Biosynthesis in citrus

Lewinsohn E, Britsch L, Mazur Y, Gressel J (1989) Flavanone glycoside biosynthesis in Citrus chalcone synthase, UDP-glucose flavanone-7-0-glucosy-transferase and -rhamnosyltransferase activities in cell-free extracts. Plant Physiol 91 1321-1328... 

Citrus leaves were shown to be the site of limonoic acid A-ring lactone biosynthesis in citrus (55). The lactone accumulated to the level of 2000 ppm in very small leaves but as the leaf grew, the lactone content declined. The lactone content of the fruit increased as the level in the leaves declined. Hasegawa and Hoagland (55) also showed that limonoic acid A-ring lactone was not synthesized in the fruit but in the leaves. The radioactive labeled lactone was isolated from a fruit adjacent to a leaf actively synthesizing it from labeled acetate, indicating that the lactone was synthesized in the leaves and transported to the fruit (55). 

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. 

Natural Products as Inducers of Insect Resistance. Plant growth regulators have been shown to increase the biosynthesis of certain secondary plant constituents that in turn decrease plant attack by insects. -Naphthaleneacetic acid, for example, elicits increased terpene biosynthesis in citrus, thus decreasing attack by fruit flies. The approach of using both natural and synthetic plant growth regulators may continue to find applications in insect control. 

Hasegawa, S., V. P. Maier, Z. Herman, and P. Ou, Phytohormone bioregulation of nomilin biosynthesis in Citrus limon seedlings. Phytochemistry, 25, 1323-1325 (1986). 

McIntosh C, Mansell R (1990) Biosynthesis of naringin in Citrus paradisi UDP-glucosyltransferase activity in grapefruit seedlings. Phytochemistry 29 1533-1538... 

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. 

The biosynthesis the /3-hydroxylated compound synephrine has been studied in Citrus species (325). An elegant experiment carried out in Cleopatra mandarin seedlings showed that tyramine is rapidly methylated to N-methyltyramine... 

Fig. 1. Distribution of radioactivity among phenolic amines during 3 months after feeding [l-14C]tyramine to a Cleopatra mandarin seedling. ( — ) Hordenine, (0—0) synephrine, (O—O) AJ-methyltyramine, and (O—O) tyramine. (Reprinted with permission from Phytochemistry, Vol. 8, T. A. Wheaton and 1. Stewart, Biosynthesis of synephrine in citrus. Copyright 1969, Per-gamon Journals Ltd.)...

Belajova, E. Suhaj, M. 2004. Determination of phenolic constituents in citrus juices Method of high performance liquid chromatography. Food Chem. 86 339-343. Berhow, M.A. Vandercook, C.E. 1989. Biosynthesis of naringin and prunin in detached grapefruit. Phytochem. 28 1627-1630. 

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). 

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. 

The pericarp of developing fruit have been shown also to be sites of GA biosynthesis [150-152,158]. Indeed, work with Citrus indicates that the ability of fruit to set parthenocarpically depends on the levels of active GAs produced in the ovary [159]. As discussed above, seeds may stimulate GA biosynthesis in fruit such as pea, that require the presence of fertilised seeds to develop normally [124,150,151]. 

The j3-hydroxyphenethylamine synephrine (198) appears to arise in Citrus from tyrosine via tyramine and iV-methyltyramine. ° Normacromerine (199) and macromerine (200) are found in the cactus Coryphantha macromeris var. runyoniO Their genesis has also been studied.Specific incorporations of DL-[3- C]tyrosine, [l- C]tyramine, DL-[2- " C]dopa, and [l- CJdopamine into normacromerine established a pattern of biosynthesis similar to that of the other cactus alkaloids and again a C6-C2 unit is implicated. [This contrasts with the biosynthesis of the superficially similar base ephedrine (185), discussed above.]... 

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). 

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. 

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