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     

Effect of ethylene antagonists on auxin-induced inhibition of intact primary root elongation in maize (Zeamays L.)

We tested that the hypothesis that root elongation might be controlled by altering the level of ethylene in intact primary roots of maize(Zea mays L.). We measured root elongation in a short period using a computerized root auxanometer. Compounds which regulate ethylene production were applied to intact primary roots in different time periods. Root elongation was stimulated by the treatment with ethylene antagonists such as Co²⁺, aminoethoxyvinylglycine (AVG) and L-canaline. This result suggested that root elongation was closely related to ethylene level of intact primary roots. Furthermore, IAA- and 1-aminocyclopropane-1-carboxylic acid (ACC)-induced inhibition of root elongation was reversed by treatment with Co²⁺. The application of ACC to roots which have been exposed to IAA and Co²⁺ have no significant effect on root elongation. However, the inhibition of root elongation by ACC in roots previously treated with IAA and AVG became manifest when the applied IAA concentrations were lower. These results were consistent with the hypothesis that the level of ethylene in intact roots functions to moderate root elongation, and suggested that auxin-induced inhibition of root elongation results from auxin induced promotion of ethylene production.     

The influence of oxygen deficiency on ethylene synthesis, 1-aminocyclopropane-1-carboxylic acid levels and aerenchyma formation in roots of Zea mays

The relationship between ethylene production, 1-aminocyclopropane-l-carboxylic acid (ACC) concentration and aerenchyma formation (ethylene-promoted cavitation of the cortex) was studied using nodal roots of maize (Zea mays L. cv. LG11) subjected to various O₂ treatments. Ethylene evolution was 7–8 fold faster in roots grown at 3 kPa O₂ than in those from aerated solution (21 kPa O₂), and transferring roots from aerated solution to 3 kPa O₂ enhanced ethylene synthesis within less than 2 h. Ethylene production and ACC accumulation were closely correlated in different zones of hypoxic roots, regardless of whether O₂ was furnished to the roots through aerenchyma or external solution. Both ethylene production and ACC concentrations (fresh weight basis) were more than 10-fold greater in the distal 0–10 mm than in the fully expanded zone of roots at 3 kPa O₂. Aerenchyma formation occurred in the apical 20 mm of these roots. Roots transferred from air to anoxia accumulated less than 0. 1 nmol ACC (mg protein)^-1 for the first 1.75 h; no ethylene was produced in this time. The subsequent rise in ACC levels shows that ACC can reach high concentrations even in the absence of O₂, presumably due to a de-repression of ACC synthase. The hypothesis was therefore tested that anoxia in the apical region of the root caused enhanced synthesis of ACC, which was transported to more mature regions (10–20 mm behind the apex), where ethylene could be produced and aerenchyma formation stimulated. Surprisingly, exposure of intact root tips to anoxia inhibited aerenchyma formation in the mature root axis. High osmotic pressures around the growing region or excision of apices had the same effect, demonstrating that a growing apex is required for high rates of aerenchyma formation in the adjacent tissue.     

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.     

Effect of auxin and ethylene on elongation of intact primary roots of maize (Zeamays L.)

We tested that the hypothesis that root elongation might be controlled by altering the level of ethylene in intact primary roots of maize(Zea mays L.). We measured root elongation in a short period using a computerized root auxanometer. Compounds which regulate ethylene production were applied to intact primary roots in different time periods. Root elongation was stimulated by the treatment with ethylene antagonists such as Co²⁺, aminoethoxyvinylglycine (AVG) and L-canaline. This result suggested that root elongation was closely related to ethylene level of intact primary roots. Furthermore, IAA- and 1-aminocyclopropane-1-carboxylic acid (ACC)-induced inhibition of root elongation was reversed by treatment with Co²⁺. The application of ACC to roots which have been exposed to IAA and Co²⁺ have no significant effect on root elongation. However, the inhibition of root elongation by ACC in roots previously treated with IAA and AVG became manifest when the applied IAA concentrations were lower. These results were consistent with the hypothesis that the level of ethylene in intact roots functions to moderate root elongation, and suggested that auxin-induced inhibition of root elongation results from auxin induced promotion of ethylene production.     

