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First published online September 24, 2004; 10.1104/pp.104.047506 Plant Physiology 136:3058-3069 (2004) © 2004 American Society of Plant Biologists The Arabidopsis phenylalanine insensitive growth Mutant Exhibits a Deregulated Amino Acid Metabolism1Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824–1312 (L.M.V., E.E.A., A.P.M.W.); and Botanisches Institut der Universität zu Köln, D–50931 Koln, Germany (G.F.)
Amino acids and amino acid analogs have been used in numerous genetic screens to isolate mutants deficient in amino acid biosynthetic pathways or in the regulation of amino acid metabolism. Several of these mutants exhibit relaxed feedback control of branched amino acid biosynthetic pathways and are thus resistant to accumulation of pathway end products. For example, feedback-regulated enzymes of the shikimate pathway are anthranilate synthase on the branch leading to Trp and chorismate mutase on the branch leading to Phe and Tyr. A feedback-insensitive mutant of anthranilate synthase  , trp5-1, is resistant to toxic Trp analogs. Mutants resistant to Phe have not previously been reported, and this article describes the isolation of the recessive Arabidopsis Phe insensitive growth mutant pig1-1 by a forward genetic screen. pig1-1 was not only tolerant to Phe, Tyr, and Trp, but also to other, not biosynthetically related amino acids. Amino acid contents in pig1-1 were significantly elevated with respect to wild-type controls but, in contrast to the wild type, dramatically decreased when plants were supplemented with 2 mM Phe. Protein contents were similar in the mutant and the wild type at all tested conditions. Phe catabolism was similar to the wild type in pig1-1 roots but was significantly increased in pig1-1 shoots. Phenylalanine uptake into the root, its root-to-shoot translocation, and Phe and phenylpropanoid contents were unaltered in pig1-1, indicating that pig1-1 is not affected in amino acid translocation or the shikimate pathway. Instead, the response of pig1-1 toward amino acid feeding indicates that amino acid metabolism is generally deregulated in pig1-1.
End product inhibition is frequently observed at branching points of branched amino acid biosynthetic pathways to balance the flux between the different amino acid end products in one pathway. External supply of feedback-competent amino acids to the plant growth medium disturbs amino acid homeostasis by inhibiting feedback-regulated enzymes and thus causes shortages of other amino acids resulting from these biosynthetic pathways. Naturally, such induced amino acid shortages are toxic to the plant.
These toxic effects of externally applied amino acids have been employed to isolate feedback-insensitive Asp kinase mutants (Rognes et al., 1983
Feedback regulation in the plant shikimate pathway occurs at the branching point chorismate (Schmid and Amrhein, 1995
Elevated contents of particular free amino acids could also be attained by introducing feedback-insensitive isoforms into plants. For example, overexpressing feedback-insensitive orthologs of AS, Asp kinase, acetohydroxy acid synthase, or dihydrodipicolinate synthase from plants or bacteria (Falco et al., 1995  ; Cho et al., 2000; Galili et al., 2000; Lee and Duggleby, 2002; Zhu and Galili, 2003) have resulted in substantially increased free Trp, Thr, Val, and Lys contents, respectively. Here, we describe the isolation of an Arabidopsis Phe insensitive growth (pig) mutant, which is able to germinate in the presence of 10 mM Phe, while 6 mM Phe completely inhibits the germination of wild-type Arabidopsis. When grown on Murashige and Skoog (MS) agar plates, free amino acids in pig1-1 are elevated by 1.4- to 2.3-fold compared to the wild type. However, when grown on media supplemented with 2 mM Phe, free amino acid contents, except Phe and Tyr, are up to 4-fold lower in pig1-1 than in the wild type. In addition to this concomitant decrease of all free amino acids upon external Phe supply, pig1-1 displays an increased tolerance to a range of externally applied, feedback-competent amino acids. This indicates that a regulatory mechanism of amino acid metabolism, which may resemble or correspond to the general control in yeast (Saccharomyces cerevisiae), is deregulated in pig1-1. To the best of our knowledge, pig1-1 is the first Arabidopsis mutant that is characterized by constitutively increased foliar free amino acid contents.
Isolation of Phe-Insensitive Arabidopsis Mutants To establish a robust screen for Phe-resistant Arabidopsis mutants, we assessed the impact of aromatic amino acids on seed germination in dose-response curves for the wild-type accessions Col-0 (data not shown) and Ws-2 (Fig. 2A). Aromatic amino acid concentrations higher than 6 mM completely inhibited germination of the Arabidopsis wild types (for Ws-2, see Fig. 2A). Seedlings were not viable on soil if they had been preincubated on aromatic amino acids for more than 14 d before they were transplanted to soil (data not shown). When intermediate concentrations of 2 to 4 mM aromatic amino acid were applied, seedling lethality was most pronounced in media containing Trp but less severe in media containing Tyr (Fig. 2), irrespective of the accession tested. The calculated I50 values for the Ws-2 accession were 2.3 mM Trp, 3.7 mM Tyr, and 4.0 mM Phe. Remarkably, the lethality of all three amino acids to Arabidopsis seedlings decreased with increasing Suc supplements, indicating a cross-talk between carbon availability and aromatic amino acid toxicity.
