Ethylene inhibitors enhance in vitro root formation from apple shoot cultures

Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and three ethylene inhibitors, AgNO₃, aminoethoxyvinyglycine (AVG) and CoCl₂, on root formation were tested in vitro using shoot cultures of the apple (Malus×domestica Borkh.) cultivar Royal Gala. ACC inhibited root formation by delaying root emergence and increasing callus formation at the bases of shoots. In contrast, ethylene inhibitors promoted root formation. Both AgNO₃ and AVG at the appropriate concentrations increased the percentage of shoots producing roots and reduced callus formation at the base of these shoots. AgNO₃ stimulated root emergence and enhanced root growth, while AVG increased the number of roots per shoot. CoCl₂ slightly increased root number and rooting efficiency. These promotive effects may result from a reduction in ethylene concentration or inhibition of ethylene action. The results found in this study may be used to improve the rooting efficiency of other apple cultivars and rootstocks, and possibly of other plant species. Received: 2 March 1997 / Revision received: 1 July 1997 / Accepted: 18 July 1997     

Ethylene inhibitors enhance in vitro root formation on faba bean shoots regenerated on medium containing thidiazuron

The possible involvement of ethylene in in vitrorooting of faba bean (Vicia faba L.) shootsregenerated on medium containing thidiazuron wasinvestigated. The effects of the ethylene precursor1-aminocyclopropane-1-carboxylic acid (ACC) and threeethylene inhibitors, silver nitrate (AgNO₃),acetyl salicylic acid (ASA) and cobalt chloride(CoCl₂) on root formation were tested in vitrousing TDZ-induced shoots of faba bean accession 760.ACC inhibited root formation. In contrast, ethyleneinhibitors promoted root formation, AgNO₃ at theappropriate concentrations enhanced root emergence andincreased root number per shoot, root growth rate, androot length. Both CoCl₂ and ASA at theappropriate concentrations increased rootingefficiency. These promotive effects may result from areduction in ethylene concentration or inhibition ofethylene action. The results offer a new approach toimprove the rooting efficiency of TDZ-induced shootsof faba bean and possibly of other plant species.     

1-Methylcyclopropene and ethylene as regulators of in vitro organogenesis in kiwi explants

In this paper, we study the influence of ACC, AVG and 1-MCP on in vitro organogenesis of kiwi (Actinidia deliciosa) explants and on ethylene metabolism. Results indicated that increasing ethylene production and accumulation in the head space of the culture vessel by adding ACC to the culture medium inhibited organogenesis, except for rooting, which increased and stimulated ACC oxidase activity threefold, whereas AVG increased the length and number of shoots and leaves and inhibited about 80% ACC synthase activity compared with the reference explants. 1-MCP exerts a stimulatory effect analogous to AVG, increasing ACC synthase activity and organogenesis of kiwi explants. This effect is not reverted by the addition of ACC to the culture medium. Therefore, ethylene production and its accumulation in the headspace of the culture vessels seems to be responsible for the inhibition of shoot development as well as increasing rooting in in vitro cultured kiwi explants.     

Involvement of ethylene in the rooting of seedling shoot cultures of <Emphasis Type="Italic">Bixa orellana</Emphasis> L.

Using seedlings derived from the shoot apex of annatto (Bixa orellana L. cv. Bico-de-Pato) we observed the rooting frequency of B. orellana, the number and length of roots and the rate of ethylene production during 30 d in culture. The rhizogenesis response was affected by auxins (NAA or IBA) and by both the ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the inhibitor 2-aminoethoxyvinylglycine (AVG). Auxin supplementation to the medium resulted in root induction, ethylene production, and an undesirable callusing in the epidermal and cortical tissues. Irrespective of the presence of auxins, supplementing the medium with ACC promoted ethylene biosynthesis and callusing, which resulted in increased cell proliferation mainly in the cortical and vascular tissues, while the epidermis was mostly unaltered. In both ACC and auxin-supplemented medium, increased ethylene production and callusing occurred, suggesting a synergistic effect between these two responses. ACC was capable of inducing adventitious root formation, but the roots produced had a wrinkled appearance when compared to normal roots. Conversely, AVG reduced ethylene production and callusing, while the epidermis, cortex, and inner tissues remained unaltered, regardless of the presence of auxins. AVG was beneficial in these aspects, although its application led to a reduction in the number of roots and in the average root length. In conclusion, it was not possible to establish a direct relation between ethylene and rooting, but we hypothesize that, under the experimental conditions described, ethylene may enhance tissue sensitivity to auxin. However, ethylene did not seem essential to the rhizogenesis process in annatto.     

