Inhibitory action of auxin on root elongation not mediated by ethylene

The inhibitory effects of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) on elongation growth of pea (Pisum sativum L.) seedling roots were investigated in relation to the effects of these compounds on ethylene production by the root tips. When added to the growth solution both compounds caused a progressively increasing inhibition of growth within the concentration range of 0.01 to 1 micromolar. However, only ACC increased ethylene production in root tips excised from the treated seedlings after 24 hours. High auxin concentrations caused a transitory increase of ethylene production during a few hours in the beginning of the treatment period, but even in 1 micromolar IAA this increase was too low to have any appreciable effect on growth. ACC, but not IAA, caused growth curvatures, typical of ethylene treatment, in the root tips. IAA caused conspicuous swelling of the root tips while ACC did not. Cobalt and silver ions reversed the growth inhibitory effects induced by ACC but did not counteract the inhibition of elongation or swelling caused by IAA. The growth effects caused by the ACC treatments were obviously due to ethylene production. We found no evidence to indicate that the growth inhibition or swelling caused by IAA is mediated by ethylene. It is concluded that the inhibitory action of IAA on root growth is caused by this auxin per se.     

Cytokinin effects on root growth and possible interactions with ethylene and indole-3-acetic acid

Etiolated pea seedlings ( Pisum sativum L. cv. Weibull's Marma) were used to investigate the effects of exogenous cytokinins on root growth. Benzylaminopurine (BAP) added to the growth solution inhibited the elongation and formation of lateral roots and stimulated swelling of the root tips. Similar effects were obtained with zeatin. The effects were obtained over a wide concentration range down to 0.01 μ M . Growth responses appeared only after treatment for several hours, and the duration of treatment had an important influence on the degree of the effects. BAP caused a moderate increase in ethylene production as measured in excised 10-mm-long root tips. Lowering ethylene production by treatment with cobalt ions counteracted both the inhibition and swelling caused by BAP. Treatment with silver ions also reversed the effect to some extent, indicating that ethylene is involved in the response of the roots to BAP. To further study the involvement of the increased ethylene production in the elongation and swelling response, the effects were compared with those obtained after application of 1-aminocyclopropane-1-carboxylic acid (ACC) in relation to the ethylene produced from this compound. This comparison showed that the increase in ethylene production caused by BAP was too low to explain the response of the roots. However, ACC treatment caused a considerable lowering of the content of indole-3-acetic acid (IAA) in the root tips, whereas BAP did not; instead, BAP increased the amount of IAA per root tip. It is concluded that cytokinins influence growth processes in roots via several mechanisms. A synergistic interaction between endogenous IAA, maintained at a high level by the cytokinin treatment, and the increased ethylene levels appears to explain most of the cytokinin effects during the first day of treatment.     

Auxin and the response of pea roots to auxin transport inhibitors: morphactin

The auxin transport inhibitor methyl-2-chloro-9-hydroxyfluorene-9-carboxylate (CFM), a morphactin, inhibits negative geotropism, causes cellular swelling, and induces root hair formation in roots of intact Pisum sativum L. seedlings. In excised pea root tips, CFM inhibits elongation more than increase in fresh weight (swell ratio = 1.3 at 20 mum CFM). CFM growth inhibition was expressed in the presence of ethylene. Indoleacetic acid (IAA) prevented the expression of CFM growth inhibition possibly because IAA inhibited the accumulation of CFM into the tissue sections. CFM inhibited the accumulation of IAA and 2,4-dichlorophenoxyacetic acid into excised root tips. Applying Leopold's (1963. Brookhaven Symp. Biol. 16: 218-234) model for polar auxin transport, this result suggests a possible explanation for CFM inhibition of geotropism in pea roots, i.e. disruption of auxin transport by interfering with auxin binding.     

