Limonoids, biosynthesis

Inhibition of Limonoid Biosynthesis by Auxins. Auxins are potent inhibitors of nomilin biosynthesis in citrus seedlings (31). For instance, up to 91% inhibition was observed when 10 ppm of indoleacetic acid was fed to the stem of a lemeon seedling two days prior to and two days following feeding of 25 pCi of 14-C acetate (Table 1). Other auxins tested include 1-naphthaleneacetic acid (NAA), indolepropionic acid, indolebutyric acid, 3-indole acetonitrile, ethyl indole-3-acetate, 3-indoleacrylic acid, 3-(2-hydroxyethyl)indole, indole-2-carboxylic acid and 2,3,4-trichlorophenoxyacetic acid. They were all very effective. 

Limonin and nomilin cause bitter flavor in citrus products. The bitterness is a major problem for the industry. Hasegawa et al. (this volume) have identified three target enzymes involved in limonoid biosynthesis for development of transgenic citrus free of limonin and nomilin bitterness. They are linoleate dehydrogenase, UDP glucose transferase and nomilin deacetylesterase. The isolation of the genes for these enzymes is currently being conducted in order to eventually insert them into cultured citrus cells where they will convert the bitter compounds to non-bitter derivatives. From the cultured cells mature citrus plants will be produced and these plants should produce fhiit free of bitter flavor. 

Radioactive tracer work has shown that nomilin (3) is biosynthesized by the terpenoid pathway from acetate in the phloem region of stems and translocated to other parts of plants such as leaves, fruit tissues and seeds (13,14). At those locations, nomilin is further biosynthesized to other limonoids. Limonoid biosynthesis occurs at each location independently, thus the composition of limonoids in fruit tissues, seeds and leaves are different from each of the others. Limonin is biosynthesized from nomilin via obacunone (4), obacunoate (5) and ichangin (6) (15-17) (Fig. 2)... 

No evidence of limonoid biosynthesis in fruit or seed tissues exists, despite the fact that most of the limonins are found in the seeds of mature fruits. Limonoid synthesis occurs in the leaves and limonoids are transported into the fruits (Maier, 1983). In citrus tissues, the naturally occurring precursor of limonin is a salt of limonoic acid A-ring lactone (60) (Fig. 25.13) in which the A ring is closed and the D ring is open. This tasteless compound is stable only in the salt form (Maier, 1983). In the presence of acid or the enzyme citrus limonoate D-ring hydrolase, the D-ring lacton-izes to form limonin (19). The rate of lactonization is accelerated by pasteurization of the juice. In the fruit, the precursor appears to be located in a compartment of the cell where the pH is neutral or alkaline, probably the cytoplasm (Maier, 1983). 

Nishiura M, Kamiya S, Esaki S (1971b) Flavonoids in Citrus and related genera. III. flavonoid pattern and Citrus taxonomy. Agric Biol Chem 35 1691-1706 Ou P, Hasegawa S, Herman Z, Fong CH (1988) Limonoid biosynthesis in the stem of Citrus limon. Phytochemistry 27 115-118... 

Ethylphenoxy)triethylamine and 2-(3,4-dimethoxyphenoxy)triethylamine markedly reduce the biosynthesis of limonoids in citrus leaves, presumably by inhibition of cyclase activity. Radio-tracer studies have revealed that limonoids are synthesized in the leaves of citrus and transported to the fruit. The fruit tissue does not appear to be capable of the de novo synthesis of limonoids from acetate or mevalonate. 

Inhibition of Biosynthesis. Triethylamine derivatives such as 2-(4-ethylphenoxy)triethylamine and 2-(3,4-dimethylphenoxy)-triethylamine markedly inhibit the accumulation of limonoids in citrus leaves (15). For example, young lemon leaves sprayed with 500 ppm of 2-(4-ethylphenoxy)triethylamine contained only 27 ppm of XIV 8 days after the treatment, whereas the control contained 344 ppm. Similarly, those sprayed with 300 ppm of the compound contained 0.3 times as much XIV as the control. 

During the past several years, significant progress has been made in studies on the biosynthesis of citrus limonoids. Based on recent radioactive tracer work, the biosynthetic pathways of the major limonoids in common citrus are well established (Fig. 1). 

Thus, nomilin acetyl-lyase appears to play a key role in the regulatory system which controls the biosynthesis and accumulation of limonoids in citrus. 

Stems are the major site of nomilin biosynthesis from acetate in citrus (26). Analysis of the phloem, the cortex and the inner core regions of the stem showed that the phloem region is the site of nomilin biosynthesis from acetate (27). Root tissues also have this capacity. Leaves, fruits and seeds are either incapable of biosynthesizing limonoids from acetate or have a very low capacity. However, these tissues are capable of biosynthesizing limonoids from nomilin. Nomilin is translocating from the stem to other locations, where it is further biosynthesized to other limonoids (26). 

Biosynthesis and metabolism of limonin and its closely related limonoids... 

Hasegawa, S., R. D. Bennett, and V. P. Maier, Biosynthesis of limonoids in Citrus seedlings. Phytochemistry, 23, 1601-1603... 

Hasegawa and Hoagland (1977) have recently carried out tracer studies on the biosynthesis of limonoids in citrus. These studies showed that young leaves were capable of synthesizing limonoids and su ested that the limonoids were translocated from this biosynthetic site to the fruits. 

Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40 347-369 Hasegawa S, Herman Z (1992) Biosynthesis of limonoids in Citrus. In Petroski RJ, McCormick SP (eds) Secondary-metabolite biosynthesis and metabolism. Plenum Press, New York, pp 305-317... 

Hasegawa S, Herman Z, Orme E, Ou P (1986) Biosynthesis of limonoids in Citrus sites and translocation. Phytochemistry 25 2783-2785... 

The common precursor in the biosynthesis of limonoids and cucurbitacins is squalene-2,3-oxide (IV). Based on some identified intermediary compounds, the biosynthetic pathway is probably as postulated in Reaction 18.7. 

The partial synthesis of the furan ring in havanensin-type compounds has been accomplished by sodium metaperiodate oxidation of turreanthin in the presence of perchloric acid (Scheme 4). Execution of the apo rearrangement as previously described then led to the partial synthesis of a simple havanensin derivative 36, 39). It is not known if the biosynthesis goes by this route or by an alternative following the more usual limonoid pattern of an oxide rearrangement 81). 

Further work on the biosynthesis of limonoids is required. Experiments aimed at laboratory simulation of production of phragmalin and prie-urianin are in hand, but experiments using labelled precursors in biological systems are badly needed. Research on the probable relationship of limonoids and quassinoids is also required. 

Figure 3.34 Outline biosynthesis of the dammarane triterpenoids including ginsenosides and limonoids.

---