Higher plants use aromatic amino acids not only as building blocks in proteosynthesis, but also, and in even greater quantities, as precursors to a large number of secondary metabolites. In addition, different plants also use the intermediates of the shikimate pathway as precursors to aromatic compounds (Figure 1). Under normal growth conditions, a significant amount of carbon fixed by plants flows through the shikimate pathway (Haslam, 1993; Connelly and Conn, 1985).
2.1 DAHP Synthase
The main trunk of the shikimate pathway, also called the prechorismate pathway, consists of seven enzymes. 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyses the first reaction—the condensation of erythrose 4-phosphate and phosphoenol pyruvate (Figure 2). The product of this reaction is a seven-carbon six- membered heterocylic compound, DAHP.
Two isoenzymes of DAHP synthase have been identified in Vigna radiata (Rubin and Jensen, 1985), showing Mn2 +-stimulated and Co2+-dependent activities. This pair
has been isolated from leaves and cell suspension cultures of Nicotiana silvestris and from various monocotyledonous and dicotyledonous plants (Ganson et al., 1986). In chloroplasts prepared from tobacco and spinach leaves by sucrose gradient centrifugation only the Mn2+-stimulated isoform was identified. The Co2+-dependent isoform of DAHP synthase was present in a soluble fraction.
Recently the cytosolic, Co2 +-dependent isoform has been purified to electrophoretic homogeneity from cultured carrot cells (Suzuki et al., 1996). The molecular mass of this enzyme was established to be 115kDa. The molecular masses of this isoform from other plants were reported to be around 400 kDa (Doong et al., 1992). The pH optimum of the purified Co2 +-dependent isoform was 9.0 and the Km for erythrose- 4-phosphate was 3.3 mM.
The Mn2+-stimulated isoform of DAHP synthase was purified to electrophoretic homogeneity from potato tubers (Pinto et al., 1986) and carrot (Suzich et al., 1985). Figure 1 A schematic diagram showing different classes of secondary metabolites that arise from aromatic amino acids and from intermediates of the shikimate pathway
To identify the first cDNA coding for plant DAHP synthase (Dyer et al., 1990), a cDNA expression library from potato cell suspension cultures (Dyer et al., 1989) was screened by antibodies (Pinto et al., 1988) specific to the Mn2+-stimulated isoform of DAHP synthase. This potato cDNA was than used to clone a second cDNA from potato (Zhao and Herrmann, 1992) and the homologues from Arabidopsis (Keith et al., 1991), tobacco (Wang et al., 1991) and tomato (Görlach et al.,1993a). Functional complementation of yeast (Keith et al., 1991) and E. coli (Weaver et al., 1993) both defective in DAHP synthase with cDNA from potato and Arabidopsis demonstrated that these cDNA encode polypeptides with DAHP synthase activity. Polypeptides obtained by translation of cDNA for DAHP synthase have N-terminal sequences resembling the transit sequences targeting the enzymes in chloroplasts (Gavel and von Heijne, 1990). Therefore, it seems likely that chloroplasts contain two Mn2+- stimulated isoenzymes. Gene coding for these Mn2+ -stimulated isoenzymes (Shk1
and Shk2) in potato (Dyer et al., 1989; Keith et al., 1991) and DHS1 and DHS2 in Arabidopsis (Keith et al., 1991) responded differently to wounding and pathogen attack. In the tomato these two genes also code for plastidic enzymes and exhibit strikingly different organ-specific expressions and sensitivity to elicitation and pathogen attack (Görlach et al., 1994, 1995a,b).