We employed half-strength MS plates supplemented with 10 mM Phe and 0.5% Suc to screen an M2 population of 200,000 ethyl methanesulfonate-treated Arabidopsis (cv Ws-2) individuals as described in more detail in "Materials and Methods." In this screening, eight mutant lines were isolated. Hence, the pig mutation appears at a frequency of 1/25,000. Among the isolated mutants, the growth of pig1-1 was least affected on medium containing 10 mM Phe; therefore, pig1-1 was selected for further analysis. The Phe-insensitive phenotype of pig1-1 was confirmed in the M3 and M4 generations before pig1-1 was backcrossed three times to the wild type to clean the genetic background from second site mutations. Backcrosses of pig1-1 pollen to wild-type stigmata demonstrated that the pig1-1 phenotype is caused by a single nuclear recessive mutation because of two observations: (1) the F1 progeny of backcrosses to the wild type was uniformly Phe intolerant when grown on MS-Phe, and (2) the ratio of Phe-susceptible to Phe-tolerant F2 individuals of these backcrosses was 204 to 55, which best fits a 3:1 segregation according to  2 tests.
While 10 mM Phe is lethal to both tested wild-type accessions, pig1-1 seedlings displayed a germination rate of 81.2% ± 3.2% on 10 mM Phe containing MS. In addition, pig1-1 also tolerated elevated Tyr and Trp concentrations (Fig. 2). Eight millimolar of either Tyr or Trp were 100% lethal to the wild type, whereas 66% of the pig1-1 seedlings survived on 8 mM Tyr and 59% survived on 8 mM Trp. The values for 8 mM Tyr are extrapolated because Tyr solubility in the medium was limited to slightly less than 8 mM.
While the dose-response curves of Phe, Tyr, and Trp for wild-type seeds did not indicate pronounced differences between the treatments (Fig. 2), specific effects for individual aromatic amino acids could be observed upon feeding intermediate concentrations (4 mM) to wild-type (cv Ws-2) seedlings (Fig. 3). To demonstrate that the degree of aromatic amino acid toxicity depended on carbon supply, Figure 3 depicts growth effects observed on half-strength MS, 4 mM Phe, and 2% Suc, i.e. milder selection conditions than in the mutant screen where 0.5% Suc and 10 mM Phe were employed. Interestingly, feeding of 4 mM Phe or Tyr to the wild type induced a reticulate leaf phenotype with darker paraveinal tissue and light green to chlorotic intercostal regions (Fig. 3, A and C). A similar phenotype has been reported for the cue1 mutant that is deficient in a plastidic phosphoenolpyruvate/phosphate translocator PPT (Li et al., 1995
pig1-1 Does Not Share Phenotypic Features of trp Mutants
The pathway for aromatic amino acid biosynthesis is bifurcated with one branch leading to Trp and the other one leading to Phe and Tyr. The loci encoding enzymes of the biosynthetic branch toward Trp, TRP1 to TRP5, are covered with mutants (for summary, see Li and Last, 1996 Because Phe and Trp are biosynthetically related, it was investigated whether pig1-1 shared one of the already described trp phenotypes. pig1-1 and the trp mutants were subjected to the screening conditions that had been previously employed to isolate the individual trp mutants (Table I). Alternatively, pig1-1 was grown alongside the Trp auxotrophic mutants trp2-1 and trp3-1 on Trp-deplete medium (Table I). pig1-1 did not share any of the examined traits with the individual trp mutants, and, vice versa, the trp mutants trp1-100, trp2-1, trp3-1, and trp5-1 did not display tolerance to 10 mM Phe. In summary, this suggests that the mutation in pig1-1 differs from the mutations in trp1-100, trp2-1, trp3-1, and trp5-1.
pig1-1 Displays a Deregulated Amino Acid Biosynthesis Because of its Phe-insensitive growth phenotype, examining amino acid contents in pig1-1 was of paramount interest. Therefore, the free amino acid contents of wild type and pig mutants grown on MS agar plates with and without 2 mM Phe were determined (Table II). An external Phe concentration of 2 mM was chosen to elucidate the physiological effects of Phe supply, as 2 mM Phe did not cause severe growth retardation in the wild type (Fig. 2). It should be noted that the free amino acid pools of plants grown on MS closely resembled that of plants cultivated on soil at standard growth conditions (at a photon flux density [PFD] of 100 µmol m–2 s–1, 12/12-h light/dark cycle, 21°/18°C; data not shown for simplicity).
