Ethylene-Enhanced 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Ripening Apples

Apples (Malus sylvestris Mill, cv Golden Delicious) were treated before harvest with aminoethoxyvinylglycine (AVG). AVG is presumed to reversibly inhibit 1-aminocyclopropane-1-carboxylic acid (ACC) activity, but not the formation of ACC synthase. AVG treatment effectively blocked initiation of autocatalytic ethylene production and ripening of harvested apples. Exogenous ethylene induced extractable ACC synthase activity and ripening in AVG-treated apples. Removal of exogenous ethylene caused a rapid decline in ACC synthase activity and in CO₂ production. The results with ripened, AVG-treated apples indicate (a) a dose-response relationship between ethylene and enhancement of ACC synthase activity with a half-maximal response at approximately 0.8 μl/l ethylene; (b) reversal of ethylene-enhanced ACC synthase activity by CO₂; (c) enhancement of ACC synthase activity by the ethylene-activity analog propylene.

Induction of ACC synthase activity, autocatalytic ethylene production, and ripening of preclimacteric apples not treated with AVG were delayed by 6 and 10% CO₂, but not by 1.25% CO₂. However, each of these CO₂ concentrations reduced the rate of increase of ACC synthase activity.

     

The effect of CO2 on ethylene evolution and elongation rate in roots of sunflower (Helianthus annuus) seedlings

Both carbon dioxide and ethylene can affect the rate of root elongation. Carbon dioxide can also promote ethylene biosynthesis by enhancing the activity of 1-aminocylopropane-1-carboxylic acid (ACC) oxidase. Since the amount of CO₂ in the soil air, and in the atmosphere surrounding roots held in enclosed containers, is known to vary widely, we investigated the effects of varying CO₂ concentrations on ethylene production by excised and intact sunflower roots (Helianthus annuus L. cv. Dahlgren 131). Seedlings were germinated in an aeroponic system in which the roots hung freely in a chamber and were misted with nutrient solution. This allowed for treatment, manipulation and harvest of undamaged and minimally disturbed roots. While exposure of excised roots to 0.5% CO₂ could produce a small increase in ethylene production (compared to roots in ambient CO₂), CO₂ concentrations of 2% and above always inhibited ethylene evolution. This inhibition of ethylene production by CO₂ was attributed to a reduction in the availability of ACC: however, elevated CO₂ had no effect on ACC oxidase activity. ACC levels in excised roots were depressed by CO₂ at a concentration of 2% (as compared to ambient CO₂), but n-malonyl-ACC (MACC) levels were not affected. Treating intact roots with 2% CO₂ inhibited elongation by over 50%. Maximum inhibition of elongation occurred 1 h after the CO₂ treatment began, but elongation rates returned to untreated values by 6 h. Supplying these same intact roots with 2% CO₂ did not alter ethylene evolution. Thus, in excised sunflower roots 2% CO₂ treatment reduces ethylene evolution by lowering the availability of ACC. Intact seedlings respond differently in that 2% CO₂ does not affect ethylene production in roots. These intact roots also temporarily exhibit a significantly reduced rate of elongation in response to 2% CO₂.     

The roles of carbon dioxide and abscisic acid in the production of ethylene

Since CO₂ is liberated in the conversion of ACC to ethylene, the evidence that ethylene production by plant tissues is actually promoted by CO₂ calls for an explanation. Accordingly, the formation of ethylene by oat (Avena sativa L. cv. Victory) leaves and by apple (Golden Delicious) and pear (Pyrus communis L. cv. Anjou) tissue in very low levels of CO₂ has been studied. The gas chromatograph was modified to measure CO₂ and ethylene at the same time, by reducing both to methane. (Response of the gas chromatograph to CO₂ concentrations is linear.) The work establishes a clear difference between the endogenous production of ethylene and its production from applied ACC, a difference which holds about equally for leaves and for fruit tissue. The difference is in the CO₂ requirement, namely, lowering the CO₂ level by 99% or more decreases the production of ethylene from applied ACC by 50–60%, but it does not decrease, or even slightly increases, its production from endogenous precursors. Thus, while the need for CO₂ has not been explained, it has at least been delimited. The responses to abscisic acid (ABA) also differ, but in the reverse direction, the endogenous production of ethylene being decreased up to 70% by ABA. while the liberation from ACC is promoted about 20%. ABA also promoted the respiratory CO₂ production by 30% or, in presence of 1-aminocyclopropane-1-carboxylic acid (ACC), by over 50%. Inhibition of ethylene production by cobalt or EDTA shows no such differentiation, while inhibition by Na catechol-4,6-disulfonate (Tiron) shows a small difference. It is concluded either that endogenous ethylene is formed by an enzyme system different from that reacting with ACC, or (more likely) that when ethylene arises from endogenous precursors the reaction does not proceed by way of free ACC, but by some activated form of it.     

