Inhibition of in Vivo Conversion of Methionine to Ethylene by l-Canaline and 2,4-Dinitrophenol

109

The rate of C2H, production in plant tissue appears to be limited by the level of endogenous 1-aminocyclopropane-1-carboxylic acid (ACC). Exogenous ACC stimulated C2H4 production considerably in plant tissues, but this required 10 to 100 times the endogenous concentrations of ACC before significant increases in C2H4 production were observed. This was partially due to poor penetration of ACC into the tissues. Conversion of ACC to C2H4 was inhibited by free radical scavengers, reducing agents, and copper chelators, but not by inhibitors of pyridoxal phosphate-mediated reactions. The system for converting ACC to C2H4 may be membrane-associated, for it did not survive treatment with surface-active agents and cold or osmotic shock reduced the capacity of the system to convert ACC to C2H4. The reaction rate was sensitive to temperatures above 29 and below 12 C, which suggests that the system may be associated with membrane-bound lipoproteins. The data presented support the possibility that the conversion of exogenous ACC to C2H4 proceeds via the natural physiological pathway.Considerable evidence has accumulated in the past 14 years to show that C2H4 derives from methionine (10, 1 1). However, until recently, the pathway from methionine to C2H4 remained obscure.The report by Adams and Yang (1) that implicated SAM4 as intermediate between methionine and C2H4 led to their further discovery that the unique amino acid l-aminocyclopropane-lcarboxylic acid is the immediate precursor of C2H4 (2). This discovery established the following biosynthetic sequence for C2H4 production: methionine -* SAM --ACC --C2H4.Knowledge of the intermediates in the biosynthetic pathway to C2H4 has shifted emphasis in this research area to identifying the enzymes and establishing the control and regulation of the reactions involving these intermediates. The enzyme which converts SAM to ACC has recently been shown to be soluble (6,17). However, the enzyme system which converts ACC to C2H4 has not been sufficiently characterized and may be associated with cellular particulates (8,13 4 Abbreviations: SAM, S-adenosyl-L-methionine; ACC, I-aminocyclopropane-l-carboxylic acid; AVG, aminoethoxyvinylglycine.presence of a mercury-catalyzed NaOCl system (12), or simply by reaction with high concentrations of H202 (9). Since there appears to be a number of possible reactions which can convert ACC to C2H2, the question arises as to whether or not, at high concentrations, all ACC exogenously applied to plant tissues is converted to C2H4 by the same enzymic system that utilizes native ACC to form endogenous C2H4. Here, we report some characteristics of the conversion of ACC to C2H4. Plant Material. Apple fruits (Malus sp. Cultivar Golden Delicious) were harvested in the Beltsville orchard at a preclimacteric stage and stored at 0 C until used. Discs 1.0 cm in diameter and 2 mm thick were cut from friits at various stages of ripening. Six discs (1 g) were incubated in a 25-ml Erlenmeyer flask containing 3 ml incubation medium consisting of 600 mm sorbi...

Section: Resultsmentioning

confidence: 99%

Some Characteristics of the System Converting 1-Aminocyclopropane-1-carboxylic Acid to Ethylene

Apelbaum¹,

Burgoon²,

Anderson³

et al. 1981

109

“…As indicated below, ethylene is generally synthesized from methionine, which is the most common precursor of this hormone in plants (16), through a series of steps involving S-adenosylmethionine (SAM) and ACC (4): methionine -+ SAM ACC --ethylene (4,15). Several inhibitors acting at different points in this pathway have been described, notably rhizobitoxine and its analogs (17), L-canaline (20), and benzylisothiocyanate (21). Because of this knowledge, experiments were designed with the purpose of elucidating the role of endogenous ethylene in the accumulation of HRGP, into the cell wall, in diseased plants.…”

Section: Discussionmentioning

confidence: 99%

“…L-Can (Sigma), benzylisothiocyanate (Fluka), and aminoethoxyvinylglycine (Hoffman-La Roche) were used as inhibitors (17,20,21) of ethylene synthesis, and I-aminocyclopropane-l-carboxylic acid (Sigma) as a precursor of ethylene (4). LCan was prepared from L-Can dipicrate before each experiment by filtration through a column (5 x I cm) of Dowex AG 2 x 8, 200-400 mesh, Cl-form, in water as reported by Murr and Yang (20). The recovered L-Can was measured according to Yemm and Cocking (29) and adjusted to a suitable concentration with H20.…”

