Cyanide metabolism in relation to ethylene production in plant tissues

HCN is the putative product of C-1 and amino moieties of 1-aminocyclopropane-1-carboxylic acid (ACC) during its conversion to ethylene. In apple (Malus sylvestrus Mill.) slices or auxin-treated mungbean (Vigna radiata L.) hypocotyls, which produced ethylene at high rates, the steady state concentration of HCN was found to be no higher than 0.2 micromolar, which was too low to inhibit respiration (reported Ki for HCN to inhibit respiration was 10-20 micromolar). However, these tissues became cyanogenic when treated with ACC, the precursor of ethylene, and with 2-aminoxyacetic acid, which inhibits β-cyanoalanine synthase, the main enzyme to detoxify HCN; the HCN levels in these tissues went up to 1.7 and 8.1 micromolar, respectively. Although ethylene production by avocado (Persea gratissima) and apple fruits increased several hundred-fold during ripening, β-cyanoalanine synthase activity increased only one- to two-fold. These findings support the notion that HCN is a co-product of ethylene biosynthesis and that the plant tissues possess ample capacity to detoxify HCN formed during ethylene biosynthesis so that the concentration of HCN in plant tissues is kept at a low level.     

The effect of inorganic phosphate on the ethylene production in tomato and apple fruits

Tomato fruits on stems immersed in phosphate solution 0.2 M K₂HPO₄ produced less ethylene than control fruits on stems immersed in water. Phosphate mediated inhibition of ethylene production was found to be the highest in fruits in the pink stage of maturity, which produced ethylene at the highest rate. Phosphate also inhibited ethylene production in slices prepared from maturing fruits, both apple and tomato. We suggest that phosphate is an inhibitor of ethylene biosynthesis in sufficiently mature tomato and apple fruits in which the rise of ethylene production is already very rapid. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

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.     

Stimulation of ethylene production in tomato tissue by propionic Acid

Propionic acid (10⁻³m) increases ethylene production by about 30 to 60% in tissue from green and half-ripe tomatoes (Lycopersicon esculentum Mill. var. Homestead) but does not increase ethylene production in tissue from ripe fruit. Stimulation is not due to the conversion of propionic acid to ethylene but appears to be secondary in nature and to operate on the endogenous ethylene-forming system. Thus conversion of methionine to ethylene in green and half-ripe tomato tissue is increased in the presence of propionic acid. Inhibitors which affect the normal endogenous ethylene-forming system similarly affect the propionic acid-stimulated system. Endogenous propionic acid may play a role in the regulation of ethylene production in tomato tissues.     

Biosynthesis of ethylene: the effect of phosphate*

Abstract Biosynthesis of ethylene in tomato and avocado fruit slices, carrot root, pea seedling and tomato shoot segments, Penicillium expansum and Escherichia coli was found to be inhibited by inorganic phosphate. Compared with microbial systems, relatively high concentrations of phosphate in the incubating medium were necessary to bring about a significant inhibition of ethylene production in higher plants. The degree of inhibition in higher plants correlated with the increased internal cellular concentration of phosphate and not with that of the incubating medium. Phosphate concentrations inhibitory for ethylene biosynthesis did not affect the respiration of tomato fruit slices. The phosphate effect was reversible, confined to only the biological systems and was not due to a change in the ionic strength. The differential inhibitory effects of aminoethoxyvinylglycine on ethylene biosynthesis in tomato fruit slices of various stages of ripening, were markedly influenced by high phosphate concentrations. The data indicate a biological significance to the phosphate control of ethylene biosynthesis.     

Inhibition of ethylene production by cobaltous ion

The effect of Co²⁺ on ethylene production by mung bean (Phaseolus aureus Roxb.) and by apple tissues was studied. Co²⁺, depending on concentrations applied, effectively inhibited ethylene production by both tissues. It also strongly inhibited the ethylene production induced by IAA, kinetin, IAA plus kinetin, Ca²⁺, kinetin plus Ca²⁺, or Cu²⁺ treatments in mung bean hypocotyl segments. While Co²⁺ greatly inhibited ethylene production, it had little effect on the respiration of apple tissue, indicating that Co²⁺ does not exert its inhibitory effect as a general metabolic inhibitor. Ni²⁺, which belongs to the same group as Co²⁺ in the periodic table, also markedly curtailed both the basal and the induced ethylene production by apple and mung bean hypocotyl tissues.     

