Mutations in the Diageotropica (Dgt) gene uncouple patterned cell division during lateral root initiation from proliferative cell division in the pericycle

In angiosperms, root branching requires a continuous re-initiation of new root meristems. Through some unknown mechanism, in most eudicots pericycle cells positioned against the protoxylem change identity and initiate patterned division, leading to formation of lateral root primordia that further develop into lateral roots. This process is auxin-regulated. We have observed that three mutations in the Diageotropica (Dgt) gene in tomato prevent primordium formation. Detailed analysis of one of these mutants, dgt1-1, demonstrated that the mutation does not abolish the proliferative capacity of the xylem-adjacent pericycle in the differentiated root portion. Files of shortened pericycle cells found in dgt1-1 roots were unrelated to primordium formation. Auxin application stimulated this unusual proliferation, leading to formation of a multi-layered xylem-adjacent pericycle, but did not rescue the primordium formation. In contrast to wild type, auxin could not induce any cell divisions in the pericycle of the most distal dgt1-1 root-tip portion. In wild-type roots, the Dgt gene promoter was expressed strongly in lateral root primordia starting from their initiation, and on auxin treatment was induced in the primary root meristem. Auxin level and distribution were altered in dgt1-1 root tissues, as judged by direct auxin measurements, and the tissue-specific expression of an auxin-response reporter was altered in transgenic plants. Together, our data demonstrate that the Dgt gene product, a type-A cyclophilin, is essential for morphogenesis of lateral root primordia, and that the dgt mutations uncouple patterned cell division in lateral root initiation from proliferative cell division in the pericycle.     

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.     

Cytokinins act directly on lateral root founder cells to inhibit root initiation

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.     

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.     

Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato

In this study we investigated the role of ethylene in the formation of lateral and adventitious roots in tomato ( Solanum lycopersicum ) using mutants isolated for altered ethylene signaling and fruit ripening. Mutations that block ethylene responses and delay ripening – Nr ( Never ripe ), gr ( green ripe ), nor ( non ripening ), and rin ( ripening inhibitor ) – have enhanced lateral root formation. In contrast, the epi ( epinastic ) mutant, which has elevated ethylene and constitutive ethylene signaling in some tissues, or treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC), reduces lateral root formation. Treatment with ACC inhibits the initiation and elongation of lateral roots, except in the Nr genotype. Root basipetal and acropetal indole-3-acetic acid (IAA) transport increase with ACC treatments or in the epi mutant, while in the Nr mutant there is less auxin transport than in the wild type and transport is insensitive to ACC. In contrast, the process of adventitious root formation shows the opposite response to ethylene, with ACC treatment and the epi mutation increasing adventitious root formation and the Nr mutation reducing the number of adventitious roots. In hypocotyls, ACC treatment negatively regulated IAA transport while the Nr mutant showed increased IAA transport in hypocotyls. Ethylene significantly reduces free IAA content in roots, but only subtly changes free IAA content in tomato hypocotyls. These results indicate a negative role for ethylene in lateral root formation and a positive role in adventitious root formation with modulation of auxin transport as a central point of ethylene–auxin crosstalk.     

ARL1, a LOB-domain protein required for adventitious root formation in rice

Adventitious roots constitute the bulk of the fibrous root system in cereals. Compared with the current understanding of shoot development, knowledge of the molecular mechanisms of development of the adventitious roots of cereals is limited. We have isolated and characterized a novel gene controlling the initiation of adventitious root primordia in rice (Oryza sativa L.). The gene, designated Adventitious rootless1 (ARL1), encodes a protein with a LATERAL ORGAN BOUNDARIES (LOB) domain. It is expressed in lateral and adventitious root primordia, tiller primordia, vascular tissues, scutellum, and young pedicels. ARL1 is a nuclear protein and can form homodimers. ARL1 is an auxin- and ethylene-responsive gene, and the expression pattern of ARL1 in roots parallels auxin distribution. Our findings suggest that ARL1 is an auxin-responsive factor involved in auxin-mediated cell dedifferentiation, and that it promotes the initial cell division in the pericycle cells adjacent to the peripheral vascular cylinder in the stem.     

