Ethylene methionine

Buffer and chemicals pH 7.8 phosphate buffer (16.29g Na2HP04-2H20, 1.17g NaH2P04H20, 1000 ml distilled water), Ethylene Diamine Tetra Acetic Acid (EDTA), Methionine (Met), Polyvinylpyrrolidone (PVP), Triton X-100, Phenylmethylsulphonylfluoride (PMSF), Riboflavin, Nitro Blue Tetrazolium (NBT). 

The nonprotein amino acid, 1-aminocyclopropane-l-carboxylic acid, is an intermediate of ethylene biosynthesis in plants. This amino acid is synthesized from the L-a-amino acid methionine through the intermediate 5 -adenosyl-L-methionine (SAM) (Scheme 8). ... 

The possibility that many organic compounds could potentially be precursors of ethylene was raised, but direct evidence that in apple fruit tissue ethylene derives only from carbons of methionine was provided by Lieberman and was confirmed for other plant species. The pathway of ethylene biosynthesis has been well characterized during the last three decades. The major breakthrough came from the work of Yang and Hoffman, who established 5-adenosyl-L-methionine (SAM) as the precursor of ethylene in higher plants. The key enzyme in ethylene biosynthesis 1-aminocyclopropane-l-carboxylate synthase (S-adenosyl-L-methionine methylthioadenosine lyase, EC 4.4.1.14 ACS) catalyzes the conversion of SAM to 1-aminocyclopropane-l-carboxylic acid (ACC) and then ACC is converted to ethylene by 1-aminocyclopropane-l-carboxylate oxidase (ACO) (Scheme 1). 

In addition to ACC, ACS produces 5 -methylthioadenosine (MTA), which is recycled through methionine cycle to methionine (see Scheme 1). Recycling of MTA back to methionine requires only the available ATP. A constant concentration of cellular methionine is maintained even when ethylene is rapidly synthesized or when the pool of free methionine is small. The methionine cycle involves the following subsequent intermediates MTA, 5-methylthioribose (MTR), 5-methylthioribose-1-phosphate (MTR-l-P), 2-keto-4-methylthiobutyrate (KMB), and then the recycled methionine. ... 

Scheme 1 The ethylene biosynthetic pathway. The enzymes catalyzing each step are shown above the arrows. SAM S-adenosyl-L-methionine SAMS S-adenosyl-i-methionine synthetase ACC 1-aminocyclopropane-1-carboxylic acid ACS 1-aminocyclopropane-1-carboxylate synthase ACO 1-aminocyclopropane-1-carboxylate oxidase Ade adenine MTA methylthioadenosine. The atoms of SAM recycled to methionine through methionine cycle are marked in red and the atoms of methionine converted to ethylene are marked in bold. For details see text.

The pathway of ethylene biosynthesis in higher plants is from l-methionine4 (Figure 5.9). Methionine is an intermediate in other metabolic processes and the control of ethylene biosynthesis via the interference of methionine production is not realistic. The ACC synthase step from S-adenosyl methionine to ACC appears more susceptible to chemical modification auxin promotes ethylene production by increasing the activity of ACC synthase. Subsequent steps from ACC are less controlled and ethylene is readily produced from the conversion of ACC in most tissues. 

A reduction and activation of HjOj by other one-electron donors, like semiquinones, has also to be considered. This follows from a study of the ethylene production from methionine in the presence of pyridoxal phosphate, a reaction characteristic for OH radicals or for Fenton-type oxidants. The ethylene production in the presence of dioxygen, anthraquinone-2-sulfonate, and an NADPH-generating system in phosphate buffer pH 7.6 was inhibited by SOD and by catalase, but stimulated by scavengers of OH radicals, like 0.1 mM mannitol, a-tocopherol, and formiate... 

Ethylene is now considered to be one of the main plant-hormones involved in fruit development. Many responses formerly believed to result from the presence of auxins are now ascribed to induced ethylene production.425 The biosynthetic pathway for formation of ethylene from methionine, in a wide variety of plant tissues, including shoots of mung bean,426 tomato,427 and pea427 carrot427 and tomato428 roots and the fruits of apple,429,430 tomato,427 and avocado,427 has been elucidated, and is as follows. 

The formation of ethylene is often induced by the hormone auxin (Chapter 30), which stimulates activity of the synthase that forms 1-aminocyclopropane-1-carboxylate (ACC) from S-adenosyl methionine (Eq. 14-27, step j Fig. 24-16).320a/b Although ACC has... 

The most recent discoveries in the methionine to ethylene pathway are the demonstration of S-adenosylmethionine as the intermediate and the existence of the multigene family of ACC synthases that convert S-adenosylmethionine to ACC (for review see Kende, 1993). The expression of the different genes in different tissues is determined by different stimuli such as ripening, tissue wounding or the status of cell growth responses. The isolation of the oxidase enzyme that converts ACC to liberate the free ethylene molecule in vitro (Ververidis and John, 1991) was another breakthrough, particularly because for many years it was thought that the enzymes concerned would operate only on an intact membrane system in vivo. 

