream of nitrogen gas, and re-suspended in 0.1 ml ethylacetate. Extracts were shielded from strong light during the entire preparation. The separation system consisted of an HPLC (Waters 2690; Waters Chromatography; http://www.waters.com/) coupled to a photo diode array detector (Waters 2996), and a YMC-Pack C30 column (250 × 4.6 mm; 5 μm), coupled to a 4 × 3 mm C18 guard (Phenomenex; http://www.phenomenex.com/), maintained at 30°C. The mobile-phase composition, gradient and flow rate were as described by Fraser et al. (2000). The UV spectra were monitored between 200 and 750 nm. Data were collected and analyzed using waters millennium32 software. The absorbance spectra and retention times of eluting peaks were compared with those of commercially available standards [δ-tocopherol and γ-tocopherol (Sigma-Aldrich; http://www.sigmaaldrich.com/), α-tocopherol (Sigma-Aldrich), α-tocotrienol, γ-tocotrienol and δ-tocotrienol (Cayman Chemical; http://www.caymanchem.com) and to the spectra reported by Fraser et al. (2000). Peak areas of the compounds were determined at the wavelength providing maximum absorbance.
Metabolomics analysis using LC-qTOF-MS and GC-MS
Non-targeted metabolic analysis was performed using aerial tissues of 10-day-old wild-type Arabidopsis seedlings (100 mg frozen powder) and seedlings expressing PheA* (n = 5), extracted in 450 μl of 80% methanol. Sample preparation and injection conditions were as previously described (Mintz-Oron et al., 2008). Analysis of the raw UPLC-qTOF-MS data was performed using XCMS software, which performs chromatogram alignment, mass signal detection and peak integration (Smith et al., 2006), from the Bioconductor package (version 2.1) for the R statistical language (version 2.6.1). XCMS was used with the following parameters: fwhm = 10.8, step = 0.05, steps = 4, mzdiff = 0.07, snthresh = 8, max = 1000. Injections of samples in the positive and negative ionization modes and pre-processing was performed independently for each ionization mode. Differential mass ions were determined using Student’s t test (JMP software, SAS Institute Inc., http://www.jmp.com), and 18 differential metabolites were subsequently assigned. The GC-MS analysis was performed as previously described (Malitsky et al., 2008) on the same plant material as for LC-MS (n = 4–6). Xcalibur software version 1.4 (Thermo Finnigan; http://www.thermo.com/) was used for data analysis, and compounds were identified by comparison of their retention index and mass spectrum to those generated for authentic standards analyzed on the same instrument. In cases when standards were not available, compounds were putatively identified by comparison of their retention index and mass spectrum to those present in the mass spectra library of the Max-Planck Institute for Plant Physiology, Golm, Germany (Q_MSRI_ID, http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/msri/gmd_msri.html) and the commercial mass spectra library NIST05 (http://www.nist.gov). The response values for metabolites resulting from the Xcalibur processing method were normalized to the ribitol internal standard. A Student’s t test analysis was performed for metabolites with significant level changes in all three transformed PheA* genotypes using the JMP software. For PCA, the XCMS software was first applied to the GC-MS dataset with the following parameters: fwhm = 4, step = 0.05, steps = 4, mzdiff = 0.5, snthresh = 4, max = 1000 (Smith et al., 2006). Then, PCA plots were generated using tmev4 software (Saeed et al., 2003; Scholz et al., 2004).
Microarray and bioinformatics analysis
Total RNA was extracted from two pools of 100 mg of seedlings for the control and PheA* plants (PheA5 genotype), using an RNeasy Plant Mini Kit (Qiagen, http://www.qiagen.com/) and treated with DNAse RQ-1 (Promega, http://www.promega.com/). Hybridization to the GeneChip Arabidopsis Genome Array (AtH1, Affymetrix, http://www.affymetrix.com) and data extraction were performed according to standard Affymetrix protocols. Transcriptome analysis and removal of the batch effect were performed using Partek Genome Suite software (Partek, http://www.partek.com) and the robust microarray averaging (RMA) algorithm (Irizarry et al., 2003). Changes in expression level were determined by anova analysis. A false discovery rate (FDR) was used to correct for multiple comparisons (Hochberg and Benjamini, 1990). Differentially expressed genes were chosen based on an FDR <0.05 and a two-fold change between genotypes. Functional annotation analysis was performed using the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/index.jsp).
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- Supporting Information
We thank Hanna Levanony, Merav Yativ, Liran Shkop and Clarita BenDayan for excellent technical assistance, and Ester Feldmesser (Department of Biological Sciences) for assistance with the bioinformatics analysis. We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. This study was supported by a Magnet Program of the Israeli Ministry of Industry, Trade and Labor and the Israeli Bio-TOV Consortium including Evogene Ltd, Frutarom Ltd, Hazera Genetics Ltd, Rahan Meristem (1998) Ltd and Zeraim Gedera Ltd. G.G. is the incumbent of the Bronfman Chair in Plant Sciences. A.A. is the incumbent of the Adolpho and Evelyn Blum Career Development Chair. The work in the Aharoni laboratory was supported by research grants from Sir Harry Djanogly CBE, Mrs Louise Gartner (Dallas, TX) and Mr and Mrs Mordechai Segal (Israel). Arye Tishbee is thanked for operating the LC-MS instrument.
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- Experimental procedures
- Supporting Information
Table S1. List of putative metabolites identified in aerial tissues of 10 days old Arabidopsis seedlings by GC-MS analyses.
Table S2. List of putative metabolites identified in aerial tissues of 10 days old Arabidopsis seedlings by UPLC-qTOF-MS and MS-MS analyses.
Table S3. List of mRNA transcripts which were significantly induced or repressed (fold change >2 or <−2) between the control and pPheA* plants.
Figure S1. Effect of pPheA* expression on the shoot anthocyanins content. Anthocyanins content were measured in aerial tissues of 18 days old seedlings of the two genotypes: pap1-D and PheA5::pap1-D. Bars on top of the histograms represent the standard error. Different letters on top of the histograms represent statistically significant changes (P < 0.05, using Student’s t-test).
Figure S2. Microarray data analysis of the PheA5 and control genotypes. a, Hierarchical clustering dendrogram of the different samples, each presented in two duplicates (Ward’s algorithm). The two biological replicates of the pPheA* plants (blue) were group together and separated from the control (red). The separation was according to plants genotype and not according to datasets (set 1 – green and set 2 – purple). b, Volcano plot for differentially expressed genes. Differentially expressed genes appear above the thick horizontal lines (Step-up P value <0.05). Genes induced >2-fold are outside of the right vertical line, and those repressed >2-fold are outside the left vertical line.
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