When grown on MS, the total amino acid content in pig1-1 was 1.8-fold increased as compared to the Ws-2 controls (Table II). Major amino acids were elevated between 1.4- and 2.3-fold in pig1-1, whereas some minor amino acids, including Phe, were essentially unaltered in comparison to the wild type (Table II). By contrast, free amino acid contents except for Phe and Tyr decreased up to 4-fold in pig1-1 but not in the wild type, when plants were grown on 2 mM Phe (Table II). In pig1-1, Phe and Tyr decreased 5- and 11-fold under these conditions in comparison to the wild type, respectively. Taken together, Phe, Gln, and Asn accounted for more than 85% of the total amino acid discrepancy between pig1-1 and the wild type. However, the protein content in pig1-1 (5.0 ± 0.4 mg protein g fw–1) was similar to wild type (5.1 ± 0.4 mg protein g fw–1), indicating a specific effect on the free amino acid pool. Compared to plantlets grown on MS, the usually low-abundant amino acid Phe accumulated 340-fold on 2 mM Phe in the wild type but only 30-fold in pig1-1 (Table II). This suggests that Phe uptake and/or catabolism could be altered in pig1-1. Nevertheless, both the mutant and the wild type showed a 25% increase of soluble protein when Phe was provided (from approximately 3.8 mg protein g fw–1 on MS to 5.0 mg protein g fw–1 on 2 mM Phe), indicating that organic nitrogen in form of Phe was allocated into protein in both lines. Expressing the data for 2 mM Phe as molar percentages to correct for the large differences in total amino acid contents between pig1-1 and the wild type evinced that the relative compositions of the amino acid pools in pig1-1 and the control were similar, except for a massive decrease of Phe in pig1-1 (Table II). Nevertheless, the total free amino acid pool in pig1-1 apparently decreased relative to the control on 2 mM Phe (see above). To express the different effects of Phe feeding on pig1-1 and the wild type in numbers, a repression ratio was calculated that is indicative of the relative decrease of a particular amino acid content in pig1-1 in comparison to the wild type upon Phe supply (Table II). Phe displayed the strongest repression ratio, 10.5, on 2 mM Phe (Table II). Gln and Asn displayed repression ratios of 6.3 and 4.5, respectively, which is close to the average repression ratio (5.2) for all amino acids (Table II). Most of the other amino acids were only up to 4-fold deregulated in pig1-1, underlining the fact that changes in Phe, Gln, and Asn account for the largest portion of the decrease of the total amino acid pool in pig1-1. Importantly, the Glu-to-Gln and Asp-to-Asn ratios remained constant in pig1-1 in all experimental conditions, while these ratios exhibited a considerable variation in the wild type (Table III). In consequence, a nitrogen overflow with a more than 5-fold increase in total amino acid content, as observed in the wild type, is prevented in pig1-1 (Table II). In milder conditions, i.e. MS or MS + 2 mM Phe at low light (LL; 50 µmol m–2 s–1), pig1-1 exhibited a 3- to 4-fold lower Glu-to-Gln ratio, whereas the Pro-to-Glu and the Asp-to-Asn ratios were similar to the wild type (Table III). This suggests a specific imbalance between Glu and Gln and a nitrogen surplus in pig1-1 at LL. In these conditions, the Ser-to-Gly ratio was decreased by 30% to 40% in pig1-1 (Table III), indicating a lower flux through the photorespiratory C2 cycle in LL, especially as Ser and Gly contents were substantially diminished compared to pig1-1 or wild type at all other conditions (data not shown).
The effects of 2 mM Phe feeding at a PFD of 50 µmol m–2 s–1 were similar to the results at a PFD of 150 µmol m–2 s–1 (data not shown). However, three interesting aspects were exacerbated by low incident light:
As outlined above, pig1-1 was not only resistant to high concentrations of all three aromatic amino acids but also displayed a general deregulation of amino acid homeostasis. This raised the question as to whether pig1-1 would also display increased tolerance to nonaromatic amino acids. We therefore tested the effects of all proteinogenic amino acids on germination and growth of pig1-1 and the wild type. The dose-response curves of individual nonaromatic amino acids (data not shown) resembled those of aromatic amino acids (Fig. 2). Pronounced and characteristic effects were usually observed at amino acid concentrations of 10 mM, hence all further experiments were carried out at this concentration (Table IV).
Similar to aromatic amino acids, 10 mM Asp, Lys, and Arg were toxic to the wild type (Table IV). Although pig1-1 is insensitive toward aromatic amino acids, it exhibited a similar susceptibility to Asp, Lys, and Arg as the wild type (Table IV). At concentrations up to 10 mM, all other amino acids did not inhibit germination but either positively or negatively affected the fresh weight production of seedlings. The major amino acids Glu, Gln, and Ala enhanced the fresh weight production of both the wild type and pig1-1 to a similar extent, whereas Asn or Gly were beneficial only to the mutant (Table IV). Asn had no effect on the wild type, and Gly significantly reduced its fresh weight production by 40%. Similar to the wild type, the fresh weight production of pig1-1 was impeded by most minor amino acids (Table IV) but significantly less so than the wild type (for example, see the effects of Thr, Ser, and His; Table IV). In summary, pig1-1 grew equally well or, in most cases, better than the wild type on all tested amino acids.