Light inhibition of the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in leaves is mediated through carbon dioxide

The mechanism of light-inhibited ethylene production in excised rice (Oryza sativa L.) and tobacco (Nicotiana tabacum L.) leaves was examined. In segments of rice leaves light substantially inhibited the endogenous ethylene production, but when CO₂ was added into the incubation flask, the rate of endogenous ethylene production in the light increased markedly, to a level which was even higher than that produced in the dark. Carbon dioxide, however, had no appreciable effect of leaf segments incubated in the dark. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, was not significantly affected by lightdark or CO₂ treatment, indicating that dark treatment or CO₂exerted its effect by promoting the conversion of ACC to ethylene. This conclusion was supported by the observations that the rate of conversion of exogenously applied ACC to ethylene was similarly inhibited by light, and this inhibition was relieved in the presence of CO₂. Similar results were obtained with tobacco leaf discs. The concentrations of CO₂ giving half-maximal activity was about 0.06%, which was only slightly above the ambient level of 0.03%. The modulation of ACC conversion to ethylene by CO₂ or light in detached leaves of both rice and tobacco was rapid and fully reversible, indicating that CO₂ regulates the activity, but not the synthesis, of the enzyme converting ACC to ethylene. Our results indicate that light inhibition of ethylene production in detached leaves is mediated through the internal level of CO₂, which directly modulates the activity of the enzyme converting ACC to ethylene.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid Recipient of a Republic of China National Science Council Fellowship     

Light Stimulation of Ethylene Release from Leaves of Gomphrena globosa L

The effect of light and CO₂ on both the endogenous and 1-aminocyclopropane-1-carboxylic acid (ACC)-dependent ethylene evolution from metabolically active detached leaves and leaf discs of Gomphrena globosa L. is reported. Treatment with varying concentrations of ACC did not appear to inhibit photosynthesis, respiration, or stomatal behavior. In all treatments, more ethylene was released into a closed flask from ACC-treated tissue, but the pattern of ethylene release with respect to light/dark/CO₂ treatments was the same.

Leaf tissue in the light with a source of CO₂ sufficient to maintain photosynthesis always generates 3 to 4 times more ethylene than tissue in the dark. Conversely, the lowest rate of ethylene release occurs when leaf tissue is illuminated and photosynthetic activity depletes the CO₂ to the compensation point. Ethylene release in the dark is also stimulated by CO₂ either added to the flask as bicarbonate or generated by dark respiration. Ethylene release increases dramatically and in parallel with photosynthesis at increasing light intensities in this C₄ plant. Ethylene release appears dependent on CO₂ both in the light and in the dark. Therefore, it is suggested that the important factor regulating the evolution of ethylene gas from leaves of Gomphrena may be CO₂ metabolism rather than light per se.

     

Effects of turgor potential on 1-aminocyclopropane-1-carboxylic acid uptake into the vacuolar compartment and ethylene production in tomato pericarp slices

While solute transport and ethylene production by plant tissue are sensitive to the osmotic concentration of the solution bathing the tissue, the influence of tissue water relations and specifically tissue turgor potential on the kinetics of 1-aminocyclopropane-1-carboxylic acid (ACC) uptake into the vacuolar compartment and ethylene production have not been examined. 1-Aminocyclopropane-1-carboxylic acid transport and ethylene production were examined in tomato (Lycopersicon esculentum Mill. cv. Liberty) pericarp slices incubated in solutions having a range of mannitol, polyethylene glycol 3350 and ethylene glycol concentrations known to affect tissue water relations. Tissue osmotic and turgor potentials were derived from osmolality measurements of cell saps recovered by freeze-thawing and corrected for the contribution of the free-space solution. When relatively nonpermeable (mannitol or polyethylene glycol 3350) osmotica were used, both ACC uptake and ethylene production were greatest at a solution osmolality of 230 milliosmolal where tissue turgor potential ranged between 120 and 140 kPa. At higher and lower turgor potentials, the high-affinity saturating component of ACC uptake and ethylene production were inhibited, and ACC efflux from the vacuolar compartment was increased. The inhibition of ACC uptake was evident as a decrease in V_max with no effect on K_m. Turgor potential changes caused by adjusting solution osmolality with mannitol or polyethylene glycol 3350 were accompanied by changes in the osmotic potential and water potential of the tissue. The effects of turgor potential vs the osmotic and water potentials of tomato pericarp slices were differentiated by comparing responses to nonpermeable osmotica and mixtures of nonpermeable and permeable osmotica. Ethylene glycol-mannitol mixtures had effects on the osmotic potential and water potential of the tissue similar to those of nonpermeable osmotica but had less effect on tissue turgor, ACC transport and ethylene production. Incubating tissue in solutions without nonpermeable osmotica osmotically shocked the tissue. Increasing solution osmolality with ethylene glycol in the absence of nonpermeable osmotica increased tissue turgor and ethylene production. The present study indicates that tissue turgor is an important factor affecting the kinetics of ACC uptake into the vacuolar compartment and ethylene production in tomato pericarp slices.     

