Ethylene Promotes Root Hair Growth Through Coordinated EIN3/EIL1 and RHD6/RSL1 Activity in Arabidopsis

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

Ying Fenga,b, Ping Xub, Bosheng Lia, Pengpeng Lib, Xing Wena, Fengying Anb, Yan Gongb, Yi Xinb, Ziqiang Zhub,c, Yichuan Wanga,1, and Hongwei Guoa,d,1

aInstitute of Plant and Food Science, Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; bThe State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China; cCollege of Life Sciences, Nanjing Normal University, Nanjing 210023, China; and dPeking-Tsinghua Center of Life Sciences, Beijing 100871, China

Edited by José M. Alonso, North Carolina State University, Raleigh, NC, and accepted by Editorial Board Member Joseph R. Ecker November 14, 2017 (received for review July 7, 2017)

Root hairs are an extensive structure of root epidermal cells and before hair cells enter the elongation stage, with the amount of are critical for nutrient acquisition, soil anchorage, and environ- accumulated RSL4 mRNA proportional to the final root hair mental interactions in sessile plants. The phytohormone ethylene length (13). Recently, phytohormone application, such as auxin (ET) promotes root hair growth and also mediates the effects of and cytokinin treatment, and nutrient starvation were found to different signals that stimulate hair cell development. However, stimulate root hair growth in an RSL4-dependent manner (12, the molecular basis of ET-induced root hair growth remains poorly 14, 15), revealing that RSL4 is an essential node that integrates understood. Here, we show that ET-activated transcription factor multiple root hair elongation signals. ETHYLENE-INSENSITIVE 3 (EIN3) physically interacts with ROOT The gaseous phytohormone ethylene (ET) is a well-known stress HAIR DEFECTIVE 6 (RHD6), a well-documented positive regulator hormone that helps plants to survive under various biotic and of hair cells, and that the two factors directly coactivate the hair abiotic stresses (16, 17). Upon ET binding, ET receptors deactivate length-determining gene RHD6-LIKE 4 (RSL4) to promote root hair the Ser/Thr kinase CONSTITUTIVE TRIPLE RESPONSE 1

elongation. Transcriptome analysis further revealed the parallel (CTR1), leading to the hypophosphorylation and proteolytic acti- PLANT BIOLOGY vation of ETHYLENE INSENSITIVE 2 (EIN2) (18). Down- roles of the regulator pairs EIN3/EIL1 (EIN3-LIKE 1) and RHD6/ stream of EIN2, EIN3 and its functionally redundant homolog RSL1 (RHD6-LIKE 1). EIN3/EIL1 and RHD6/RSL1 coordinately en- EIN3-LIKE 1 (EIL1) are two master transcription factors that hance root hair initiation by selectively regulating a subset of core activate numerous signaling cascades and ET responses (16, 17). root hair genes. Thus, our work reveals a key transcriptional com- Activated EIN2 protects EIN3/EIL1 from proteasomal degrada- plex consisting of EIN3/EIL1 and RHD6/RSL1 in the control of root tion, at least in part, by repressing the E3 ligases EIN3-BINDING hair initiation and elongation, and provides a molecular frame- F-BOX 1 (EBF1) and EBF2 (19, 20). Thus, EIN3/EIL1 proteins work for the integration of environmental signals and intrinsic accumulate in the nuclei upon ET treatment (21, 22). One notable regulators in modulating plant organ development. role of ET is to stimulate root hair growth. Exogenous application of the ET biosynthesis precursor 1-aminocyclopropane-1-carboxylic ethylene | root hair | EIN3 | RHD6 | RSL4 Significance lants, unlike animals, are sessile organisms constantly exposed Pto various environmental changes. Organ morphogenesis in Root hairs are unicellular extensions of root epidermal cells that response to different challenges is critical for plant survival. Root help plants increase water and nutrient uptake and improve soil hairs are unicellular extensions of root epidermal cells that help anchorage, both of which are crucial for the globally recognized increase water and nutrient uptake, and improve soil anchorage. goal of yield improvement with reduced fertilizer use. Previous As the outermost plant surface in the soil, root hairs rapidly and studies have implicated numerous genes and phytohormones in effectively respond to environmental stimuli. Their flexible nature the control of root hair development. This work uncovers the makes them an exceptional model system for studying the orga- molecular mechanism of ethylene (ET)-promoted root hair growth nization and coordination of gene regulatory networks modulated and identifies a transcriptional complex consisting of EIN3/ by endogenous and exogenous signals (1, 2). EIL1 and RHD6/RSL1 as the key regulator of root hair initiation In the angiosperm model plant Arabidopsis thaliana, the two root and elongation. As ET mediates the effects of various root hair epidermal cell fates are determined by the relative positioning of stimuli, this work also elucidates a convergent signaling network cells above the inner layer of cortical cells. Hair (H) cells overlie that integrates diverse environmental cues and intrinsic signals to the junction between two cortical cells, whereas nonhair (N) cells modulate plant organ development. overlie only one cortical cell (3). Although numerous genes influ-

