Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana

Lateral root branching is a genetically defined and environmentally regulated process. Auxin is required for lateral root formation, and mutants that are altered in auxin synthesis, transport or signaling often have lateral root defects. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in the regulation of Arabidopsis lateral root formation are not well characterized. This study utilized Arabidopsis mutants altered in ethylene signaling and synthesis to explore the role of ethylene in lateral root formation. We find that enhanced ethylene synthesis or signaling, through the eto1-1 and ctr1-1 mutations, or through the application of 1-aminocyclopropane-1-carboxylic acid (ACC), negatively impacts lateral root formation, and is reversible by treatment with the ethylene antagonist, silver nitrate. In contrast, mutations that block ethylene responses, etr1-3 and ein2-5 , enhance root formation and render it insensitive to the effect of ACC, even though these mutants have reduced root elongation at high ACC doses. ACC treatments or the eto1-1 mutation significantly enhance radiolabeled indole-3-acetic acid (IAA) transport in both the acropetal and the basipetal directions. ein2-5 and etr1-3 have less acropetal IAA transport, and transport is no longer regulated by ACC. DR5-GUS reporter expression is also altered by ACC treatment, which is consistent with transport differences. The aux1-7 mutant, which has a defect in an IAA influx protein, is insensitive to the ethylene inhibition of root formation. aux1-7 also has ACC-insensitive acropetal and basipetal IAA transport, as well as altered DR5-GUS expression, which is consistent with ethylene altering AUX1-mediated IAA uptake, and thereby blocking lateral root formation.     

Ethylene–auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana

Plant root systems display considerable plasticity in response to endogenous and environmental signals. Auxin stimulates pericycle cells within elongating primary roots to enter de novo organogenesis, leading to the establishment of new lateral root meristems. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in root branching are not well characterized. We find that enhanced ethylene synthesis, resulting from the application of low concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), promotes the initiation of lateral root primordia. Treatment with higher doses of ACC strongly inhibits the ability of pericycle cells to initiate new lateral root primordia, but promotes the emergence of existing lateral root primordia: behaviour that is also seen in the eto1 mutation. These effects are correlated with decreased pericycle cell length and increased lateral root primordia cell width. When auxin is applied simultaneously with ACC, ACC is unable to prevent the auxin stimulation of lateral root formation in the root tissues formed prior to ACC exposure. However, in root tissues formed after transfer to ACC, in which elongation is reduced, auxin does not rescue the ethylene inhibition of primordia initiation, but instead increases it by several fold. Mutations that block auxin responses, slr1 and arf7 arf19, render initiation of lateral root primordia insensitive to the promoting effect of low ethylene levels, and mutations that inhibit ethylene-stimulated auxin biosynthesis, wei2 and wei7 , reduce the inhibitory effect of higher ethylene levels, consistent with ethylene regulating root branching through interactions with auxin.     

Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation

Ethylene represents an important regulatory signal for root development. Genetic studies in Arabidopsis thaliana have demonstrated that ethylene inhibition of root growth involves another hormone signal, auxin. This study investigated why auxin was required by ethylene to regulate root growth. We initially observed that ethylene positively controls auxin biosynthesis in the root apex. We subsequently demonstrated that ethylene-regulated root growth is dependent on (1) the transport of auxin from the root apex via the lateral root cap and (2) auxin responses occurring in multiple elongation zone tissues. Detailed growth studies revealed that the ability of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid to inhibit root cell elongation was significantly enhanced in the presence of auxin. We conclude that by upregulating auxin biosynthesis, ethylene facilitates its ability to inhibit root cell expansion.     

Ethylene mediates dichromate‐induced inhibition of primary root growth by altering AUX1 expression and auxin accumulation in Arabidopsis thaliana