2.2 3-Dehydroquinate Synthase
The second enzyme of the prechorismate pathway catalyzes the cyclization step in this pathway. For catalytic activity the enzyme requires NAD+ and a divalent cation. By density-gradient methods 3-dehydroquinate synthase has been localized in chloroplasts from pea seedlings (Mousdale and Coggins, 1985). The purified enzyme has been obtained from the mung bean (Yamamoto, 1980) and pea (Pompliano et al., 1989). Analyses of organ-specific expression of the gene encoding this enzyme in tomato plants showed that the abundance of the corresponding transcripts was relatively high in tomato flowers. A cDNA library was constructed from tomato flowers (Bischoff et al., 1996) and used for complementation of E. coli cells defective in dehydroquinate synthase. A cDNA clone encoding dehydroquinate synthase was isolated. The N-terminal region of the deduced amino acid sequence contained a putative plastidic transit peptide, that is 3-dehydroquinate synthase appears to be a plastidic enzyme. The level of dehydroquinate synthase transcripts was highest in the roots (with an eightfold difference between roots and leaves) and lower in the flowers and stems. Strong induction of the dehydroquinate synthase transcripts was observed after elicitation of cultured tomato cells by a cell wall preparation of Phytophthora megasperma f. sp. glycinea.
2.3 3-Dehydroquinate Synthase-Shikimate Dehydrogenase
The third step of the prechorismate pathway is catalyzed by 3-dehydroquinate dehydratase. In the course of conversion of 3-dehydroquinate to dehydroshikimate the process of aromatization is initiated, i.e. the first of the three double bonds is introduced. The elements of water are released by a syn (cis) elimination (Bentley, 1990). 3-dehydroquinate dehydratase and the next enzyme of the prechorismate pathway, the shikimate NADP oxidoreductase, reside on a single polypeptide (Fiedler and Schultz, 1985; Mausdale et al., 1987; Mousdale and Coggins, 1984). Within this bifunctional enzyme, the turnover of the first reaction is only one-ninth of the second reaction, that is dehydroshikimate does not accumulate during the conversion of dehydroquinate to shikimate. Pea enzymes were purified to near homogeneity (Deka et al., 1994) and used to prepare antibodies against the pure bifunctional enzyme. Partial cDNA encoding the bifunctional enzymes of the pea (Deka et al., 1994) and tobacco (Bonner and Jensen, 1994) have been cloned. This near full length cDNA from tobacco complemented the aroD and aroE mutants of E. coli.
Partially purified shikimate dehydrogenase was prepared from dark-grown three- day old poppy seedlings ( Šmogrovièová et al., 1981). The kinetic data of this enzyme (pH optimum 9.8; Km values for shikimate and NADP 0.59 mM and 0.018 mM respectively) are similar to those from other plants (Balinsky et al., 1961; Koshiba, 1978). The activity of the enzyme was not affected by aromatic amino acids,
phenylpyruvic and chorismic acids or thebaine, codeine and morphine (up to 1 mM final concentration). Four isoforms of shikimate dehydrogenase were identified in dry poppy seeds and three isoforms were determined in individual organs of etiolated poppy seedlings.
2.4 Shikimate Kinase
Shikimate kinase catalyses an unexceptional phosphate transfer from ATP to C-3- OH group of shikimate. This enzyme has been described in different plants (Koshiba, 1979; Bowen and Kosuge, 1979; Mousdale and Coggins, 1985). A near homogeneous enzyme preparation has been obtained from spinach chloroplasts (Schmidt et al., 1990) and the first cDNA encoding this enzyme has been isolated from tomato (Schmid et al., 1992). Southern blot analysis showed the presence of only one gene for shikimate kinase in haploid tomato genome. When tomato cells were treated with elicitors, the abundance of shikimate kinase specific transcripts was found to increase dramatically with time (Görlach et al., 1995a, b), i.e. the expression of the gene for shikimate kinase is sensitive to environmental stimuli. The pattern of organ-specific expression of the shikimate kinase gene in tomato is similar to the chorismate synthase gene and the 5-enolpyruvylshikimate 3-phosphate synthase gene (Görlach et al., 1994).