ence root epidermal cell morphology after cell fate determination, Author contributions: Y.F., Z.Z., and H.G. designed research; Y.F., P.X., P.L., X.W., Y.G., the core regulatory network is driven by a transcriptional cascade Y.X., and Y.W. performed research; X.W., F.A., and Y.W. contributed new reagents/ana- (2, 4). GLABRA 2 (GL2), a homeodomain-leucine zipper tran- lytic tools; Y.F. and B.L. analyzed data; and Y.F. and H.G. wrote the paper. scription factor, plays a vital role in N cell differentiation (5, 6). The authors declare no conflict of interest. GL2 maintains N cell fate mainly through direct repression of a This article is a PNAS Direct Submission. J.M.A. is a guest editor invited by the group of basic helix–loop–helix (bHLH) family transcription factors Editorial Board. that positively regulate root hair initiation and elongation in H cells Published under the PNAS license. (7). Among them, ROOT HAIR DEFECTIVE 6 (RHD6) plays a – Data deposition: The sequencing data reported in this paper have been deposited in the major role in promoting H cell development (8 10). RHD6 and its Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession closest homolog, RHD6-LIKE 1 (RSL1), form class I of the bHLH no. GSE107699). group VIII subfamily. RHD6/RSL1 positively regulate four class II 1To whom correspondence may be addressed. Email: [email protected] or guohw@ members of group VIII, RSL2–5, which also positively regulate root sustc.edu.cn. hair growth (11). However, only RSL4 has been shown to be directly This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. regulated by RHD6 (12). RSL4 transcripts rapidly accumulate 1073/pnas.1711723115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1711723115 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 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) Quantifica- tion of root hair length in A.(C) Representative root hairs from 6-d-old iEIN3 ein3 eil1.(D) Quan- tification 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.