The hexavalent form of chromium [Cr(VI)] causes a major reduction in yield and quality of crops worldwide. The root is the first plant organ that interacts with Cr(VI) toxicity, which inhibits primary root elongation, but the underlying mechanisms of this inhibition remain elusive. In this study, we investigate the possibility that Cr(VI) reduces primary root growth of Arabidopsis by modulating the cell cycle‐related genes and that ethylene signalling contributes to this process. We show that Cr(VI)‐mediated inhibition of primary root elongation was alleviated by the ethylene perception and biosynthesis antagonists silver and cobalt, respectively. Furthermore, the ethylene signalling defective mutants (ein2‐1 and etr1‐3) were insensitive, whereas the overproducer mutant (eto1‐1) was hypersensitive to Cr(VI). We also report that high levels of Cr(VI) significantly induce the distribution and accumulation of auxin in the primary root tips, but this increase was significantly suppressed in seedlings exposed to silver or cobalt. In addition, genetic and physiological investigations show that AUXIN‐RESISTANT1 (AUX1) participates in Cr(VI)‐induced inhibition of primary root growth. Taken together, our results indicate that ethylene mediates Cr(VI)‐induced inhibition of primary root elongation by increasing auxin accumulation and polar transport by stimulating the expression of AUX1.     

Apoplastic alkalinization is instrumental for the inhibition of cell elongation in the Arabidopsis root by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid

In Arabidopsis (Arabidopsis thaliana; Columbia-0) roots, the so-called zone of cell elongation comprises two clearly different domains: the transition zone, a postmeristematic region (approximately 200-450 μm proximal of the root tip) with a low rate of elongation, and a fast elongation zone, the adjacent proximal region (450 μm away from the root tip up to the first root hair) with a high rate of elongation. In this study, the surface pH was measured in both zones using the microelectrode ion flux estimation technique. The surface pH is highest in the apical part of the transition zone and is lowest at the basal part of the fast elongation zone. Fast cell elongation is inhibited within minutes by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid; concomitantly, apoplastic alkalinization occurs in the affected root zone. Fusicoccin, an activator of the plasma membrane H(+)-ATPase, can partially rescue this inhibition of cell elongation, whereas the inhibitor N,N'-dicyclohexylcarbodiimide does not further reduce the maximal cell length. Microelectrode ion flux estimation experiments with auxin mutants lead to the final conclusion that control of the activity state of plasma membrane H(+)-ATPases is one of the mechanisms by which ethylene, via auxin, affects the final cell length in the root.     

Auxin and ethylene promote root hair elongation in Arabidopsis

Genetic and physiological studies implicate the phytohormones auxin and ethylene in root hair development. To learn more about the role of these compounds, we have examined the root hair phenotype of a number of auxin- and ethylene-related mutants. In a previous study, Masucci and Schiefelbein (1996) showed that neither the auxin response mutations aux1 and axr1 nor the ethylene response mutations etr1 and ein2 have a significant effect on root hair initiation. In this study, we found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length. Treatment of wild-type, axr1 and etr1 seedlings with the synthetic auxin, 2,4-D, or the ethylene precursor ACC, led to the development of longer root hairs than untreated seedlings. Furthermore, axr1 seedlings grown in the presence of ACC produce ectopic root hairs and an unusual pattern of long root hairs followed by regions that completely lack root hairs. These studies indicate that both auxin and ethylene are required for normal root hair elongation.     

Interactions between jasmonates and ethylene in the regulation of root hair development in Arabidopsis

Root hair formation is an important model with which to study cell patterning and differentiation in higher plants. Ethylene and auxin are critical regulators of root hair development. The role of jasmonates (JAs) was examined in Arabidopsis root hair development as well as their interactions with ethylene in this process. The results have shown that both methyl jasmonate (MeJA) and jasmonic acid (JA) have a pronounced effect on promoting root hair formation. However, the effect of MeJA and JA on root hair formation was blocked by ethylene inhibitors Ag+ or aminoethoxyvinylglycine (AVG). The stimulatory effects of MeJA and JA were also diminished in ethylene-insensitive mutants etr1-1 and etr1-3. Furthermore, the JA biosynthesis inhibitors ibuprofen and salicylhydroxamic acid (SHAM) suppressed 1-aminocyclopropane-1-carboxylic acid (ACC)-induced root hair formation, and decreased the root hairs in seedlings of the ethylene over-producing mutant eto1-1. These results suggested that JAs promote root hair formation, through an interaction with ethylene.     