However, it should be pointed out here that not all plants follow the same synthetic pathway for ethylene. Lower plants (liverworts, mosses, ferns, lycopods) do not produce it from methionine, nor from ACC—an alternative ethylene pathway therefore exists (Osborne et al., 1996). In evolutionary terms this is very significant and it remains to be established whether any cells in higher plants still retain this earlier primitive route for ethylene synthesis as part of their metabolic repertoire. 

Adams, D.O. Yang, S.F. (1979). Ethylene biosynthesis identification of 1-aminocyclopropane-l-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA 76, 170-174. 

Lieberman, M., Kunishi, A., Mapson, L.W., Wardale, D.A. (1966). Stimulation of ethylene production in apple tissue slices by methionine. Plant Physiol. 41, 376-382. 

Fig. 1. Ethylene biosynthesis. The numbered enzymes are (1) methionine adenosyltransferase, (2) ACC (l-aminocyclopropane-l-carboxylic acid) synthase, (3) ethylene forming enzyme (EFE), (4) 5 -methylthio-adenosine nucleosidase, (5) 5 -methylthioribose kinase. Regulation of the synthesis of ACC synthase and EFE are important steps in the control of ethylene production. ACC synthase requires pyridoxal phosphate and is inhibited by aminoethoxy vinyl glycine EFE requires 02 and is inhibited under anaerobic conditions. Synthesis of both ACC synthase and EFE is stimulated during ripening, senescence, abscission, following mechanical wounding, and treatment with auxins.

Ethylene plays an important role in a number of plant developmental processes, including senescence and abscission of leaves and flowers, responses to wounding, and the ripening of climacteric fruits (Abeles, 1973). In each case ethylene is produced from methionine (Fig. 1). The two enzymes specific to the pathway, ACC synthase and ethylene forming enzyme, increase in activity in response to wounding and during ripening,... 

Methionine is the major precursor in the biochemical pathway to ethylene (9). Ethylene is formed from carbons 3 and 4 of methionine which is degraded in reactions possibly involving free radicals and oxygen (9). Recently Adams and Yang (10,11) identified S-adenosylmethionine (SAM) and 1-aminocyclopropane-l-carboxylic acid (ACC) as intermediates in the pathway from methionine to ethylene. The sequence of reactions in the pathway... 

Adams and Yang (10) have suggested that the S atom of methionine is recycled in the ethylene reaction pathway, as shown in Fig. 2. In this scheme, 5 -methylthioadenosine, the residual molecule which derives from the reaction converting SAM to ACC, is further metabolized to 5 -methylthioribose, which then transfers the S-methyl group to homoserine to form methionine. This scheme is hypothetical, and the enzymes necessary for all these reactions have not as yet been demonstrated. 

Figure 1. Reactions from methionine to ethylene showing intermediates and inhibitors of each step in the pathway and the possible direct conversion of...

Figure 2. Proposed pathway from methionine to ethylene indicating recycling of the S atom according to Adams and Yang (10J...

In recent studies protoplasts prepared from apple tissue did not produce ethylene. The loss of ethylene-producing ability by tissue slices incubated in cell wall-digesting enzymes during preparation of protoplasts is shown in Fig. 9. Methionine, the precursor of ethylene, delayed the loss of ethylene production somewhat during preparation of the protoplasts. 

Ethylene production was restored to some extent when the protoplasts were cultured for 3 or more days (Fig. 10) (58). Restoration of ethylene producing ability by culturing was correlated with regeneration of some cell-wall material, as shown by incorporation of myoinositol in the ethanol-insoluble fraction of the protoplasts and by increased fluorescence with calcafluor-white (58). Regeneration of cell-wall material was correlated with ethylene production in response to methionine added to the cultured protoplasts. Production of ethylene by these cultured protoplasts was not only dependent on addition of... 

Figure 9. Loss of ethylene-producing ability in apple slices treated with a mixture of cell-wall-digesting enzymes in presence and absence of methionine (58) (0,9), preclimacteric (A, A), climacteric.

Advances in ethylene biochemistry and physiology have preceded along a number of fronts. Firstly the biosynthetic pathway from methionine to ethylene has been further clarified and intermediates identified. Secondly some progress has been made in recognising two possible receptor sites which are inhibited by Ag ions and C0 , respectively. Thirdly the localization of ethylene production has been shown to be associated with membranes in studies with protoplasts. 

Production and Inhibition of Ethylene. Now I would like to illustrate how knowledge about a plant hormone can be used to control and regulate its action. Methionine is the precursor of ethylene in plant tissues (30). Therefore, any compound which blocks methionine metabolism might be expected to inhibit ethylene biosynthesis. Rhizobitoxine was recognized as an inhibitor of methionine biosynthesis (31) as were its analogues shown in Figure 6 (32). 9... 

Figure 6. Enol ether substituted amino acid analogues of methionine which are inhibitors of ethylene production in plants...

Rhizobitoxine. Various Rhizoblum. laponlcum strains produce rhizobitoxine [75], whose structure was reported In 1972 (2691. Although the host of this bacterium Is soybean, this compound Is also phytotoxic to many other plant species. This phytotoxin Is an analog of cystathionine and acts as an Irreversible Inhibitor of -cystathlonase which catalyzes production of homoserlne from cystathionine (2701. Rhizobitoxine also Inhibits ethylene production from methionine (2711. as does a similar phytotoxin, 2-am1no-4-methoxy-3-eno1c acid (28). 

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