Metabolism of avenanthramide phytoalexins in oats

Oat leaves produce phytoalexins, avenanthramides, in response to infection by pathogens or treatment with elicitors. The metabolism of avenanthramides was investigated using low molecular weight, partially deacetylated chitin as an elicitor. When oat leaf segments are floated on the elicitor solution, avenanthramides accumulate in the solution. The transfer of elicited oat leaves to solutions containing stable-isotope-labeled avenanthramides resulted in a rapid decrease in the labeled avenanthramides, suggesting the metabolism of avenanthramides. The rate of decrease was enhanced by elicitor treatment, and was dependent on the species of avenanthramides, with avenanthramide B decreasing most rapidly. The rates of biosynthesis and metabolism of avenanthramides A and B were measured using a model of isotope-labeling dynamics. Avenanthramide B was found to be more actively biosynthesized and metabolized than avenanthramide A. Radiolabeled avenanthramide B was incorporated into the cell wall fraction and 99% of incorporated activity was released by alkaline treatment. Gel filtration indicated that high-molecular-weight compounds derived from avenanthramide B were released by alkaline treatment. The decrease in stable-isotope-labeled avenanthramides was suppressed by catalase, salicylhydroxamic acid, and sodium ascorbate, suggesting the involvement of peroxidase in the metabolism. Consistent with this, peroxidase activity that accepts avenanthramide B as a substrate was induced in apoplastic fractions by elicitor treatment. The appearance of multiple basic isoperoxidases was observed by activity staining with 3-amino-9-ethylcarbazole coupled with isoelectric focusing of proteins from elicitor-treated leaves. These findings suggest that accumulated avenanthramides are further metabolized in apoplasts in oat leaves by inducible isoperoxidases.     

Differential induction of chorismate mutase isoforms by elicitors in oat leaves

Avenanthramides, a series of substituted cinnamic acid amides with anthranilate, are phytoalexins in oats (Avena sativa L.). The precursors of avenanthramides, cinnamate and anthranilate, are biosynthesized via the shikimate pathway that branches at chorismate. Chorismate mutase (CM, EC 5.4.99.5) is the first enzyme on the branch that provides the cinnamate part of avenanthramides. The induction of CM was investigated in primary oat leaves using oligo-N-acetylchitooligosaccharides as elicitors. The CM activity started to increase 6 h after elicitation, and reached a maximum by 9 h, being around twice as large as that in control leaves. Among the oligo-N-acetylchitooligosaccharides tested, tetra-, penta-, and hexasaccharides effectively induced the CM activity in a dose-dependent manner. The activity was separated into two major peaks on anion exchange chromatography with Mono Q, indicating that at least two CM isoforms are present in oat leaves. A comparison of elution profiles of CM activity in intact and elicitor-treated leaves revealed that only one CM isoform is responsive to the elicitor. Two CM isoforms in oat leaves were partially purified and characterized. Both CM isoforms were insensitive to l-phenylalanine, l-tyrosine, l-tryptophan, and caffeate. The fractionation of oat cells indicated that both CM isoforms localized in plastids.     

Elicitor induced secondary metabolism in Ruta graveolens L.