Ethylene as a possible mediator of light-induced inhibition of root growth

Eliasson, L. and Bollmark, M. 1988. Ethylene as a possible mediator of light-induced inhibition of root growth. - Physiol. Plant. 72: 605–609.
Pea seedlings ( Pisum sativum L. cv. Weibull's Marma) were used to investigate the possible role of ethylene in light-induced inhibition of root elongation. Illumination of the roots with white light inhibited root elongation by 40–50% and increased ethylene production by the roots about 4-fold. Our main approach was to use exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), supplied in the growth solution, to monitor ethylene production of the roots independent of light treatment. Ethylene production of excised root tips increased with increasing ACC concentrations. The rate of ethylene production in dark-grown roots treated with 0.1 μ M ACC was similar to that caused by illumination. Low ACC concentrations (0.01–0.1 μ M ) decreased the rate of root elongation, especially in seedlings grown in the dark, and 0.1 μ M ACC inhibited elongation to about the same extent as light. In light the roots curved and grew partly plagiogravitropically. This effect was also simulated by the 0.1 μ M ACC treatment. At 1 μ M and higher concentrations, ACC inhibited root growth almost completely and caused conspicuous curvatures of the root tips both in light and darkness. Inhibitors of ethylene synthesis and action partially counteracted the inhibition of root elongation caused by light. These observations suggest that the increase in ethylene production caused by light is at least partly responsible for the decreased growth of light-exposed roots.     

Effects of 1-aminocyclopropane-1-carboxylic acid and oxygen concentrations on in vivo and in vitro activity of ACC oxidase of sunflower hypocotyl segments

In vivo ethylene production by hypocotyl segments of sunflower seedlings and in vitro activity of 1-aminocyclopropane-1-carboxylic acid oxidase (formerly ethylene-forming enzyme) extacted from the same tissues increase with increasing concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and oxygen. ACC oxidase activity follows Michaelis-Menten kinetics. The apparent Km values of the enzyme towards ACC, estimated in vivo and in vitro, are respectively 219 M and 20.6 M. Both Km values towards O₂ are similar, ca 10.6–11.4%. A decrease in concentration in one of the substrates (ACC or O₂) results in an increase in in vivo apparent Km of ACC oxidase for the other substrate. On the contrary, Km values of the enzyme towards ACC or O₂ estimated in vitro are not dependent upon the concentration of the other substrate (ACC or O₂).Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - MACC malonylate 1-aminocyclopropane-1-carboxylic acid - SD standard deviation     

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.

     

Ca2+ effects on ethylene, carbon dioxide and 1-aminocyclopropane-1-carboxylic acid synthase activity

The response of pericarp disks from ripening tomato (Lycopersicon esculentum Mill. cv. Traveler‘76) to CaCl₂, additions was studied to determine the effect of Ca²⁺ on ethylene and CO₂ production. Application of 5 mM CaCl₂ resulted in a 2, 20, 33, 39, and 50% increase in ethylene production in disks obtained from preclimacteric minimum, climacteric rise, climacteric peak, one, and two days postclimacteric fruit, respectively. CaCl₂ concentrations of 10 and 50 mM gave no additional stimulation of ethylene production; CO₂ production at 5 mM CaCl₂ was not different from controls, but is increased at 10 and 50mM CaCl₂. CaCl₂ also increased ethylene production in disks treated with 1-aminocyclopropane-1-carboxylic acid (ACC) or aminoethoxy-vinylglycine. Chloride salts of K⁺, Na⁺, Mg²⁺, Sr²⁺ and La³⁺ did not stimulate ethylene production. SrCl₂ stimulated ethylene production to a lesser degree than CaCl₂. Disks from potato (Solanum tuberosum L. cv. Katahdin) tubers produced greater quantities of ethylene and ACC when 5 mM CaCl₂ was included in the incubation medium (K. B. Evensen, 1983. Physiol. Plant. 60:125–128). Ca²⁺-treated disks had more than three times as much ACC synthase activity as control disks after 18 to 24 h incubation, when ethylene and ACC were maximal. The apparent K_m for S-adenosylmethionine was 13 μM at 29°C, pH 8.0 in extracts from both Ca²⁺-treated and control disks. Inclusion of 1 to 50 mM CaCl₂ in the assay medium did not significantly affect enzyme activity. ACC synthase extracted from control and Ca²⁺-treated disks had a pH optimum of 8.5 and an apparent molecular weight of 72 kdalton, estimated by gel filtration. It is likely that the presence of Ca²⁺ in the buffer allows greater synthesis of ACC synthase as part of the wound-healing response in potato, while in tomato the predominant effect is on membrane stabilization.     

Ethylene and carbon dioxide in the growth and development of cultured radish roots

Ethylene is produced by cultured radish roots in amounts large enough to be physiologically important. When roots were grown in controlled atmospheres, applied ethylene was generally inhibitory to elongation, lateral root initiation, and cambial activity. 1% CO₂ similarly affected roots not given ethylene. In contrast, elongation and lateral root production of ethylene-treated roots were stimulated by 1% CO₂. The results suggest that the often-observed stimulation of root growth by CO₂ is due to an interaction with endogenous ethylene.     

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.     