Factors increasing ethylene production enhance the sensitivity of root growth to auxins

Light inhibits root elongation, increases ethylene production and enhances the inhibitory action of auxins on root elongation of pea ( Pisum sativum L. cv. Weibulls Marma) seedlings. To investigate the role of ethylene in the interaction between light and auxin, the level of ethylene production in darkness was increased to the level produced in light by supplying 1-aminocyclopropane-1-carboxylic acid (ACC) or benzylaminopurine (BAP). Ethylene production was measured in excised root tips after treatment of intact seedlings for 24 h, while root growth was measured after 48 h. Auxin, at a concentration causing a partial inhibition of root elongation, did not increase ethylene production significantly. A 4-fold increase in ethylene production, caused either by light, 0.1 μ M ACC or 0.1 μ M BAP, inhibited root elongation by 40–50%. The auxins 2,4-dichlorophenoxyacetic acid and indolebutyric acid applied at 0.1 μ M inhibited root elongation by 15–25% in darkness but by 50–60% in light. Supply of ACC or BAP in darkness enhanced the inhibitory effects of auxins to about the same extent as in light. The inhibition caused by the auxins as well as by the BAP was associated with swelling of the root tips. ACC and BAP treatment synergistically increased the swelling caused by auxins. We conclude that auxin and ethylene, when applied or produced in partially inhibitory concentrations, act synergistically to inhibit root elongation and increase root diameter. The effect of light on the response of the roots to auxins is mediated by a light-induced increase in ethylene production.     

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.     

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.     

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.     

Detailed quantitative analysis of architectural traits of basal roots of young seedlings of bean in response to auxin and ethylene

Vertical placement of roots within the soil determines their efficiency of acquisition of heterogeneous belowground resources. This study quantifies the architectural traits of seedling basal roots of bean (Phaseolus vulgaris), and shows that the distribution of root tips at different depths results from a combined effect of both basal root growth angle (BRGA) and root length. Based on emergence locations, the basal roots are classified in three zones, upper, middle, and lower, with each zone having distinct architectural traits. The genotypes characterized as shallow on BRGA alone produced basal roots with higher BRGA, greater length, and more vertically distributed roots than deep genotypes, thereby establishing root depth as a robust measure of root architecture. Although endogenous indole-3-acetic acid (IAA) levels were similar in all genotypes, IAA and 1-N-naphthylphthalamic acid treatments showed different root growth responses to auxin because shallow and deep genotypes tended to have optimal and supraoptimal auxin levels, respectively, for root growth in controls. While IAA increased ethylene production, ethylene also increased IAA content. Although differences in acropetal IAA transport to roots of different zones can account for some of the differences in auxin responsiveness among roots of different emergence positions, this study shows that mutually dependent ethylene-auxin interplay regulates BRGA and root growth differently in different genotypes. Root length inhibition by auxin was reversed by an ethylene synthesis inhibitor. However, IAA caused smaller BRGA in deep genotypes, but not in shallow genotypes, which only responded to IAA in the presence of an ethylene inhibitor.     

Effect of indoleacetic acid,abscisic acid,root tips and coleoptile tips on growth and curvature of maize roots

The effect of externally applied indoleacetic acid (IAA) and abscisic acid (ABA) on the growth of roots of Zea mays L. was measured. Donor blocks of agar with IAA or ABA were placed laterally on the roots and root curvature was measured. When IAA was applied to vertical roots, a curvature directed toward the donor block was observed. This curvature corresponded to a growth inhibition at the side of the root where the donor was applied. When IAA was applied to horizontal roots from the upper side, normal geotropic downward bending was delayed or totally inhibited. The extent of retardation and the inhibition of curvature were found to depend on the concentration of IAA in the donor block. ABA neither induced curvature in vertical roots nor inhibited geotropic curvature in horizontal roots; thus the growth of roots was not inhibited by ABA. However, when, instead of donor blocks, root tips or coleoptile tips were placed onto vertical roots, a curvature of the roots was observed.Abbreviations ABA abscisic acid - IAA 3-indoleacetic acid     

Ribonuclease Activity and Auxin Effects in the Lens Root

In Lens root tips, a direct proportionality between RNA and auxin levels and inverse proportionality between RNA content and RNase activity were found. IAA treatment of the Lens seedlings causes, in the root, both an increase of RNA and auxin content and a decrease of RNase activity. Addition of IAA to the excised roots produces an inhibition of both the decrease of the RNA levels and an inhibition of the increase of the RNase activity. The action of IAA on growth, related to the control of the RNA metabolism is discussed.     

