Ethylene promotes root hair growth through coordinated EIN3/EIL1 and RHD6/RSL1 activity in Arabidopsis

Abstract

Root hairs are an extensive structure of root epidermal cells and are critical for nutrient acquisition, soil anchorage, and environmental interactions in sessile plants. The phytohormone ethylene (ET) promotes root hair growth and also mediates the effects of different signals that stimulate hair cell development. However, the molecular basis of ET-induced root hair growth remains poorly understood. Here, we show that ET-activated transcription factor ETHYLENE-INSENSITIVE 3 (EIN3) physically interacts with ROOT HAIR DEFECTIVE 6 (RHD6), a well-documented positive regulator of hair cells, and that the two factors directly coactivate the hair length-determining gene RHD6-LIKE 4 (RSL4) to promote root hair elongation. Transcriptome analysis further revealed the parallel roles of the regulator pairs EIN3/EIL1 (EIN3-LIKE 1) and RHD6/RSL1 (RHD6-LIKE 1). EIN3/EIL1 and RHD6/RSL1 coordinately enhance root hair initiation by selectively regulating a subset of core root hair genes. Thus, our work reveals a key transcriptional complex consisting of EIN3/EIL1 and RHD6/RSL1 in the control of root hair initiation and elongation, and provides a molecular framework for the integration of environmental signals and intrinsic regulators in modulating plant organ development.

Keywords: EIN3; RHD6; RSL4; ethylene; root hair.

Fig. 1.

EIN3/EIL1 mediate ET-induced root hair elongation. (A) Representative root hairs from 6-d-old Col-0 and ein3 eil1 on ACC media. (B) Quantification of root hair length in A. (C) Representative root hairs from 6-d-old iEIN3 ein3 eil1. (D) Quantification of root hair length in C. (Scale bars: A and C, 200 μm.) Data are means ± SD (n = 10 roots). One-way ANOVA with a post hoc Tukey honest significant difference (HSD) test (**P < 0.01) was used. NS, not significantly different.

Fig. 2.

ET-promoted root hair elongation requires RSL4/RSL2. (A) Representative root hairs from 6-d-old Col-0, ein3 eil1, rhd6 rsl1, and rsl4 rsl2 on ACC media. (Scale bar: 200 μm.) (B) Quantification of root hair length in A. Data are means ± SD (n = 10 roots). One-way ANOVA with a post hoc Tukey HSD test (**P < 0.01) was used. ND, not detectable; NS, not significantly different.

Fig. 3.

RSL4 is a direct target of EIN3. (A) Relative (Rel.) expression levels of RSL class II genes in Col-0 and ein3 eil1 (ee) in response to ET. Six-day-old seedlings were treated with air or ET at 10 ppm for 4 h. (B) Rel. expression levels of RSL class II genes with EIN3 overexpression induction. Six-day-old iEIN3 ein3 eil1 ebf1 ebf2 (iEqm) seedlings were transferred to Murashige and Skoog medium containing 2 μM β-estrogen for the amount of time indicated on the x axis (hours). (C) In vitro EMSA showing EIN3 directly binding to the RSL4 promoter. (Upper) EIN3-binding site (ATGTAT, boldfaced) and the mutated form (GAAGCC) are shown. The GST-fused EIN3 N terminus (GST-EIN3^141-352) was purified from E. coli. Unlabeled cold probe (200-fold) was added for competition. Probe containing the mutated binding site (m) was used to assess binding specificity. (D) ChIP-qPCR shows that EIN3 binds to the RSL4 promoter in vivo. The roots of 6-d-old iEqm were collected after incubation in 10 nM β-estrogen for 4 h to induce EIN3-FLAG overexpression. Fragmented chromatin was precipitated with anti-FLAG antibody. (E and F) RSL4 overexpression restored hair elongation in ein3 eil1. (E) Representative root hairs from Col-0, ein3 eil1, RSL4ox, and RSL4ox ein3 eil1. (Scale bar: 200 μm.) (F) Quantification of root hair length in E. Data are means ± SD (n = 10 roots). R4ox, RSL4ox. One-way ANOVA with a post hoc Tukey HSD test (**P < 0.01, *P < 0.05) was used.