Effect of chlorsulfuron on ethylene evolution from sunflower seedlings

The effect of the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy - 6 - methyl -1, 3,5 - triazin - 2 - yl)aminocarbonyl]benzenesulfonamide) on ethylene production in light-grown sunflower (Helianthus annuus L.) seedlings was examined. Application of chlorsulfuron to the apex stimulated ethylene production in all tissues examined: cotyledons, hypocotyls, and roots. The greatest stimulation occurred in the upper portion of the hypocotyl adjacent to, and including, the cotyledonary node. Ethylene evolution from hypocotyls excised from treated seedlings was stimulated over control levels 1 day after herbicide application and reached a maximum (approx. 75 x control or 17 nl/g f wt/h) 2 to 3 days after treatment. Labeling and inhibitor studies indicated that the ethylene produced was derived primarily from methionine. Chlorsulfuron treatment stimulated the rate of accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), as well as the ability of the tissue to convert exogenous ACC to ethylene. Chlorsulfuron had little effect on ethylene production when administered to the hypocotylsin vitro. Removal of the cotyledons from treated seedlings reduced the rate of ethylene evolution from the hypocotyls. These results suggest that stimulation of ethylene production in sunflower hypocotyls by chlorsulfuron is not a wound response but rather is dependent on factors derived from the cotyledons.     

Involvement of ethylene in the morphological and developmental response of rice to elevated atmospheric CO2 concentrations

We tested the hypothesis that increased carbohydrate flux under elevatedCO₂ regulates accelerated development using rice (Oryzasativa L. cv. Jarrah). Plants were grown either in flooded soil orsolution culture at either 360 or 700 L CO₂L^–1. Total dry mass, shoot elongation rates (SER),tiller appearance rates (TAR) and ethylene release from intact rice seedlingswere measured from 5 to 42 days after planting (DAP). At maturity, shoot andsheath length, tiller number and grain mass were also measured. ElevatedCO₂ had a profound effect on growth, morphology and development andthe effects were more pronounced during the early growth phase. Total aboveground biomass increased at elevated CO₂ and this was accounted for by enhanced tiller number. Grain yield was increased by 56% under elevated CO₂mainly due to increased tiller number and hence panicle number. TAR and SERwereenhanced at elevated CO₂ but SER increased only untill 25 DAP.Elevated CO₂ stimulated a 2-3-fold increase in endogenous andACC-mediated ethylene release but the ACC concentration in the leaves waslittleaffected showing that rates of ACC synthesis matched its oxidation. Inhibitionof ethylene action by 1-aminocyclopropane (1-MCP) had a more pronouncedinhibitory effect on ethylene release in plants that were grown at 700 ascompared to 360 L CO₂ L^–1. Feedingsucrose to intact plants enhanced ethylene synthesis and these results areconsistent with the hypothesis that increased accumulation of sucrose atelevated CO₂ may enhance expression of genes in the ethylenebiosynthetic pathway. We conclude that increase in ethylene release may becentral in promoting accelerated development under elevated CO₂ andthis coincides with the release of auxiliary buds and accelerated rates oftiller appearance hence increased grain yield at elevated CO₂.     

Enhanced ethylene production by primary roots of Zea mays L. in response to sub-ambient partial pressures of oxygen

Ethylene production by primary roots of 72–h-old intact seedlings of Zea mays L. cv. LG11 was studied under ambient and sub-ambient oxygen partial pressures (pO₂) using a gas flow-through system linked to a photoacoustic laser detector. Despite precautions to minimize physical perturbation to seedlings while setting-up, ethylene production in air was faster during the first 6h than later, in association with a small temporary swelling of the roots. When roots were switched from air (20–8kPa O₂) to 3 or 5kPa O₂ after 6h, ethylene production increased within 2—3 h. When, the roots were returned to air 16 h later, ethylene production decreased within 2—3 h. The presence of 10kPa CO₂ did not interfere with the effect of 3kPa O₂. Transferring roots from air to 12–5kPa did not change ethylene production, while a reduction to 1 kPa O₂ induced a small increase. The extra ethylene formed in 3 and 5 kPa O₂ was associated with plagiotropism, swelling, root hair production, and after 72 h, increased amounts of intercellular space (aerenchyma) in the root cortex. Root extension was also slowed down, but the pattern of response to oxygen shortage did not always match that of ethylene production. On return to air, subsequent growth patterns became normal within a few hours. In the complete absence of oxygen, no ethylene production was detected, even when anaerobic roots were returned to air after 16 h.     

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.     

Effect of carbon dioxide and light on ethylene production in intact sunflower plants

High CO₂ concentration (0.5%) increased the rate of ethylene production, measured in a continuous flow system, in intact sunflower (Helianthus annuus L.) plants. However, the rate of ethylene production subsided to near control levels after approximately 24 hours. The effect of high CO₂ could only be observed in light. Although high CO₂ concentration had no effect on the rate of ethylene production in darkness, prolonged exposure (approximately 16 hours) of plants to high CO₂ in the dark prevented the increase in ethylene production when the plants were exposed to light and high CO₂.