Mutations in Arabidopsis multidrug resistance-like ABC transporters separate the roles of acropetal and basipetal auxin transport in lateral root development

Auxin affects the shape of root systems by influencing elongation and branching. Because multidrug resistance (MDR)-like ABC transporters participate in auxin transport, they may be expected to contribute to root system development. This reverse genetic study of Arabidopsis thaliana roots shows that MDR4-mediated basipetal auxin transport did not affect root elongation or branching. However, impaired acropetal auxin transport due to mutation of the MDR1 gene caused 21% of nascent lateral roots to arrest their growth and the remainder to elongate 50% more slowly than the wild type. Reporter gene analyses indicated a severe auxin deficit in the apex of mdr1 but not mdr4 lateral roots. The mdr1 deficit was explained by 40% less acropetal auxin transport within the mdr1 lateral roots. The slow elongation of mdr1 lateral roots was rescued by auxin and phenocopied in the wild type by an inhibitor of polar auxin transport. Confocal microscopy analysis of a functional green fluorescent protein-MDR1 translational fusion showed the protein to be auxin inducible and present in the tissues responsible for acropetal transport in the primary root. The protein also accumulated in lateral root primordia and later in the tissues responsible for acropetal transport within the lateral root, fully supporting the conclusion that auxin levels established by MDR1-dependent acropetal transport control lateral root growth rate to influence root system architecture.     

Resumption of DNA Synthesis and Cell Division in Wheat Roots as Related to Lateral Root Initiation

The arrest of DNA synthesis and termination of cell division in basal meristematic cells as well as the resumption of these processes as related to the initiation of lateral root primordia (LRP) were studied in tissues of Triticum aestivumroots incubated with ³H-thymidine. All cells of the stelar parenchyma and cortex as well as most endodermal and pericycle cells left the mitotic cycle and ceased proliferative activity at the basal end of the meristem and at the beginning of the elongation zone. Some endodermal and pericycle cells started DNA synthesis in the basal part of the meristem and completed it later on during their elongation, but they did not divide. In the cells of these tissues, DNA synthesis resumed above the elongation zone, the cells being located much closer to the root tip than the first newly dividing cells. Thus, the initiation of LRP started much closer to the root tip than it was previously believed judging from the distance of the first dividing pericycle cells from the root tip. DNA synthesizing and dividing cells first appeared in the stelar parenchyma, then, in the pericycle, and later, in the endodermis and cortex. It seems likely that a release from the inhibition of DNA synthesis allows the cells that completed mitotic cycle in the basal part of meristem in the G₁phase to cease the proliferative arrest above the elongation zone and to continue their cycling. The location of the first DNA synthesizing and dividing cells in the stelar parenchyma and pericycle did not strictly correspond to the LRP initiation sites and proximity to the xylem or phloem poles. This indicates that LRP initiation results from the resumption of DNA synthesis in all pericycle and stelar parenchyma cells that retained the ability to synthesize DNA and occurs only in the pericycle sector situated between the two tracheal protoxylem strands, all cells of which terminated their mitotic cycles in the G₁phase.     

Stage-specific crosstalk between light, auxin, and ethylene during low-pH-induced root hair formation in lettuce (Lactuca sativa L.) seedlings

In the light, transfer of lettuce seedlings precultured on liquid medium at pH 6.0 to fresh medium at pH 4.0 induces root hair formation. However, no root hairs form in the dark. Here, we investigated how light induces root hair formation. Randomization of the transverse cortical microtubule (CMT) arrays which occurs in root epidermal cells in the light prior to root hair initiation was not observed in the dark. However, addition of indole-3-acetic acid (IAA) or 1-aminocyclopropane-1-carboxylic acid (ACC) induced CMT randomization and root hair formation. In these cases, CMT randomization occurred in almost the same time-dependent manner as under light. However, root hair initiation was delayed for several hours in the dark. These results suggest that light promotes CMT randomization and root hair initiation via auxin and ethylene signaling but light additionally influences root hair initiation independently of these signaling mechanisms. Furthermore, addition of a microtubule-depolymerizing drug in the dark disrupted the transverse CMT arrays and initiated root hair formation; however, root hair elongation was still suppressed. Root hairs elongated when IAA or ACC was applied with the drug. These results suggest that light also promotes root hair elongation via auxin and ethylene signaling.     

Pericycle cell proliferation and lateral root initiation in Arabidopsis

In contrast with other cells generated by the root apical meristem in Arabidopsis, pericycle cells adjacent to the protoxylem poles of the vascular cylinder continue to cycle without interruption during passage through the elongation and differentiation zones. However, only some of the dividing pericycle cells are committed to the asymmetric, formative divisions that give rise to lateral root primordia (LRPs). This was demonstrated by direct observation and mapping of mitotic figures, cell-length measurements, and the histochemical analysis of a cyclin-GUS fusion protein in pericycle cells. The estimated duration of a pericycle cell cycle in the root apical meristem was similar to the interval between cell displacement from the meristem and the initiation of LRP formation. Developmentally controlled LRP initiation occurs early, 3 to 8 mm from the root tip. Thus the first growth control point in lateral root formation is defined by the initiation of primordia in stochastic patterns by cells passing through the elongation and young differentiation zones, up to where lateral roots begin to emerge from the primary root. Therefore, the first growth control point is not restricted to a narrow developmental window. We propose that late LRP initiation is developmentally unrelated to the root apical meristem and is operated by a second growth control point that can be activated by environmental cues. The observation that pericycle cells divide and lateral root primordia form without intervening mitotic quiescence suggests that lateral organ formation in roots and shoots might not be as fundamentally different as previously thought.     

