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.     

The rhd6 Mutation of Arabidopsis thaliana Alters Root-Hair Initiation through an Auxin- and Ethylene-Associated Process

Root-hair initiation in Arabidopsis thaliana provides a model for studying cell polarity and its role in plant morphogenesis. Root hairs normally emerge at the apical end of root epidermal cells, implying that these cells are polarized. We have identified a mutant, rhd6, that displays three defects: (a) a reduction in the number of root hairs, (b) an overall basal shift in the site of root-hair emergence, and (c) a relatively high frequency of epidermal cells with multiple root hairs. These defects implicate the RHD6 gene in root-hair initiation and indicate that RHD6 is normally associated with the establishment of, or response to, root epidermal cell polarity. Similar alterations in the site of root-hair emergence, although less extreme, were also discovered in roots of the auxin-, ethylene-, abscisic acid-resistant mutant axr2 and the ethylene-resistant mutant etr1. All three rhd6 mutant phenotypes were rescued when either auxin (indoleacetic acid) or an ethylene precursor (1-aminocyclopropane-1-carboxylic acid) was included in the growth medium. The rhd6 root phenotypes could be phenocopied by treating wild-type seedlings with an inhibitor of the ethylene pathway (aminoethoxyvinylglycine). These results indicate that RHD6 is normally involved in directing the selection or assembly of the root-hair initiation site through a process involving auxin and ethylene.     

Genetic Analysis of Ethylene Signal Transduction in Arabidopsis Thaliana: Five Novel Mutant Loci Integrated into a Stress Response Pathway

The response of Arabidopsis thaliana etiolated seedlings to the plant hormone ethylene is a conspicuous phenotype known as the triple response. We have identified genes that are required for ethylene perception and response by isolating mutants that fail to display a triple response in the presence of exogenous ethylene. Five new complementation groups have been identified. Four of these loci, designated ein4, ein5, ein6 and ein7, are insensitive to ethylene. The fifth complementation group, eir1, is defined by a novel class of mutants that have agravitropic and ethylene-insensitive roots. Double-mutant phenotypes have allowed the positioning of these loci in a genetic pathway for ethylene signal transduction. The ethylene-response pathway is defined by the following loci: ETR1, EIN4, CTR1, EIN2, EIN3, EIN5, EIN6, EIN7, EIR1, AUX1 and HLS1. ctr1-1 is epistatic to etr1-3 and ein4, indicating that CTR1 acts after both ETR1 and EIN4 in the ethylene-response pathway. Mutations at the EIN2, EIN3, EIN5, EIN6 and EIN7 loci are all epistatic to the ctr1 seedling phenotype. The EIR1 and AUX1 loci define a root-specific ethylene response that does not require EIN3 or EIN5 gene activity. HLS1 appears to be required for differential cell growth in the apical hook. The EIR1, AUX1 and HLS1 genes may function in the interactions between ethylene and other plant hormones that occur late in the signaling pathway of this simple gas.     

Arabidopsis ROOT HAIR DEFECTIVE3 is involved in nitrogen starvation-induced anthocyanin accumulation

Anthocyanin accumulation is a common phenom-enon seen in plants under environmental stress. In this study, we identified a new allele of ROOT HAIR DEFECTIVE3 (RHD3) showing an anthocyanin overaccumulat...     