Translocation of Shoot-Applied Indolylacetic Acid into the Roots of Populus tremula

Application of 10 to 100 μg indol-3-ylacetic acid to the leaves of rooted cuttings of aspen caused inhibition of root growth after three hours. Root growth recovered within 24 hours after IAA treatment. Swelling of the root tips occurred during the period of inhibition. The roots responded in the same way if IAA was applied in solution to the cut stem surface above the mature leaves. IAA-1-¹⁴C applied through a cut stem surface or to mature leaves was translocated downwards in the plants and labelled IAA could be isolated from the roots 3 to 24 hours after application. The ethanol-soluble activity decreased rapidly indicating a rapid metabolism or binding of IAA. IAA-1-¹⁴C applied to growing leaves was not translocated. From the rapid response of root growth it was concluded that IAA was translocated into the roots at a rate of about 7 cm per hour. This rate of translocation indicates that the sieve tubes are involved in the translocation. Implications of the results for the translocation of endogenous auxin into the roots are discussed.     

Does Ethylene Mediate Root Growth Inhibition by Indole-3-Acetic Acid?

The effects of ethylene and of indole-3-acetic acid (IAA) on growth of excised pea root sections have been compared under a variety of conditions. After 16 hours treatment the inhibitory action of IAA is fully reversible on transfer of the root sections to IAA-free solutions. In contrast, inhibition by ethylene is almost totally irreversible. IAA inhibits growth from zero time; ethylene is generally without effect during the first 3 to 6 hours. The inhibitory action of ethylene is dependent on factors such as tissue age and solution composition which have no major effect on IAA inhibition. Ethylene production is enhanced by 100 μm IAA, but conditions which reduce the rate of ethylene evolution 2 to 3-fold at the same IAA concentration fail to affect the inhibitory action of IAA on elongation. It seems unlikely that ethylene can play more than a minor role in mediating inhibition of pea root growth by IAA.     

The effects of 3,5-diiodo-4-hydroxybenzoic acid on the oxidation of IAA and auxin-induced ethylene production by cress root segments

Summary In previous research here, 3,5-diiodo-4-hydroxybenzoic acid (DIHB) was shown to promote the elongation of roots of cress (Lepidium sativum) seedlings growing in light, and to inhibit the auxin-induced production of ethylene in this tissue. Although DIHB is a cofactor for the oxidation of indole-3-acetic acid (IAA) by horse-radish peroxidase, it inhibits the decarboxylation of [1-¹⁴C]IAA by segments excised from cress roots. The inhibition by DIHB of ethylene production by this tissue does not, therefore, arise from a reduction of IAA levels. These findings are discussed in relation to the effects of DIHB on cress root growth.Abbreviations IAA indole-3-acetic acid - DIHB 3,5-diiodo-4-hydroxybenzoic acid - DCP 2,4-dichlorophenol - 2,4-D 2,4-dichlorophenoxyacetic acid This study forms part of a research project to be submitted by M.L.R. for PhD degree and supported by a grant from Consejo Nacional de Ciencia y Tecnología (México).     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. IV. The role of changes in endogenous free and conjugated indole‐3‐acetic acid