In this work, we have demonstrated that external supply of 10 mM Phe caused a substantial concomitant decrease of all free amino acid contents in pig1-1 (Table II). An impaired amino acid uptake or a compromised long-distance amino acid transport in pig1-1 could explain this observation. Therefore, it was tested whether uptake or long-distance transport of Phe are impaired in pig1-1. To this end, [U-14C]-labeled Phe was fed to roots of 14-d-old intact pig1-1 and wild-type plantlets, and the incorporation of 14C into the root and shoot was determined. The experiment was conducted in the light to stimulate the transpiration stream. Phe accumulation in roots (Fig. 4A) and shoots (Fig. 4B) were comparable in pig1-1 and the wild type, irrespective of whether the plantlets had been adapted to Phe on Phe-replete medium (Fig. 4, solid bars) or not (Fig. 4, hatched bars), indicating that the uptake of Phe into pig1-1 roots, rather by active uptake than by diffusion, and the transport of Phe with the transpiration stream in pig1-1 were not altered. It should be noted that Phe uptake by pig1-1 and wild-type roots was stimulated when the plantlets had been grown on 1 mM Phe before the experiment (Fig. 4A), while the root-to-shoot transport was not subject to adaptation (Fig. 4B). Based on these results, we conclude that the uptake and long-distance transport of Phe in pig1-1 is unaltered with respect to the wild type.
Phe Catabolism Is Increased in pig1-1 Shoots After ruling out that Phe allocation in the cormus was altered in pig1-1, it was examined if an increased Phe catabolism could account for the tolerance of pig1-1 toward externally applied amino acids. To this end, ethanol extracts of [14C]Phe-fed wild-type and mutant plants were separated into soluble and insoluble fractions, and the soluble fraction was subsequently further separated into neutral (e.g. sugars; Fig. 5A), acidic (e.g. phosphorylated intermediates, organic acids; Fig. 5B), and basic (e.g. amino acids; Fig. 5C) fractions by ion-exchange chromatography.
The incorporation of label from [14C]Phe into the ethanol insoluble fraction was negligible (data not shown), indicating that carbon derived from externally applied Phe was not allocated to structural carbohydrates, lignin, or starch. In roots of both mutant and wild type, a predominant portion of the 14C label (>98%) was retained in the cationic fraction, and the allocation of label to the acidic and neutral fractions was low and not significantly different between mutant and wild type. However, a significantly (P < 0.02) larger portion of the label was allocated to the acidic and neutral fractions in pig1-1 shoots but not in the wild type (Fig. 5, A and B). Accordingly, a smaller portion of the label was retained in the basic fraction of pig1-1 shoots (Fig. 5C). The 10-fold increase of 14C found in the neutral and acidic fractions of pig1-1 shoots indicates a 10-fold higher catabolism of the Phe carbon backbone in pig1-1 shoots in comparison to the wild type.
As reasoned initially, tolerance to high external Phe concentrations may involve a Phe-feedback insensitive shikimate pathway. As it has been observed previously that the flux through Phe ammonia lyase (PAL), the committed step of phenylpropanoid biosynthesis, can be restricted by Phe availability (Da Cunha, 1987 To test this hypothesis, the flavonoid content was measured in crude extracts of plantlets grown on 2 mM Phe and in standard growth conditions on soil. Although the contents of UV-absorbing phenylpropanoids were increased 3-fold in the wild type when Phe was supplied, this increase was less pronounced (1.75-fold) in pig1-1 (Fig. 6A). Furthermore, phenylpropanoid contents were similar in wild type and pig1-1 at standard growth conditions (Fig. 6).
Surprisingly, anthocyanin contents were generally higher in soil-grown plants as when 2 mM Phe was supplied. In addition, pig1-1 mutants grown on 2 mM Phe displayed the lowest anthocyanin contents (Fig. 6B). These results will be discussed in more detail; however, it seems clear that phenylpropanoid metabolism is not stimulated in pig1-1.