In vitro cultures of Ruta graveolens L. respond with rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins, when challenged with autoclaved homogenate of the yeast Rhodotorula rubra. A transient increase of several enzymes of the respective biosynthetic pathways was measured but we still look for the key regulatory enzymes. We investigated whether the branch point enzymes of the shikimic acid pathway anthranilate synthase (AS) and chorismate mutase (CM) possibly play such a role. The two enzymes compete for chorismate. AS forms anthranilate, the precursor amino acid of acridone and furoquinoline alkaloids. CM channels chorismate into phenylalanine, tyrosine and phenylpropanoid biosynthesis. Elicitation resulted in a transient increase of the activity of both enzymes. Relative induction rates were 2–4 fold for AS and about 1.5 fold for CM. Constitutive CM activity, however, is about 1000 fold higher than AS activity. As in other plants 2 isoforms of CM are expected to be present in R. graveolens. A differential determination of the activity of the isoforms via the tryptophan activation rate proved to be ambiguous. Some evidence for the specific induction of a plastidic form of CM was obtained by inhibition of translation. The time courses of CM induction show CM not to be a key enzyme in elicitor induction of furanocoumarin accumulation. In comparison to other enzyme activities induction of anthranilate synthase activity corresponds closest to inducible acridone epoxide accumulation indicating a key role in its regulation. Induction of AS and CM was inhibited by actinomycin D and chloramphenicol while cycloheximid inhibited AS induction only.Abbreviations ACT actinomycin D - AS anthranilate synthase - CAP chloramphenicol - CHX cycloheximid - 4-CL 4-coumarate CoA ligase - CM chorismate mutase - DTT dithiothreitol - NMT S-adenosyl-L-methionine:anthranilic acid N-methyltransferase - PAL phenylalanine ammonia lyase - XOMT S-adenosylmethionine: xanthotoxol-O-methyltransferase     

Occurrence of avenanthramides and hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyltransferase activity in oat seeds

Oat phytoalexins, avenanthramides, occur as constitutive components in seeds. The amounts of each avenanthramide were analyzed. The composition of avenanthramides in dry seeds was different from that in elicitor-treated leaves. In seeds, avenanthramide C was most abundant with an amount two times larger than that of avenanthramide A or B. On the other hand, avenanthramide A was the major component in elicitor-treated leaves. The total amount of avenanthramides in seeds increased 2.5 times during imbibition for 48 h although the composition did not change. The hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyltransferase (HHT, EC 2.3.1.-) activity, which is responsible for the final condensation step in the avenanthramide biosynthesis, was detected in dry seeds. The activity was localized in endosperm and scutellum, and slightly increased during 48-h imbibition. The enzyme was partially purified by anion exchange chromatography from both dry seeds and elicitor-treated leaves The activity was separated into two peaks by chromatography, indicating that HHT consists of at least two isoforms. The substrate specificities of HHT isoforms from seeds were different from each other.     

Induction of biosynthetic enzymes for avenanthramides in elicitor-treated oat leaves

The accumulation of oat (Avena sativa L.) phytoalexins, avenanthramides, occurred in leaf segments treated with oligo-N-acetylchitooligosaccharides. The amount of avenanthramide A, the major oat phytoalexin, reached a maximum 36–48 h after elicitor treatment. This accumulation was preceded by a marked increase in enzyme activities of phenylpropanoid pathway members, including phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamate 4-hydroxylase (EC 1.14.13.11) and 4-coumarate:CoA ligase (EC 6.2.1.12). These enzyme activities reached a maximum 6–12 h after elicitor treatment, when the avenanthramides were produced most rapidly. Both phenylalanine ammonia-lyase and 4-coumarate:CoA ligase activities decreased thereafter to undetectable levels 72 h after treatment, while cinnamate 4-hydroxylase activity showed a second increase 48 h after treatment. Among the chitooligosaccharides tested, tetra- and pentasaccharides most effectively induced these enzyme activities in a dose-dependent manner. The elicitor-induced 4-coumarate: CoA ligase accepted all hydroxycinnamic acids occurring in the avenanthramides as substrates, with the exception of avenalumic acid. These findings indicate that accumulation of the avenanthramides results from de-novo synthesis through the general phenylpropanoid pathway and that early biosynthetic enzymes function as regulatory points of carbon flow to the avenanthramides. Received: 3 December 1998 / Accepted: 27 January 1999     

Modification of Serratia marcescens anthranilate synthase with pyridoxal 5′-phosphate

