Regulation of soybean nodulation independent of ethylene signaling

Leguminous plants regulate the number of Bradyrhizobium- or Rhizobium-infected sites that develop into nitrogen-fixing root nodules. Ethylene has been implicated in the regulation of nodule formation in some species, but this role has remained in question for soybean (Glycine max). The present study used soybean mutants with decreased responsiveness to ethylene, soybean mutants with defective regulation of nodule number, and Ag⁺ inhibition of ethylene perception to examine the role of ethylene in the regulation of nodule number. Nodule numbers on ethylene-insensitive mutants and plants treated with Ag⁺ were similar to those on wild-type plants and untreated plants, respectively. Hypernodulating mutants displayed wild-type ethylene sensitivity. Suppression of nodule numbers by high nitrate was also similar between ethylene-insensitive plants, wild-type plants, and plants treated with Ag⁺. Ethylene insensitivity of the roots of etr1-1 mutants was confirmed using assays for sensitivity to 1-aminocyclopropane-1-carboxylic acid and for ethylene-stimulated root-hair formation. Additional phenotypes of etr1-1 roots were also characterized. Ethylene-dependent pathways regulate the number of nodules that form on species such as pea and Medicago truncatula, but our data indicate that ethylene is less significant in regulating the number of nodules that form on soybean.     

Exogenous Ethylene Inhibits Nodulation of Pisum sativum L. cv Sparkle

Exogenous ethylene inhibited nodulation on the primary and lateral roots of pea, Pisum sativum L. cv Sparkle. Ethylene was more inhibitory to nodule formation than to root growth; nodule number was reduced by half with only 0.07 μL/L ethylene applied continually to the roots for 3 weeks. The inhibition was overcome by treating roots with 1 μm Ag⁺, an inhibitor of ethylene action. Exogenous ethylene also inhibited nodulation on sweet clover (Melilotus alba) and on pea mutants that are hypernodulating or have ineffective nodules. Exogenous ethylene did not decrease the number of infections per centimeter of lateral pea root, but nearly all of the infections were blocked when the infection thread was in the basal epidermal cell or in the outer cortical cells.     

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     

Ethylene as an effector of wound-induced resistance to cellulase in oat leaves

Peeling the abaxial epidermis from oat leaves (Avena sativa var. Victory) induces the formation of wound ethylene and the development of resistance to cellulolytic digestion of mesophyll cell walls. Ethylene release begins between 1 and 2 hours after peeling in the light or dark. Aminoethoxyvinylglycine (AVG, 0.1 millimolar), CoCl₂ (1.0 millimolar), propyl gallate (PG, 1.0 millimolar) or aminooxyacetic acid (AOA, 1.0 millimolar) inhibits, whereas AgNO₃ stimulates wound ethylene formation. Incubation on inhibitors of ethylene biosynthesis (AVG, CoCl₂, PG, AOA) or action (AgNO₃, hypobaric pressure or the trapping of ethylene with HgClO₄) also prevents the development of wound-induced resistance to enzymic cell wall digestion. 1-Aminocyclopropane-1-carboxylic acid (ACC, 1.0 millimolar) reverses AVG (0.1 millimolar) inhibition of the development of resistance. Exogenous ethylene partially induces the development of resistance in unwounded oat leaves.

These results suggest that peeling of oat leaves induces ethylene biosynthesis, which in turn effects changes in the mesophyll cells resulting in the development of resistance to cellulolytic digestion.

     

Nitrate-induced ethylene biosynthesis and the control of nodulation in alfalfa

We previously reported that inhibition of ethylene biosynthesis with aminoethoxyvinylglycine (AVG) eliminated the inhibitory effect of NO₃^– on nodulation of alfalfa (Medicago sativa L. cv. Aragon) plants grown aeroponically. In this work, the effect of Ag⁺, as an inhibitor of ethylene action, has been studied in plants growing aeroponically or in darkened tubes with vermiculite, and low-nitrate or high-nitrate solution. Vermiculite-grown plants developed up to 3 times as many nodules as did those growing aeroponically. Nodule formation was mirrored by dry-matter accumulation. High (10 mol m^–3) NO₃^– applied from planting inhibited nodulation to an equal extent (c. 50%) in the two growth conditions. In contrast, Ag⁺ treatment increased nodule formation at all NO₃^– concentrations assayed under the two growth conditions, with the stimulation being higher in plants grown aeroponically. Finally, no effect of Ag⁺ (10 mmol m^–3) on plant growth was observed in either of the growth conditions. The effectiveness of NO₃^– as a nodulation inhibitor and enhancer of ethylene biosynthesis in roots of alfalfa was also studied. Within 24 h after inoculation, 10 mol m^–3 NO₃^– exerted most of its inhibitory effect on nodulation. At the same time, both 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity and ethylene evolution rates markedly increased in inoculated and uninoculated alfalfa roots treated with NO₃^–. Support for a role of endogenous ethylene in the control of nodule formation in legumes is discussed.     