An ethylene-related cDNA from ripening apples

We report the isolation of a ripening-related apple cDNA which is complementary to a mRNA which may be involved in ethylene production. Poly(A)⁺ RNA was extracted from cortical tissue of ripe apple fruit (Malus domestica Borkh cv. Golden Delicious) and a cDNA library constructed in the plasmid vector pSPORT. The library was screened with pTOM13, a tomato cDNA clone thought to code for ACC oxidase in that fruit. An apple cDNA clone (pAP4) was isolated and sequenced. The 1182 bp cDNA insert includes an open reading frame of 942 bp, and shows strong homology with reported tomato and avocado sequences, both at the nucleic acid and amino acid levels. The polypeptide has a calculated molecular mass of 35.4 kDa and a calculated pI of 5.15. In apple cortical tissue, expression of pAP4-complementary RNA increased with ethylene production by the fruit during ripening. Expression was also enhanced in both ethylene-treated and wounded fruit.     

Biosynthesis of ethylene in higher plants: the metabolic site of inhibition by phosphate*

Abstract. Phosphate inhibited endogenous as well as 1-aminocyclopropane-1-carboxylic acid (ACC)-stimulated ethylene synthesis in slices of tomato fruit, segments of carrot root and pea hypocotyls. ACC concentrations of up to 10 mol m^?3 did not overcome this inhibition. Phosphate inhibited the conversion of ¹⁴C ACC to ethylene in tomato fruit and vegetative tissue. Enzymatic conversion of ACC to ethylene by pea seedling homogenate was also inhibited by phosphate with a linear concentration dependency. The formation of ACC from S-adenosylmethionine (SAM) by extracts of pink tomatd fruit was slightly, but not significantly, affected by phosphate. However, the SAM to ACC conversion was greater when extracts from tomato fruit were made in phosphate rather than in HEPES-KOH buffer. Non-enzymatic ethylene synthesis from ACC in a model system was stimulated by phosphate. We suggest that phosphate is an inhibitor of ethylene biosynthesis in higher plants and that one site of its control is the conversion of ACC to ethylene.     

Influence of Plant Hormones on Ethylene Production in Apple, Tomato, and Avocado Slices during Maturation and Senescence

Ethylene production by tissue slices from preclimacteric, climacteric, and postclimacteric apples was significantly reduced by isopentenyl adenosine (IPA), and by mixtures of IPA and indoleacetic acid, and of IPA, indoleacetic acid, and gibberellic acid after 4 hours of incubation. Ethylene production by apple (Pyrus malus L.) slices in abscisic acid was increased in preclimacteric tissues, decreased in climacteric peak tissues, and little affected in postclimacteric tissues. Indoleacetic acid suppressed ethylene production in tissues from preclimacteric apples but stimulated ethylene production in late climacteric rise, climacteric, and postclimacteric tissue slices. Gibberellic acid had less influence in suppressing ethylene production in preclimacteric peak tissue, and little influenced the production in late climacteric rise, climacteric peak, and postclimacteric tissues. IPA also suppressed ethylene production in pre- and postclimacteric tissue of tomatoes (Lycopersicon esculentum) and avocados (Persea gratissima). If ethylene production in tissue slices of ripening fruits is an index of aging, then IPA would appear to retard aging in ripening fruit, just as other cytokinins appear to retard aging in senescent leaf tissue.     