The Shikimate Pathway and Phenylpropanoid Metabolism Are Not Positively Affected in pig1-1
In this article, we have described the Arabidopsis pig1-1 mutant that is insensitive to high external Phe concentrations. The aromatic amino acid Phe serves as building block for secondary metabolites of the phenylpropanoid class and Phe supply to the committed step of phenylpropanoid biosynthesis (PAL) can be rate limiting (Da Cunha, 1987
Although pig1-1 exhibits tolerance to high external Phe, Phe did not accumulate in pig1-1 when grown on MS (Table II) or soil (data not shown). Accordingly, phenylpropanoid contents were similar in pig1-1 and the wild type on soil (Fig. 6). However, the contents of UV-absorbing phenylpropanoids were increased 3-fold in the wild type on 2 mM Phe as compared to standard growth conditions (Fig. 6), indicating that the provided Phe can partially be funneled into phenylpropanoids as observed previously (Da Cunha, 1987
Many approaches to enhance phenylpropanoid production in plants have demonstrated that anthocyanins could be regarded as an overfall destination for excess carbon directed into phenylpropanoid metabolism in different angiosperm species as tomato (Lycopersicon esculentum), maize (Zea mays), and Arabidopsis (Borevitz et al., 2000
The Shikimate Pathway If the chorismate mutase branch was affected in pig1-1, Tyr levels would predominantly be altered together with Phe in the mutant. Indeed, we observed that Phe (11-fold) and Tyr (5-fold) contents declined strongest in pig1-1 on 2 mM Phe (Table II). However, the calculated repression ratio, an indicator for the deregulation of free amino acid abundance, for Tyr was comparable to the majority of the amino acids, while the repression ratio for Phe was much greater (Table II). This indicates the absence of a specific effect of Phe supply on Tyr contents in pig1-1. Phe feeding also had no specific effect on Tyr contents in the wild type. Furthermore, it is unlikely that a locus promoting phenylpropanoid biosynthesis downstream of Phe is affected in pig1-1 because phenylpropanoid contents were not increased in pig1-1 (see above).
Phe Uptake or Long-Distance Transport
We found that total free amino acid contents were 2-fold elevated when Ws-2 was grown on 2 mM Phe at 150 µmol m–2 s–1 as compared to 50 µmol m–2 s–1 (data not shown). It has been observed previously that sugar abundance enhanced amino acid uptake in Scots pine (Pinus sylvestris; Persson and Näsholm, 2003 It might thus be hypothesized that amino acid transport in pig1-1 is disturbed in such a fashion that the externally supplied amino acids are either not taken up from the medium or not transported throughout the plant body as in the wild type and hence do not exert the same effects on amino acid metabolism in pig1-1. Therefore, we tested as to whether amino acid (i.e. [14C]Phe) uptake by the root or amino acid translocation from roots to the shoot are altered in pig1-1, but both were comparable in pig1-1 and the wild type (Fig. 4). When root extracts were further separated into neutral, acidic, and basic fractions, 98% of the label in both wild-type and pig1-1 roots were retained in the cationic fraction (Fig. 5), indicating that Phe metabolization rates are similar in pig1-1 and wild-type roots. However, Phe metabolization in pig1-1 shoots was elevated 10-fold compared to the wild type (Fig. 5) in plants grown in permissive Phe-replete conditions. As discussed below, we assume that not only Phe catabolism but also free amino acid metabolization in general are increased in pig1-1.
Amino Acid Metabolization Likewise, the observed effects of Phe feeding on amino acid metabolism in pig1-1 are nonspecific (Table II). Ammonium (data not shown) and free amino acid contents mutually declined in the mutant upon Phe supply (Table II), while soluble protein contents even increased when Phe was abundant in the medium (from approximately 3.8–5.0 mg protein g fw–1). This broad effect argues against a specific elevation of Phe breakdown in pig1-1.
However, we found a 10-fold increased Phe metabolization rate in pig1-1 shoots during a 6-h pulse with [14C]Phe, while the partitioning of 14C label was similar in pig1-1 and wild-type roots (Fig. 5). Physiological counterbalances in response to excess amounts of one particular amino acid are frequently observed in genetic engineering approaches that aim at modifying amino acid breakdown or biosynthesis (e.g. Karchi et al., 1994 In conclusion, the mechanism by which pig1-1 achieves tolerance toward Phe apparently involves a general stimulation of amino acid breakdown, which is presumably more relaxed in pig1-1 at standard growth conditions in comparison to Phe-replete conditions.
Amino Acid Homeostasis
A flexible response seems to be affected by the pig1-1 mutation because the total free amino acid pool in pig1-1 is almost 2-fold elevated on MS medium and substantially decreased upon Phe supply when compared to the wild type (Table II). Secondly, this flexible response toward exogenous amino acid supply is nonspecific, as pig1-1 displays a higher tolerance toward a number of exogenously supplied nonbiosynthetically related minor and major amino acids (Table IV). In addition, amino acid catabolism seems to be affected in pig1-1. Hence, we hypothesize that the integration of amino acid catabolism into a superseding regulatory network that governs amino acid homeostasis is deregulated or defective in pig1-1. Such a control mechanism, the general amino acid control, has been reported in Escherichia coli and yeast (Hinnebusch, 1988
The response of free amino acid contents in pig1-1 to Phe feeding exhibits four important features: (1) The Phe-induced decline of free amino acid contents is not specific to aromatic amino acids but extends to all proteinogenic amino acids, (2) it is light-dependent, (3) soluble protein contents remain unaffected, and (4) Phe and likely amino acid catabolism in general are increased in pig1-1 shoots but not in pig1-1 roots. In addition, pig1-1 is resistant to a large number of biosynthetically nonrelated amino acids, and the uptake of Phe by roots and its translocation to the shoot is similar to the wild type. These results suggest that the deregulated amino acid metabolism in pig1-1 might be caused by a defect in a regulatory mechanism that integrates into a network comparable to the general amino acid control in yeast. Testing this hypothesis will require the molecular identification of the defective gene in pig1-1 that is currently in progress.
