Biosynthesis of wound ethylene in morning-glory flower tissue

Production of wound ethylene was investigated in rib segments excised from flower buds of morning-glory (Ipomoea tricolor). Segments of the ribs were cut from buds 2 days before flower opening, floated overnight on 5 mm KCl solution, and transferred to agar the following morning. These immature segments evolved only a small quantity of ethylene during incubation on agar, with most of the production occurring in the morning. When such segments were wounded mechanically early in the afternoon, the rate of ethylene production rose more than 10-fold within 1 hour and returned to a low rate after about 3 hours.Production of ethylene by both untreated and wounded rib segments was inhibited more than 95% by overnight pretreatment with the ethoxy analog of rhizobitoxine (3 x 10(-5) and 10(-4)m). After overnight exposure of segments to 9 muml-methionine-U-(14)C, the specific radioactivity of the ethylene evolved by untreated and wounded tissue was determined and compared to the specific radioactivities of carbon atoms 3 plus 4 of methionine and S-methylmethionine (SMM) extracted from the segments. The specific radioactivity of methionine was about one-half that of SMM; neither value was significantly affected by wounding. The specific radioactivity of ethylene evolved by untreated tissue was close to that of SMM. In wounded tissue the specific radioactivity of the ethylene evolved was lower, but still above that of methionine. These results are consistent with the interpretations that wound ethylene is synthesized from carbon atoms 3 plus 4 of either SMM or methionine. On the basis of earlier experiments with senescing rib segments, it is suggested that methionine serves as the precursor of the wound ethylene.     

Methionine metabolism and ethylene formation in etiolated pea stem sections

Stem sections of etiolated pea seedlings (Pisum sativum L. cv. Alaska) were incubated overnight on tracer amounts of l-[U-(14)C]methionine and, on the following morning, on 0.1 millimolar 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 S-methylmethionine (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 millimolar methionine enhanced indoleacetic acid-induced ethylene formation by 5 to 10%. Under the same conditions, 1 millimolar homocysteine thiolactone increased ethylene synthesis by 20 to 25%, while SMM within a concentration range of 0.1 to 10 millimolar did not influence ethylene production. When unlabeled methionine or homocysteine thiolactone was applied to stem sections which had been incubated overnight in l-[U-(14)C]methionine, the specific radioactivity of the ethylene evolved was considerably lowered. Application of unlabeled SMM reduced the specific radioactivity of ethylene only slightly.     

Ethylene production and respiration in aging leaf segments and in disks of fruit tissue of normal and mutant tomatoes

Leaf segments of tomato plants (Lycopersicon esculentum Mill.) of a normal strain and of two nonripening mutants rin and nor were aged in darkness. Respiration in leaf segments of all strains followed a climacteric-like pattern which was accompanied by a similar pattern of ethylene production. l-Methionine-U-(14)C vacuum-infiltrated into leaf segments at the beginning of the climacteric-like rise in respiration was metabolized to ethylene and CO(2) during the subsequent 48 hours to about the same extent in all strains. Pericarp disks of immature fruits of all strains also metabolized l-methionine-U-(14)C to ethylene and CO(2) to about the same extent during the first 48 hours following cutting and vacuum infiltration. Conversion of methionine to ethylene in disks was much more efficient than in aging leaf segments. The apparent capacity for increased production of ethylene in aging leaf segments and in response to wounding in pericap disks of rin and nor is contrasted with the absence of a respiratory climacteric and an associated large increase in ethylene production during natural aging of intact fruits of these two strains.     

Auxin-induced ethylene biosynthesis in subapical stem sections of etiolated seedlings of Pisum sativum L.

1-Aminocyclopropane-1-carboxylic acid (ACC) stimulated the production of ethylene in subapical stem sections of etiolated pea (cv. Alaska) seedlings in the presence and absence of indole-3-acetic acid (IAA). No lag period was evident following application of ACC, and the response was saturated at a concentration of 1 mM ACC. Levels of endogenous ACC paralleled the increase in ethylene production in sections treated with different concentrations of IAA and with selenoethionine or selenomethionine plus IAA. The IAA-induced formation of both ACC and ethylene was blocked by the rhizobitoxine analog aminoethoxyvinylglycine (AVG). Labelling studies with L-[U-¹⁴C]methionine showed an increase in the labelling of ethylene and ACC after treatment with IAA. IAA had no specific effect on the incorporation of label into S-methylmethionine or homoserine. The specific radioactivity of ethylene was similar to the specific radioactivity of carbon atoms 2 and 3 of ACC after treatment with IAA, indicating that all of the ethylene was derived from ACC. The activity of the ACC-forming enzyme was higher in sections incubated with IAA than in sections incubated with water alone. These results support the hypothesis that ACC is the in-vivo precursor of ethylene in etiolated pea tissue and that IAA stimulates ethylene production by increasing the activity of the ACC-forming enzyme.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine - IAA indole-3-acetic acid - SAM S-adenosylmethionine - SMM S-methylmethionine     