The glutamine-dependent activity of Serratia marcescens anthranilate synthase was inactivated by pyridoxal 5′-phosphate and sodium cyanide. The reaction was specific in that the ammonia-dependent activity of the enzyme was unaffected. The inactivation was stable to dilution or dialysis but was reversed by dithiothreitol. The enzyme contains dissimilar subunits designated anthranilate synthase components I (AS I) and II (AS II). Incorporation of [¹⁴C]NaCN demonstrates that modification was limited to one to two residues per AS I · AS II protomer. An active site cysteine is involved in the glutamine-dependent activity. Modification by pyridoxal 5′-phosphate and NaCN blocked affinity labeling of the active site cysteine by the glutamine analog 6-diazo-5-oxo-l-norleucine and reduced alkylation of the active site cysteine by iodoacetamide. These results suggest modification is at the glutamine active site. Initial modification by iodoacetamide did not prevent pyridoxal 5′-phosphate-dependent incorporation of ¹⁴CN showing that the pyridoxal 5′-phosphate modification did not involve the essential cysteinyl residue. These results suggest that modification of a lysyl residue in the glutamine active site of anthranilate synthase reduces the reactivity of the essential cysteinyl residue resulting in the loss of the amidotransferase activity.     

Metabolic changes in Arabidopsis thaliana expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D

Anthranilate synthase (AS) is a key enzyme in tryptophan (Trp) biosynthesis. Metabolic changes in transgenic Arabidopsis plants expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D were investigated with respect to Trp synthesis and effects on secondary metabolism. The Trp content varied depending on the transgenic line, with some lines showing an approximately 200-fold increase. The levels of AS activity in crude extracts from the transgenic lines were comparable to those in the wild type. On the other hand, the enzyme prepared from the lines accumulating high levels of Trp showed a relaxed feedback sensitivity. The AS activity, determined in the presence of 50 microM L-Trp, correlated well with the amount of free Trp in the transgenic lines, indicating the important role of feedback inhibition in control of Trp pool size. In Arabidopsis, Trp is a precursor of multiple secondary metabolites, including indole glucosinolates and camalexin. The amount of indol-3-ylmethyl glucosinolate (I3 M) in rosette leaves of the high-Trp accumulating lines was 1.5- to 2.1-fold greater than that in wild type. The treatment of the leaves with jasmonic acid resulted in a more pronounced accumulation of I3 M in the high-Trp accumulating lines than in wild type. The induction of camalexin formation after the inoculation of Alternaria brassicicola was not affected by the accumulation of a large amount of Trp. The accumulation of constitutive phenylpropanoids and flavonoids was suppressed in high-Trp accumulating lines, while the amounts of Phe and Tyr increased, thereby indicating an interaction between the Trp branch and the Phe and Tyr branch in the shikimate pathway.     

Induction of ajmalicine formation and related enzyme activities in Catharanthus roseus cells: effect of inoculum density

In Catharanthus roseus cell cultures the time courses of four enzyme activities, tryptophan decarboxylase (TDC), strictosidine synthase (SSS), geraniol-10-hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid accumulation were compared under two different culture conditions (low-inoculum density and high-inoculum density on induction medium) and a control on growth medium. In growth medium a transient increase in TDC activity was first observed after which G10H reached its maximum activity; only tryptamine accumulated, no ajmalicine could be detected. Apparently, a concerted induction of enzyme activities is required for ajmalicine formation. Cells inoculated in induction medium showed such a concerted induction of AS, TDC and G10H activities. After 30 days the low-density culture had accumulated six times more ajmalicine (in moles/g) than the high-density culture. Thus, increase in biomass concentration (high-density cultures) did not enhance the total alkaloid production. The major differences observed in enzyme levels between high-and low-density cultures were in the AS and TDC activities, which were two to three times higher in the low-density culture, indicating that there is a positive correlation between ajmalicine formation and AS and TDC activities.Biotechnology Delft Leiden, Project Group Plant Cell Biotechnology Correspondence to: R. Verpoorte     

Anthranilate synthase forms in plants and cultured cells of Nicotiana tabacum L.