Plant Material A population of 200,000 ethyl methanesulfonate-mutagenized M2 individuals (cv Ws-2) was obtained from Lehle Seeds (Round Rock, TX) and screened for the Phe-tolerant phenotype as described below. Seed material for the lines trp1-100, trp2-1, trp3-1, and trp5-1 was obtained from Arabidopsis Biological Resource Center stock center (Ohio State University, Columbus, OH).
Aliquots of the seed populations were vapor sterilized according to Clough and Bent (1998) Selected individuals of the uniform M4 pig mutant populations were backcrossed to the wild type three times prior to further physiological analysis.
To evaluate cross-tolerance of pig mutants to Trp and Tyr, pig1-1 and control seeds were sterilized and dispersed on sterile plates as outlined in "Screening Procedures," except that 10 mM Phe was substituted by other aromatic amino acids as indicated. Germination rates on the individual plates were scored after 14 d of growth at a PFD of 50 µmol m–2 s–1 in a 12/12-h light/dark cycle. Germination was judged to have failed when growth of the plantlets was aborted after the cotyledons and the radicle had emerged, and the plants subsequently died. To better visualize growth effects of aromatic amino acid feeding to the wild type, seeds were also germinated and grown on half-strength MS, 2% Suc, and 4 mM of the respective aromatic amino acid.
Concentration-dependent growth inhibition effectuated by nonaromatic amino acids was essentially determined as the germination rates on aromatic amino acids described in the section above, except for two differences. First, 2.5 mM MES, pH 5.7, was added to the plates to avoid pH-dependent effects that might be induced by acidic or basic amino acids. Secondly, the fresh weight of the four to six biggest individuals from populations consisting of 60 individuals per replicate was scored after 14 d of growth at the same conditions indicated above. Table IV only displays the results for 10 mM amino acid supplements because this concentration was judged to be the most representative among the tested amino acid concentrations.
Surface-sterilized pig1-1, trp1-100, trp2-1, trp3-1, trp5-1, and Ws-2 control seeds (see "Screening Procedures") were sown on plates of four different compositions as indicated in Table I. After 21 d at a PFD of 50 µmol m–2 s–1 in a 12/12-h light/dark cycle, the individual plant lines were tested for the growth or fluorescence responses indicated in Table I. The observed effects were compared to plants sown on half-strength MS, 2% Suc control plates.
Free amino acid and protein contents were assayed in Arabidopsis plantlets grown on sterile agar plates. Vapor-sterilized seeds were germinated on half-strength MS, 0.5% Suc, and 2 mM Phe, and whole plantlets were harvested after 14 d of growth at a PFD of 150 µmol m–2 s–1 in a 12/12-h light/dark cycle.
For the determination of free amino acids, the plant samples were extracted in distilled water as described previously (Voll et al., 2003
To assess total soluble proteins, plant samples were extracted in a buffer containing 50 mM HEPES/NaOH, pH 7.5, 5 mM MgCl2, 1 mM EDTA, and 0.5% (v/v) Triton X-100 with a rotating pestle and quantified according to Bradford (1976)
The contents of UV-absorbing compounds and anthocyanins were determined in methanol extracts of plant material grown on 2 mM Phe (see paragraph above) and in leaves of soil-grown plants as described by Voll et al. (2003) To obtain soil-grown material, seeds of pig1-1 and the Ws-2 wild-type control were germinated on half-strength MS plates, transplanted to soil after 18 d, and fully expanded leaves were harvested 4 h before the end of the light period after 7 d of growth on soil at a PFD of 100 µmol m–2 s–1 in a 12/12-h light/dark cycle.
Arabidopsis seeds were germinated and grown on half-strength MS, 0.5% Suc at a PFD of 50 µmol m–2 s–1 for 14 d, either in the presence or absence of 1 mM Phe. For the uptake experiment, individual wells of a 48-well plate were loaded with 800 µL of liquid medium (half-strength MS, 0.5% Suc, 1 mM [U-14C]Phe; 23.1 MBq mmol–1 specific activity). The roots of two plantlets were submerged in each well with the rosettes attaching the edges of the wells, not shading each other, and the plate was illuminated at room temperature at a PFD of 60 µmol m–2 s–1. Three replicate samples for each pretreatment (+Phe, –Phe) were harvested after 3 and 6 h as follows: The roots were rinsed twice with distilled water to remove adhering label and root and rosettes were then separated by cutting the hypocotyls with a razor blade. The samples were snap-frozen in liquid nitrogen, and the fresh weight was recorded in the frozen state. The samples were extracted by boiling the seedlings in 600 µL of 80% ethanol at 95°C for 45 min. The ethanol extracts were transferred to fresh tubes, and 50-µL aliquots were subjected to (1) liquid scintillation counting and (for rosette samples) to (2) spectrophotometric chlorophyll determination according to Lichtenthaler (1987)
We thank Momoko Minakawa and Samuel Vandenberg for plant culture work and the Michigan State University Macromolecular Structure Analysis Facility for help with amino acid analysis. Received June 2, 2004; returned for revision August 17, 2004; accepted August 20, 2004.
