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First published online May 21, 2004; 10.1104/pp.104.039347 Plant Physiology 135:828-839 (2004) © 2004 American Society of Plant Biologists Glucosinolate and Amino Acid Biosynthesis in Arabidopsis1Department of Metabolic Biology, John Innes Centre, Norwich NR4 7UH (B.F.); BayerCrop Science N.V., B–9000 Gent, Belgium (G.C., J.B., G.V.); and Nutrition Division, Institute of Food Research, Norwich NR4 7UA, United Kingdom (M.T., R.M.)
Enzymes that catalyze the condensation of acetyl coenzyme A and 2-oxo acids are likely to be important in two distinct metabolic pathways in Arabidopsis. These are the synthesis of isopropylmalate, an intermediate of Leu biosynthesis in primary metabolism, and the synthesis of methylthioalkylmalates, intermediates of Met elongation in the synthesis of aliphatic glucosinolates (GSLs), in secondary metabolism. Four Arabidopsis genes in the ecotype Columbia potentially encode proteins that could catalyze these reactions. MAM1 and MAML are adjacent genes on chromosome 5 at the Gsl-elong locus, while MAML-3 and MAML-4 are at opposite ends of chr 1. The isopropylmalate synthase activity of each member of the MAM-like gene family was investigated by heterologous expression in an isopropylmalate synthase-null Escherichia coli mutant. Only the expression of MAML-3 restored the ability of the mutant to grow in the absence of Leu. A MAML knockout line (KO) lacked long-chain aliphatic GSLs, which were restored when the KO was transformed with a functional MAML gene. Variation in expression of MAML did not alter the total levels of Met-derived GSLs, but just the ratio of chain lengths. MAML overexpression in Columbia led to an increase in long-chain GSLs, and an increase in 3C GSLs. Moreover, plants overexpressing MAML contained at least two novel amino acids. One of these was positively identified via MS/MS as homo-Leu, while the other, with identical mass and fragmentation patterns, was likely to be homo-Ile. A MAML-4 KO did not exhibit any changes in GSL profile, but had perturbed soluble amino acid content.
Arabidopsis contains a series of glucosinolates (GSLs) derived from elongated forms of Met (Table I; Haughn et al., 1991  ). Elongation of Met is by a process analogous to the synthesis of Leu from 2-oxo-3-methylbutanoate (Fig. 1, a and b). Met is initially transaminated to an oxo acid. The oxo acid undergoes condensation with acetyl coenzyme A (CoA) to result in 2-methylthiopropylmalate, which then undergoes isomerization and oxidative decarboxylation to result in the net gain of a single methyl unit to the oxo acid. This elongated oxo acid can be transaminated to an amino acid and enter core GSL biosynthesis, or else undergo further rounds of acetyl CoA condensation (Fig. 1, a and b; Chisholm and Wetter, 1964; Graser et al., 2000). In Arabidopsis leaves, the major GSLs are derived from Met that has either undergone a single round of elongation to produce 3C GSLs, such as 3-methylsulphinylpropyl, 3-hydroxypropyl, or 2-propenyl GSLs, or two rounds of elongation to produce 4C GSLs, such as 4-methylsulphinylbutyl and 3-butenyl GSLs. The 3C GSLs predominate in the ecotype Landsberg erecta (Ler), while 4C GSLs predominate in ecotype Columbia (Col-0). In addition, all ecotypes produce similar amounts of long chain GSLs in leaves and seeds, derived from four, five, and six rounds of Met elongation. The variation in 3C to 4C ratio is determined by alleles at the Gsl-elong locus on chromosome 5 (de Quiros et al., 2000; Kroymann et al., 2001). In Col-0, this locus contained two genes, MAM1 (At5g23020) and MAML (At5g23010), which have high levels of homology to isopropylmalate synthase (IPMS; EC 2.3.3.13) that catalyzes acetyl CoA-oxo acid condensation in Leu biosynthesis. Of these, the MAM1 gene has been shown to determine the production of 4C GSLs (Kroymann et al., 2001). Variation in MAM1 has no effect on the expression of 6C, 7C, and 8C GSLs. Hence it is likely that there are other genes in Arabidopsis responsible both for 3C GSL synthesis, and for 5C, 6C, 7C, and 8C GSL biosynthesis.
