Isochorismate Synthase Activity Research Articles

Effects of PAL and ICS on the production of total flavonoids, daidzein and puerarin in Pueraria thomsonii Benth. suspension cultures under low light stress

Under stress, some secondary metabolites are synthesized and accumulated in plants. Salicylic acid, which is synthesized through phenylalanine ammonia-lyase and isochorismate synthase, is involved in this response to stress. However, the effects of phenylalanine ammonia-lyase and isochorismate synthase on the total flavonoids and desirable isoflavonoids in plants under stress remain unknown. In this study, the effects of phenylalanine ammonia-lyase and isochorismate synthase on total flavonoids, daidzein and puerarin in Pueraria thomsonii Benth. under low light stress were investigated. When the phenylalanine ammonia-lyase activity was inhibited by l-AOPP, the salicylic acid content was not reduced, but gradually increased along with an increase of isochorismate synthase activity. The trends of salicylic acid and total flavonoid content were similarly consistent, but there was no significant positive correlation between phenylalanine ammonia-lyase suppression and total flavonoid production in P. thomsonii under low light stress. However, daidzein synthesis was correlated with the isochorismate synthase pathway, and puerarin with the phenylalanine ammonia-lyase pathway.

The Pathway and Regulation of Salicylic Acid Biosynthesis in Probenazole-Treated Arabidopsis

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is a highly effective chemical inducer of systemic-acquired resistance (SAR). It has been used widely to protect rice plants against the rice blast fungus Magnaporthe grisea. Previous studies have shown that PBZ induces SAR through enhanced accumulation of salicylic acid (SA). Plants synthesize SA by either a pathway that uses phenylalanine as substrate or another that involves isochorismate. To clarify how SA is produced in PBZ-treated Arabidopsis, we examined the expression patterns and enzyme activities of phenylalanine ammonia lyase (PAL) and isochorismate synthase (ICS), which are the main components of the phenylalanine and isochorismate pathways, respectively. PBZ exposure significantly improved the accumulation of SA and increased ICS activity. In the sid2–2 mutant, which has a defect in ICS1, PBZ had no effect on the level of endogenous SA or activity of ICS. In contrast, PAL activity and the expression of most PAL genes were down-regulated by such treatment in wild-type plants. These results suggest that SA is mainly synthesized via the ICS-mediated pathway in Arabidopsis.

The isochorismate pathway is negatively regulated by salicylic acid signaling in O3-exposed Arabidopsis

Ozone (O3), a major photochemical oxidant, causes leaf injury in plants. Plants synthesize salicylic acid (SA), which is reported to greatly affect O3 sensitivity. However, the mechanism of SA biosynthesis under O3 exposure remains unclear. Plants synthesize SA either by a pathway involving phenylalanine as a substrate or another involving isochorismate. To clarify how SA is produced in O3-exposed Arabidopsis, we examined the activities of phenylalanine ammonia lyase (PAL) and isochorismate synthase (ICS), which are components of the phenylalanine and isochorismate pathways, respectively. Exposure of Arabidopsis to O3 enhanced the accumulation of SA and the increase of ICS activity but did not affect PAL activity. In sid2 mutants, which have a defect in ICS1, the level of SA and the activity of ICS did not increase in response to O3 exposure. These results suggest that SA is mainly synthesized from isochorismate in Arabidopsis. Furthermore, the level of ICS1 expression and the activity of ICS during O3 exposure elevated in plants deficient for SA signaling (npr1 and eds5 mutants and NahG transgenics). Treatment of plants with SA also suppressed the enhancement of ICS1 expression by O3. These results suggest that SA synthesis is negatively regulated by SA signaling.

The phenylalanine pathway is the main route of salicylic acid biosynthesis in Tobacco mosaic virus-infected tobacco leaves

In plants, salicylic acid (SA), a molecule important for resistance to pathogens, is synthesized from phenylalanine or isochorismate. Although SA is reportedly synthesized via the isochorismate pathway in pathogen-infected Arabidopsis, the predominant pathway in pathogen-infected tobacco has not been known. To determine the pathway in tobacco, we studied the gene expression and enzyme activity of phenylalanine ammonia-lyase (PAL) and isochorismate synthase (ICS) in tobacco leaves infected with Tobacco mosaic virus (TMV). Two days after TMV inoculation, necrotic lesions had appeared, and the levels of SA, PAL activity, and transcripts of PAL A and PAL B had increased substantially. In contrast, no ICS activity was detected, and the levels of ICS transcript did not increase, after the formation of necrotic lesions caused by TMV infection. These results suggest that SA is synthesized mainly by the phenylalanine pathway in TMV-infected and necrotic-lesion-bearing tobacco leaves.

