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.     

Root Formation in Ethylene-Insensitive Plants

Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia x hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more below-ground root mass but fewer above-ground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated tap-roots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli.     

Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.     

A Mutation Altering Auxin Homeostasis and Plant Morphology in Arabidopsis

Many aspects of plant development are associated with changing concentrations of the phytohormone auxin. Several stages of root formation exhibit extreme sensitivities to exogenous auxin and are correlated with shifts in endogenous auxin concentration. In an effort to elucidate mechanisms regulating development of adventitious roots, an ethyl methanesulfonate-mutagenized M2 population of Arabidopsis was screened for mutants altered in this process. A recessive nuclear mutant, rooty (rty), displayed extreme proliferation of roots, inhibition of shoot growth, and other alterations suggesting elevated responses to auxin or ethylene. Wild-type Arabidopsis seedlings grown on auxin-containing media phenocopied rty, whereas rty seedlings were partially rescued on cytokinin-containing media. Analysis by gas chromatography-selected ion monitoring-mass spectrometry showed endogenous indole-3-acetic acid concentrations to be two to 17 times higher in rty than in the wild type. Dose-response assays with exogenous indole-3-acetic acid indicated equal sensitivities to auxin in tissues of the wild type and rty. Combining rty with mutations conferring resistance to auxin (axr1-3) or ethylene (etr1-1) suggested that root proliferation and restricted shoot growth are auxin effects, whereas other phenotypic alterations are due to ethylene. Four mutant alleles from independently mutagenized populations were identified, and the locus was mapped using morphological and restriction fragment length polymorphism markers to 3.9 centimorgans distal to marker m605 on chromosome 2. The wild-type RTY gene product may serve a critical role in regulating auxin concentrations and thereby facilitating normal plant growth and development.     

The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling

The rate and plane of cell division and anisotropic cell growth are critical for plant development and are regulated by diverse mechanisms involving several hormone signaling pathways. Little is known about peptide signaling in plant growth; however, Arabidopsis thaliana POLARIS (PLS), encoding a 36-amino acid peptide, is required for correct root growth and vascular development. Mutational analysis implicates a role for the peptide in hormone responses, but the basis of PLS action is obscure. Using the Arabidopsis root as a model to study PLS action in plant development, we discovered a link between PLS, ethylene signaling, auxin homeostasis, and microtubule cytoskeleton dynamics. Mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered microtubule sensitivity to inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. PLS expression is repressed by ethylene and induced by auxin. Our results suggest a mechanism whereby PLS negatively regulates ethylene responses to modulate cell division and expansion via downstream effects on microtubule cytoskeleton dynamics and auxin signaling, thereby influencing root growth and lateral root development. This mechanism involves a regulatory loop of auxin-ethylene interactions.     

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.     

Ethylene Evolution from Maize (Zea mays L.) Seedling Roots and Shoots in Response to Mechanical Impedance

The effect of mechanical impedance on ethylene evolution and growth of preemergent maize (Zea mays L.) seedlings was investigated by pressurizing the growth medium in triaxial cells in a controlled environment. Pressure increased the bulk density of the medium and thus the resistance to growth. The elongation of maize primary roots and preemergent shoots was severely hindered by applied pressures as low as 10 kilopascals. Following a steep decline in elongation at low pressures, both shoots and roots responded to additional pressure in a linear manner, but shoots were more severely affected than roots at higher pressures. Radial expansion was promoted in both organs by mechanical impedance. Primary roots typically became thinner during the experimental period when grown unimpeded. In contrast, pressures as low as 25 kilopascals caused a 25% increase in root tip diameter. Shoots showed a slight enhancement of radial expansion; however, in contrast to roots, the shoots increased in diameter even when growing unimpeded. Such morphological changes were not evident until at least 3 hours after initiation of treatment. All levels of applied pressure promoted ethylene evolution as early as 1 hour after application of pressure. After 1 hour, ethylene evolution rates had increased 10, 32, 70, and 255% at 25, 50, 75, and 100 kilopascals respectively, and continued to increase linearly for at least 10 hours. When intact corn seedlings were subjected to a series of hourly cycles of pressure, followed by relaxation, ethylene production rates increased or decreased rapidly, illustrating tight coupling between mechanical impedance and tissue response. Seedlings exposed to 1 microliter of ethylene per liter showed symptoms similar to those shown by plants grown under mechanical impedance. Root diameter increased 5 times as much as the shoot diameter. Pretreatment with 10 micromolar aminoethoxyvinyl glycine plus 1 micromolar silver thiosulfate maintained ethylene production rates of impeded seedlings at basal levels and restored shoot and root extension to 84 and 90% of unimpeded values, respectively. Our results support the hypothesis that ethylene plays a pivotal role in the regulation of plant tissue response to mechanical impedance.     