Ethylene and auxin-ethylene interaction in adventitious root formation in mung bean (Vigna radiata) cuttings

The role of ethylene in adventitious root formation and its involvement in auxin-induced rooting were investigated in cuttings ofVigna radiata (L.). Treatment with 30 M indole-3-acetic acid (IAA) for 24 h slightly inhibited rooting, whereas the same concentration of indole-3-butyric acid (IBA) significantly stimulated it. Ethylene derived from 1-aminocyclopropane-1-carboxylic acid (ACC) increased the number of adventitious roots but inhibited their emergence and elongation. Endogenous levels of ethylene, ACC, and malonyl-ACC (MACC) were initially higher in cuttings treated with IAA. This trend was quickly reversed, and cuttings, particularly hypocotyls, treated with IBA produced higher levels of ethylene and had more ACC and MACC during most of the rooting process. Aminoethoxyvinylglycine significantly inhibited rooting, but its inhibitory effect could not be reversed by ACC. The data suggest that the stimulating effect of IBA on rooting is closely associated with its induction of ACC and ethylene biosynthesis.     

Promotion ofin vitro shoot formation from excised roots of silktree (Albizzia julibrissin) by an oxime ether derivative and other ethylene inhibitors

Summary This report describes the regeneration response of excised seedling roots of silktree (Albizzia julibrissin) to added ethylene precursors/generators (1-amino-cyclopropane-1-carboxylic acid [ACC], 2-chloroethylphosphonic acid [CEPA]), biosynthesis inhibitors (aminoethoxyvinylglycine [AVG], an oxime ether derivative [OED={[(ispropylidene)-amino]oxy}-acetic acid-2-(methoxy)-2-oxoethyl ester], CoCl² [Co⁺⁺]), and an ethylene action inhibitor (AgNO₃ [Ag⁺]). When placed on B5 medium, about 50% of the control explants formed shoot buds within 15 days. Addition of ACC or CEPA (1–10 µM) to the culture medium decreased both the percentage of cultures forming shoots and the number of shoots formed per culture. In contrast, AVG and OED (1–10 µM) increased shoot formation to almost 100% and increased the number of shoots formed per culture. Likewise, both Co⁺⁺ and Ag⁺ (1–10 µM) increased shoot regeneration, but the number of shoots produced after 30 days was less than with AVG or OED. The inhibitors of ethylene biosynthesis were partially effective in counteracting the inhibitory effect of ACC on shoot formation. These results suggest that modulation of ethylene biosynthesis and/or action can strongly influence the formation of adventitious shoots from excised roots of silktree.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CEPA 2-chloroethylphosphonic acid - OED oxime ether derivative     

The Effect of the Ethylene Action Inhibitor 1-Cyclopropenylmethyl Butyl Ether on Early Plant Growth

Increased levels of ethylene in plants are responsible for many deleterious effects such as early senescence, fruit deterioration and inhibition of root elongation. Several cyclopropene derivatives have previously been studied as inhibitors of ethylene action in plants. This study focuses on one such compound, 1-cyclopropenylmethyl butyl ether and its effect on the growth of roots and shoots of canola plants as well as rooting of mung bean seedlings 1-cyclopropenylmethyl butyl ether increased root length in canola plants, but had no significant effect on shoot length. In rooting studies, mung bean seedlings treated with 1-cyclopropenylmethyl butyl ether prior to root excision had fewer numbers of roots than control plants that were not treated with the ethylene action inhibitor. The same rooting study, when repeated in the presence of 1-aminocyclopropane-1-carboxylic acid (ACC), demonstrated an overall increase in the number of roots of inibitor-treated and non-treated plants, however, the inhibitor was still effective in decreasing the number of roots, compared to its non-treated conterpart. Online publication: 7 April 2005     