Tissue-specific expression of stabilized SOLITARY-ROOT/IAA14 alters lateral root development in Arabidopsis

Auxin is important for lateral root (LR) initiation and subsequent LR primordium development. However, the roles of tissue-specific auxin signaling in these processes are poorly understood. We analyzed transgenic Arabidopsis plants expressing the stabilized mutant INDOLE-3 ACETIC ACID 14 (IAA14)/SOLITARY-ROOT (mIAA14) protein as a repressor of the auxin response factors (ARFs), under the control of tissue-specific promoters. We showed that plants expressing the mIAA14-glucocorticoid receptor (GR) fusion protein under the control of the native IAA14 promoter had the solitary-root/iaa14 mutant phenotypes, including the lack of LR formation under dexamethasone (Dex) treatment, indicating that mIAA14-GR is functional in the presence of Dex. We then demonstrated that expression of mIAA14-GR under the control of the stele-specific SHORT-ROOT promoter suppressed LR formation, and showed that mIAA14-GR expression in the protoxylem-adjacent pericycle also blocked LR formation, indicating that the normal auxin response mediated by auxin/indole-3 acetic acid (Aux/IAA) signaling in the protoxylem pericycle is necessary for LR formation. In addition, we demonstrated that expression of mIAA14-GR under either the ARF7 or the ARF19 promoter also suppressed LR formation as in the arf7 arf19 double mutants, and that IAA14 interacted with ARF7 and ARF19 in yeasts. These results strongly suggest that mIAA14-GR directly inactivates ARF7/ARF19 functions, thereby blocking LR formation. Post-embryonic expression of mIAA14-GR under the SCARECROW promoter, which is expressed in the specific cell lineage during LR primordium formation, caused disorganized LR development. This indicates that normal auxin signaling in LR primordia, which involves the unknown ARFs and Aux/IAAs, is necessary for the establishment of LR primordium organization. Thus, our data show that tissue-specific expression of a stabilized Aux/IAA protein allows analysis of tissue-specific auxin responses in LR development by inactivating ARF functions.     

The Control of Lateral Root Development in Cultured Pea Seedlings. II. Root Fasciation Induced by Auxin Inhibitors

Lateral root development in cultured seedlings of Pisum sativum (cv. Alaska) was modified by the application of auxin transport inhibitors or antagonists. When applied either to replace the root tip or beneath the cotyledonary node, two auxin transport inhibitors, 2,3,5-triiodobenzoic acid (TIBA) and 3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5,1-α]isoindol-8-one (DPX-1840), increased cell division activity opposite the protoxylem poles. This resulted in the formation of masses of cells, which we are calling root primordial masses (RPMs), 2 to 3 days after treatment. RPMs differed from lateral root primordia in that they lacked apical organization. Some roots however developed both RPMs and lateral roots indicating that both structures were similar in terms of the timing and location of cell division in the pericycle and endodermis leading to their initiation. Removal of the auxin transport inhibitors allowed many of the RPMs to organize later into lateral root primordia and to emerge in clusters. When the auxin, indoleacetic acid (IAA) was added to the growth medium along with DPX-1840, 3 ranks of RPMs now in the form of fasciated lateral roots emerged from the primary root. The auxin antagonist, p-chlorophenoxy-isobutyric acid (PCIB), also induced RPM formation. In contrast to DPX-1840 treatment, the addition of IAA during PCIB treatment caused normal lateral root development.     

An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin in pericycle cell activation

Arabidopsis (Arabidopsis thaliana) plants display a number of root developmental responses to low phosphate availability, including primary root growth inhibition, greater formation of lateral roots, and increased root hair elongation. To gain insight into the regulatory mechanisms by which phosphorus (P) availability alters postembryonic root development, we performed a mutant screen to identify genetic determinants involved in the response to P deprivation. Three low phosphate-resistant root lines (lpr1-1 to lpr1-3) were isolated because of their reduced lateral root formation in low P conditions. Genetic and molecular analyses revealed that all lpr1 mutants were allelic to BIG, which is required for normal auxin transport in Arabidopsis. Detailed characterization of lateral root primordia (LRP) development in wild-type and lpr1 mutants revealed that BIG is required for pericycle cell activation to form LRP in both high (1 mm) and low (1 microm) P conditions, but not for the low P-induced alterations in primary root growth, lateral root emergence, and root hair elongation. Exogenously supplied auxin restored normal lateral root formation in lpr1 mutants in the two P treatments. Treatment of wild-type Arabidopsis seedlings with brefeldin A, a fungal metabolite that blocks auxin transport, phenocopies the root developmental alterations observed in lpr1 mutants in both high and low P conditions, suggesting that BIG participates in vesicular targeting of auxin transporters. Taken together, our results show that auxin transport and BIG function have fundamental roles in pericycle cell activation to form LRP and promote root hair elongation. The mechanism that activates root system architectural alterations in response to P deprivation, however, seems to be independent of auxin transport and BIG.     