Lithium,aminoethoxyvinylglycine and cobalt reversal of the cotyledonary pricking-induced growth inhibition in the hypocotyl of Bidens pilosus in relation to ethylene and peroxidases

Pretreatment of young Bidens pilosus plants with lithium (Li), aminoethoxyvinylglycine (AVG) or cobalt (Co) prevents the cotyledonary pricking-induced growth inhibition of the hypocotyl. The effect is correlated with parallel prevention of the pricking-induced enhancement of peroxidase and ethylene production in the hypocotyl. Only Co prevents the increased capacity of hypocotyl segments or microsomes to convert aminocyclopropane carboxylic acid to ethylene. Li, AVG, and Co do not interfere with peroxidase and ethylene metabolism in pricked cotyledons. Although Li, AVG as well as Co are known to interfere with ethylene biosynthesis and action, they could well remove the wounding effect by different modes of action.     

Nitrate inhibition of nodulation can be overcome by the ethylene inhibitor aminoethoxyvinylglycine

Previously, we reported (a) a positive correlation between the nitrate concentrations in growth medium and ethylene evolved from uninoculated and inoculated alfalfa (Medicago sativa) roots and (b) a negative correlation between ethylene evolution and nodulation. Here, we report that the inhibitory effect of NO₃⁻ on nodulation of alfalfa can be eliminated by the ethylene inhibitor aminoethoxyvinylglycine (AVG). This effect was probably related to the strong inhibition (90%) of ethylene biosynthesis caused by AVG in these inoculated and NO₃⁻-treated roots. These results support our hypothesis that the inhibitory effect of NO₃⁻ is mediated through the phytohormone ethylene. A possible role of endogenous ethylene in the autoregulation of nodulation also is discussed. AVG at 10 micromolar significantly (P < 0.05) increased total nitrogenase activity (acetylene reduction) in 2.5 and 5 millimolar NO₃⁻-fed plants probably as a result of the very high stimulation of nodulation.     

Axillary bud development of passionfruit as affected by ethylene precursor and inhibitors

Summary The effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and two inhibitors, silver thiosulfate (STS) and aminoethoxyvinylglycine (AVG), were tested in yellow passionfruit (Passiflora edulis f. flaricarpa Degener) acillary cultured in vitro. The organogenic responses were assessed by the number of buds per explant, mean leaf area per explant, and shoot length. ACC-supplemented medium significantly inhibited all evaluated responses at both concentrations tested. When ethylene action and biosynthesis were inhibited, a significant increase in the number of developed buds and average leaf area was observed. Accumulated ethylene and its accumulation rate were significantly greater at 10 μM ACC, with a maximum production rate deteeted: at the 14th day and a decline at the 21st day. The results suggest beneficial effects of ethylene inhibitors on in vitro development of axillary buds and their reliability for use as an alternative approach to evaluate sensitivity of Passiflora species to ethylene. Even though shoot elongation did not differ from that of the control, the inhibition of the ethylene action and its biosynthesis by AVG and STS, respectively, significantly enhanced the number of buds per explant and leaf area.     

Biosynthesis of stress ethylene in soybean seedlings: Similarities to endogenous ethylene biosynthesis

The similarity of stress ethylene biosynthesis in whole plants to endogenous ethylene biosynthesis was investigated using two inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) and cobalt chloride (Co²⁺); and the intermediates, methionine, S -adenosylmethionine (SAM), and 1-aminocyclopropane-1-carboxylic acid (ACC), of basal ethylene biosynthesis. Stress ethylene production induced by ozone, cadmium, or 2,4-dichlorophenoxyacetic acid was inhibited in hydroponically-grown soybean seedlings in a concentration-dependent manner by both AVG and CO²⁺. The ethylene intermediates evoked responses in intact seedlings similar to that described for endogenous ethylene production in isolated vegetative tissue. The addition of SAM to the hydroponic system relieved AVG inhibition of stress ethylene production. Feeding ACC to the seedlings resulted in increased ethylene production independent of stress application or prior AVG inhibition. Cobalt inhibition of stress ethylene production was relieved by increasing concentrations of ACC. A short lag period of 12–18 min was observed in stress ethylene production following a 30-min ozone exposure. Addition of cycloheximide partially inhibited ozone-induced ethylene production.
These results suggest a common pathway in whole plants for stress ethylene production and endogenous ethylene biosynthesis.     