In addition to determining the ratio of 3C to 4C chain lengths, the Gsl-elong locus functions as a quantitative trait locus determining overall levels of Met-derived GSLs (de Quiros et al., 2000 ; Kroymann et al., 2001), similar to a quantitative trait locus in Brassica that determines both 3C to 4C ratio and total amounts (Mithen et al., 2003). Fine mapping and sequence analysis in several Arabidopsis ecotypes reveal that the Gsl-elong locus comprises, in addition to MAM1 and MAML, a further gene designated MAM2. Whereas all ecotypes appear to have a functioning MAML gene, only some ecotypes have both MAM1 and MAM2, while others have either a functioning MAM1 or a functioning MAM2. The relationship between MAM1 and MAM2 is complex; putative reciprocal deletion of MAM2 in Col-0 and MAM1 in Ler has resulted in the remaining MAM1 and MAM2 genes segregating as alleles of each other, and, in other ecotypes, there is evidence for genetic interchange between MAM1 and MAM2 (Kroymann et al., 2003). Ecotypes that have a functioning MAM1 gene, such as Col-0, make 4C GSLs, whereas those in which MAM1 is impaired in function synthesize 3C GSLs. Whether 3C synthesis is due to MAM2 expression, or to other genes, has not been functionally demonstrated. In addition to these three genes, Arabidopsis contains two more genes in this family, designated MAML-3 (At1g74040) and MAML-4 (At1g18500), both found on chromosome 1.
The predicted proteins of all five MAM genes suggest that they may catalyze the condensation of acetyl CoA with 2-oxo acids (Fig. 1c); they all have highly conserved domains near the amino terminus which are signatures of the active site for oxo acid condensation reactions (Evans et al., 1991
Within secondary metabolism, all or some of these genes are likely to function in the synthesis of GSLs, and one or more must function as an IPMS. Currently, a functional analysis has only been undertaken for MAM1 (Kroymann et al., 2001
The Arabidopsis Col-0 MAM-Like Gene Family Within this paper, we will refer to the four Col-0 members of the gene family as MAM1 (At5g23020), MAML (At5g23010), MAML-3 (At1g74040), and MAML-4 (At1g18500). The predicted protein for each MAM synthase contains a LeuA domain and a chloroplast leader-peptide as predicted by TargetP, P > 0.9 (Fig. 1c). MAM1 and MAML form a subgroup of the Arabidopsis MAM synthase family sharing 78% amino acid identity, while MAML-3 and MAML-4 form another subgroup sharing 90% identity (Fig. 1c).
Expression constructs were designed to heterologously express the predicted mature peptide encoded by each MAM-like Arabidopsis gene, with the addition of an N-terminal 6x His tag. MAM1, MAML, MAML-3, and MAML-4 lacking their predicted leader-peptide sequences were amplified from total Arabidopsis cDNA and cloned into the E. coli expression vector pQE-30 to give pQE-MAM1, pQE-MAML, pQE-MAML-3, and pQE-MAML-4. The E. coli strain CV512 has a nonfunctional IPMS (
Characterization of MAM-Like Insertion Lines As neither MAML nor MAML-4 exhibited IPMS activity, they may be involved in GSL biosynthesis. We obtained homozygous knockout (KO) lines for MAML and MAML-4. MAML::En1 (Col-0 background) contains a single, stable En-1 insertion in the 1st exon of MAML and Garlic1175 (Col-0 background) contains a single T-DNA insertion in the 10th intron of MAML-4 (Fig. 3a). These two lines are referred to as the MAML KO and MAML-4 KO lines, respectively. Reverse transcription (RT)-PCR analysis of MAML KO cDNA showed that no MAML transcript was produced (data not shown), while analysis of MAML-4 KO cDNA showed that chimeric MAML-4::T-DNA fusion transcripts were produced. However, the chimeric transcript in MAML-4 KO was present at considerably reduced levels compared to the normal transcript in Col-0 (Fig. 3b). No compensatory increase in expression was observed for the other MAM synthase genes. The growth and appearance of the MAML KO line did not differ significantly from the wild-type controls. However, the MAML-4 KO line showed a 20% reduction in germination compared to Col-0. Subsequent growth appeared normal.