Auxin transport inhibitors act through ethylene to regulate dichotomous branching of lateral root meristems in pine

Many soil fungi colonize the roots of pines to form symbiotic organs known as ectomycorrhizas. Dichotomous branching of short lateral roots and the formation of coralloid organs are diagnostic of ectomycorrhizas in many pine species, although the regulation of these changes in root morphology is not well understood. We used axenic root cultures of six pine species to examine the role of auxin, cytokinin, ethylene and nutrients in the regulation of root architecture. Surprisingly, extensive dichotomous and coralloid branching of lateral roots occurred spontaneously in Pinus taeda , P. halepensis and P. muricata . In P. sylvestris , P. ponderosa and P. nigra , treatment with auxin transport inhibitors (ATIs), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or the ethylene-releasing compound 2-chloroethylphosphonic acid (CEPA or ethephon) induced extensive dichotomous branching and coralloid organ formation. Formation of both spontaneous and ATI-induced coralloid structures was blocked by treatment with an ethylene synthesis inhibitor L-α-(2-aminoethoxyvinyl)glycine; this inhibition was reversed by either ACC or CEPA. In addition, the induction of this unique morphogenetic pattern in pine root cultures was regulated by nutrient levels. The morphology and anatomical organization of the chemically induced dichotomous and coralloid structures, as well as the regulation of their formation by nutrient levels, show a striking similarity to those of ectomycorrhizas.     

Histological analysis of adventitious rooting in Arabidopsis thaliana (L.) Heynh seedlings

ABSTRACT

Adventititous rooting is essential for the post-embryonic growth of the root apparatus in various species. In Arabidopsis thaliana, adventitious rooting has been reported in some mutants, and auxin seems to be the inducer of the process. The objective of the study was to identify the tissues involved in adventitious rooting in the most commonly used ecotypes for molecular and genetic studies (i.e. Columbia, Wassilewskija and Landsberg erecta) both in the presence and absence of exogenous auxin. Seedlings of the three ecotypes were grown under various conditions. When grown under 16 hours light/day for 11 days, all seedlings showed adventitious roots, both with and without auxin, however, both adventitious and lateral rooting were enhanced by exogenous auxin (2 µM naphthaleneacetic acid). Independently of the presence of auxin and of the ecotype, the hypocotyl pericycle produced adventitious roots directly (i.e., according to the same pattern of lateral root formation by the pericycle cells in the primary root). However, in the presence of auxin, roots of indirect origin also, and mainly, formed and their formation was preceded by the exfoliation of the tissues external to the stele. Exfoliation was caused by cell hypertrophy, separation, and disintegration, which mainly involved the endodermis. At the exfoliation site, the pericycle, with a minor contribution of a few endodermal cells, produced the callus from which indirect roots arose. The finding that adventitious rooting occurs in the absence of auxin (all ecotypes) indicates that this process is part of the normal root apparatus in Arabidopsis, with the hypocotyl pericycle as the target tissue of the process. Exogenous auxin alters adventitious rhizogenesis mainly affecting the endodermis response.     

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.     

Lateral Root Initiation or the Birth of a New Meristem

Root branching happens through the formation of new meristems out of a limited number of pericycle cells inside the parent root. As opposed to shoot branching, the study of lateral root formation has been complicated due to its internal nature, and a lot of questions remain unanswered. However, due to the availability of new molecular tools and more complete genomic data in the model species Arabidopsis, the probability to find new and crucial elements in the lateral root formation pathway has increased. Increasingly more data are supporting the idea that lateral root founder cells become specified in young root parts before differentiation is accomplished. Next, pericycle founder cells undergo anticlinal asymmetric, divisions followed by an organized cell division pattern resulting in the formation of a new organ. The whole process of cell cycle progression and stimulation of the molecular pathway towards lateral root initiation is triggered by the plant hormone auxin. In this review, we aim to give an overview on the developmental events taking place from the very early specification of founder cells in the pericycle until the first anticlinal divisions by combining the knowledge originating from classical physiology studies with new insights from genetic-molecular analyses. Based on the current knowledge derived from recent genetic and developmental studies, we propose here a hypothetical model for LRI.     

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.