Methionine metabolism and ethylene biosynthesis in senescing petals of Tradescantia

The endogenous content of methionine in isolated petals of Tradescantia was found to increase during petal senescence while the levels of S-methylmethionine and protein were found to decline. The increase in free methionine was, at least in part, the result of protein degradation. Methionine and homocysteine were shown to be intermediates in ethylene biosynthesis while S-methylmethionine was not involved. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to all floral tissues resulted in large stimulations of ethylene production. ACC was shown to be an endogenous amino acid the internal levels of which correlated positively with the rate of ethylene production. Application of l-methionine-[U-¹⁴C] led to a rapid appearance of radioactivity in both ethylene and ACC. The specific radioactivity of C-2 and C-3 of ACC and that of ethylene were found to be nearly identical which indicated that ACC was the immediate precursor of ethylene in senescing petals of Tradescantia.     

Enhancement of ethylene formation by selenoamino acids

Selenomethionine and selenoethionine enhanced ethylene production in senescing flower tissue of Ipomoea tricolor Cav. and in auxin-treated pea (Pisum sativum L.) stem sections. This enhancement was fully inhibited by the aminoethoxy analog of rhizobitoxine. Methionine did not have a comparable promotive effect, and ethionine partly inhibited ethylene production. When [¹⁴C]methionine was applied to flower or pea stem tissue followed by treatment with unlabeled selenomethionine or selenoethionine, the specific radioactivity of the ethylene evolved was considerably reduced. The dilution of the specific radioactivity of ethylene by selenomethionine, and in pea stem sections also by selenoethionine, was greater than the dilution by nonradioactive methionine at the same concentration. These results indicate that both selenoamino acids serve as precursors of ethylene and that they are converted to ethylene more efficiently than is methionine.     

Effect of methionine and its related compounds on rumen bacterial activity

The effect of several methionine sources (L‐methionine = L‐MET; DL‐ methionine = DL‐MET, DL‐S‐methyl‐methionine‐sulphonium‐chloride = SMM; N‐hydroxymethyl‐DL‐methionine‐Ca = NHM; methionine‐hydroxy‐analog free acid=MHA; methionine‐sulphoxyde=MSO) on rumen bacterial growth was studied using a new methodical approach which utilises a methionine free assay medium (Bacto Methionine Assay Media, Difco) supplemented by increasing quantities of the methionine sources and inoculated with one drop of diluted rumen bacteria. The optical density was measured after 18 h incubation on 39 °C.

L‐ and DL‐MET promoted the highest growth response, while SMM and NHM exerted significantly (p < 0.05) lower optical densities. MHA and MSO showed no growth response.

The methodical approach and the possible bacterial strains, which might have contributed to the growth response have been discussed.     

Ethylene formation in Pisum sativum and Vicia faba protoplasts

Protoplasts isolated from leaves of peas (Pisum sativum L.) and of Vicia faba L. produced 1-aminocyclopropane-1-carboxylic acid (ACC) from endogenous substrate. Synthesis of ACC and conversion of ACC to ethylene was promoted by light and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and carbonyl cyanide m-chlorophenylhydrazone. Aminoethoxyvinylglycine inhibited ethylene synthesis to a minor extent when given during incubation of the protoplasts but was very effective when added both to the medium in which the protoplasts were isolated and to the incubation medium as well. Radioactivity from [U-¹⁴C]methionine was incorporated into ACC and ethylene. However, the specific radioactivity of the C-2 and C-3 atoms of ACC, from which ethylene is formed, increased much faster than the specific radioactivity of ethylene. It appears that ACC and ethylene are synthesized in different compartments of the cell and that protoplasts constitute a suitable system to study this compartmentation.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

The origin of the sulfur atom of thiamin

The incorporation of the sulfur atom of 35S-labeled amino acids into thiamin in Escherichia coli and Saccharomyces cerevisiae was studied. The specific radioactivity of the S atoms was incorporated at similar levels into thiamin and cysteine residues in cell proteins. However, the specific radioactivity of the S atoms from [35S]methionine was not incorporated into thiamin but into methionine residues in cell proteins. Thus, the origin of the S atom of thiamin was established as being the S atom of cysteine. No activity from [U-14C]cysteine was recovered in thiamin, proving that the carbon skeleton of this amino acid was not utilized in synthesizing the thiazole moiety of thiamin.     