Ethylene as an endogenous inhibitor of root regeneration in tomato leaf discs cultured in vitro

We examined ethylene effects on root regeneration in tomato leaf discs cultured in vitro. Applied ethylene or Ethephon did not stimulate rooting in the leaf discs. In the presence of indoleacetic acid. 5 × 10^-6M, these substances significantly inhibited root formation. Ethylene production (nl C₂H₄· (24 h)^-1. flask^-1) was positively correlated with increased IAA concentrations at various times during the culture period and, as a consequence, with the rooting response after 168 h. However, separate testing of equimolar concentrations of seven different auxins and auxin-like compounds showed no positive correlation between the rate of ethylene production and subsequent rooting response. Aeration of gas-tight flasks containing leaf discs and absorption of ethylene evolved from the discs by mercuric perchlorate in gas-tight flasks or pre-treatment of leaf discs with AgNO₃ significantly enhanced IAA induced root regeneration. Thus, these studies indicate that ethylene is not a rooting hormone per se. Furthermore, ethylene (whether applied externally or synthesized by the tissue) does not appear to account for the ability of auxin to stimulate rooting.     

Polyamines and ethylene in relation to adventitious root formation in Prunus avium shoot cultures

An attempt was made to identify some of the hormonal factors that control adventitious root formation in our Prunus avium micropropagation system in order to improve rooting in difficult-to-root genotypes. Changes in endogenous contents of free polyamines were determined at intervals during auxin-induced rooting of shoot cultures. Accumulation of putrescine and spermidine peaked between days 9 and 11. Spermine was only present in traces, Exogenously supplied putrescine or spermine (50-500 μM), in the presence of optimal or suboptimal levels of indolebutyric acid (IBA), had no effect on rooting percentage or root density, except for spermine at 500 μM. At this external concentration spermine caused a substantial accumulation in both free spermine and putrescine. The use of several inhibitors of polyamine biosynthesis, namely α-difluoromethylornithine (DFMO), α-difluoromethylarginine (DFMA), dicyclohexylammonium sulphate (DCHA) and methylglyoxal-bis-guanyl-hydrazone (MGBG) alone or in combination in the 0.1 to 5 μM range, resulted in an inhibition of rooting that was partially reversed by the addition of the corresponding polyamine. Cellular polyamine levels were significantly reduced by DFMO and DFMA but not by DCHA and MGBG, Labeled putrescine incorporation into spermidine increased somewhat in the presence of the ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG). A system based on [3,4-¹⁴C]methionine incorporation was used to measure ethylene synthesis by the in vitro cultured shoots. Label incorporation was drastically reduced by 10 μM AVG and increased 3.5-fold in the presence of 50 μM IBA with respect to controls (no IBA). Labeled methionine incorporation into spermidine increased to some extent when ethylene synthesis was inhibited by AVG. Adding the ethylene precursor 1-aminocyclopropane-l-carboxylic acid (ACC) to the rooting medium significantly inhibited rooting percentage; AVG caused the formation of a greater number of roots per shoot but delayed their growth. Supplying the shoots with both compounds resulted in an intermediate rooting response, in which both rooting percentage and root density were affected. These results indicate that polyamines may play a significant role at least in some stages of root formation. The polyamine and ethylene biosynthetic pathways seem to be competitive but under our conditions, the enhancement of one pathway when the other was inhibited, was not dramatic. Although IBA promoted ethylene synthesis, AVG, which drastically reduced it, also promoted root formation. Thus, the auxin effect on root induction cannot be directly related to its ability to enhance ethylene synthesis.     