Auxin and ethylene response interactions during Arabidopsis root hair development dissected by auxin influx modulators

The plant hormones auxin and ethylene have been shown to play important roles during root hair development. However, cross talk between auxin and ethylene makes it difficult to understand the independent role of either hormone. To dissect their respective roles, we examined the effects of two compounds, chromosaponin I (CSI) and 1-naphthoxyacetic acid (1-NOA), on the root hair developmental process in wild-type Arabidopsis, ethylene-insensitive mutant ein2-1, and auxin influx mutants aux1-7, aux1-22, and double mutant aux1-7 ein2. Beta-glucuronidase (GUS) expression analysis in the BA-GUS transgenic line, consisting of auxin-responsive domains of PS-IAA4/5 promoter and GUS reporter, revealed that 1-NOA and CSI act as auxin uptake inhibitors in Arabidopsis roots. The frequency of root hairs in ein2-1 roots was greatly reduced in the presence of CSI or 1-NOA, suggesting that endogenous auxin plays a critical role for the root hair initiation in the absence of an ethylene response. All of these mutants showed a reduction in root hair length, however, the root hair length could be restored with a variable concentration of 1-naphthaleneacetic acid (NAA). NAA (10 nM) restored the root hair length of aux1 mutants to wild-type level, whereas 100 nM NAA was needed for ein2-1 and aux1-7 ein2 mutants. Our results suggest that insensitivity in ethylene response affects the auxin-driven root hair elongation. CSI exhibited a similar effect to 1-NOA, reducing root hair growth and the number of root hair-bearing cells in wild-type and ein2-1 roots, while stimulating these traits in aux1-7and aux1-7ein2 roots, confirming that CSI is a unique modulator of AUX1.     

Loss-of-function mutations of ROOT HAIR DEFECTIVE3 suppress root waving, skewing, and epidermal cell file rotation in Arabidopsis

Wild-type Arabidopsis (Arabidopsis thaliana L. Heynh.) roots growing on a tilted surface of impenetrable hard-agar media adopt a wave-like pattern and tend to skew to the right of the gravity vector (when viewed from the back of the plate through the medium). Reversible root-tip rotation often accompanies the clockwise and counterclockwise curves that form each wave. These rotations are manifested by epidermal cell file rotation (CFR) along the root. Loss-of-function alleles of ROOT HAIR DEFECTIVE3 (RHD3), a gene previously implicated in the control of vesicle trafficking between the endoplasmic reticulum and the Golgi compartments, resulted in an almost complete suppression of epidermal CFR, root skewing, and waving on hard-agar surfaces. Several other root hair defective mutants (rhd2-1, rhd4-1, and rhd6-1) did not exhibit dramatic alterations in these root growth behaviors, suggesting that a generalized defect in root hair formation is not responsible for the surface-dependent phenotypes of rhd3. However, similar alterations in root growth behavior were observed in a variety of mutants characterized by defects in cell expansion (cob-1, cob-2, eto1-1, eto2-1, erh2-1, and erh3-1). The erh2-1 and rhd3-1 mutants differed from other anisotropic cell expansion mutants, though, by an inability to respond to low doses of the microtubule-binding drug propyzamide, which normally causes enhanced left-handed CFR and right skewing. We hypothesize that RHD3 may control epidermal CFR, root skewing, and waving on hard-agar surfaces by regulating the traffic of wall- or plasma membrane-associated determinants of anisotropic cell expansion.     

Susceptibility to the sugar beet cyst nematode is modulated by ethylene signal transduction in Arabidopsis thaliana

Previously, we identified Arabidopsis thaliana mutant rhd1-4 as hypersusceptible to the sugar beet cyst nematode Heterodera schachtii. We assessed rhd1-4 as well as two other rhd1 alleles and found that each exhibited, in addition to H. schachtii hypersusceptibility, decreased root length, increased root hair length and density, and deformation of the root epidermal cells compared with wild-type A. thaliana ecotype Columbia (Col-0). Treatment of rhd1-4 and Col-0 with the ethylene inhibitors 2-aminoethoxyvinylglycine and silver nitrate and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid suggests that the rhd1-4 hypersusceptibility and root morphology phenotypes are the result of an increased ethylene response. Assessment of known ethylene mutants further support the finding that ethylene plays a role in mediating A. thaliana susceptibility to H. schachtii because mutants that overproduce ethylene (eto1-1, eto2, and eto3) are hypersusceptible to H. schachtii and mutants that are ethylene-insensitive (etr1-1, ein2-1, ein3-1, eir1-1, and axr2) are less susceptible to H. schachtii. Because the ethylene mutants tested show altered susceptibility and altered root hair density and length, a discrimination between the effects of altered ethylene signal transduction and root hair density on susceptibility was accomplished by analyzing the ttg and gl2 mutants, which produce ectopic root hairs that result in greatly increased root hair densities while maintaining normal ethylene signal transduction. The observed normal susceptibilities to H. schachtii of ttg and g12 indicate that increased root hair density, per se, does not cause hypersusceptibility. Furthermore, the results of nematode attraction assays suggest that the hypersusceptibility of rhd1-4 and the ethylene-overproducing mutant eto3 may be the result of increased attraction of H. schachtii-infective juveniles to root exudates of these plants. Our findings indicate that rhd1 is altered in its ethylene response and that ethylene signal transduction positively influences plant susceptibility to cyst nematodes.     