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.     

Ethylene regulates arabidopsis development via the modulation of DELLA protein growth repressor function

Phytohormones regulate plant development via a poorly understood signal response network. Here, we show that the phytohormone ethylene regulates plant development at least in part via alteration of the properties of DELLA protein nuclear growth repressors, a family of proteins first identified as gibberellin (GA) signaling components. This conclusion is based on the following experimental observations. First, ethylene inhibited Arabidopsis root growth in a DELLA-dependent manner. Second, ethylene delayed the GA-induced disappearance of the DELLA protein repressor of ga1-3 from root cell nuclei via a constitutive triple response-dependent signaling pathway. Third, the ethylene-promoted "apical hook" structure of etiolated seedling hypocotyls was dependent on the relief of DELLA-mediated growth restraint. Ethylene, auxin, and GA responses now can be attributed to effects on DELLA function, suggesting that DELLA plays a key integrative role in the phytohormone signal response network.     

Ethephon and auxin induce mycorrhiza-like changes in the morphology of root organ cultures of Mugo pine

The effects of ethylene and auxin on the morphology and anatomy of root organ cultures of Pinus mugo Turra var. mugo were investigated to test the hypothesis that changes in root morphology associated with formation of ectomycorrhizae may be related to ethylene produced by ectomycorrhizal fungi or by host plant roots in response to fungus-produced auxin. Morphological changes characteristic of mycorrhizal infection include dichotomous branching of lateral roots, inhibition of root hair formation and enlargement of cortical cells. Lateral roots on non-mycorrhizal root organ cultures, grown in a defined medium, underwent dichtotomous branching while root hair formation was inhibited in response to the ethylene released by 50 and 100 μ M ethephon (2-chloroethylphosphonic acid), but no effect on cortical cell dimensions was observed. The auxin, naphthaleneacetic acid (1 and 10 μ M ) also stimulated dichotomous branching and inhibited root hair formation, but to a lesser extent and with a greater lag time than ethephon. Auxin-stimulated ethylene production by root organ cultures was demonstrated. This appeared to be responsible, at least in part, for the auxin-induced dichotomous branching since the ethylene action inhibitor, silver thiosulfate (0.1 m M ) inhibited the response to auxin by 35%.     

The influence of mechanical impedance on the growth of maize roots

Summary Maize roots were grown between 1 mm glass beads on which a pressure of 40 kPa was applied. The roots were supplied with a constant flow of aerated nutrient solution. Compared with roots grown in a nutrient solution, the impeded crown roots showed a reduction in length of about 75%, whereas the diameter was about 50% increased.These changes in root morphology have been attributed to changes in cell wall structure of the cortex cells, which also occur as a result of the influence of ethylene.It is suggested that ethylene acts as an intermediate factor in the effect of mechanical impedance on root growth.     

植物激素ABA调控植物根系生长的研究进展

脱落酸（abscisic acid,ABA）是一种重要的植物激素,在调控种子发育、种子休眠与萌发、抑制生长、促进落花落果、参与植物应对外界环境胁迫等过程中发挥着重要的生理功能。ABA还能与其他植物激素（如生长素、乙烯等）互作进而精细调控植物根系的生长。本文以模式植物拟南芥（Arabidopsis thaliana（L.）Heynh）为主要对象,对近年来国内外在ABA调控植物根系生长方面的研究成果、ABA与其他植物激素（如GA等）互作调控根系生长及调控非生物逆境下根系发育的机理等进行综述,并对其未来的研究方向进行了展望。     

HPS4/SABRE regulates plant responses to phosphate starvation through antagonistic interaction with ethylene signalling

The phytohormone ethylene plays important roles in regulating plant responses to phosphate (Pi) starvation. To date, however, no molecular components have been identified that interact with ethylene signalling in regulating such responses. In this work, an Arabidopsis mutant, hps4, was characterized that exhibits enhanced responses to Pi starvation, including increased inhibition of primary root growth, enhanced expression of Pi starvation-induced genes, and overproduction of root-associated acid phosphatases. Molecular cloning indicated that hps4 is a new allele of SABRE, which was previously identified as an important regulator of cell expansion in Arabidopsis. HPS4/SABRE antagonistically interacts with ethylene signalling to regulate plant responses to Pi starvation. Furthermore, it is shown that Pi-starved hps4 mutants accumulate more auxin in their root tips than the wild type, which may explain the increased inhibition of their primary root growth when grown under Pi deficiency.     

