The general phenylpropanoid pathway

TEUTSCH, H.G., HASENFRATZ, M.P., LESOT, A., STOLTZ, C., GARNIER, J.M., JELTSCH, J.M., DURST, F., WERCK-REICHHART, D., Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway, Proc. Natl. Acad. Sci. USA, 1993,90,4102-4106. 

The tightly regulated pathway specifying aromatic amino acid biosynthesis within the plastid compartment implies maintenance of an amino acid pool to mediate regulation. Thus, we have concluded that loss to the cytoplasm of aromatic amino acids synthesized in the chloroplast compartment is unlikely (13). Yet a source of aromatic amino acids is needed in the cytosol to support protein synthesis. Furthermore, since the enzyme systems of the general phenylpropanoid pathway and its specialized branches of secondary metabolism are located in the cytosol (17), aromatic amino acids (especially L-phenylalanine) are also required in the cytosol as initial substrates for secondary metabolism. The simplest possibility would be that a second, complete pathway of aromatic amino acid biosynthesis exists in the cytosol. Ample precedent has been established for duplicate, major biochemical pathways (glycolysis and oxidative pentose phosphate cycle) of higher plants that are separated from one another in the plastid and cytosolic compartments (18). Evidence to support the hypothesis for a cytosolic pathway (1,13) and the various approaches underway to prove or disprove the dual-pathway hypothesis are summarized in this paper. 

In a recent study (54), we showed increased activities of two enzymes of the general phenylpropanoid pathway, PAL and 4-coumarate CoA lig-ase, as well as one enzyme of the specific pathway of lignin biosynthesis, cinnamy 1-alcohol dehydrogenase (CAD), in resistant plants at the time of the hypersensitive host cell death. On the other hand, decreased activities were observed at the same time with susceptible host plants (54). Furthermore, we showed that the well known increase in peroxidase activities, which is strong in resistant and only weak in susceptible plants (55-58), is at least partly due to the increased activity of the lignin biosynthetic pathway (54,59). 

Coumaroyl-CoA is produced from the amino acid phenylalanine by what has been termed the general phenylpropanoid pathway, through three enzymatic conversions catalyzed by phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate CoA ligase (4CL). Malonyl-CoA is formed from acetyl-CoA by acetyl-CoA carboxylase (ACC) (Figure 3.2). Acetyl-CoA may be produced in mitochondria, plastids, peroxisomes, and the cytosol by a variety of routes. It is the cytosolic acetyl-CoA that is used for flavonoid biosynthesis, and it is produced by the multiple subunit enzyme ATP-citrate lyase that converts citrate, ATP, and Co-A to acetyl-CoA, oxaloacetate, ADP, and inorganic phosphate. ... 

The general phenylpropanoid pathway links the shikimate pathway to the lignin branch pathway. The latter pathway leads to the formation of a series of hydroxycinnamic acids and hydroxycinnamoyl-CoA esters varying in their degrees of hydroxylation and methylation [5]. 

The general phenylpropanoid pathway begins with the deamination of L-phenylalanine to cinnamic acid catalyzed by phenylalanine ammonia lyase (PAL), Fig. (1), the branch-point enzyme between primary (shikimate pathway) and secondary (phenylpropanoid) metabolism [5-7]. Due to the position of PAL at the entry point of phenylpropanoid metabolism, this enzyme has the potential to play a regulatory role in phenolic-compound production. The importance of this is illustrated by the high degree of regulation both during development as well as in response to environmental stimuli. 

Studies have shown that phenylpropanoid metabolism can be stimulated by ozone. The activity of PAL increased in soybean [91], Scots pine (Pinus sylvestris L.) [92], and parsley (Petroselinum crispum L.) [93] soon after treatment with 150-200 nmol O3 mol 1. Rapid increases in transcript levels for PAL in response to ozone have been observed in parsley [93], Arabidopsis thaliana L. Heynhold [94] and tobacco (Nicoticma tabacum L.) [95]. Transcript levels for 4-coumarate CoA ligase (4CL), the last enzyme in the general phenylpropanoid pathway, increased commensurately with PAL transcripts in ozone-treated parsley seedlings [93]. Phenolic compunds reported to accumulate in leaf tissue in response to ozone include hydroxycinnamic acids, salicylic acid, stilbenes, flavonoids, furanocoumarins, acetophenones, and proanthocyanidins [85, 92, 93, 96, 97]. 