Exploiting the triple response of Arabidopsis to identify ethylene-related mutants.

Alterations in the response of dark-grown seedlings to ethylene (the "triple response") were used to isolate a collection of ethylene-related mutants in Arabidopsis thaliana. Mutants displaying a constitutive response (eto1) were found to produce at least 40 times more ethylene than the wild type. The morphological defects in etiolated eto1-1 seedlings reverted to wild type under conditions in which ethylene biosynthesis or ethylene action were inhibited. Mutants that failed to display the apical hook in the absence of ethylene (his1) exhibited reduced ethylene production. In the presence of exogenous ethylene, hypocotyl and root of etiolated his1-1 seedlings were inhibited in elongation but no apical hook was observed. Mutants that were insensitive to ethylene (ein1 and ein2) produced increased amounts of ethylene, displayed hormone insensitivity in both hypocotyl and root responses, and showed an apical hook. Each of the "triple response" mutants has an effect on the shape of the seedling and on the production of the hormone. These mutants should prove to be useful tools for dissecting the mode of ethylene action in plants.     

Auxin-induced inhibition of lateral root initiation contributes to root system shaping in Arabidopsis thaliana

The hormone auxin is known to inhibit root elongation and to promote initiation of lateral roots. Here we report complex effects of auxin on lateral root initiation in roots showing reduced cell elongation after auxin treatment. In Arabidopsis thaliana, the promotion of lateral root initiation by indole-3-acetic acid (IAA) was reduced as the IAA concentration was increased in the nanomolar range, and IAA became inhibitory at 25 nM. Detection of this unexpected inhibitory effect required evaluation of root portions that had newly formed during treatment, separately from root portions that existed prior to treatment. Lateral root initiation was also reduced in the iaaM-OX Arabidopsis line, which has an endogenously increased IAA level. The ethylene signaling mutants ein2-5 and etr1-3, the auxin transport mutants aux1-7 and eir1/pin2, and the auxin perception/response mutant tir1-1 were resistant to the inhibitory effect of IAA on lateral root initiation, consistent with a requirement for intact ethylene signaling, auxin transport and auxin perception/response for this effect. The pericycle cell length was less dramatically reduced than cortical cell length, suggesting that a reduction in the pericycle cell number relative to the cortex could occur with the increase of the IAA level. Expression of the DR5:GUS auxin reporter was also less effectively induced, and the AXR3 auxin repressor protein was less effectively eliminated in such root portions, suggesting that decreased auxin responsiveness may accompany the inhibition. Our study highlights a connection between auxin-regulated inhibition of parent root elongation and a decrease in lateral root initiation. This may be required to regulate the spacing of lateral roots and optimize root architecture to environmental demands.     

Cell wall integrity controls root elongation via a general 1-aminocyclopropane-1-carboxylic acid-dependent, ethylene-independent pathway

Cell expansion in plants requires cell wall biosynthesis and rearrangement. During periods of rapid elongation, such as during the growth of etiolated hypocotyls and primary root tips, cells respond dramatically to perturbation of either of these processes. There is growing evidence that this response is initiated by a cell wall integrity-sensing mechanism and dedicated signaling pathway rather than being an inevitable consequence of lost structural integrity. However, the existence of such a pathway in root tissue and its function in a broader developmental context have remained largely unknown. Here, we show that various types of cell wall stress rapidly reduce primary root elongation in Arabidopsis (Arabidopsis thaliana). This response depended on the biosynthesis of 1-aminocyclopropane-1-carboxylic acid (ACC). In agreement with the established ethylene signaling pathway in roots, auxin signaling and superoxide production are required downstream of ACC to reduce elongation. However, this cell wall stress response unexpectedly does not depend on the perception of ethylene. We show that the short-term effect of ACC on roots is partially independent of its conversion to ethylene or ethylene signaling and that this ACC-dependent pathway is also responsible for the rapid reduction of root elongation in response to pathogen-associated molecular patterns. This acute response to internal and external stress thus represents a novel, noncanonical signaling function of ACC.     