Influence of calcium and magnesium on ethylene production by apple tissue slices

The decline in ethylene production in apple (Pyrus malus L. cv. Golden Delicious) tissue slices during 24 hours incubation in 600 millimolar sorbitol and 10 millimolar 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) is recognized as a senescent phenomenon. The inclusion of very high concentrations (100 millimolar) of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ severely inhibited ethylene production during the first 6 hours of incubation. However, after 6 hours and up to 24 hours the ethylene-forming system was stablized. These high concentrations of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ virtually eliminated lipid peroxidation and protein leakage from these slices. Also conversion of 1-aminocyclopropane-1-carboxylic-1-acid to ethylene and the influence of indoleacetic acid on ethylene production was stabilized after 24 hours of incubation by these high concentrations of Ca²⁺, Mg²⁺, and Ca²⁺ plus Mg²⁺. Addition of divalent ionophores severely inhibited ethylene production, but this inhibition was prevented by Ca²⁺ in concentrations greater than the ionophore. These data suggest that the loss of ethylene production by aging tissue slices results from degradation of membranes. They support previous work that indicates that the ethylene-forming system, perhaps the segment of the pathway from 1-aminocyclo-propane-1-carboxylic-1-acid to ethylene, resides in the plasma membrane.     

Decarboxylative metabolism of [1′-14C]indole-3-acetic acid by tomato pericarp discs during ripening

The rate of decarboxylation of [1′-¹⁴C]indole-3-acetic acid (IAA) infiltrated into tomato (Lycopersicon esculentum Mill.) pericarp discs was much more rapid in green than in breaker and pink tissues. Studies were carried out in order to determine whether the decarboxylative catabolism occurring in the green pericarp discs was associated with ripening or was a consequence of wound-induced peroxidase activity and/or ethylene production. After a 2-h lag, the decarboxylative capacity of the green pericarp discs increased exponentially during a 24-h incubation period. This increase was accompanied by increases in IAA-oxidase activity in cell-free preparations from the intercellular space and cut surface of the discs. Although higher IAA-oxidase activity was detected in extracts from the tissue residue, which comprises mainly intracellular peroxidases, this activity did not increase during the 24-h incubation period. Analysis of the cell-free preparations by isoelectric focusing revealed the major component in all samples was a highly anionic peroxidase (pI=3.5) the levels of which did not increase during incubation. However, the intercellular and cut-surface preparations contained additional anionic and cationic peroxidases which increased in parallel with the increases in both the IAA-oxidase activity of the preparations and the decarboxylative capacity of the green pericarp discs from which they were derived. Treatment of green discs with the ethylene-biosynthesis inhibitors aminooxyacetic acid and CoCl₂, inhibited the development of an enhanced capacity to decarboxylate [1′-¹⁴C]IAA but the inhibition was not counteracted by exogenous ethylene. Another ethylene-biosynthesis inhibitor, aminoethoxyvinyl glycine, also reduced ethylene levels but did not affect IAA decarboxylation, indicating that the decarboxylation was not a consequence of wound-induced ethylene production. The data obtained thus demonstrate that the enhanced capacity to decarboxylate [1′-¹⁴C]IAA that develops in green tomato pericarp discs following excision is not associated with ripening but instead is attributable to a wound-induced increase in anionic and cationic peroxidase activity in the intercellular fluid and at the cut surface of the excised tissues.     

Insensitivity of the diageotropica tomato mutant to auxin

The sensitivity of excised hypocotyl segments to indoleacetic acid (IAA) in two assays, ethylene production and elongation, was determined in the ethylene-requiring tomato (Lycopersicon esculentum Mill.) mutant, diageotropica (dgt), and its isogenic parent, cv VFN8. Endogenous (uninduced) ethylene synthesis rates were slightly lower in dgt hypocotyls than in VFN8 hypocotyls. Ethylene production was essentially unaffected by IAA in dgt, but was stimulated up to 10-fold by 10 micromolar IAA in VFN8. Elongation of dgt hypocotyls was also insensitive to concentrations of IAA as high as 100 micromolar, as compared to significant elongation of VFN8 hypocotyls in response to 0.1 micromolar IAA. A range of IAA analogs active in VFN8 was also ineffective in stimulating elongation of dgt hypocotyls, suggesting that the differences were not due to rapid metabolism of IAA by dgt tissues. Auxin-induced elongation of VFN8 hypocotyls was unaffected by 2,3,5-triiodobenzoic acid and naphthylphthalamic acid, indicating that polar auxin transport was not a factor in these experiments. Exogenous and auxin-induced ethylene had no effect on the elongation respone of either genotype, nor did exogenous ethylene restore the sensitivity of dgt hypocotyls to IAA. Despite their apparent insensitivity to auxin, dgt hypocotyls elongated dramatically and synthesized ethylene rapidly in response to 1.2 micromolar fusicoccin. These results suggest that the primary effect of the dgt mutation is to reduce the sensitivity of the tissue to auxin. As altered regulation of ethylene synthesis is only one symptom of this fundamental deficiency, dgt should more properly be considered to be the auxin-insensitive tomato mutant.     