Section: )mentioning

confidence: 99%

Cell Surfaces in Plant-Microorganism Interactions

Toppan¹,

Roby²,

Esquerré-Tugayé³

1982

MATERIALS AND METHODSEthylene production and cell wail hydroxyproline-rich glycoprotein (HRGP) biosynthesis are greatly enhanced in melon (Cucumin melo cv.Cantaloup charentais) seedlings infected with CoUetotrichum lagenariwm.Short-term experiments performed in the presence of specific inhibitors of the ethylene pathway from methionine, namely L-canaline and aminoethoxyvinylglycine, indicate that under non-toxic conditions, both ethylene and I'4Clhydroxyproline deposition in the cell wall of infected tissues are significantly lowered. On the contrary, treatment of healthy tissues with 1-aminocyclopropane 1-carboxylic acid, a natural precursor of ethylene, stimulates both the production of the hormone and the incorporation of I'Clhydroxyproline into cell wall proteins.The data provide the first evidence of the in vivo effect of ethylene on the cell wall hydroxyproline-rich glycoprotein biosynthesis in plants.It has been reported in previous papers (10, 11) that melon seedlings respond to a fungal attack by the accumulation of HRGP2 in their cell walls. Special attention has been paid to this modification of the cell surface for it involves glycoproteins, a class of components often mediating cell to cell interactions (5). Availability of plants with higher or lower than normal amounts of cell wall hydroxyproline allowed us to ascertain that the enrichment of diseased plants in HRGP is closely associated to their defense against microorganisms (9). Indirect evidence suggests a role for ethylene in the regulation of this mechanism. When supplied exogenously to intact plants, this hormone promotes an enrichment of the cell wall in hydroxyproline (24) corresponding to an enrichment in HRGP (9). A similar hydroxyproline response occurs after wounding of plant tissues (7), a process otherwise well-known to stimulate the production ofethylene (13). Inasmuch as large amounts of this hormone are often released by infected plants (1,22) Patil and Tang (21), and then diluted to appropriate concentrations with 50 mm phosphate buffer (pH 6.0). AVG and ACC were each dissolved and adjusted to I x lo-3 M in 50 mm phosphate buffer (pH 6.0). Appropriate dilutions were then made with the same buffer.Ethylene Measurement. The production of ethylene was measured from excised seedlings, petiole of the first leaf, and from C. lagenarium, as follows. Four excised seedlings (without roots) were enclosed for 24 h in 570-ml serum flasks stopped with serum caps, and containing 30 ml of the usual growth medium (28). Newly excised seedlings were used everyday. The petioles were cut into 5-mm segments as previously indicated (9), and divided into lots of 2 g. The segments of each lot were incubated for 4 h under the light in 13 ml serum vials, stopped with serum vaccine caps, containing 6 ml of a medium consisting of 2% sucrose in 50 mm K-phosphate buffer (pH 6.0). A CO2 trap made of filter paper soaked with 200 td of 1 N KOH was inserted into each vial.Measurements of ethylene from C. lagenarium were performed on a 6-d-old culture i...

“…1 and 10). Recently, S-adenosylmethionine was proposed as an intermediate in the conversion of methionine to ethylene in apple tissue (3,9). Results of Hanson and Kende (5) indicated that ethylene production in flower tissue of Ipomoea tricolor might be dependent on methionine derived from SMM3 and homocysteine.…”

mentioning

confidence: 99%

Methionine Metabolism and Ethylene Formation in Etiolated Pea Stem Sections

Schilling¹,

Kende²

1979

Stem sections of etiolated pea seedlings (Pisum sativum L. cv. Alaska) were incubated overnight on tracer amounts of L-IU-'4Clmethionine and, on the following morning, on 0.1 mnllmlar indoleacetic acid to induce ethylene formation. Following the overnight incubation, over 70% of the radioactivity in the soluble fraction was shown to be associated with Smethylmethionine (SMM). The specific radioactivity of the ethylene evolved closely paralleled that of carbon atoms 3 and 4 of methionine extracted from the tissue and was always higher than that determined for carbon atoms 3 and 4 of extracted SMM.Overnight incubation of pea stem sections on 1 milimolar methionine enhanced indoleacetic acid-induced ethylene formation by 5 to 10%. Under the same conditions, 1 mil;imoar homocysteine thiolactone increased ethylene synthesis by 20 to 25%, while SMM within a concentration range of 0.1 to 10 miilimlar did not influence ethylene production. When unlabeled methionine or homocysteine thiolactone was applied to stem sections which had been incubated overnight in L-IU-_4Clmethlonine, the specific radioactivity of the ethylene evolved was considerably lowered. Application of unlabeled SMM reduced the specific radioactivity of ethylene only slightly.Tissues of higher plants produce ethylene when subjected to stress, during ripening and senescence or after application of auxin (for a review see ref. 1). Methionine has been shown to be the precursor of ethylene in a number of higher plants (for reviews see refs. 1 and 10). Recently, S-adenosylmethionine was proposed as an intermediate in the conversion of methionine to ethylene in apple tissue (3, 9). Results of Hanson and Kende (5) indicated that ethylene production in flower tissue of Ipomoea tricolor might be dependent on methionine derived from SMM3 and homocysteine.This investigation using etiolated pea stem sections was carried out to determine: (a) whether all ethylene evolved as a result of IAA treatment is derived from carbon atoms 3 and 4 of methionine; (b) whether SMM is a metabolite of methionine; and (c) whether induction of ethylene synthesis is accompanied by formation of methionine from SMM and homocysteine as found earlier in flower tissue of Ipomoea tricolor (5).