1 This work was supported by the Deutsche Forschungsgemeinschaft (postdoctoral fellowship to L.M.V.), by the National Science Foundation (REU-supplement MCB–0348074 to A.P.M.W.), and by the Michigan State University Center for Plant Products and Technologies (grant to A.P.M.W.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.047506. * Corresponding author; e-mail aweber{at}msu.edu; fax 517–432–5294.
Arruda P, Bright SWJ, Kueh JSH, Lea PJ, Rognes SE (1984) Regulation of aspartate kinase isoenzymes in barley mutants resistant to lysine plus threonine—construction and analysis of combinations of the LT1A, LT1B and LT2 mutant-genes. Plant Physiol 76: 442–446
Bender J, Fink GR (1998) A Myb homologue, ATR1, activates tryptophan gene expression in Arabidopsis. Proc Natl Acad Sci USA 95: 5655–5660 Bonner CA, Jensen RA (1997) Recognition of specific patterns of amino acid inhibition of growth in higher plants, uncomplicated by glutamine-reversible ‘general amino acid inhibition’. Plant Sci 130: 133–143[CrossRef]
Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12: 2383–2394
Bovy A, de Vos R, Kemper M, Schijlen E, Pertejo MA, Muir S, Collins G, Robinson S, Verhoeyen M, Hughes S, et al (2002) High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1. Plant Cell 14: 2509–2526 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254[CrossRef][Web of Science][Medline]
Bruce W, Folkerts O, Garnaat C, Crasta O, Roth B, Bowen B (2000) Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P. Plant Cell 12: 65–79
Cho HJ, Brotherton JE, Song HS, Widholm JM (2000) Increasing tryptophan synthesis in a forage legume Astragalus sinicus by expressing the tobacco feedback-insensitive anthranilate synthase (ASA2) gene. Plant Physiol 123: 1069–1076 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743[CrossRef][Web of Science][Medline] Da Cunha A (1987) The estimation of L-phenylalanine ammonia-lyase shows phenylpropanoid biosynthesis to be regulated by L-phenylalanine supply and availability. Phytochemistry 26: 2723–2727[CrossRef] Falco SC, Guida T, Locke M, Mauvais J, Sanders C, Ward RT, Webber P (1995) Transgenic canola and soybean seeds with increased lysine. Biotechnology (N Y) 13: 577–582[CrossRef][Medline] Galili G, Guenoune D, Wininger S, Hana B, Schupper A, Ben-Dor B, Kapulnik Y (2000) Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene. Transgenic Res 9: 137–144[CrossRef][Web of Science][Medline]
Guyer D, Patton D, Ward E (1995) Evidence for cross-pathway regulation of metabolic gene-expression in plants. Proc Natl Acad Sci USA 92: 4997–5000 Heremans B, Jacobs M (1995) Threonine accumulation in a mutant of Arabidopsis thaliana (L.) Heynh. with an altered aspartate kinase. J Plant Physiol 146: 249–257 Heremans B, Jacobs M (1997) A mutant of Arabidopsis thaliana (L) Heynh with modified control of aspartate kinase by threonine. Biochem Genet 35: 139–153[CrossRef][Web of Science][Medline] Hervieu F, Vaucheret H (1996) A single amino acid change in acetolactate synthase confers resistance to valine in tobacco. Mol Gen Genet 251: 220–224[Medline]
Hinnebusch AG (1988) Mechanisms of gene regulation and general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev 52: 248–273 Jander G, Norris SR, Joshi V, Fraga M, Rugg A, Yu SX, Li LL, Last RL (2004) Application of a high-throughput HPLC-MS/MS assay to Arabidopsis mutant screening; evidence that threonine aldolase plays a role in seed nutritional quality. Plant J 39: 465–475[CrossRef][Web of Science][Medline]
Karchi H, Shaul O, Galili G (1994) Lysine synthesis and catabolism are coordinately regulated during tobacco seed development. Proc Natl Acad Sci USA 91: 2577–2581
Kreps JA, Ponappa T, Dong WQ, Town CD (1996) Molecular basis of
Last RL, Bissinger PH, Mahoney DJ, Radwanski ER, Fink GR (1991) Tryptophan mutants in Arabidopsis: the consequences of duplicated tryptophan synthase Lee YT, Duggleby RG (2002) Regulatory interactions in Arabidopsis thaliana acetohydroxyacid synthase. FEBS Lett 512: 180–184[CrossRef][Web of Science][Medline] Li H, Culligan K, Dixon RA, Chory J (1995) CUE1: a mesophyll cell-specific positive regulator of light-controlled gene expression in Arabidopsis. Plant Cell 7: 1599–1610[Abstract]