Bicarbonate/CO(2)-Facilitated Conversion of 1-Amino-cyclopropane-1-carboxylic Acid to Ethylene in Model Systems and Intact Tissues

Bicarbonate markedly enhances ethylene production from 1-aminocyclopropane-1-carboxylic acid (ACC) in model chemical systems where the conversion is free radical-mediated, in thylakoid membrane suspensions of Phaseolus vulgaris L. cv Kinghorn where the reaction is light-dependent, and in microsomal membrane suspensions and intact tissues where the reaction is enzymically mediated. In two model systems generating free radicals—the Fenton reaction and a reaction mixture containing xanthine/xanthine oxidase, NaHCO₃ (200 millimolar) increased the formation of ethylene from ACC by 84-fold and 54-fold, respectively. Isolated thylakoid membranes also proved capable of ACC-dependent ethylene production, but only upon illumination, and this too was enhanced by added NaHCO₃. As well, light-induced inhibition of ACC-dependent ethylene production by leaf discs was relieved by adding 200 millimolar NaHCO₃. Finally, NaHCO₃ (200 millimolar) augmented ACC-dependent ethylene production from young carnation flowers by about 4-fold, and the conversions of ACC to ethylene by microsomes isolated from carnation flowers and etiolated pea epicotyls were higher by 1900 and 62%, respectively, in the presence of 200 millimolar NaHCO₃.

This increased production of ethylene appears not to be due to bicarbonate or CO₂-induced release of the gas from putative receptor sites, since the addition of NaHCO₃ to sealed reaction mixtures after the ACC to ethylene conversion had been terminated had no effect. Spin-trapping studies have confirmed that bicarbonate does not facilitate the formation of free radicals thought to be involved in the conversion of ACC to ethylene. Nor did bicarbonate alter the physical properties of the membrane bilayer, which might indirectly modulate the activity of the membrane-associated enzyme capable of converting ACC to ethylene. Rather, bicarbonate appears to directly facilitate the conversion of ACC to ethylene, and the data are consistent with the view that CO₂ derived from bicarbonate is the active molecular species.

     

Effect of low oxygen and high carbon dioxide on the levels of ethylene and 1-aminocyclopropane-1-carboxylic acid in ripening apple fruits

The association of the level of ACC and the ethylene concentration in ripening apple fruit (Malus sylvestris Mill, var. Ben Davis) was studied. Preclimacteric apple contained small amounts of ACC and ethylene. With the onset of the climacteric and a concomitant decrease in flesh firmness, the level of ACC and ethylene concentration both increased markedly. During the postclimacteric period, ethylene concentration started to decline, but the level of ACC continued to increase. Ethylene production and loss of flesh firmness of fruits during ripening were greatly suppressed by treatments with low O₂ (O₂ 1–3%, CO₂ O%) or high CO₂ (CO₂ 20–30%, O₂ 15–20%) at the preclimacteric stage. However, after 4 weeks an accumulation of ACC was observed in treated fruits when control fruit was at the postclimacteric stage. Treatment of fruit with either low O₂ or high CO₂ at the climacteric stage resulted in a decrease of ethylene production. However, the ACC level in fruit treated with low O₂ was much higher than both control and high CO₂ treated fruit; it appears that low O₂ inhibits only the conversion of ACC to ethylene, resulting in an accumulation of ACC. Since CO₂ inhibits ethylene production but does not result in an accumulation of ACC, it appears that high CO₂ inhibits both the conversion of ACC to ethylene and the formation of ACC.     