Growth regulators and the control of nucleotide sugar flux

A small number of plant growth regulators are involved in the control of cell expansion. Despite knowledge of some of their signal transduction cascades, surprisingly little is known of how basic cell expansion-related processes, such as cell wall biosynthesis, are affected during growth. The Arabidopsis (Arabidopsis thaliana) mutant root hair defective1 (rhd1) lacks a functional UDP-glucose 4-epimerase gene, UGE4, which is involved in channeling UDP-D-galactose (UDP-D-Gal) into cell wall polymers. Here, we use rhd1 as a genetic model to analyze the physiological and genetic controls of nucleotide sugar flux. We find that ethylene specifically suppresses all visible aspects of the rhd1 phenotype. The ethylene-triggered suppression of rhd1 is negatively regulated by CONSTITUTIVE TRIPLE RESPONSE1 and requires the function of the wild-type genes ETHYLENE INSENSITIVE2 (EIN2), EIN4, AUXIN-RESISTENT1, and ETHYLENE-INSENSITIVE ROOT1 but does not depend on the activity of wild-type ETHYLENE RECEPTOR1 or EIN3 genes, highlighting the nonlinearity of ethylene signal transduction. Ethylene does not induce the expression of alternative UGE genes but, instead, suppresses the expression of two isoforms, UGE1 and UGE3, in a tissue-specific manner. Ethylene restores the biosynthesis of galactose-containing xyloglucan and arabinosylated galactan cell wall polymers in rhd1 back to wild-type levels. However, the dependence on UGE4 of pectic (1-->4)-beta-D-galactan and glucuronosyl-modified AGP biosynthesis is exacerbated. Our data suggest that ethylene and auxin together participate in the flux control of UDP-D-Gal into cell wall polymers and that the genetic control of this process is qualitatively distinct from previously described responses to ethylene.     

Root hair defective4 encodes a phosphatidylinositol-4-phosphate phosphatase required for proper root hair development in Arabidopsis thaliana

Polarized expansion of root hair cells in Arabidopsis thaliana is improperly controlled in root hair-defective rhd4-1 mutant plants, resulting in root hairs that are shorter and randomly form bulges along their length. Using time-lapse fluorescence microscopy in rhd4-1 root hairs, we analyzed membrane dynamics after labeling with RabA4b, a marker for polarized membrane trafficking in root hairs. This revealed stochastic loss and recovery of the RabA4b compartment in the tips of growing root hairs, consistent with a role for the RHD4 protein in regulation of polarized membrane trafficking in these cells. The wild-type RHD4 gene was identified by map-based cloning and was found to encode a Sac1p-like phosphoinositide phosphatase. RHD4 displayed a preference for phosphatidylinositol-4-phosphate [PI(4)P] in vitro, and rhd4-1 roots accumulated higher levels of PI(4)P in vivo. In wild-type root hairs, PI(4)P accumulated primarily in a tip-localized plasma membrane domain, but in rhd4-1 mutants, significant levels of PI(4)P were detected associated with internal membranes. A fluorescent RHD4 fusion protein localized to membranes at the tips of growing root hairs. We propose that RHD4 is selectively recruited to RabA4b-labeled membranes that are involved in polarized expansion of root hair cells and that, in conjunction with the phosphoinositide kinase PI-4Kbeta1, RHD4 regulates the accumulation of PI(4)P on membrane compartments at the tips of growing root hairs.     

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.     

Chromosaponin I specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots

We have found that chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea (Pisum sativum L. cv Alaska), specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots. Application of 60 microM CSI disrupts the vertically oriented elongation of wild-type roots grown on agar plates but orients the elongation of agravitropic mutant aux1-7 roots toward the gravity. The CSI-induced restoration of gravitropic response in aux1-7 roots was not observed in other agravitropic mutants, axr2 and eir1-1. Because the aux1-7 mutant is reduced in sensitivity to auxin and ethylene, we examined the effects of CSI on another auxin-resistant mutant, axr1-3, and ethylene-insensitive mutant ein2-1. In aux1-7 roots, CSI stimulated the uptake of [(3)H]indole-3-acetic acid (IAA) and induced gravitropic bending. In contrast, in wild-type, axr1-3, and ein2-1 roots, CSI slowed down the rates of gravitropic bending and inhibited IAA uptake. In the null allele of aux1, aux1-22, the agravitropic nature of the roots and IAA uptake were not affected by CSI. This close correlation between auxin uptake and gravitropic bending suggests that CSI may regulate gravitropic response by inhibiting or stimulating the uptake of endogenous auxin in root cells. CSI exhibits selective influence toward IAA versus 1-naphthaleneacetic acid as to auxin-induced inhibition in root growth and auxin uptake. The selective action of CSI toward IAA along with the complete insensitivity of the null mutant aux1-22 toward CSI strongly suggest that CSI specifically interacts with AUX1 protein.     