MAML Is Required for Long Chain GSLs GSLs were analyzed in the seeds and leaves of the MAML KO line. There was a complete absence of 6C, 7C, and 8C GSLs (Fig. 4, a and b). Despite the loss of these compounds, the total level of Met-derived GSLs in the knockout and controls was not significantly different (Table II), due to enhanced levels of 4C GSLs. To test whether these alterations in GSL composition were controlled by a nonfunctional MAML allele, both the MAML KO line and wild-type Col-0 were transformed with vector pTKC28, containing a wild-type genomic copy of the MAML gene with the endogenous promoter replaced by the cauliflower mosaic virus (CAMV) 35S promoter. Production of 6C, 7C, and 8C GSLs was restored in the MAML KO line and enhanced in wild-type Col-0 (Fig. 4c; Table II). Segregation analysis of T2 transformants and subsequent analysis of T3 families confirmed that the enhanced levels of long chain GSLs in transgenic Col-0 was due to the presence of the transgene (Fig. 4). The total level of aliphatic GSLs was the same in the seeds of T3 plants whether they possessed the transgene or had lost it through segregation (70.2 ± 1.31 µmol g–1, n = 15 and 75.9 ± 4.79 µmol g–1, n = 5, respectively), and also in leaves (20.5 ± 1.95 µmol g–1 and 17.4 ± 2.41 µmol g–1, respectively). However, the ratio of chain lengths varied in both tissues; those plants that contained the transgene had higher levels of long chain GSLs and 3C GSLs, but lower levels of 4C GSLs compared to wild-type plants and outsegregants in both seeds and leaves (Fig. 4).
Overexpression of MAML Results in the Synthesis of Novel Amino Acids As acetyl CoA-oxo acid condensation is also an important component of amino acid biosynthesis, we investigated the amino acid content of 35S::MAML. Soluble amino acids were extracted from 9-d-old seedlings of 35S::MAML and derivatized with the AccQ Tag reagent. Amino acids were separated and detected by liquid chromatography-fluorescence detection (LC-FLD), and quantified using standards. All amino acids identified in 35S::MAML seedlings were at equivalent levels to wild type (Col-0) with the exception of Tyr. Soluble Tyr decreased more than 2-fold in 35S::MAML seedlings (37 ± 2.5 pmol mg–1 in 35S::MAML and 100 ± 7.4 pmol mg–1 in Col-0, n = 4, P < 0.005; Fig. 5). Furthermore, two new peaks, not seen in the wild type, were observed in the LC-FLD chromatograms of 35S::MAML extracts (Fig. 6a). These compounds were investigated by liquid chromatography-mass spectrometry (LC-MS) in derivatized 35S::MAML extracts. The most abundant ion in both the new peaks had a mass of 316, which corresponds to a prederivatization mass of 145 (Fig. 6b). The second of the two peaks has an identical retention time to derivatized homoleucine (11.6 min; Fig. 6, a and b).
To obtain ionization spectra for the novel amine compounds an LC-MS/MS method was developed for underivatized extracts. Two novel peaks of mass 146 (i.e. M+H+) coeluted between 6.2 and 6.6 min in 35S::MAML extracts (Fig. 6d). Homoleucine also eluted at 6.5 min. Ionization spectra were obtained for both novel peaks and the authentic standard (Fig. 6e). All three mass spectra were identical, with a major transition from 146 to 100 amu. This transition is likely to be due to the loss of an HCOOH group. In summary, the underivatized compound eluting at 6.5 min is homoleucine. The earlier peak represents an isomer of homoleucine and is almost certainly homo-Ile. Further analysis suggested the occurrence of a further novel amino acid in 35S::MAML with derivatized M+H+ mass of 330, which is consistent with dihomoleucine and/or dihomoisoleucine (Fig. 6c).
MAML-4 KO plants had similar GSLs to their wild-type control (data not shown), but had perturbed amino acid content. There were highly significant (P < 0.005) increases in His, Val, Asn, and Gln when compared to wild-type Col-0, and less significant decreases in Leu (P = 0.057; Fig. 7). The less significant decrease in Leu was due to just one of the four replicates that had similar levels to wild-type Col-0 (MAML-4 KO: 26.6, 27.5, 18.2, and 36.7 pmol mg–1; Col-0: 40.0, 38.0, 38.0, and 32.0 pmol mg–1). If this anomalous result in excluded, the level of probability of a decrease in Leu in MAM4-L KO compared to wild type decreases to P = 0.01.
Heterologous expression in an E. coli IPMS null mutant indicated that MAML-3 is likely to function as an IPMS gene in planta (Fig. 2). No IPMS activity was detected for MAM1, MAML-4, or MAML. While this is not definite proof for lack of IPMS activity, as eukaryotic posttranslational processing may be required, it suggests that these genes are less likely to function as IPMS in planta compared to MAML-3 and may therefore be involved in GSL biosynthesis. This result is contrary to a previous report that claimed that heterologously expressed MAM1 and MAML possessed IPMS activity, although no supporting data were provided (Junk and Mourad, 2002 ). As the activity of MAM1 had been previously studied (Kroymann et al., 2001), we obtained knockout lines for MAML and MAML-4. As part of characterization of these lines, it was interesting to note the relatively poor expression of MAM1 in Col-0 compared to the other members of this gene family (Fig. 3b).