Tobacco (N. tabacum cv. Xanthi) cell lines contained two forms of anthranilate synthase (AS; EC 4.1.3.27) which could be partially separated by gel-filtration chromatography. One form was resistant to feedback inihibition by 10 M tryptophan (trp) while the other form was almost completely inhibited by trp at the same concentration. Cell lines selected as resistant to 5-methyltryptophan (5MT) had more of the trp-resistant AS form. Only the trp-sensitive form was detected in plants regenerated from both normal and 5MT-resistant cell lines. Overexpression of the trp-resistant form in 5MT-resistant tobacco cells disappeared during plant regeneration but reappeared when callus was initiated from the leaves of these plants. The trp-sensitive form was localized in the particulate fraction and the trp-resistant form in the cytosol of tobacco cultured cell protoplasts. The trp-resistant form of AS from tobacco had an estimated MW of 200 000, determined by Sephacryl S-200 chromatography, compared to an estimated MW of 150 000 for the trp-sensitive form. The estimated molecular weights of AS from carrot and corn were 160 000 and 150 000, respectively. Analysis of AS activity from the diploid Nicotiana species Nicotiana otophora (chromosome number 2n=24) by high-performance liquid chromatography showed two activity peaks identical in elution time and trp inhibition characteristics to the activity from N. tabacum (chromosome No. 48). Thus the two enzyme forms found in tobacco did not appear to have originated individually from the progenitor species genomes which combined to make up the tobacco genome.Abbreviations AS anthranilate synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - HPLC high-performance liquid chromatography - 5MT D1-5-methyltryptophan - trp L-tryptophan     

Chorismate aminations: partial purification of Escherichia coli PABA synthase and mechanistic comparison with anthranilate synthase

Chorismate is converted by regiospecific amination/aromatization sequences to o-aminobenzoate and p-aminobenzoate (PABA) by anthranilate synthase (AS) and PABA synthase (PABS), respectively. We report here the first partial purification of the large subunit of Escherichia coli PABA synthase, previously reported to be quantitatively inactivated in purification attempts. The subunit encoded by the pabB gene was overexpressed from a T7 promoter and purified 9-fold to 25-30% homogeneity. The pabB subunit appears unusually sensitive to inactivation by glycerol so this cosolvent is contraindicated. The Km for chorismate is 42 microM in the ammonia-dependent conversion to PABA, and we estimate a turnover number of 2.6 min-1. A variety of chorismate analogues have been prepared and examined. Of these compounds, cycloheptadienyl analogue 11 has been found to be the most potent inhibitor of Serratia marcescens anthranilate synthase (Ki = 30 microM for an RS mixture) and of the E. coli pabB subunit of PABA synthase (Ki = 226 microM). Modifications in the substituents at C-3 [enolpyruyl ether, (R)- or (S)-lactyl ether, glycolyl ether] or C-4 (O-methyl) of chorismate lead to alternate substrates. The Vmax values for (R)- and (S)-lactyl ethers are down 10-20-fold for each enzyme, and V/K analyses show the (S)-lactyl chorismate analogue to be preferred by 12/1 over (R)-lactyl for anthranilate synthase while a 3/1 preference was observed for (R)-/(S)-lactyl analogues by PABA synthase. The glycolyl ether analogue of chorismate shows 15% Vmax vs. chorismate for anthranilate synthase but is actually a faster substrate (140%) than chorismate with PABA synthase, suggesting the elimination/aromatization step from an aminocyclohexadienyl species may be rate limiting with AS but not with PABS. Indeed, studies with (R)-lactyl analogue 14 and anthranilate synthase led to accumulation of an intermediate, isolable by high-performance liquid chromatography and characterized by NMR and UV-visible spectroscopy as 6-amino-5-[(1-carboxyethyl)oxy]-1,3-cyclohexadiene-1-carboxylic acid (17). This is the anticipated intermediate predicted by our previous work with conversion of synthetic trans-6-amino-5-[(1-carboxyethenyl)oxy]-1,3-cyclohexadiene-1-carbo xylic acid (2) to anthranilate by the enzyme. Compound 17 is quantitatively converted to anthranilate on reincubation with enzyme, but at a 1.3-10-fold lower Vmax than starting lactyl substrate 14 under the conditions investigated; the basis for this kinetic variation is not yet determined.     

Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots

In plants, the indole pathway provides precursors for a variety of secondary metabolites. In Catharanthus roseus, a decarboxylated derivative of tryptophan, tryptamine, is a building block for the biosynthesis of terpenoid indole alkaloids. Previously, we manipulated the indole pathway by introducing an Arabidopsis feedback-insensitive anthranilate synthase (AS) alpha subunit (trp5) cDNA and C. roseus tryptophan decarboxylase gene (TDC) under the control of a glucocorticoid-inducible promoter into C. roseus hairy roots [Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004a. Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol. Bioeng. 86, 718-727; Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004b. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metabol. Eng. 6, 268-276]. Inducible expression of either or both transgenes did not lead to significant increases in overall alkaloid levels despite the considerable accumulation of tryptophan and tryptamine. In an attempt to more successfully engineer the indole pathway, a wild type Arabidopsis ASbeta subunit (ASB1) cDNA was constitutively expressed along with the inducible expression of trp5 and TDC in C. roseus hairy roots. Transgenic hairy roots expressing both trp5 and ASB1 show a significantly greater resistance to feedback inhibition of AS activity by tryptophan than plants expressing only trp5. In fact, a 4.5-fold higher concentration of tryptophan is required to achieve 50% inhibition of AS activity in plants overexpressing both genes than in plants expressing only trp5. In addition, upon a 3 day induction during the exponential phase, a trp5:ASB1 hairy root line produced 1.8 times more tryptophan (specific yield ca. 3.0 mg g(-1) dry weight) than the trp5 hairy root line. Concurrently, tryptamine levels increase up to 9-fold in the induced trp5:ASB1 line (specific yield ca. 1.9 mg g(-1) dry weight) as compared with only a 4-fold tryptamine increase in the induced trp5 line (specific yield ca. 0.3 mg g(-1) dry weight). However, endogenous TDC activities of both trp5:ASB1 and trp5 lines remain unchanged irrespective of induction. When TDC is ectopically expressed together with trp5 and ASB1, the induced trp5:ASB1:TDC hairy root line accumulates tryptamine up to 14-fold higher than the uninduced line. In parallel with the remarkable accumulation of tryptamine upon induction, alkaloid accumulation levels were significantly changed depending on the duration and dosage of induction.     

Biosynthesis of avenanthramides in suspension cultures of oat (<Emphasis Type="Italic">Avena sativa</Emphasis>)

Oats produce a group of secondary metabolites termed avenanthramides (avn). These compounds are biosynthesized through the action of the enzyme hydroxycinnamoyl CoA: hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) which catalyzes the condensation of one of several cinnamate CoA thioesters with the amine functionality of anthranilic acid, 4-hydroxy- or 5-hydroxy-anthranilic acid. In oat leaf tissue the biosynthesis of avenanthramides appears to result from elicitation by fungal infection. Here we demonstrate the biosynthesis of several avenanthramides in suspension cultures of oat apical meristem callus tissue. This phenomenon appears as a generalized pathogen response, evidenced by the production of PR-1 mRNA, in response to elicitation with chitin (poly-N-acetyl glucosamine). The suspension cultures also produce relatively large quantities of avnA and G in response to chitin elicitation. Under certain culture conditions avnB and C are also produced as well as three additional metabolites tentatively identified as avnH, O and R. These findings portend the utility of oat suspension culture as a tool for more detailed investigation of the mechanisms triggering their biosynthesis as well as the factors dictating the particular types of avenanthramides biosynthesized.     

The Arabidopsis thaliana trp5 mutant has a feedback-resistant anthranilate synthase and elevated soluble tryptophan.