Dependence of in vivo ethylene production rate on 1-aminocyclopropane-1-carboxylic Acid content and oxygen concentrations

1-Aminocyclopropane-1-carboxylic acid (ACC) is aerobically oxidized in plant tissues to form ethylene by ethylene-forming enzyme (EFE). The effect of substrate (ACC and oxygen) concentrations on ethylene production rate by plant tissues was investigated. The K_m value for O₂ in ethylene production varied greatly depending on the internal ACC content. When ACC levels in the tissue were low (below its K_m value), the concentration of O₂ giving half-maximal ethylene production rate ([S]_0.5) ranged between 5 and 7%, and was similar among different tissues. As the concentration of ACC was increased (greater than its K_m value), [S]_0.5 for O₂ decreased markedly. In contrast, the K_m value for ACC was not much dependent on O₂ concentration, but varied greatly among different plant tissues, ranging from 8 micromolar in apple (Malus sylvestris Mill.) tissue to 120 micromolar in etiolated wheat (Triticum aestivum) leaf. Such a great variation was thought to be due to the different compartmentation of ACC within the cells in different tissues. These kinetic data are consistent with the view that EFE follows an ordered binding mechanism in which EFE binds first to O₂ and then to ACC.     

Deferral of senescence and abscission by chemical inhibition of ethylene synthesis and action in bean explants

Three compounds known to inhibit ethylene synthesis and/or action were compared for their ability to delay senescence and abscission of bean explants (Phaseolus vulgaris L. cv Contender). Aminoethoxyvinyl-glycine (AVG), AgNO₃, and sodium benzoate were infiltrated into the petiole explants. Their effect on abscission was monitored by measuring the force required to break the abscission zone, and their effect on senescence was followed by measuring chlorophyll and soluble protein in the distal (pulvinus) sections. AVG at concentrations between 1 and 100 micromolar inhibited ethylene synthesis by about 80 to 90% compared to the control during sampling periods of 24 and 48 hours after treatment. This compound also delayed the development of abscission and senescence. Treatment with AgNO₃ at concentrations between 1 and 100 micromolar progressively reduced ethylene production, but to a lesser extent than AVG. The effects of AgNO₃ on senescence and abscission were quite similar to those of AVG. Sodium benzoate at 50 micromolar to 5 millimolar did not inhibit ethylene synthesis during the first 24 hours, but appreciably inhibited ethylene synthesis 48 hours after treatment. It also delayed the development of abscission and senescence. The effects of AVG, Ag⁺, and sodium benzoate suggest that ethylene could play a major role in both the senescence induction phase and the separation phase in bean explants.     

Ethylene Biosynthesis and Cadmium Toxicity in Leaf Tissue of Beans (Phaseolus vulgaris L.)

Stress ethylene production in bean (Phaseolus vulgaris L., cv. Taylor's Horticultural) leaf tissue was stimulated by Cd²⁺ at concentrations above 1 micromolar. Cd²⁺-induced ethylene biosynthesis was dependent upon synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Activity of ACC synthase and ethylene production rate peaked at 8 h of treatment. The subsequent decline in enzyme activity was most likely due to inactivation of the enzyme by Cd²⁺, which inhibited ACC synthase activity in vitro at concentrations as low as 0.1 micromolar. Decrease in ethylene production rate was accompanied by leakage of solutes and increasing inhibition of ACC-dependent ethylene production. Ca²⁺, present during a 2-hour preincubation, reduced the effect of Cd²⁺ on leakage and ACC conversion. This suggests that Cd²⁺ exerts its toxicity through membrane damage and inactivation of enzymes. The possibility of an indirect stimulation of ethylene biosynthesis through a wound signal from injured cells is discussed.     