Brassinosteroid-induced epinasty in tomato plants

The effects of root treatments of brassinosteroid (BR) on the growth and development of hydroponically grown tomato plants (Lycopersicon esculentum Mill cv Heinz 1350) were evaluated. There was a dramatic increase in petiole bending when the plants were treated with 0.5 to 1.0 micromolar BR. The leaf angle of the treated plants was almost three times that of untreated controls. BR-induced epinasty appeared to be due to stimulation of ethylene production. Excised petioles from BR-treated plants produced more than twice as much ethylene as did untreated controls. As ethylene production increased, the degree of petiole bending also increased, and inhibition of ethylene production by AOA or CoCl₂ also inhibited epinasty. BR-treated plants had increased levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaf tissue. ACC appeared to accumulate primarily in the petioles with the greatest amount of ACC accumulating in the youngest petioles. Time course evaluations revealed that BR treatment stimulated ACC production. As ACC accumulated, ethylene increased, resulting in epinasty. Little or no ACC was found in the xylem sap, indicating that there was a signal transported from the roots which stimulated ACC synthesis in the leaf tissue.     

Effects of 1-MCP and exogenous ethylene on fruit ripening and antioxidants in stored mango

Mango (Mangifera indica L. cv. Tainong) fruits were harvested at the green-mature stage in Hainan and air-freighted to the laboratory at Peking. The fruits were treated with either 1 μl l⁻¹ 1-MCP or 5 μl l⁻¹ ethylene for 24 h and stored at 20°C for up to 16 days. 1-MCP maintained fruit firmness, whereas exogenous ethylene decreased fruit firmness. Exogenous ethylene accelerated the increase in ethylene and 1-aminocyclopropane-1-carboxylate (ACC) oxidase, whereas 1-MCP reduced both. Exogenous ethylene stimulated and 1-MCP inhibited the production of H₂O₂ of mango fruit during storage. Ascorbic acid was maintained at a high concentration in 1-MCP-treated fruit but was low in ethylene-treated fruit. 1-MCP inhibited activities of antioxidant enzymes including catalase, superoxide dismutase and ascorbate peroxidase. These results suggest that 1-MCP could play a positive role in regulating the activated oxygen metabolism balance. Baogang Wang and Jianhui Wang contributed equally to this work.     

Endogenous ethylene requirement for adventitious root induction and growth in tomato cotyledons and lavandin microcuttings in vitro

The role of ethylene in the formation of adventitious roots in vitro was studied in tomato (Lycopersicon esculentum Mill. cv. UC 105) cotyledons and lavandin (Lavandula officinalis Chaix × Lavandula latifolia microshoots. Both systems were able to form roots on hormone-free medium evolving low amounts of ethylene. The addition of 20–50 M indole-3-acetic acid (IAA) inhibited root formation in tomato cotyledons while increasing ethylene production. Naphthaleneacetic acid (NAA, 3 M) stimulated root number in lavandin explants and induced a transient rise in ethylene evolution. Enhanced ethylene levels via the endogenous precursors 1-aminocyclopropane-1-carboxylic acid (ACC, 25–50 M) drastically impaired root regeneration and growth in tomato. In lavandin, 10 M ACC stimulated ethylene production and significantly inhibited the rooting percentage and root growth. Conversely, ACC enhanced the root number in the presence of NAA only. Severe inhibition of rooting was also caused by ethylene reduction via biosynthetic inhibitors, aminoethoxyvinylglycine (AVG, 5–10 M) in tomato, and salicylic acid (SA, 100 M) in lavandin. A strict requirement of endogenous ethylene for adventitious root induction and growth is thus suggested.Abbreviations LS Linsmaier and Skoog medium - BA N⁶-benzyladenine - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - AVG Aminoethoxyvinylglycine - SA Salicylic acid - ACC 1-aminocyclopropane-1-carboxylic acid     

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.     

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.     