Ethylene and wound-induced cyanide-resistant respiration climaxes in potato: The synergistic role of CO2 and the selective role of lycorine

When treated with ethylene in O₂, conditioned potato (Solanum tuberosum L. cv. Russet Burbank) tubers – that is, tubers kept at room temperature for 10 days or more – yield slices that are CN^? resistant. Ten % CO₂ in the gas mixture not only synergizes the effect of ethylene, but replaces the need for conditioning as well. The response to CO₂ is more pronounced with increasing time from harvest. By contrast fresh slices from untreated tubers are CN^? sensitive, as are slices from tubers incubated in O₂ or O₂ plus CO₂. The suggestion is made that CN^? resistance is constitutive, and that treatment with ethylene/CO₂ in O₂ confers on potato tuber tissue a resistance to the extensive degradation of membrane phospholipids that normally attends slicing and leads to the loss of CN^? resistance. In this connection respiration inhibition by imidazole, an inhibitor of fatty acid α-oxidation, is extensive in slices of untreated tubers, and sharply diminished in slices of ethylene-treated tubers in proportion to their CN^? resistance. The coextensive rise of respiration rate and CN^? resistance in aged potato slices has led to the presumption that the CN^?-resistant path mediates the respiration climax. Accordingly the alkaloid, lycorine, has been considered to inhibit the development of CN^? resistance in aging potato slices because it curtails the wound-induced respiration. A comparison was carried out on the effect of lycorine on CN^?-sensitive and CN^?-resistant fresh slices – the latter obtained from ethylene/CO₂-treated tubers. Lycorine suppressed the development of the wound-induced respiration without restricting the development of CN^? resistance.     

Ethylene-promoted conversion of 1-aminocyclopropane-1-carboxylic Acid to ethylene in peel of apple at various stages of fruit development

Internal ethylene concentration, ability to convert 1-amino-cyclopropane-1-carboxylic acid (ACC) to ethylene (ethylene-forming enzyme [EFE] activity) and ACC content in the peel of apples (Malus domestica Borkh., cv Golden Delicious) increased only slightly during fruit maturation on the tree. Treatment of immature apples with 100 microliters ethylene per liter for 24 hours increased EFE activity in the peel tissue, but did not induce an increase in ethylene production. This ability of apple peel tissue to respond to ethylene with elevated EFE activity increased exponentially during maturation on the tree. After harvest of mature preclimacteric apples previously treated with aminoethoxyvinyl-glycine, 0.05 microliter per liter ethylene did not immediately cause a rapid increase of development in EFE activity in peel tissue. However, 0.5 microliter per liter ethylene and higher concentrations did. The ethylene concentration for half-maximal promotion of EFE development was estimated to be approximately 0.9 microliter per liter. CO₂ partially inhibited the rapid increase of ethylene-promoted development of EFE activity. It is suggested that ethylene-promoted CO₂ production is involved in the regulation of autocatalytic ethylene production in apples.     

Ethylene Release from Leaves of Xanthium strumarium L. and Zea mays L.

The release of ethylene into sealed Erlenmeyer flasks by intactleaves and leaf discs of Xanthium strumarium L. a C₃ plant andZea mays L. a C₄ plant were compared both in white light andin darkness. The effects of the presence or absence of addedCO₂ (in the form of sodium bicarbonate) the photosynthetic inhibitor3-[3,4-dichlorophenyl]-l, l-dimethyl urea (DCMU) and 1-aminocyclopropane-1-carboxylicacid (ACC), the precursor of ethylene in higher plants, werealso investigated. The rate of ethylene release from leaf tissue of Xanthium inthe absence of added CO₂ was markedly reduced in the light (i.e.at the CO₂ compensation point). Treatments that would enhancethe CO₂ availability to the tissue (i.e. added bicarbonate,darkness, treatment with DCMU) allowed higher levels of ethylenerelease. Incubation of the tissue with ACC considerably enhancedthe release of ethylene compared to that from the correspondingcontrol tissue without ACC. However, the pattern of ethylenerelease induced by the various treatments was similar with orwithout added ACC. When tissue, in the absence of added CO₂, was transferred fromlight to darkness, and back to light for 90 min periods, theethylene release rates Increased during the interposed darkperiod but resumed the lower rate during the final light period.The addition of CO₂ in the light resulted in a similar rateof ethylene release to that found in the dark. The overall pattern of ethylene release from Zea leaf tissuesubjected to light and dark in the presence or absence of addedCO₂ was similar to that of Xanthium. However, two or three timesmore ethylene was released from maize leaves in the light whenCO² was added compared to that generated in the dark. This isin marked contrast to Xanthium, where, under the light conditionsused, the ethylene release rate in the dark equalled or exceededthat occurring in the light, even in the presence of high levelsof CO₂. A very low rate of ethylene release was observed atthe CO₂ compensation point of maize. A speculative model is presented to explain how photosyntheticactivity might act as a key factor in regulating ethylene evolutionfrom leaf tissue in these experiments. It invokes the conceptof an inhibition by CO₂ of ethylene retention or breakdown thuspermitting more ethylene to be released from the leaves.     