Incorporation of 5-methylorcylaldehyde and methionine into the acetogenin (polyketide) gliorosein in Gliocladium roseum I.M.I. 93065

1. methyl-(14)C-labelled 1,3-dihydroxy-4,5-dimethylbenzene, 5-methylorcylaldehyde and 5-methylorsellinic acid were synthesized from orcinol and sodium [(14)C]cyanide and tested for activity as precursors of gliorosein. ring-(14)C-labelled orcylaldehyde was also prepared. 5[(14)C]-Methylorcylaldehyde was incorporated into gliorosein (36% conversion); all the radioactivity was located in the C-methyl groups. 5-Methylorsellinic acid was decarboxylated by Gliocladium roseum and the resulting phenol was secreted into the medium. 2. The formation of an enzyme-bound derivative of 5-methylorsellinic acid as the first aromatic compound in the biosynthesis of gliorosein is suggested to explain these results. 3. ring-(14)C-labelled 3,4-dihydroxy-6-methyltoluquinone was also effectively incorporated into gliorosein and related products (20% conversion). 4. Sodium [(14)C]formate and [Me-(14)C]-methionine were incorporated into gliorosein and related products (15.4 and 22.2% conversion respectively). Isolation and estimation of the radioactivity in the O-methyl and C-methyl groups in the (14)C-labelled gliorosein thus formed showed an appreciable difference in the specific activities of the two types of methyl group (14 and 15% respectively). The results in the doubly-labelled methionine experiment indicate that the C-methyl group arises in the same manner as that in ergosterol; one of the original hydrogen atoms of the methyl group is lost. This confirms that C-methylation occurs at an ethylenic group at the aliphatic level. 5. The sequence of reactions at the aromatic level leading to the formation of gliorosein is proposed as 5-methylorsellinyl-enzyme-->3-hydroxy-5-methylorsellinyl-enzyme-->3,4-dihydroxy-6-methyltoluquinol-->3,4-dimethoxy-6-methyltoluquinol-->gliorosein.     

Biosynthesis of 3-dimethylsulfoniopropionate in Wollastonia biflora (L.) DC. Evidence that S-methylmethionine is an intermediate.

The compatible solute 3-dimethylsulfoniopropionate (DMSP) is accumulated by certain salt-tolerant flowering plants and marine algae. It is the major biogenic precursor of dimethylsulfide, an important sulfur-containing trace gas in the atmosphere. DMSP biosynthesis was investigated in Wollastonia biflora (L.) DC. [= Wedelia biflora (L.) DC., Melanthera biflora (L.) Wild, Asteraceae]. After characterizing DMSP and glycine betaine accumulation in three diverse genotypes, a glycine betaine-free genotype was chosen for radiotracer and stable isotope-labeling studies. In discs from young leaves, label from [U-14C]methionine was readily incorporated into the dimethylsulfide and acrylate moieties of DMSP. This establishes that DMSP is derived from methionine by deamination, decarboxylation, oxidation, and methylation steps, without indicating their order. Five lines of evidence indicated that methylation is the first step in the sequence, not the last. (a) In pulse-chase experiments with [14C]methionine, S-methylmethionine (SMM) had the labeling pattern expected of a pathway intermediate, whereas 3-methylthiopropionate (MTP) did not. (b) [14C]SMM was efficiently converted to DMSP but [14C]MTP was not. (c) The addition of unlabeled SMM, but not of MTP, reduced the synthesis of [14C]DMSP from [14C]methionine. (d) The dimethylsulfide group of [13CH3,C2H3]SMM was incorporated as a unit into DMSP. (e) When [C2H3,C2H3]SMM was given together with [13CH3]methionine, the main product was [C2H3,C2H3]DMSP, not [13CH3,C2H3]DMSP or [13CH3,13CH3]DMSP. The stable isotope labeling results also show that the SMM cycle does not operate at a high level in W. biflora leaves.     

Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine and by polyamines shunts label from 3,4-[C]methionine into spermidine in aged orange peel discs

The flux of radioactivity from 3,4-[(14)C]methionine into S-adenosyl-l-methionine (SAM), 1-aminocyclopropane-1-carboxylic acid (ACC), spermine, and spermidine while inhibiting conversion of ACC to ethylene by 100 millimolar phosphate and 2 millimolar Co(2+) was studied in aged peel discs of orange (Citrus sinensis L. Osbeck) fruit. Inhibition up to 80% of ethylene production by phosphate and cobalt was accompanied by a 3.3 times increase of label in ACC while the radioactivity in SAM was only slightly reduced. Aminoethoxyvinylglycine (AVG) increased the label in SAM by 61% and reduced it in ACC by 47%. Different combinations of standard solution, in which putrescine or spermidine were administered alone or with AVG, demonstrated clearly that inhibition of ethylene biosynthesis-at the conversion of SAM to ACC-by AVG, exogenous putrescine or exogenous spermidine, stimulated the incorporation of 3,4-[(14)C]methionine into spermidine.     

Biosynthesis of phytoquinones. Incorporation of l-[Me-14C3H]methionine into terpenoid quinones and chromanols in maize shoots

1. Radioactivity from l-[Me-(14)C,(3)H]methionine is incorporated into phylloquinone, plastoquinone, gamma-tocopherol, alpha-tocopherol, alpha-tocopherolquinone and ubiquinone in maize shoots. 2. Comparative studies with other terpenoids (squalene and beta-carotene) and chemical degradation of selected quinones (ubiquinone and plastoquinone) established that all the radioactivity is confined to nuclear methyl substituents. 3. In ubiquinone 76% of the radioactivity is in the methoxyl groups and 24% in the ring C-methyl group. 4. Taking the phytosterols as an internal reference and accepting the atomic ratio of (14)C/(3)H transferred from l-[Me-(14)C,(3)H]methionine to the supernumerary group at C(24) to be 1:2 the ratio of all the quinones and chromanols examined approached 1:3. After allowing for the fact that for plastoquinone, gamma-tocopherol, alpha-tocopherol and alpha-tocopherolquinone one nuclear methyl group is formed from the beta-carbon of tyrosine, these results show that one nuclear C-methyl group for phylloquinone, plastoquinone and gamma-tocopherol, two nuclear methyl groups for alpha-tocopherol and alpha-tocopherolquinone and one nuclear methyl and two methoxyl groups for ubiquinone are formed by the transfer of intact methyl groups from methionine. 5. From a comparison of the incorporation of (14)C radioactivity into these compounds it would appear that the methylation reactions involved in phylloquinone and plastoquinone biosynthesis take place in the chloroplast, whereas those involved with ubiquinone biosynthesis occur else-where within the cell.     

Precursors of the pyrimidine moiety of thiamine

1. A method was devised for obtaining the pyrimidine moiety of thiamine in a pure form after its excretion into the medium by de-repressed washed-cell suspensions of mutants of Salmonella typhimurium LT2. 2. By using amino acid-requiring mutants, this excretion of pyrimidine moiety was shown to be dependent on the presence of both methionine and glycine. 3. In the presence of either [Me-¹⁴C]methionine or [G-¹⁴C]methionine, methionine-requiring mutants did not incorporate radioactivity into the pyrimidine moiety. 4. In contrast, both [1-¹⁴C]glycine and [2-¹⁴C]glycine were incorporated into the pyrimidine moiety excreted by glycine-requiring mutants, and this occurred with little or no dilution of specific radioactivity. 5. The possible requirement for methionine as a cofactor and the significance of the incorporation of both carbon atoms of glycine are discussed.     

The formation of sulphur-containing amino acids in germinating seeds of rape (Brassica napus L.)

Sodium [(35)S]sulphide was fed to batches of germinating rapeseed, in some instances with the addition of unlabelled cysteine. Both the total radioactivity and specific radioactivity of the free sulphur-containing amino acids were examined. Cysteine and homocysteine were rapidly labelled; label subsequently appeared in cystathionine and methionine. The results obtained indicated that both the sulphydration and trans-sulphuration pathways were operating. This conclusion was reinforced by the results of experiments in which batches of rapeseed were incubated with l-[(14)C]homoserine. These showed the formation of labelled homocysteine, cystathione and methionine. It was thought the trans-sulphuration pathway was making the greater contribution to the biosynthesis of methionine in germinating rapeseed.     