We have studied the role of endogenous auxin on adventitious rooting in hypocotyls of derooted sunflower (Helianthus annuus L. var. Dahlgren 131) seedlings. Endogenous free and conjugated indole-3-acetic acid (IAA) were measured in three segments of hypocotyls of equal length (apical, middle, basal) by using gas chromatography-mass spectrometry with [¹³C₆]-IAA as an internal standard. At the time original roots were excised (0 h), the free IAA level in the hypocotyls showed an acropetally decreasing gradient, but conjugated IAA level increased acropetally; i.e. free to total IAA ratio was highest in the basal portion of hypocotyls. The basal portion is the region where most of root primordia were found. Some primordia were seen in this region within 24 h after the roots were excised. The quantity of free IAA in the middle portion of the hypocotyl increased up to 15 h after excision and then decreased. In this middle region there were fewer root primordia, and they could not be seen until 72 h. In the apical portion the amount of free IAA steadily increased and no root primordia were seen by 72 h. Surgical removal of various parts of the hypocotyl tissues caused adventitious root formation in the hypocotyl regions where basipetally transported IAA could accumulate. Reduction in the basipetal flow of auxin by N-1-naphthylphthalamic acid and 2,3,5-tri-iodobenzoic acid resulted in fewer adventitious roots. The fewest root primordia were seen if the major sources of endogenous auxin were removed by decapitation of the cotyledons and apical bud. Exogenous auxins promoted rooting and were able to completely overcome the inhibitory effect of 2,3,5-tri-iodobenzoic acid. Exogenous auxins were only partially able to overcome the inhibitory effect of decapitation. We conclude that in sunflower hypocotyls endogenously produced auxin is necessary for adventitious root formation. The higher concentrations of auxin in the basal portion may be partially responsible for that portion of the hypocotyl producing the greatest number of primordia. In addition to auxins, other factors such as wound ethylene and lowered cytokinin levels caused by excision of the original root system cuttings must also be important.     

Auxin is a positive regulator for ethylene-mediated response in the growth of Arabidopsis roots

The requirement of auxin for the ethylene-mediated growth response in the root of Arabidopsis thaliana seedlings was investigated using two ethylene-resistant mutants, aux1-7 and eir1-1, whose roots have been shown to have a defect in the auxin influx and efflux carriers, respectively. A 50% inhibition of growth (I(50)) was achieved with 0.84 microl liter(-1) ethylene in wild-type roots, but 71.3 microl liter( -1) ethylene was required to induce I(50) in eir1-1 roots. In aux1-7 roots, I(50) was not obtained even at 1,000 microl liter(-1) ethylene. By contrast, in the presence of 10 nM 1-naphthaleneacetic acid (NAA), the concentrations of ethylene required to induce I(50) in eir1-1 and aux1-7 roots were greatly reduced nearly to the level required in wild-type roots. Since the action of NAA to restore the ethylene response in aux1-7 roots was not replaced by IAA, an increase in the intracellular level of auxin is likely to be the cause for the restoration of ethylene response. NAA at 10 nM did not inhibit root growth when applied solely, but it was the optimum concentration to recover the ethylene response in the mutant roots. These results suggest that auxin is a positive regulator for ethylene-induced inhibition in root elongation.     

Exogenous auxin effects on growth and phenotype of normal and hairy roots of Pueraria lobata (Willd.) Ohwi

Pueraria lobata hairy roots have faster elongationand more branches than normal roots. The responses of hairy roots and normalroots to treatment with three auxins, indole-3-acetic acid (IAA),indole-3-butyric acid (IBA), and naphthalene acetic acid (NAA) were different.In normal roots, all three auxins strongly stimulated lateral root formation atall tested concentrations. Responses to IAA and IBA in primary root growth andlateral root elongation were similar and depended on concentration; promotionat0.1 M, no effect at 1.0 M, and inhibition at2.5 M. In hairy roots, lateral root formation varied inresponseto the different auxins, i.e. depressed by NAA, unaffected by IAA, and promotedby IBA. Primary root growth was slightly inhibited by IBA and was unaffected byIAA. However, mean lateral root length was reduced in response to IAA and IBA.Only NAA exerted strong inhibition on primary and lateral root elongation inboth root types. The similar free IAA and conjugated IAA content but quitedifferent basal ethylene production and biosynthesis in hairy and normal rootssuggested different mechanisms of response to exogenous auxins in the two roottypes.     