Through both analysis of MAML KO and overexpression of MAML in MAML KO and wild-type Col-0, we have shown that the MAML gene is required for 6C, 7C, and 8C GSL synthesis, suggesting that its product can catalyze the condensation of 2-oxo-8-methylthiooctanoate and longer homologs with acetyl CoA (Fig. 8). The ability of MAML to catalyze multiple condensation reactions is analogous to an enzyme in Methanococcous jannaschii that can catalyze condensation of a series of elongated 2-oxo acids (Howell et al., 1998
Knocking out MAML led to the loss of 6C, 7C, and 8C GSLs, and an equivalent increase in 4C and 5C GSLs (Fig. 3b; Table II), resulting in no overall change in total Met-derived GSL content. Likewise, overexpression of MAML led to an increase in 3C, 6C, 7C, and 8C GSLs and a reduction in 4C and 5C GSLs, again with no change in overall amounts. This indicates that MAML does not alter the flux of Met homologs into GSL biosynthesis (possibly by 2-oxo-3-methylthiobutanoate/acetyl CoA condensation) but only the extent of subsequent 2-oxo acid elongation (Fig. 8). The total levels of Met-derived GSLs found in the analyses of the wild types and primary transgenics (Table II), and those found in subsequent analyses of transgenic plants and outsegregants (Fig. 5), are quite different, but are within the range of variation observed previously. When MAML was overexpressed in Arabidopsis Col-0 two novel amino acids were detected in 35S::MAML seedlings with mass 145 (Fig. 6, a and b). On the basis of HPLC and MS we positively identified one of these as homoleucine, and it is highly likely that the other, which has an identical mass and fragmentation pattern but a slightly different retention time, is isohomoleucine (Fig. 6, d and e). The presence of these novel amino acids strongly indicates that MAML is capable of initiating elongation of the Leu intermediate, 2-oxo-4-methylpentanoate, and the Ile intermediate, 2-oxo-3-methylpentanoate (Fig. 8). Indeed, both intermediates are 2-oxo acids and possess five-carbon backbones and proximal methyl groups, as does 2-oxo-5-methylthiopentanoate in Met elongation for GSL biosynthesis (Fig. 1, a and b). Moreover we detected low levels of a third amino acid, with a mass consistent with dihomoleucine and dihomoisoleucine indicating that MAML can initiate multiple rounds of Leu/Ile elongation, in an analogous manner to Met elongation (Fig. 6c). The absence of homoleucine and homoisoleucine from wild-type plants indicates that under normal conditions MAML is confined to GSL biosynthesis, but when spatially, temporally, and/or quantitatively misexpressed MAML may promiscuously initiate elongation of oxo acids, leading to aberrant metabolism (Fig. 8). Overexpression or misexpression of MAM1 may also result in novel amino acid biosynthesis. Overexpression of MAML also caused a significant decrease in soluble Tyr, without a concomitant decrease in the related amino acid Phe. This change cannot be explained by current models of amino acid biosynthesis.
Although we can now ascribe functions in GSL synthesis to MAM1 (Kroymann et al., 2001 There remains the question of which enzymes in Arabidopsis catalyze the initial 2-oxo-3-methylthiobutanoate condensation in Arabidopsis ecotypes with impaired MAM1 function. While MAM2 is a strong candidate, MAML-3 and MAML-4 may also be involved. As we show that the MAML gene can function both in long-chain GSL biosynthesis and in the synthesis of long-chain forms of Leu, it is possible that both MAML-3 and MAML-4, in addition to MAM2, can also function in short chain GSL biosynthesis and in amino acid biosynthesis (Fig. 8). A degree of redundancy in genes determining this reaction may explain why no null Met-derived GSL mutants have been described in Arabidopsis.
Bioinformatic Analyses
The CD search at the National Center for Biotechnology Information (Altschul et al., 1997
Plants were routinely grown in Arabidopsis mix (2 parts Levington's M3 potting compost to 1 part grit/sand) under standard glasshouse conditions at approximately 20°C. For aseptic growth, seeds were surface-sterilized and plated on growth medium (1x Murashige and Skoog salts plus vitamins [Duchefa, Haarlem, the Netherlands], 1% Suc, 0.8% agar [BACTOAGAR, Fisher Chemicals, Loughorough, UK], and 2.5 mM MES, pH 5.7). The seeds were stratified for 2 d at 4°C in the dark before germination in a growth room (16 h light/8 h dark, 20°C).