Physiology of a choline-requiring strain of Torulopsis pintolopesii

Summary A choline-requiring strain of Torulopsis pintolopesii when growing on choline-methyl-¹⁴C or choline-methyl-³H excretes the radioactivity incorporated in the first 24–48 h of incubation up to ca. 85–95% of the radioactivity added at the beginning of the incubation. The addition of non-radioactive methionine did not interfere with the uptake and excretion of radioactivity from choline-Me-¹⁴C. Radioactive methyl group of methionine previously incorporated by growing cells of T. pintolopesii on varying concentrations of choline was not excreted in appreciable amounts. No evidence was obtained for the oxidation of choline to betaine, degradation to trimethylamine, or net incorporation of labelled choline into lecithin. The occurrence of a new pathway for the utilization of choline in yeasts is suggested. The requirement of choline by T. pintolopesii is explained tentatively by the formation and excretion of a compound containing the carbon and hydrogen atoms from the methyl groups of choline and whose chemical structure still under study, may comprise a heteroside containing mannitol as the polyhydroxylated moiety.     

Biosynthesis of s-methylcysteine in radish leaves

Investigation on the biosynthesis of S-methyl-L-cysteine in radish leaves has shown that it is formed by the methylation of cysteine. This conclusion is based on: A) the relatively high recovery of radioactivity in methylcysteine sulfoxide after the administration of cysteine or methyl-labeled methionine to radish leaves; B) the nearly complete recovery of label from methyl-labeled methionine in the methyl group of methylcysteine sulfoxide; and C) the similarity in the ratio of tritium to ¹⁴C in methylcysteine sulfoxide and in its methyl group to this ratio in the methyl group of methionine given to radish leaves. Direct evidence for the synthesis of methylcysteine in radishes was obtained for the first time.     

Biosynthesis of ethylene from methionine in aminoethoxyvinylglycine-resistant avocado tissue

This study was conducted to determine if aminoethoxyvinylglycine (AVG) insensitivity in avocado (Persea americana Mill., Lula, Haas, and Bacon) tissue was due to an alternate pathway of ethylene biosynthesis from methionine. AVG, at 0.1 millimolar, had little or no inhibitory effect on either total ethylene production or [(14)C] ethylene production from [(14)C]methionine in avocado tissue at various stages of ripening. However, aminoxyacetic acid (AOA), which inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (the AVG-sensitive enzyme of ethylene biosynthesis), inhibited ethylene production in avocado tissue. Total ethylene production was stimulated, and [(14)C]ethylene production from [(14)C]methionine was lowered by treating avocado tissue with 1 millimolar ACC. An inhibitor of methionine adenosyltransferase (EC 2.5.1.6), l-2-amino-4-hexynoic acid (AHA), at 1.5 millimolar, effectively inhibited [(14)C]ethylene production from [(14)C]methionine in avocado tissue but had no effect on total ethylene production during a 2-hour incubation. Rates of [(14)C]AVG uptake by avocado and apple (Malus domestica Borkh., Golden Delicious) tissues were similar, and [(14)C]AVG was the only radioactive compound in alcohol-soluble fractions of the tissues. Hence, AVG-insensitivity in avocado tissue does not appear to be due to lack of uptake or to metabolism of AVG by avocado tissue. ACC synthase activity in extracts of avocado tissue was strongly inhibited (about 60%) by 10 micromolar AVG. Insensitivity of ethylene production in avocado tissue to AVG may be due to inaccessibility of ACC synthase to AVG. AVG-resistance in the avocado system is, therefore, different from that of early climacteric apple tissue, in which AVG-insensitivity of total ethylene production appears to be due to a high level of endogenous ACC relative to its rate of conversion to ethylene. However, the sensitivity of the avocado system to AOA and AHA, dilution of labeled ethylene production by ACC, and stimulation of total ethylene production by ACC provide evidence for the methionine --> SAM --> ACC --> ethylene pathway in avocado and do not suggest the operation of an alternate pathway.     

Radioactive methionine: determination, and distribution of radioactivity in the sulfur, methyl and 4-carbon moieties

A simple and inexpensive method is described for isolation and determination of [14C]methionine in the non-protein fraction of tissues extensively labeled with 14C. The effectiveness of the method was demonstrated by isolation of non-protein [14C]methionine (as the carboxymethylsulfonium salt) of proven radiopurity from the plant Lemna which had been grown for a number of generations on [U-14C]sucrose and contained a 2000-fold excess of 14C in undefined non-protein compounds. To our knowledge, this is the first reported assay for radioactive methionine under these demanding conditions. This method also offers an attractive alternative to the use of more expensive and sophisticated equipment for assay of radioactive methionine under less demanding conditions. An advantage is that the isolated methioninecarboxymethylsulfonium salt is readily degraded to permit separate determination of radioactivity in the 4-carbon, methyl and sulfur moieties of methionine. During this work, a facile labilization of 3H attached to the (carboxy)methylene carbon of methioninecarboxymethylsulfonium salt was observed. This labilization is ascribed to formation of a sulfur ylid.