Salicylic Acid Accumulation Under O3 Exposure is Regulated by Ethylene in Tobacco Plants

Ozone (O3), a major photochemical oxidant, induces leaf injury concomitant with salicylic acid (SA) synthesis. In pathogen-infected leaves, SA is synthesized via two pathways, involving phenylalanine or isochorismate. SA biosynthesis under O3 fumigation is not well understood. When we applied 14C-labeled benzoic acid (a precursor of SA in the pathway via phenylalanine) to O3-exposed tobacco leaves, it was effectively metabolized to SA. However, the activity and mRNA level of isochorismate synthase (ICS) were not increased. In contrast, ICS activity was increased in O3-exposed Arabidopsis thaliana L. These results suggest that SA is synthesized via benzoic acid from phenylalanine in O3-exposed tobacco leaves but via isochorismate in Arabidopsis. Ethylene is a plant hormone that promotes leaf damage in O3-exposed plants. During O3 exposure, transgenic plants with a phenotype of reduced O3-induced ethylene production accumulated less SA than did wild-type plants. O3 increased the activity of phenylalanine ammonia-lyase (PAL) and the transcript levels of the chorismate mutase (CM) and PAL genes in wild-type tobacco, but their induction was suppressed in the transgenic plants. These results indicate that ethylene promotes SA accumulation by regulating the expression of the CM and PAL genes in O3-exposed tobacco.

Regulation of anthraquinone biosynthesis in cell cultures of Morinda citrifolia

Cell cultures of Morinda citrifolia L. are capable of accumulating substantial amounts of anthraquinones. Chorismate formed by the shikimate pathway is an important precursor of these secondary metabolites. Isochorismate synthase (EC 5.4.99.6), the enzyme that channels chorismate into the direction of the anthraquinones, is involved in the regulation of anthraquinone biosynthesis. Other enzymes of the shikimate pathway such as deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) and chorismate mutase (EC 5.4.99.5) do not play a regulatory role in the process. The accumulation of anthraquinones is correlated with isochorismate synthase activity under a variety of conditions, which indicates that under most circumstances the concentration of the branchpoint metabolite chorismate is not a rate-limiting factor. Anthraquinone biosynthesis in Morinda is strongly inhibited by 2,4-D, but much less by NAA. Both auxins inhibit the activity of isochorismate synthase proportionally to the concomitant reduction in the amount of anthraquinone accumulated. However, the correlation between enzyme activity and rate of biosynthesis is less clear when the activity of the enzyme is very high. In this case, a limiting concentration of precursor may determine the extent of anthraquinone accumulation. Partial inhibition of chorismate biosynthesis by glyphosate leads to less anthraquinone accumulation, but also to a reduction in ICS activity. The complexity of the interference of glyphosate with anthraquinone biosynthesis is illustrated by the effect of the inhibitor in cell cultures of the related species Rubia tinctorum L. in these cells, glyphosate leads to an increase in anthraquinone content and a concomitant rise in ICS activity. All data indicate that the main point of regulation in anthraquinone biosynthesis is located at the entrance of the specific secondary route.

Clustering of isochorismate synthase genes menF and entC and channeling of isochorismate in Escherichia coli

There are two isochorismate synthase genes entC and menF in Escherichia coli. They encode enzymes (isochorismate synthase, EC 5.4.99.6) which reversibly synthesize isochorismic acid from chorismic acid. The genes share a 24.2% identity but are differently regulated. Activity of the MenF isochorismate synthase is significantly increased under anaerobic conditions whereas the activity of the EntC isochorismate synthase is greatly stimulated during growth in an iron deficient medium. Isochorismic acid synthesized by EntC is mainly channeled into enterobactin synthesis whereas isochorismic acid synthesized by MenF is mainly channeled into menaquinone synthesis. When menF or entC were separately placed onto overexpression plasmids and the plasmids introduced into a menF −/ entC − double mutant in two separate experiments, the isochorismate formed was fed into both, the menaquinone and the enterobactin pathway. Moreover, in spite of a high isochorismate synthase activity menaquinone and enterobactin formation were not fully restored, indicating that isochorismate was lost by diffusion. Thus, under these conditions channeling was not observed. We conclude that in E. coli the chromosomal position of both menF and entC in their respective clusters is a prerequisite for channeling of isochorismate in both pathways.