Disruption of the Polar Auxin Transport System in Cotton Seedlings following Treatment with the Defoliant Thidiazuron

The effect of the defoliant thidiazuron (TDZ) on basipetal auxin transport in petiole segments isolated from cotton (Gossypium hirsutum L. cv LG102) seedlings was examined using the donor/receiver agar block technique. Treatment of intact seedlings with TDZ at concentrations of 1 micromolar or greater resulted in a dose-dependent inhibition of ¹⁴C-IAA transport in petiole segments isolated 1 or 2 days after treatment. Using 100 micromolar TDZ, the inhibition was detectable 19 hours after treatment and was complete by 27 hours. Both leaves and petiole segments exhibited a marked increase in ethylene production following treatment with TDZ at concentrations of 0.1 micromolar or greater. The involvement of ethylene in this TDZ response was evaluated by examining the effects of two inhibitors of ethylene action: silver thiosulfate, 2,5-norbornadiene. One day after treatment, both inhibitors effectively antagonized the TDZ-induced inhibition of auxin transport. Two days after TDZ treatment both inhibitors were ineffective. The decrease in IAA transport in TDZ treated tissues was associated with increased metabolism of IAA. The transport of ¹⁴C-2,4-dichlorophenoxyacetic acid was also inhibited by TDZ treatment. This inhibition was not accompanied by increased metabolism. Incorporation of TDZ into the receiver blocks had no effect on auxin transport. The ability of the phytotropin N-1-naphthylphthalamic acid to stimulate IAA uptake from a bathing medium was reduced in TDZ-treated tissues. This reduction is thought to reflect a decline in the auxin efflux system following TDZ treatment.     

Intermediates in the recycling of 5-methylthioribose to methionine in fruits

The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [¹⁴CH₃]MTR was not metabolized in cell free extract from avocado fruit. Either [¹⁴CH₃]MTR plus ATP or [¹⁴CH₃]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as α-keto-γ-methylthiobutyric acid (α-KMB) and α-hydroxy-γ-methylthiobutyric acid (α-HMB) by chromatography in several solvent systems. [³⁵S]α-KMB was found to be further metabolized to methionine and α-HMB by these extracts, whereas α-HMB was not. However, α-HMB inhibited the conversion of α-KMB to methionine. Both [U-¹⁴C]α-KMB and [U-¹⁴C]methionine, but not [U-¹⁴C]α-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of α-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR → MTR-1-P → α-KMB → methionine → S-adenosylmethionine → 1-aminocyclopropane-1-carboxylic acid → ethylene.     

Inhibition of ethylene production by rhizobitoxine

Rhizobitoxine, an inhibitor of methionine biosynthesis in Salmonella typhimurium, inhibited ethylene production about 75% in light-grown sorghum seedlings and in senescent apple tissue. Ethylene production stimulated by indoleacetic acid and kinetin in sorghum was similarly inhibited. With both apple and sorghum, the inhibition could only be partially relieved by additions of methionine. A methionine analogue, α-keto-γ-methylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition.     

Metabolism of 5-methylthioribose to methionine

During ethylene biosynthesis, the H₃CS- group of S-adenosylmethionine is released as 5′-methylthioadenosine, which is recycled to methionine via 5-methylthioribose (MTR). In mungbean hypocotyls and cell-free extracts of avocado, [¹⁴C]MTR was converted into labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB), as intermediates. Incubation of [ribose-U-¹⁴C]MTR with avocado extract resulted in the production of [¹⁴C]formate, indicating the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR. Tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract requires an amino donor. Among several potential donors examined, l-glutamine was the most efficient. Anaerobiosis inhibited only partially the oxidation of MTR to formate, KMB/HMB, and methionine by avocado extract. The role of O₂ in the conversion of MTR to methionine is discussed.     

5-Methylthioribose kinase activity in plants

The presence of a previously unidentified plant enzyme, 5-methylthioribose kinase, has been demonstrated to exist in cell-free extracts from several fruit tissues. The enzyme catalyzes the ATP-dependent phosphorylation of 5-methylthioribose to 5-methylthioribose-1-phosphate. Enzyme activity has been found in avocado, pear, apple, strawberry and tomato tissues. The significance of the presence of this enzyme in relation to ethylene biosynthesis is discussed.