Fig. 4.

EIN3 physically interacts with RHD6. (A) Pull-down analysis shows direct interaction between EIN3 and RHD6 in vitro. EIN3-HA and His-TF-RHD6 (TF-RHD6) were incubated for 4 h before precipitation by nickel-nitrilotriacetic acid agarose. TF, trigger factor. (B) Colocalization of EIN3 and RHD6 in H cells (white arrows). Six-day-old F1 progeny of pRHD6::GFP:RHD6 × pEIN3::EIN3:RFP were treated with ET for 2 h. (C) Coimmunoprecipitation of EIN3 and RHD6. Whole-protein extraction from root tips of pRHD6::GFP:RHD6 was immunoprecipitated by GFP-trap agarose, and endogenous anti-EIN3 antibody was used for blotting. IP, immunoprecipitation. (D) Relative (Rel.) expression level of RSL4 in ein3 eil1 rhd6 rsl1 (ee61). Seedlings were treated with air or ET for 4 h. (E) Dual-luciferase reporter assay shows the coactivation of RSL4 by EIN3 and RHD6. The indicated vectors were transformed into Arabidopsis root protoplasts. The activity ratio of firefly luciferase relative to Renilla luciferase was calculated as a metric of transcriptional activity. Error bars indicate ±SD of three biological replicates. One-way ANOVA with a post hoc Tukey HSD test (**P < 0.01, *P < 0.05) was used.

Fig. 5.

ET promotes root hair initiation through EIN3/EIL1 and RHD6/RSL1. (A) Representative roots show hair bulge formation induced by 2 μM ACC in 6-d-old hairless mutants. (Scale bar: 200 μm.) (B) Root hair/bulge percentage in H cells. The percentage was calculated based on bulge and hair numbers per H cell. Two hundred cells per sample were counted. ee, ein3 eil1; ee42, ein3 eil1 rsl4 rsl2; ee61, ein3 eil1 rhd6 rsl1; 42, rsl4 rsl2; 61, rhd6 rsl1; ND, not detectable; NS, not significantly different. Error bars indicate ±SD of three biological replicates. Statistical significance was determined by a Student’s t test (***P < 0.001). (C) Summary diagram for the identification of root hair initiation genes. RNA sequencing and differential expression analysis were performed using roots of Col-0, ee, 61, and ee61 after air or ET treatment. Among the 43 H genes, 25 were identified as RSL4-regulated genes (12, 32). Nine genes, not characterized in previous publications, are closely related to the known root hair genes based on coexpression analysis (Fig. S5). These nine genes plus 18 non–RSL4-regulated H genes comprise the group of 27 hair initiation candidate genes. EPGs, ET-promoted genes.

Fig. 6.

Relative root hair length of Col-0, ein3 eil1(ee), rhd6 rsl1(61), and ein3 eil1 rhd6 rsl1(ee61) under the following stimuli: auxin [indole-3-acetic acid (IAA)] (A), cytokinin [6-benzylaminopurine (6-BA)] (B), nitrogen (N) deficiency (C), and phosphorus (P) deficiency (D). The length of Col-0 root hair was calibrated as 1.0. Data are means ± SD (n = 10 roots). The values were compared between treated and untreated samples within the same genotype. Statistical significance was determined by a Student’s t test (***P < 0.001). ND, not detectable. (E) Working model. (Top Right) In the presence of ET in Col-0, high levels of ET-induced EIN3/EIL1 protein accumulate (E3; red ovals). EIN3/EIL1 interact with RHD6/RSL1 (R6; yellow hexagons) in H cells to directly activate the transcription of RSL4, an essential positive regulator of root hair tip growth, as well as a set of genes involved in hair initiation. (Top Left) In the absence of ET, root hair initiation and elongation are mainly regulated by R6, with E3 playing a minor role. (Bottom Right) In the rhd6 rsl1 mutant, ET-induced and stabilized E3 independently promotes hair initiation and elongation to partially restore hair growth of the hairless mutant. (Bottom Left) In the absence of both ET-induced E3 and functional R6, the hairless phenotype is observed.