The role of ethylene in the senescence of oat leaves

The evolution of ethylene, both from the endogenous source and from added 1-aminocyclopropane-1-carboxylic acid (ACC), has been followed in close relationship with the senescent loss of chlorophyll from seedling oat leaves. In white light, where chlorophyll loss is slow, the ethylene evolution increases slowly at first, but when the loss of chlorophyll becomes more rapid, ethylene evolution accelerates. CoCl₂ inhibits this increase and correspondingly maintains the chlorophyll content, with an optimum concentration of 10 micromolar. The rapid rate of chlorophyll loss in the dark is slightly decreased by 3-aminoethoxyvinyl glycine (AVG), by cobalt, and slightly stimulated by ACC. The slower chlorophyll loss in white light, however, is almost completely inhibited by silver ions, greatly decreased by cobalt and by AVG, and strongly increased by ACC. Since the chlorophyll loss is accompanied by proteolysis, it represents true senescence. Chlorophyll loss in light is also strongly antagonized by CO₂, 1% CO₂ giving almost 50% chlorophyll maintenance in controls, while in the presence of added ACC or ethylene gas, the chlorophyll loss is 50% reversed by about 3% CO₂. The ethylene system in leaves is thus more sensitive to CO₂ than that in fruits. Indoleacetic acid also clearly decreases the effect of ACC. It is shown that kinetin, CO₂, Ag⁺, and indoleacetic acid, all of which oppose the effect of ethylene, nevertheless increase the evolution of ethylene by the leaves, and it is suggested that ethylene evolution may, in many instances, mean that its hormonal metabolism is being prevented.     

Carbon dioxide enhances the development of the ethylene forming enzyme in tobacco leaf discs

Since CO₂ is known to stimulate ethylene production by promoting the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, the effect of CO₂ on the activity and the development of the ethylene forming enzyme (EFE) was studied in tobacco (Nicotiana tabacum L. cv Havana 425 and Xanthi) leaf discs. In addition to previous observations that EFE activity is dependent on CO₂ concentration and is saturable with 2% CO₂, present data show two saturation curves at 2% and 10% CO₂. Promotion of EFE development was dependent also on CO₂ concentration (saturated at 2% CO₂) and duration (maximum at 24 in the dark), and was abolished by 20 micromolar cycloheximide. Application of exogenous ethylene (20 microliters per liter) or light treatment further increased the CO₂-enhanced development of EFE, implying that these two factors can also affect EFE development via interaction with CO₂. The results suggest that CO₂ exerts its stimulatory effect on the conversion of ACC to ethylene by enhancing not only the activity but also the synthesis of EFE in leaf discs.     

Ethylene biosynthesis: 3. Evidence concerning the fate of C1-N1 of 1-aminocyclopropane carboxylic acid

Evidence has been obtained that the product of ethylene biosynthesis from 1-aminocyclopropane carboxylic acid (ACC), aside from CO₂, is cyanide. This has been accomplished by synthesis of a carbon-13-labeled ACC, feeding to apple tissue, and isolation of the free amino acids. By enzymatic degradation to CO₂, followed by mass spectrometry, label is shown to be incorporated into the 4-carbon of asparagine. This occurs via well-known pathways for the metabolism of cyanide, and is confirmed by incorporation experiments using labeled cyanide. In conjunction with previous stereochemical studies, evidence for a sequential single-electron transfer pathway for ethylene biosynthesis has been considerably strengthened.     

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.     