Strigolactones suppress adventitious rooting in Arabidopsis and pea

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation.     

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.     

RCN1-regulated phosphatase activity and EIN2 modulate hypocotyl gravitropism by a mechanism that does not require ethylene signaling

The roots curl in naphthylphthalamic acid1 (rcn1) mutant of Arabidopsis (Arabidopsis thaliana) has altered auxin transport, gravitropism, and ethylene response, providing an opportunity to analyze the interplay between ethylene and auxin in control of seedling growth. Roots of rcn1 seedlings were previously shown to have altered auxin transport, growth, and gravitropism, while rcn1 hypocotyl elongation exhibited enhanced ethylene response. We have characterized auxin transport and gravitropism phenotypes of rcn1 hypocotyls and have explored the roles of auxin and ethylene in controlling these phenotypes. As in roots, auxin transport is increased in etiolated rcn1 hypocotyls. Hypocotyl gravity response is accelerated, although overall elongation is reduced, in etiolated rcn1 hypocotyls. Etiolated, but not light grown, rcn1 seedlings also overproduce ethylene, and mutations conferring ethylene insensitivity restore normal hypocotyl elongation to rcn1. Auxin transport is unaffected by treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid in etiolated hypocotyls of wild-type and rcn1 seedlings. Surprisingly, the ethylene insensitive2-1 (ein2-1) and ein2-5 mutations dramatically reduce gravitropic bending in hypocotyls. However, the ethylene resistant1-3 (etr1-3) mutation does not significantly affect hypocotyl gravity response. Furthermore, neither the etr1 nor the ein2 mutation abrogates the accelerated gravitropism observed in rcn1 hypocotyls, indicating that both wild-type gravity response and enhanced gravity response in rcn1 do not require an intact ethylene-signaling pathway. We therefore conclude that the RCN1 protein affects overall hypocotyl elongation via negative regulation of ethylene synthesis in etiolated seedlings, and that RCN1 and EIN2 modulate hypocotyl gravitropism and ethylene responses through independent pathways.     

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.     

Bacillus megaterium rhizobacteria promote growth and alter root-system architecture through an auxin- and ethylene-independent signaling mechanism in Arabidopsis thaliana

Soil microorganisms are critical players in plant-soil interactions at the rhizosphere. We have identified a Bacillus megaterium strain that promoted growth and development of bean (Phaseolus vulgaris) and Arabidopsis thaliana plants. We used Arabidopsis thaliana as a model to characterize the effects of inoculation with B. megaterium on plant-growth promotion and postembryonic root development. B. megaterium inoculation caused an inhibition in primary-root growth followed by an increase in lateral-root number, lateral-root growth, and root-hair length. Detailed cellular analyses revealed that primary root-growth inhibition was caused both by a reduction in cell elongation and by reduction of cell proliferation in the root meristem. To study the contribution of auxin and ethylene signaling pathways in the alterations in root-system architecture elicited by B. megaterium, a suite of plant hormone mutants of Arabidopsis, including aux1-7, axr4-1, eir1, etr1, ein2, and rhd6, defective in either auxin or ethylene signaling, were evaluated for their responses to inoculation with this bacteria. When inoculated, all mutant lines tested showed increased biomass production. Moreover, aux1-7 and eir1, which sustain limited root-hair and lateral-root formation when grown in uninoculated medium, were found to increase the number of lateral roots and to develop long root hairs when inoculated with B. megaterium. The ethylene-signaling mutants etr1 and ein2 showed an induction in lateral-root formation and root-hair growth in response to bacterial inoculation. Taken together, our results suggest that plant-growth promotion and root-architectural alterations by B. megaterium may involve auxin- and-ethylene independent mechanisms.