Expression of bacterial isochorismate synthase (EC 5.4.99.6) in transgenic root cultures ofRubia peregrina

The gene coding for isochorismate synthase (EC 5.4.99.6) was amplified by the polymerase chain reaction fromEscherichia coli, cloned into a binary vector. and mobilised intoAgrobacterium rhizogenes LBA9402 which was used to transformRubia peregrina. Transgenic roots containing bacterial isochorismate synthase cDNA expressed twice as much isochorismate synthase activity (4.88 pkat/mg protein) as the control roots (2.45 pkat/mg protein) after 10 days in culture, and accumulatedca. 20% higher levels of total anthraquinones after 30 days in culture. Whilst the amount of total alizarin (free and bound) in the transgenic roots was 30% higher than in control roots, the level of free alizarin wasca. 85% higher.

The in vitro Conversion of Chorismate to Isochorismate Catalyzed by the Escherichia coli entC Gene Product: Evidence that EntA does not contribute to isochorismate synthase activity

The entC and entA genes, coding for the enzymes isochorismate synthase and 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, respectively, were subcloned behind the T7 promoter in the expression plasmid pGEM3Z. Their protein products were overproduced and partially purified for in vitro analysis of the conversion of chorismate to isochorismate. Whereas previous genetic experiments suggested that the EntA enzyme has a role in this conversion, this study clearly indicates that EntC alone catalyzes the reaction. Addition of EntA had no effect on isochorismate synthase activity. As a result, the mutation (previously designated entC401) in strain AN191 was characterized by nucleotide sequence analysis. The lesion is a single base substitution in the entA gene, resulting in a glutamic acid-for-glycine substitution at the penultimate amino acid (residue 247) of the EntA enzyme. The mutant protein was partially purified and shown to be devoid of 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase activity, whereas the entC gene product from strain AN191 exhibited normal isochorismate synthase function. These results conflict with the earlier characterization of the entC401 mutation in a different genetic background. The data presented herein establish that the EntA protein does not contribute to isochorismate synthase activity and that the mutant strain that led to this suggestion harbors a defective allele of entA rather than entC.

Origin of p-aminobenzoic acid from chorismic rather than iso-chorismic acid in Enterobacter aerogenes and Streptomyces species

Enzyme extracts from Enterobacter aerogenes (62-1), Streptomyces aminophilus, and Streptomyces coelicolor were used to investigate the biosynthesis of p-aminobenzoic acid. The enzyme preparations from E. aerogenes and S. aminophilus contained both p-aminobenzoate synthase and iso-chorismate synthase activity, and were able to convert both chorismic and iso-chorismic acid to p-aminobenzoic acid. The apparent K M for chorismic acid was, however, significantly lower than that for iso-chorismic acid, while the V max was identical for both substrates in both enzyme systems. The enzyme preparations from S. coelicolor did not contain iso-chorismate synthase activity and p-aminobenzoic acid synthesis took place in this system from chorismic acid only. It is concluded that iso-chorismic acid is not an obligatory intermediate in p-aminobenzoic acid biosynthesis in these organisms.

Nucleotide sequence of a cluster of Escherichia coli enterobactin biosynthesis genes: identification of entA and purification of its product 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase

The nucleotide sequence of a region of the Escherichia coli chromosome encoding part of a cluster of genes involved in the biosynthesis of the iron chelator enterobactin has been determined. Four closely linked open reading frames, corresponding to the coding regions of entE (carboxy-terminal 144 amino acids), entB (32,554 daltons), entA (26,249 daltons), and an unidentified gene (P15) encoding a 14,970-dalton protein, were found. The lack of intergenic sequences and promoterlike elements suggests that these genes form part of the same transcription unit. We report the purification to homogeneity of the entA product, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase. It is an octamer of native molecular weight 210,000; the amino-terminal amino acid sequence confirmed the entA coding region. No isochorismate synthase activity was associated with this polypeptide. This finding leads to the conclusion that the recent suggestion (M. S. Nahlik, T. P. Fleming, and M. A. McIntosh, J. Bacteriol. 169:4163-4170, 1987) that 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase and isochorismate synthase activities reside on a single 26,000-dalton bifunctional enzyme is incorrect, even though the entA and entC mutations map to the same genetic locus.