2.5 5-Enoylpyruvylshikimate-3-Phosphate (ESPS) Synthase
This enzyme is the most thoroughly studied of all the enzymes in the shikimate pathway. The chemical aspects of the enzyme-catalyzed reaction of phosphoenol pyruvate and shikimate-3-phosphate to ESPS have been discussed by Bentley (1990) and Anderson and Johnson (1990). ESPS synthase has been purified to electrophoretic homogeneity from seedlings of Pisum sativum (Mousdale and Coggins, 1986) and Sorghum bicolor (Ream et al., 1988). In the pea this enzyme was localized in chloroplasts and only a minor fraction was detected in the cytosolic fraction. For ESPS synthase several genes and cDNA clones have been isolated from different plants—Petunia hybrida (Sha et al., 1986), tomato (Gasser et al., 1988), Brassica napus (Gasser et al., 1990) and Arabidopsis thaliana (Klee et al., 1987). All these genes and cDNAs studied so far encode a plastidic enzyme. The abundance of ESPS synthase specific transcripts increased with time in elicited tomato cell cultures. Similar results were obtained when the abundance of ESPS synthase specific transcripts was analysed in infected tomato plants (Görlach et al., 1995a, b). The highest level of ESPS synthase mRNA was found to be in the petals and anthers of mature petunia plants. The amount of this mRNA was very low in the leaves and pistils of petunia. In the same organs of tomato plants the amounts of ESPS synthase mRNA varied only slightly (Gasser et al., 1988). The organ-specific expression of ESPS synthase gene in tomato plants is very similar to that of the shikimate kinase gene and the two chorismate synthase genes (Görlach et al., 1994). In transgenic petunia and tobacco plants the tissue specific expression of reporter genes (chloramphenicol acetyltransferase or ß-glucuronidase) was analyzed under the control of different the major target for inhibition by glyphosate, a broad spectrum non-selective herbicide (Bentley, 1990).
2.6 Chorismate Synthase
Chorismate synthase (CS) catalyzes a 1,4-trans elimination of phosphate from 5- enolpyruvylshikimate-3-phosphate (Hawkes et al., 1990). In the course of this reaction the second double bond of the benzene ring is formed. CS is unusual in requiring a reduced flavine cofactor (FMNH2 or FADH2) for catalytic activity even though the overall reaction is redox neutral. The same is true for 3-dehydroquinate synthase requiring NAD+ for a redox neutral reaction. The chorismate synthase from both N. crassa (Welch et al., 1974) and E. gracillis (Schaller et al., 1991a) is a bifunctional enzyme, containing a NADPH-driven flavine reductase activity. Chorismate synthase from higher plants is a monofunctional enzyme (Schaller et al., 1990). Chorismate synthase was first described in seedlings of Pisum sativum (Mousdale and Coggins, 1986). A homogeneous enzyme preparation was obtained from cell cultures of Corydalis serpervirens (Schaller et al., 1990b) and this enzyme preparation was used to obtain polyclonal antibodies. Screening of a cDNA library with polyclonal antibodies resulted in isolation of the first CS-specific cDNA from higher plants (Schaller et al., 1991b). Three cDNAs (CS1, CS2 and CS2Δ) corresponding to two chorismate synthase genes (LeCS1 and LeCS2) have been identified in tomato plants (Görlach et al., 1993b, 1995a). These cDNAs, as do those from C. serpervirens, encode polypeptides with N-terminal sequences resembling the peptides for chloroplast transport. Differential splicing of LeCS2-specific pre-mRNA results in the formation of two different transcripts CS2 and CS2Δ. In tomato cell cultures exposed to elicitation the abundance of CS1-specific transcripts increased dramatically with time. Under these conditions the abundance of CS2-specific transcripts remained unchanged. Identical results were obtained with infected leaves of potato plants (Görlach et al., 1995b). An analysis of the abundance of CS1- and CS2-specific transcripts in organs of tomato plants showed that the level of these transcripts is higher in the roots and flowers than the in stems, leaves and cotyledons. The abundance of transcripts of CS1 gene is consistently higher than that of CS2 gene (Görlach et al., 1994). To characterize the biochemical properties of CS isoenzymes, cDNAs for mature isoenzymes (CS1, CS2 and CS2Δ) have been expressed in E. coli (Braun et al., 1996). The isoenzyme CS2Δ appeare d to be unstable. The isoenzymes CS1 and CS2 have been purified to near homogeneity. The only difference between the two isoenzymes were their Km values for 5-enolpyruvylshikimate-3-phosphate. The Km value of CS2 is seven times higher than that of CS1.
3 BIOSYNTHESIS OF PHENYLALANINE, TYROSINE AND