Figure 3-4. The general phenylpropanoid pathway. The enzymes involved in this pathway are (a) phenylalanine ammonia lyase (PAL E.C. 4.3.1.5), (b) cinnamic acid 4-hydroxylase (C4H E.C. 1.14.13.11), and (J) 4-coumaric acid CoA ligase (4CL E.C. 6.2.1.12). (a) depicts tyrosine ammonia lyase activity in PAL of graminaceous species. The grey structures in the box represent an older version of the phenylpropanoid pathway in which the ring substitution reactions were thought to occur at the level of the hydroxycinnamic acids and/or hydroxycinnamoyl esters. The enzymes involved in these conversions are (c) coumarate 3-hydroxylase (C3H E.C. 1.14.14.1), (d) caffeate O-methyltransferase (COMT EC 2.1.1.68), (e) ferulate 5-hydroxylase (F5H EC 1.14.13), and (g) caffeoyl-CoA O-methyltransferase (CCoA-OMT EC 2.1.1.104). These enzymes are discussed in more detail in Section 10.

As discussed in Section 7, the general phenylpropanoid pathway originally included the biosynthesis of the hydroxycinnamic acids caffeic acid (3.32), femlic acid (3.33), 5-hydroxyferulic acid (3.34), and sinapic acid (3.35) from />coumaric acid (3.30), as well as the corresponding CoA-esters... 

Sinapate is synthesized via the oxidation of sinapaldehyde (3.79) by an aldehyde dehydrogenase, as described in Section 13 of this chapter. Sinapaldehyde, in turn, is derived from the amino acid phenylalanine (3.27) via the general phenylpropanoid pathway (see Section 7), followed by a number of the hydroxylation and methylation reactions described in Section 10. 

One of the major features of phenylpropanoid metabolism is the diversity of end products. The set of enzymic reactions leading from phenylalanine to 4-coumaroyl coenzyme A is common to pathways which lead to these diverse end products and is known as the general phenylpropanoid pathway (Fig. 1). Those biochemical reactions which lead to the synthesis of specialised products are known as branch pathways. 

Table 1. Genes encoding enzymes of the general phenylpropanoid pathway...

In this chapter I will focus on biochemical and molecular aspects leading to lignin production. We have studied in detail phenylalanine ammonia lyase (PAL EC 4.3.1.5), the first enzyme of the general phenylpropanoid pathway, and cinnamyl alcohol dehydrogenase (CAD EC 1.1.1.195), an enzyme specific to the branch pathway leading to lignin formation. 

The branch pathway of lignin biosynthesis is shown in Fig. 2. The first steps are shared with the general phenylpropanoid pathway. Cinnamic acid is transformed by hydroxylation and methylation to produce acids with different substitutions on the aromatic ring. The 4-coumaric, ferulic and sinapic acids are then esterified by hydroxycinnamate CoA ligase to produce cinnamyl-CoAs, which are reduced by cinnamyl-CoA reductase (CCR) to produce the three aldehydes. These in turn are reduced by CAD to the three cinnamyl alcohols which are then polymerised into lignins. 

Chappie, C.C.S., Vogt, T., Ellis, B.E. and Somerville, C.R. (1992) An Arabidopsis mutant defective in the general phenylpropanoid pathway. Plant Cell, 4,1413-24. 

A second class of defensive phytochemicals often found in poplar and aspen at substantial levels are the proanthocyanidins, or condensed tannins (CTs) (Fig. 5.1). Unlike the phenolic glycosides that are found exclusively in the Salicaceae, CTs are widespread in the plant kingdom. These flavonoid polymers consist of mostly 4,8-linked flavan-3,4-diols and flavan-3-ols, ranging in size from 1440 to over 4500 Da depending on the species. Flavonoids are derived from the general phenylpropanoid pathway by a series of enzymes beginning with the enzyme... 

CHAPPLE, C.C.S., VOGT, T., ELLIS, B.E., SOMERVILLE, C.R., An Arabidopsis Mutant Defective in the General Phenylpropanoid Pathway, Plant Cell, 1992, 4, 1413-1424. 

---