Leaf tissue samples were ground in liquid nitrogen, and total RNA was extracted using the RNeasy plant mini kit (Qiagen, Crawley, UK) and eluted in 40 µL of diethyl pyrocarbonate treated water. Contaminating DNA was removed by DNase treatment using the Ambion DNA-free kit (Huntingdon, UK). Five micrograms of total RNA was used to make first strand cDNA using SuperScript II (Invitrogen, Paisley, UK) in a 20-µL reaction with oligo(dT) primers according to the manufacturer's instructions. The completed reaction was diluted 50-fold, and in subsequent PCR 1 µL of the dilution was used per 10 µL of reaction mix.
MAM1, MAML, MAML-3, and MAML-4 lacking their predicted leader-peptide sequence were PCR amplified from Col-0 leaf cDNA in a 50-µL reaction containing 5 µL of cDNA dilution and 2 units of PfuUltra (Stratagene, Amsterdam), 1x supplied buffer, 0.3 µM each primer, and 0.2 mM dNTPs. An initial denaturation step of 96°C for 2 min was followed by 30 cycles of 94°C for 10 s, 55°C for 15 s, and 72°C for 2 min. Finally the products were extended by incubation at 72°C for 10 min. MAM1 was amplified using the primers MAM1/49SacI and MAM1/TGAXhoI; MAML with MAML/49SacI and MAML/TGAXhoI; MAML-3 with MAML-3/46BglII and MAML-3/TGAPstI; and finally MAML-4 with MAML-4/57SacI and MAML-4/TGAXhoI. After amplification the MAML-3 product was digested with BglII and PstI, while the MAM1, MAML, and MAML-4 products were digested with SacI and XhoI and gel purified. The products were then ligated into the inducible expression vector pQE30 (Qiagen) digested with BamHI and PstI in the case of MAML-3 to give pQE-MAML-3, or SacI and SalI in the case of MAM1, MAML, and MAML-4 to give pQE-MAM1, pQE-MAML and pQE-MAML-4. The four constructs and the empty vector were used to transform E. coli strain m15 (Qiagen) containing the Lac repressor plasmid pREP4. The constructs were isolated and sequenced to ensure no mutations had been introduced.
Plasmids pQE-MAM1, pQE-MAML, pQE-MAML-3, pQE-MAML-4, and an empty vector control were transferred into the Leu auxotrophic E. coli strain CV512 obtained from the CGSC E. coli Genetic Stock Centre (CGSC no. 5539) and containing pREP4. To test for complementation CV512 containing each of the constructs grown on Luria-Bertani medium was streaked onto plates of solid M9 media (1x M9 salts [48 mM Na2HPO4, 22 mM KH2PO4, 8.5 mM NaCl, and 18.7 mM NH4Cl]; 2% [w/v] Glc; 1 mM thiamine; 1 mM MgSO4; 0.1 mM CaCl2; kanamycin, 50 µg mL–1; and carbenicillin, 100 µg mL–1), M9 media supplemented with 0.1 mM isopropyl-
CV512 containing pQE-MAM1, MAML, MAML-3, MAML-4, or an empty vector control was used to inoculate 10 mL of Luria-Bertani medium and induced with 0.1 mM IPTG at mid-log phase. After 2 h total protein was extracted, separated by SDS-PAGE (Laemmli, 1970
The MAML insertion line, MAML KO, was identified by PCR screening an En-1 mutagenized population of Arabidopsis, ecotype Col-0 (Baumann et al., 1998
DNA was extracted from the leaves of each insertion line using a modified version of the cetyl-trimethyl-ammonium bromide method (Lister et al., 2000
For semiquantitative RT-PCR analysis, five microliters of cDNA dilution was used in a 50-µL PCR mixture containing 1 unit Taq polymerase (Invitrogen) and 0.15 µM each primer; the reaction was allowed to proceed for 18 cycles. The primer pairs used are shown below. The constitutively expressed housekeeping gene adenine phosphoribosyltransferase (APT) was used as an mRNA loading control (Moffatt et al., 1994
GSLs were extracted from 300 mg of seeds, converted to desulphoglucosinolates, and analyzed by LC-MS with atmospheric pressure chemical ionization, as previously described (de Quiros et al., 2000
Transgenic Arabidopsis were generated by Agrobacterium tumefaciens mediated transformation with the T-DNA vector pTKC28 through floral dipping (Clough and Bent, 1998