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.     

Defects in root development and gravity response in the aem1 mutant of rice are associated with reduced auxin efflux

The phytohormone auxin is involved in the regulation of a variety of developmental processes. In this report, we describe how the processes of lateral root and root hair formations and root gravity response in rice are controlled by auxin. We use a rice mutant aem1 (auxin efflux mutant) because the mutant is defective in these characters. The aem1 line was originally isolated as a short lateral root mutant, but we found that the mutant has a defect in auxin efflux in roots. The acropetal and basipetal indole-3-acetic acid (IAA) transports were reduced in aem1 roots compared to wild type (WT). Furthermore, gravitropic bending as well as efflux of radioactive IAA was impaired in the mutant roots. We also propose a unique distribution of endogenous IAA in aem1 roots. An immunoassay revealed a 4-fold-endogenous IAA content in the aem1 roots compared to WT, and the application of IAA to the shoot of WT seedlings mimicked the short lateral root phenotype of aem1, suggesting that the high content of IAA in aem1 roots impaired the elongation of lateral roots. However, the high level of IAA in aem1 roots contradicts the auxin requirement for root hair formation in the epidermis of mutant roots. Since the reduced development in root hairs of aem1 roots was rescued by exogenous auxin, the auxin level in the epidermis is likely to be sub-optimum in aem1 roots. This discrepancy can be solved by the ideas that IAA level is higher in the stele and lower in the epidermis of aem1 roots compared to WT and that the unique distribution of IAA in aem1 roots is induced by the defect in auxin efflux. All these results suggest that AEM1 may encode a component of auxin efflux carrier in rice and that the defects in lateral roots, root hair formation and root gravity response in aem1 mutant are due to the altered auxin efflux in roots.     

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.     

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.     

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 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.     

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.     

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.     

Ethylene Interacts with Auxin in Regulating Developmental Attenuation of Gravitropism in Flax Root

Previous research shows that gravity-sensing in flax (Linum usitatissimum) root is initiated during seed imbibition and precedes root emergence. In this study we investigated the developmental attenuation of flax root gravitropism post-germination and the involvement of ethylene. Gravity response deteriorated significantly from 3 to 11?h after root emergence, which occurred at around 19?h after imbibition (that is, from “age” 22 to 30?h). Although the root elongation rate increased from 22 to 30?h, the gravitropic curving rate declined steadily. Older roots were able to tolerate higher levels of exogenous IAA before inhibition of elongation and gravitropism occurred. The age-dependent effect of IAA on root growth and gravitropism suggests that young roots are more sensitive to auxin and respond to a smaller vertical auxin gradient than older roots upon horizontal gravistimulation. The ethylene synthesis inhibitor AVG (2-aminoethoxyvinyl glycine, 10?μM) or ethylene action inhibitor Ag⁺ (10?μM) stimulated gravitropic curvature of 30?h roots by 24 and 32%, respectively, but had no effect on 22?h roots, suggesting that as roots age, ethylene begins to play a role in root gravitropism. The auxin transport inhibitor NPA (N-naphthylphthalamic acid, 50?μM) reduced gravitropic curvature of 30?h roots by 24% but had no effect on 22?h roots. On the other hand, treating roots simultaneously with the auxin transport inhibitor and ethylene synthesis or action inhibitor stimulated gravitropic curvature of 30?h roots but not 22?h roots. Taken together, these data indicate that as roots develop, their weakened gravity response is due to decreased auxin sensitivity and possibly auxin transport regulated by ethylene.