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.     

Auxin and Ethylene-stimulated Adventitious Rooting in Relation to Tissue 9

We have previously shown that both endogenous auxin and ethylenepromote adventitious root formation in the hypocotyls of derootedsunflower (Helianthus annuus) seedlings. Experiments here showedthat promotive effects on rooting of the ethylene precursor,1-aminocyclopropane-l-carboxylic acid (ACC) and the ethylene-releasingcompound, ethephon (2-chloro-ethylphosphonic acid), dependedon the existence of cotyledons and apical bud (major sourcesof auxin) or the presence of exogenously applied indole-3-aceticacid (IAA). Ethephon, ACC, aminoethoxyvinylglycine (an inhibitorof ethylene biosynthesis), and silver thiosulphate (STS, aninhibitor of ethylene action), applied for a length of timethat significantly influenced adventitious rooting, showed noinhibitory effect on the basipetal transport of [³H]IAA. Theseregulators also had no effect on the metabolism of [³H]IAA andendogenous IAA levels measured by gas chromatography-mass spectrometry.ACC enhanced the rooting response of hypocotyls to exogenousIAA and decreased the inhibition of rooting by IAA transportinhibitor, N-1-naphthylphthalamic acid (NPA). STS reduced therooting response of hypocotyls to exogenous IAA and increasedthe inhibition of rooting by NPA. Exogenous auxins promotedethylene production in the rooting zone of the hypocotyls. Decapitationof the cuttings or application of NPA to the hypocotyl belowthe cotyledons did not alter ethylene production in the rootingzone, but greatly reduced the number of root primordia. We concludethat auxin is a primary controller of adventitious root formationin sunflower hypocotyls, while the effect of ethylene is mediatedby auxin. Key words: Auxin, ethylene, adventitious rooting, sunflower     

Elevated indole-3-acetic acid peroxidase activity is involved in the cadmium-induced hydrogen peroxide production in barley root tip

All of the analyzed heavy metals significantly inhibited root growth, but in addition to Cd exposure an elevated IAA-POD activity was detected under Co, Cu and Hg treatment, while Ni and Pb inhibited its activity. The Cd-induced IAA-POD activity increased from the root apex towards to the mature region of root tips. However similar or even more severe root growth inhibition was observed by exogenously applied IAA, IAA-POD activity was activated only at high IAA concentrations. Elevated Cd-induced H₂O₂ production was detected both in the absence or in the presence of IAA in the reaction mixture, but in the case of IAA as a possible substrate for PODs the production of H₂O₂ increased markedly just in the Cd-treated roots. Exogenously applied H₂O₂ also activated IAA-POD activity. Our results indicated that in the development of Cd toxicity syndrome the production of ROS during IAA degradation by elevated IAA-POD activity plays a crucial role, mainly under severe Cd stress.     

Regulation of root formation by auxin-ethylene interaction in pea stem cuttings

The possibility was investigated that the inhibition of rooting in pea ( Pisum sativum L. cv. Weibull's Marma) cuttings caused by low indol-3yl-acetic acid (IAA) concentrations is due to ethylene produced as a result of IAA treatment. Treatment with 10 uμ IAA reduced the number of roots to about 50% of the control and increased ethylene production in the stem bases by about 20 times the control value during the two first days of treatment. Ethylene-releasing compounds (ethephon and 1-amino-cyclopropane-1-carboxylic acid, ACC), in concentrations giving a similar ethylene release, inhibited rooting to the same extent or more strongly than IAA. These results indicate that IAA-induced ethylene is at least responsible for the negative component in IAA action on root formation in pea cuttings. A higher IAA concentration (100 μ) and indol-3yl-butyric acid efficiently counteracted the negative effect of ethylene on root formation.