Metabolism of α-aminoisobutyric acid in mungbean hypocotyls in relation to metabolism of 1-aminocyclopropane-1-carboxylic acid

1-Aminocyclopropane-1-carboxylic acid (ACC) is known to be converted to ethylene and conjugated into N-malonyl-ACC in plant tissues. When -amino[1-¹⁴C]isobutyric acid (AIB), a structural analog of ACC, was administered to mungbean (Vigna radiata L.) hypocotyl segments, it was metabolized to ¹⁴CO₂ and conjugated to N-malonyl-AIB (MAIB). -Aminoisobutyric acid inhibited the conversion of ACC to ethylene and also inhibited, to a lesser extent, N-malonylation of ACC and d-amino acids. Although the malonylation of AIB was strongly inhibited by ACC as well as by d-amino acids, the metabolism of AIB to CO₂ was inhibited only by ACC but not by d-amino acids. Inhibitors of ACC conversion to ethylene such as anaerobiosis, 2,4-dinitrophenol and Co²⁺, similarly inhibited the conversion of AIB to CO₂. These results indicate that the malonyalation of AIB to MAIB is intimately related to the malonylation of ACC and d-amino acids, whereas oxidative decarboxylation of AIB is related to the oxidative degradation of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyric acid - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid - MAIB -(malonylamino)-isobutyric acid - Mes 2-(N-morpholino)ethanesulfonic acid     

Auxin-induced Ethylene Production and Its Inhibition by Aminoethyoxyvinylglycine and Cobalt Ion

Auxin is known to stimulate greatly both C₂H₄ production and the conversion of methionine to ethylene in vegetative tissues, while amino-ethoxyvinylglycine (AVG) or Co²⁺ ion effectively block these processes. To identify the step in the ethylene biosynthetic pathway at which indoleacetic acid (IAA) and AVG exert their effects, [3-¹⁴C]methionine was administered to IAA or IAA-plus-AVG-treated mung bean hypocotyls, and the conversion of methionine to S-adenosylmethionine (SAM), 1-amino-cyclopropane-1-carboxylic acid (ACC), and C₂H₄ was studied. The conversion of methionine to SAM was unaffected by treatment with IAA or IAA plus AVG, but active conversion of methionine to ACC was found only in tissues which were treated with IAA and which were actively producing ethylene. AVG treatment abolished both the conversion of methionine to ACC and ethylene production. These results suggest that in the ethylene biosynthetic pathway (methionine → SAM → ACC → C₂H₄) IAA stimulates C₂H₄ production by inducing the synthesis or activation of ACC synthase, which catalyzes the conversion of SAM to ACC. Indeed, ACC synthase activity was detected only in IAA-treated tissues and its activity was completely inhibited by AVG. This conclusion was supported by the observation that endogenous ACC accumulated after IAA treatment, and that this accumulation was completely eliminated by AVG treatment. The characteristics of Co²⁺ inhibition of IAA-dependent and ACC-dependent ethylene production were similar. The data indicate that Co²⁺ exerts its effect by inhibiting the conversion of ACC to ethylene. This conclusion was further supported by the observation that when Co²⁺ was administered to IAA-treated tissues, endogenous ACC accumulated while ethylene production declined.     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

Inhibition of ethylene synthesis in tomato plants subjected to anaerobic root stress

Enhanced ethylene production and leaf epinasty are characteristic responses of tomato (Lycopersicon esculentum Mill.) to waterlogging. It has been proposed (Bradford, Yang 1980 Plant Physiol 65: 322-326) that this results from the synthesis of the immediate precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), in the waterlogged roots, its export in the transpiration stream to the shoot, and its rapid conversion to ethylene. Inhibitors of the ethylene biosynthetic pathway are available for further testing of this ACC transport hypothesis: aminooxyacetic acid (AOA) or aminoethoxyvinylglycine (AVG) block the synthesis of ACC, whereas CO²⁺ prevents its conversion to ethylene. AOA and AVG, supplied in the nutrient solution, were found to inhibit the synthesis and export of ACC from anaerobic roots, whereas Co²⁺ had no effect, as predicted from their respective sites of action. Transport of the inhibitors to the shoot was demonstrated by their ability to block wound ethylene synthesis in excised petioles. All three inhibitors reduced petiolar ethylene production and epinasty in anaerobically stressed tomato plants. With AOA and AVG, this was due to the prevention of ACC import from the roots as well as inhibition of ACC synthesis in the petioles. With Co²⁺, conversion of both root- and petiole-synthesized ACC to ethylene was blocked. Collectively, these data support the hypothesis that the export of ACC from low O₂ roots to the shoot is an important factor in the ethylene physiology of waterlogged tomato plants.