The Role of Endogenous Ethylene in Carbohydrate Metabolism of Medicago sativa L. Somatic Embryos in Relation to Their Regenerative Ability

Effects of the ethylene biosynthesis inhibitors salicylic acid (SA) and aminoethoxyvinylglycine (AVG) on germination of Medicago sativa L. somatic embryos and their conversion to seedlings in relation to carbohydrate content and α-amylase activity were studied. Both SA, an inhibitor of ACC oxidase, and AVG, an inhibitor of ACC synthase, when present in the regeneration medium (0.1 and 1 μM) were found to drastically reduce the embryo germination rate. In addition, SA and AVG were found to almost completely or completely, respectively, arrest the process of embryo conversion to seedlings. The inhibitory effects of SA and AVG on germination and conversion may indicate that the processes required endogenous ethylene. AVG and SA clearly slowed down starch disappearance during the 48-h imbibition in the regeneration medium prior to radicle elongation, which was correlated with inhibition of the activity of α-amylase, an enzyme responsible for starch hydrolysis. It is probable that ethylene may activate α-amylase in the germinating alfalfa somatic embryos. In contrast, the disappearance of soluble sugars in the embryos in the presence of the inhibitors tested was accelerated. The disappearance of soluble sugars (to null or almost null) in embryos was faster in the presence of SA in the regeneration medium after 24 and 48 h compared to the disappearance rate with AVG present in the medium. Only glucose was present after a 48-h incubation in the regeneration medium in the presence of the two ethylene biosynthesis inhibitors, in contrast to the control embryos in which glucose was not detected.     

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.     

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 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     

Interaction of karrikinolide and ethylene in controlling germination of dormant <Emphasis Type="Italic">Avena fatua</Emphasis> L. caryopses

Caryopses of Avena fatua L. are dormant after harvest and germinate poorly at 20 °C. Dormancy was released by after-ripening the dry caryopses in the dark at 25 °C for 3 months. Karrikinolide (butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one, KAR₁), in contrast to exogenous ethylene and the precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid (ACC), completely overcame dormancy. The effect of KAR₁ was not affected by aminoethoxyvinylglycine (AVG), α-aminoisobutyric acid (AIB) and CoCl₂, inhibitors of ACC synthase and oxidase, respectively. 2,5-Norbornadiene (NBD), a reversible inhibitor of ethylene binding to its receptor, counteracted the stimulatory effect of KAR₁. Ethylene, ethephon and ACC counteracted and AVG reinforced inhibition caused by norbornadiene. Inhibition due to norbornadiene, applied during the first 3 days of imbibition in the presence of KAR₁, disappeared after transfer to air or ethylene. The obtained results confirm that KAR₁ breaks dormancy and indicate that ethylene alone plays no role in releasing dormancy of Avena fatua caryopses. KAR₁ probably did not relieve dormancy via the stimulation of ethylene biosynthesis. Some level of endogenous ethylene is probably required for ethylene action, which might be required for releasing dormancy by KAR₁ or for subsequent germination of caryopses after removing dormancy.     

Silver Ions Increase Auxin Efflux Independently of Effects on Ethylene Response

Silver nitrate and aminoethoxyvinylglycine (AVG) are often used to inhibit perception and biosynthesis, respectively, of the phytohormone ethylene. In the course of exploring the genetic basis of the extensive interactions between ethylene and auxin, we compared the effects of silver nitrate (AgNO₃) and AVG on auxin responsiveness. We found that although AgNO₃ dramatically decreased root indole-3-acetic acid (IAA) responsiveness in inhibition of root elongation, promotion of DR5-β-glucuronidase activity, and reduction of Aux/IAA protein levels, AVG had more mild effects. Moreover, we found that that silver ions, but not AVG, enhanced IAA efflux similarly in root tips of both the wild type and mutants with blocked ethylene responses, indicating that this enhancement was independent of ethylene signaling. Our results suggest that the promotion of IAA efflux by silver ions is independent of the effects of silver ions on ethylene perception. Although the molecular details of this enhancement remain unknown, our finding that silver ions can promote IAA efflux in addition to blocking ethylene signaling suggest that caution is warranted in interpreting studies using AgNO₃ to block ethylene signaling in roots.     