Headspace ethylene accumulation on Stizolobium hassjoo hairy root culture producing L-3,4-dihydroxyphenylalanine

The addition of 1-aminocyclopropane-1-carboxylic acid (ethylene precursor), or 2-chloroethylphosphonic acid (ethephon, an ethylene-releasing compound) decreased root dry weight and l-DOPA (l-3,4-dihydroxyphenylalanine) accumulation in hairy root cultures of Stizolobium hassjoo. The inhibition caused by ethephon-mediated ethylene release was alleviated by 0.5 mg CoCl₂ l^–1 as an inhibitor of ethylene biosynthesis. The action of ethylene was inhibited by 1.5 mg AgNO₃ l^–1. Ethylene thus lowers hairy root formation and l-DOPA production; CoCl₂ decreases ethylene formation leading to a considerably improved root dry weight and l-DOPA production.     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings

Homogenates of etiolated pea (Pisum sativum L.) shoots formed ethylene upon incubation with 1-aminocyclopropane-1-carboxylic acid (ACC). In-vitro ethylene formation was not dependent upon prior treatment of the tissue with indole-3-acetic acid. When homogenates were passed through a Sephadex column, the excluded, high-molecular-weight fraction lost much of its ethylene-synthesizing capacity. This activity was largely restored when a heat-stable, low-molecular-weight factor, which was retarded on the Sephadex column, was added back to the high-molecular-weight fraction. The ethylene-synthesizing system appeared to be associated, at least in part, with the particulate fraction of the pea homogenate. Like ethylene synthesis in vivo, cell-free ethylene formation from ACC was oxygen dependent and inhibited by ethylenediamine tetraacetic acid, n-propyl gallate, cyanide, azide, CoCl₃, and incubation at 40°C. It was also inhibited by catalase. In-vitro ethylene synthesis could only be saturated at very high ACC concentrations, if at all. Ethylene production in pea homogenates, and perhaps also in intact tissue, may be the result of the action of an enzyme that needs a heat-stable cofactor and has a very low affinity for its substrate, ACC, or it may be the result of a chemical reaction between ACC and the product of an enzyme reaction. Homogenates of etiolated pea shoots also formed ethylene with 2-keto-4-mercaptomethyl butyrate (KMB) as substrate. However, the mechanism by which KMB is converted to ethylene appears to be different from that by which ACC is converted.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - KMB 2-keto-4-mercaptomethyl butyrate - SAM S-adenosylmethionine     

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.     

Responses of ethylene biosynthesis to saline stress in seedlings of eight plant species

The effect of salinity (100 mM NaCl) on ethylene metabolism in the early phase of vegetative development of several plant species has been investigated. The effects of saline treatment on shoot and root growth, ranged in sensitivity with respect to species: pepper (Capsicum annum L. cv Pairal) > tomato (Lycopersicon esculentum Mill. cv Malpica) > broccoli (Brassica oleraceae L. var. Italica Plenk. cv Marathon F1) ≅ lettuce (Lactuca sativa var. longifolia Lam. cv Inverna) ≅ melon (Cucumis melo L. cv Ruano F1, Roche type) > bean (Phaseolus vulgaris L. cv. Gator Green 15) ≅ spinach (Spinacia oleracea L. cv Boeing) > beetroot (Beta vulgaris L. var. crassa (Alef.) J. Helm. cv Detroit). After saline treatment, ethylene production increased 4.2-fold in pepper shoots. Significant increases were also found in shoots of tomato, broccoli and bean. In contrast, salinity decreased shoot ethylene production rate in melon, spinach, and beetroot. In roots, the general effect of salinity was a decrease in ethylene production, especially in broccoli and bean, except in tomato root, in which a sharp increase in ethylene production occurred. In general, saline treatment increased total ACC concentration in both shoot and root in most of the plant species examined, which was related to plant sensitivity to salinity. For example, pepper shoot was the most sensitive to saline treatment, showing the highest fresh weight inhibition and the highest increase in total ACC concentration (8.5-fold), while, beetroot was less affected by salinity and showed no effect on total ACC concentration in response to saline treatment.