Li L, Thipyapong P, Breeden DC, Steffens JC (2003) Overexpression of a bacterial branched-chain Li LY, Last RL (1996) The Arabidopsis thaliana trp5 mutant has a feedback-resistant anthranilate synthase and elevated soluble tryptophan. Plant Physiol 110: 51–59[Abstract] Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148: 350–382[Web of Science]
Mathews H, Clendennen SK, Caldwell CG, Liu XL, Connors K, Matheis N, Schuster DK, Menasco DJ, Wagoner W, Lightner J, et al (2003) Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell 15: 1689–1703 Miao S, Duncan DR, Widholm J (1988) Selection of regenerable maize callus-cultures resistant to 5-methyl-DL-tryptophan, S-2-aminoethyl-L-cysteine and high levels of L-lysine plus L-threonine. Plant Cell Tiss Org 14: 3–14 Mourad G, King J (1995) L-O-Methylthreonine-resistant mutant of Arabidopsis defective in isoleucine feedback-regulation. Plant Physiol 107: 43–52[Abstract]
Niyogi KK, Last RL, Fink GR, Keith B (1993) Suppressors of trp1 fluorescence identify a new Arabidopsis gene, TRP4, encoding the anthranilate synthase
Noctor G, Novitskaya L, Lea PJ, Foyer CH (2002) Co-ordination of leaf minor amino acid contents in crop species: significance and interpretation. J Exp Bot 53: 939–945 Persson J, Näsholm T (2003) Regulation of amino acid uptake by carbon and nitrogen in Pinus sylvestris. Planta 217: 309–315[CrossRef][Web of Science][Medline] Quick P, Siegl G, Neuhaus E, Feil R, Stitt M (1989) Short-term water stress leads to a stimulation of sucrose synthesis by activating sucrose-phosphate synthase. Planta 177: 535–546[CrossRef]
Radwanski ER, Barczak AJ, Last RL (1996) Characterization of tryptophan synthase Rognes SE, Bright SWJ, Miflin BJ (1983) Feedback-insensitive aspartate kinase isoenzymes in barley mutants resistant to lysine plus threonine. Planta 157: 32–38
Rose AB, Casselman AL, Last RL (1992) A phosphoribosylanthranilate transferase gene is defective in blue fluorescent Arabidopsis thaliana tryptophan mutants. Plant Physiol 100: 582–592 Schmid J, Amrhein N (1995) Molecular organization of the shikimate pathway in higher plants. Phytochemistry 4: 737–749[CrossRef]
Streatfield SJ, Weber A, Kinsman EA, Häusler RE, Li J, Post-Beittenmiller D, Kaiser WM, Pyke KA, Flügge U-I, Chory J (1999) The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development and plastid-dependent nuclear gene expression. Plant Cell 11: 1609–1621 van Wandelen C, Cohen SA (1997) Using quarternary high-performance liquid chromatography eluent systems for separating 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate-derivatized amino acid mixtures. J Chromatogr A 763: 11–22[CrossRef][Web of Science] Voll L, Häusler RE, Hecker R, Weber A, Weissenbock G, Fiene G, Waffenschmidt S, Flügge U-I (2003) The phenotype of the Arabidopsis cue1 mutant is not simply caused by a general restriction of the shikimate pathway. Plant J 36: 301–317[CrossRef][Web of Science][Medline] Yu O, Shi J, Hession AO, Maxwell CA, McGonigle B, Odell JT (2003) Metabolic engineering to increase isoflavone biosynthesis in soybean seed. Phytochemistry 63: 753–763[CrossRef][Web of Science][Medline]
Zhu X, Galili G (2003) Increased lysine synthesis coupled with a knockout of its catabolism synergistically boosts lysine content and also transregulates the metabolism of other amino acids in Arabidopsis seeds. Plant Cell 15: 845–853 Zor T, Selinger Z (1996) Linearization of the Bradford protein assay increases its sensitivity, theoretical and experimental studies. Anal Biochem 236: 302–308[CrossRef][Web of Science][Medline] This article has been cited by other articles:
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TOP
ABSTRACT
RESULTS
, Phe; 
, Tyr; 
, Trp) to wild-type Arabidopsis (cv Ws-2) in percent (A), dose-response curves for wild type (
= 310 nm, representing UV-absorbing compounds such as flavonols and hydroxycinnamic acid derivatives. B, Absorbance at 
genes. Plant Cell 3: 345–358