An evaluation of the role of ethylene in herbicidal injury induced by picloram or clopyralid in rapeseed and sunflower plants

The role of ethylene in herbicidal injury induced by 4-amino-3,5,6-trichloropicolinic acid (picloram) or 3,6-dichloropicolinic acid (clopyralid) was investigated in sunflower (Helianthus annuus L.) and rapeseed (Brassica napus L. cv Altex). Picloram induces herbicide injury in both species, whereas clopyralid induces injury only in sunflower. Picloram applied to the third leaf of a rapeseed plant increased ethylene evolution several-fold. Clopyralid had no effect on ethylene production in rapeseed. In sunflower, both picloram and clopyralid elevated ethylene production. Ethylene biosynthesis induced by the herbicide treatment was not restricted to treated areas. When clopyralid was applied only to the lower stem and cotyledons of sunflower, the herbicide treatment resulted in an increase in the rate of ethylene production from the true leaves. Increased ethylene production preceded or coincided with the onset of morphological responses induced by a herbicide application to a susceptible species. The contrast in ethylene production by these two plant species cannot be accounted for by differences in absorption and translocation of clopyralid and picloram.

Treatment with aminoethoxyvinylglycine (AVG) before picloram or clopyralid application prevented an increase in ethylene production. Pretreatment with AVG also delayed the development of morphological changes induced by picloram or clopyralid. It appears that enhanced ethylene biosynthesis after application of picloram or clopyralid to the susceptible plant species was a factor involved in resulting morphological changes.

     

Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula

Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia. The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor. Although the interaction is beneficial to the plant, the number of nodules is tightly regulated. The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number. The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown. Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula. We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking. This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.     

Further studies of auxin and ACC induced feminization in the cucumber plant using ethylene inhibitors

The present study was designed to establish the role of an essential hormone controlling sex expression in cucumber. A potent anti-ethylene agent, AgNO3, completely inhibited pistillate flower formation caused by IAA, ACC or ethephon. Inhibitors of ethylene biosynthesis, AVG and CoCl2 also suppressed feminization due to exogenous IAA or ACC. Though AVG also suppressed ethephon-induced feminization, this may be due to the second effect of AVG rather than the effect on ACC biosynthesis. These results confirm that ethylene is a major factor regulating feminization and that exogenous auxin induces pistillate flower formation through its stimulation of ethylene production, rather than ACC production.     

Abscission of Citrus Leaf Explants: INTERRELATIONSHIPS OF ABSCISIC ACID, ETHYLENE, AND HYDROLYTIC ENZYMES

The question whether abscisic acid (ABA) induces cellulase and polygalacturonase activity and, hence, abscission directly or whether its action is mediated by C₂H₄ was studied in citrus (Osbeck var. Shamouti) leaf explants using aminoethoxyvinyl glycine (AVG), an inhibitor of C₂H₄ biosynthesis. ABA in concentrations of 10 micromolar and higher induced C₂H₄ production and accelerated abscission. AVG inhibited C₂H₄ formation, activity of cellulase and polygalacturonase, and abscission in ABA-treated explants. AVG did not inhibit the increase in the activity of the cell-wall degrading enzymes or abscission in a saturating level of externally supplied C₂H₄. This indicates that the effect of AVG resulted from inhibition of the formation of endogenous ethylene. The data indicate that in citrus leaf explants the induction of the activity of cellulase and polygalacturonase and abscission by ABA is mediated by C₂H₄.     

Ethylene Inhibitors Partly Restore Nodulation to Pea Mutant E 107 (brz)

E107 (brz) is a pleiotropic mutant of pea (Pisum sativum L. cv Sparkle) characterized by low nodulation, leaf necrosis, excessive ion accumulation, and decreased plant size. The defective nodulation of E107 was studied by light microscopy of lateral roots. The number of infections per centimeter of lateral root was only a third that of Sparkle. Moreover, most of the infections were aborted early; i.e. in only 14% of the infections did the infection thread penetrate beyond the epidermis. Nodulation of E107 was partly restored by treating the plant with the ethylene inhibitors aminoethoxyvinylglycine (AVG) or Ag⁺. Treatment with Ag⁺ did not increase the number of infections, but half of the infections went to completion. Ag⁺ and AVG did not alter the size of the mutant, the accumulation of cations in its shoots, nor the leaf necrosis. Thus, in E107, nodule development can be uncoupled from other pleiotropic characteristics.