A total of 100 mg of 9 d-old seedlings was homogenized in 1 mL of 45°C 70% methanol and incubated at 45°C for 10 min. The supernatant was removed and the pellet extracted twice more and the supernatants pooled. The supernatants were dried under a fixed nitrogen line at 45°C and resuspended in 0.02 M HCl. The extract was then filtered through an ultrafree-MC 0.22-µm filter column (Millipore, Bedford, MA). Ten microliters of eluate was derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using the AccQ Tag system (Waters, Milford, MA). The derivatized amino acids were separated on a Waters Alliance 2695 Separation Module through a reverse phase AccQ Tag column (Waters) at 37°C using a 65-min gradient of sodium acetate buffer (0.1 M sodium acetate pH 5.80, 2.7 µM EDTA, and 6.9 mM triethylamine), acetonitrile, and water at a flow rate of 1 mL min–1. Derivatized amino acids were detected by excitation at 250 nm and emission at 395 nm using a Waters 474 scanning fluorescence detector. Millenium32 Chromatography Manager software (Waters) was used to analyze the data. Individual amino acids were identified and quantified using a calibration curve generated by the injection of standards of known concentrations. Four extractions were performed for each line analyzed to ensure reproducibility. For LC-MS/MS analysis of derivatized amino acids, derivatized extracts were separated on a Thermofinnigan Surveyor HPLC (Hemel Hempstead, UK) through a reverse phase Luna C18 column (Phenomonex, Cheshire, UK) using a gradient of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in methanol) at a flow rate of 300 µL/min throughout. Two gradients were used. Gradient 1 was as follows: 30% solvent B at start, linear gradient to 70% solvent B over 26.6 min, linear gradient to 97% solvent B over 2.4 min, linear gradient to 30% solvent B over 0.2 min, and 30% solvent B for 6.8 min. Gradient 2 was as follows: 5 min at 7% solvent B, linear gradient to 40% solvent B over 20 min, linear gradient to solvent 97% B over a further 20 min, and 97% solvent B for 5 min. Between runs the column was reequilibrated with 7% solvent B for 10 min. Derivatized amino acids were detected by UV A250. For LC MS/MS analysis of underivatized amino acids, untreated extracts were separated on a Thermofinnigan Surveyor HPLC through a reverse phase Luna C18 column using a gradient of solvent A (0.1% heptafluorobutyric acid in water; Sigma, St. Louis) and solvent B (acetonitrile) at a flow rate of 300 µL min–1 throughout. The gradient was as follows: 10% solvent B at start, linear gradient to 25% solvent B over 20 min, linear gradient to 40% solvent B over 2.5 min, linear gradient to 10% solvent B over 0.5 min. Between runs the column was reequilibrated with 10% solvent B for 10 min. After column separation, amino acids in the derivatized and underivatized extracts were detected by positive mode electrospray ionization in a Thermofinnigan LCQ DecaXP ion-trap mass spectrometer. The source conditions were 5.2 kV source voltage, 350°C capillary temperature, 50 units sheath gas, no auxiliary gas. Ions of interest were selected for fragmentation using an isolation width of 2 m/z and collision energy of 35% without wideband activation.
All primers were synthesized by Sigma Genosys (Cambridge, UK). For cDNA cloning:
Insertion site analysis:
For RT-PCR analysis:
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
We thank Lionel Hill for the LC-MS/MS analyses, and Konstantinos Petritis for help with separating underivatized amino acids. We also thank Alison Smith and Steve Rawsthorne for proof reading preliminary drafts. Received January 20, 2004; returned for revision March 1, 2004; accepted March 1, 2004.
1 This work was supported by the John Innes Foundation (BF), by the BBSRC, and by UK Bayer Crop Science.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.039347. * Corresponding author; e-mail richard.mithen{at}bbsrc.ac.uk; fax 44–1603–507–723.
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TOP
ABSTRACT
RESULTS
LeuA (IPMS-Null) E. coli Mutant
-amino-butyrate.
-D-thiogalactopyranoside (IPTG), and M9 media supplemented with 0.3 mM Leu. The plates were incubated overnight at 37°C before being photographed. This was performed at least three times. CV512 containing each construct was also used to inoculate the same liquid M9 medium to an OD600 of 0.01 and incubated at 37°C with shaking. Growth curves were obtained by measuring the OD600 at the times indicated in 