STUNTED PLANT 1, A Gene Required for Expansion in Rapidly Elongating but Not in Dividing Cells and Mediating Root Growth Responses to Applied Cytokinin

To understand the control of spatial patterns of expansion, we have studied root growth in wild type and in the stunted plant 1 mutant, stp1, of Arabidopsis thaliana. We measured profiles of cell length and calculated the distribution of elongation rate. Slow growth of stp1 results both from a failure of dividing cell number to increase and from low elongation rates in the zone of rapid expansion. However, elongation of dividing cells was not greatly affected, and stp1 and wild-type callus grew at identical rates. Thus, rapid cellular expansion differs in mechanism from expansion in dividing cells and is facilitated by the STP1 gene. Additionally, there was no difference between stp1 and wild-type roots for elongation in response to abscisic acid, auxin, ethylene, or gibberellic acid or for radial expansion in response to ethylene; however, stp1 responded to cytokinin much less than wild type. In contrast, both genotypes responded comparably to hormones when explants were cultured; in particular, there was no difference between genotypes in shoot regeneration in response to cytokinin. Thus, effects on root expansion mediated by cytokinin, but not effects mediated by other hormones or effects on other cytokinin-mediated responses, require the STP1 locus.     

Uncoupling Auxin and Ethylene Effects in Transgenic Tobacco and Arabidopsis Plants

Overproduction of auxin in transgenic plants also results in the overproduction of ethylene. Plants overproducing both auxin and ethylene display inhibition of stem elongation and growth, increased apical dominance, and leaf epinasty. To determine the relative roles of auxin and ethylene in these processes, transgenic tobacco and Arabidopsis plants expressing the auxin-overproducing tryptophan monooxygenase transgene were crossed to plants expressing an ethylene synthesis-inhibiting 1-aminocyclopropane-1-carboxylate deaminase transgene. Tobacco and Arabidopsis plants with elevated auxin and normal levels of ethylene were obtained by this strategy. Transgenic auxin-overproducing Arabidopsis plants were also crossed with the ethylene-insensitive ein1 and ein2 mutants. Analysis of these plants indicates that apical dominance and leaf epinasty are primarily controlled by auxin rather than ethylene. However, ethylene is partially responsible for the inhibition of stem elongation observed in auxin-overproducing tobacco. Finally, these data show that auxin overproduction can be effectively uncoupled from ethylene overproduction in transgenic plants to enable direct manipulation of plant morphology for agronomic and horticultural purposes.     

Auxin and ethylene are involved in the responses of root system architecture to low boron supply in Arabidopsis seedlings

Changes in root architecture are one of the adaptive strategies used by plants to compensate for nutrient deficiencies in soils. In this work, the temporal responses of Arabidopsis (Arabidopsis thaliana) root system architecture to low boron (B) supply were investigated. Arabidopsis Col-0 seedlings were grown in 10 μM B for 5 days and then transferred to a low B medium (0.4 μM) or control medium (10 μM) for a 4-day period. Low B supply caused an inhibition of primary root (PR) growth without altering either the growth or number of lateral roots (LRs). In addition, low B supply induced root hair formation and elongation in positions close to the PR meristem not observed under control conditions. The possible role of auxin and ethylene in the alteration of root system architecture elicited by low B supply was also studied by using two Arabidopsis reporter lines (DR5:GUS and EBS:GUS) and two Arabidopsis mutants with impaired auxin and ethylene signaling (aux1-22 and ein2-1). Low B supply increased auxin reporter DR5:GUS activity in PR tip, suggesting that low B alters the pattern of auxin distribution in PR tip. Moreover, PR elongation in aux1-22 mutant was less sensitive to low B treatment than in wild-type plants, which suggests that auxin resistant 1 (AUX1) participates in the inhibition of PR elongation under low B supply. From all these results, a hypothetical model to explain the effect of low B treatment on PR growth is proposed. We also show that ethylene, via ethylene-insensitive 2 (EIN2) protein, is involved in the induction of root hair formation and elongation under low B treatment.     

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.