The first step of tryptophan biosynthesis is catalyzed by anthranilate synthase (AS), which is normally subject to feedback inhibition by tryptophan. Three independent trp5 mutants defective in the Arabidopsis thaliana AS alpha subunit structural gene ASA1 were identified by selection for resistance to the herbicidal compound 6-methylanthranilate. In all three mutants these biochemical changes are caused by a single amino acid substitution from aspartate to asparagine at residue position 341. Compared with the enzyme from wild-type plants, the tryptophan concentration causing 50% inhibition of AS activity in the trp5 mutant increased nearly 3-fold, the apparent Km for chorismate decreased by approximately 50%, and the apparent Vmax increased 60%. As a consequence of altered AS kinetic properties, the trp5 mutants accumulated 3-fold higher soluble tryptophan than wild-type plants. However, even though the soluble tryptophan levels were increased in trp5 plants, the concentrations of five tryptophan biosynthetic proteins remained unchanged. These data are consistent with the hypothesis that the reaction catalyzed by A. thaliana AS is rate limiting for the tryptophan pathway and that accumulation of tryptophan biosynthetic enzymes is not repressed by a 3-fold excess of end product.     

Production of l-Tryptophan by Azaserine-resistant Mutants of Brevibacterium flavum

Azaserine-resistant mutants derived from a 5-fluorotryptophan-resistant, l-tryptophan-producing mutant of Brevibacterium flavum, accumulated 10.3 g/liter of l-tryptophan at maximum. The production increased to 11.4 g/liter when l-serine was added. In the mutant, only anthranilate synthase among enzymes of the tryptophan-specific bio synthetic pathway increased in activity to a 2-fold higher level than that in the parent strain, No. 187. Sensitivity of anthranilate synthase to the feedback inhibition was not altered by the mutation. Activity of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first common enzyme for aromatic amino acid biosynthesis, also increased 2.7-fold and was less sensitive to the feedback inhibition by phenylalanine and tyrosine. Tryptophan transport activity in strain A-100 was similar as that in the parent. Azaserine inhibited anthranilate synthase activity by 50% at 0.075 mm. The inhibition was of a mixed type with respect to both the two substrates. Anthranilate synthase of strain A-100 was inhibited in a similar manner to that of the parent.     

High-resolution spatial and temporal analysis of phytoalexin production in oats

The production of oat (Avena sativa L.) phytoalexins, avenanthramides, occurs in response to elicitor treatment with oligo-N-acetylchitooligosaccharides. In this study, avenanthramides production was investigated by techniques that provide high spatial and temporal resolution in order to clarify the process of phytoalexin production at the cellular level. The amount of avenanthramides accumulation in a single mesophyll cell was quantified by a combination of laser micro-sampling and low-diffuse nanoflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) techniques. Avenanthramides, NAD(P)H and chlorophyll were also visualized in elicitor-treated mesophyll cells using line-scanning fluorescence microscopy. We found that elicitor-treated mesophyll cells could be categorized into three characteristic cell phases, which occurred serially over time. Phase 0 indicated the normal cell state before metabolic or morphological change in response to elicitor, in which the cells contained abundant NAD(P)H. In phase 1, rapid NAD(P)H oxidation and marked movement of chloroplasts occurred, and this phase was the early stage of avenanthramides biosynthesis. In phase 2, avenanthramides accumulation was maximized, and chloroplasts were degraded. Avenanthramides appear to be synthesized in the chloroplast, because a fluorescence signal originating from avenanthramides was localized to the chloroplasts. Moreover, our results indicated that avenanthramides biosynthesis and the hypersensitive response (HR) occurred in identical cells. Thus, the avenanthramides production may be one of sequential events programmed in HR leading to cell death. Furthermore, the phase of the defense response was different among mesophyll cells simultaneously treated with elicitor. These results suggest that individual cells may have different susceptibility to the elicitor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.