acid (ACC) or a loss-of-function mutation in CTR1 leads to much RSL1 and RSL4/RSL2 positively regulate root hair differentiation longer root hairs (23, 24). In contrast, the completely abolished ET downstream of GL2 (7, 12, 23). In fact, there are no visible root response in the ein2 mutant leads to a short-hair phenotype and hairs in the rhd6 rsl1 and rsl4 rsl2 mutants (9, 12). To determine reduced sensitivity to other hair growth stimuli, such as auxin, whether ET promotes hair growth through these factors, we assessed strigolactones, and low boron (23, 25, 26). the response of these mutants to ACC treatment. In rhd6 rsl1, root Although the roles of ET in promoting root hair growth and mediating other hair growth signals are well established, the underlying molecular mechanism remains unknown. Our findings show that EIN3/EIL1 are necessary and sufficient for ET-induced root hair elongation. EIN3 and RHD6 interact and cooperatively promote the hair elongation factor RSL4. Moreover, the functions of EIN3/EIL1 and RHD6/RSL1 are coordinated during root hair initiation. These findings elucidate the molecular mechanism of ET action during root hair initiation and elongation, and provide in- sight into the coordination of environmental and developmental signals during plant organ development. Results EIN3/EIL1 Are Critical for ET-Promoted Root Hair Elongation. The ein3 eil1 mutants were previously found to have reduced root hair density and compromised hair growth induction by jasmonic acid, suggesting that EIN3/EIL1 positively regulate root hair development (27). Phenotypic analysis indicated that EIN3 and EIL1 appeared to function partially redundantly in regulating root hair length (Fig. S1). To investigate the role of EIN3/EIL1 in mediating ET-induced root hair growth, we examined root hair length in young Arabidopsis wild-type Col-0 and ein3 eil1 seedlings upon ACC treatment. In Col-0, a low concentration of ACC (20 nM) stimu- lated significant root hair elongation, with further elongation observed under a higher concentration (100 nM). The ein3 eil1 root hairs were shorter than those of Col-0 and were completely unresponsive to ACC induction, suggesting that EIN3/EIL1 are necessary for ET-induced root hair elongation (Fig. 1 A and B). We also tested the sufficiency of EIN3 protein in promoting Arabidopsis root hair elongation. In the ein3 eil1 mutant background, root hair length significantly increased with accumulated EIN3-FLAG fusion protein induced by 5 nM and 10 nM β-estrogen application (Fig. 1 C and D). These results show that EIN3/EIL1 are essential positive regulators mediating ET-promoted root hair elongation. Fig. 2. ET-promoted root hair elongation requires RSL4/RSL2.(A) Representative ET-Promoted Root Hair Growth Requires RSL4/RSL2. Arabidopsis root root hairs from 6-d-old Col-0, ein3 eil1, rhd6 rsl1,andrsl4 rsl2 on ACC media. (Scale hair formation is regulated by a series of transcription factors (2, bar: 200 μm.) (B) Quantification of root hair length in A. Data are means ± SD (n = 4). We therefore assessed the interplay between EIN3/EIL1 and 10 roots). One-way ANOVA with a post hoc Tukey HSD test (**P < 0.01) was used. these transcription factors during root hair development. RHD6/ ND, not detectable; NS, not significantly different.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1711723115 Feng et al. Downloaded by guest on September 28, 2021 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 4h.(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-EIN3141-352) 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. Frag- mented 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 = PLANT BIOLOGY 10 roots). R4ox, RSL4ox. One-way ANOVA with a post hoc Tukey HSD test (**P < 0.01, *P < 0.05) was used.

hairs grew to about 400 μminlengthwithACCtreatment(Fig.2), specific binding of Escherichia coli-purified EIN3 protein (DNA- suggesting rescue of the root hair formation defect. The expression binding region, 141–352 aa) to the RSL4 EBS but not to the mu- patternofRHD6-GFPfusionprotein driven by the native RHD6 tated EBS (5′-GGAGCC-3′)(Fig.3C). Accordingly, EIN3 induction promoter remained largely unchanged during ACC incubation (Fig. of RSL4 was significantly impaired in planta when the promoter EBS S2A), thereby excluding the regulation of RHD6 expression by ET. motif was mutated (Fig. S3A). We also conducted an in vivo chro- Furthermore, RHD6 overexpression under the constitutive 35S matin immunoprecipitation (ChIP) assay and verified the binding of promoter in the ein3 eil1 mutant background resulted in longer EIN3-FLAG fusion protein to the RSL4 EBS-containing region in hair growth, suggesting that RHD6 may function downstream of Arabidopsis roots using anti-FLAG antibody to precipitate chromatin or in parallel with EIN3/EIL1 (Fig. S2 B and C). fragments (Fig. 3D). Consistently, RSL4 was identified as an ET- In rsl4 rsl2, the roots remained hairless even with a high ACC responsive EIN3 target gene in a ChIP-sequencing assay using concentration, indicating that RSL4/RSL2 are required for ET- endogenous anti-EIN3 antibody (28). Furthermore, RSL4 over- induced hair growth (Fig. 2). RHD6/RSL1 promotes root hair dif- expression rescued the short root hair defect of ein3 eil1 (Fig. 3 E ferentiation by positively regulating the RSL class II genes RSL2–5 and F). We therefore concluded that RSL4 is a direct target of (11). Given the requirement of RSL4/RSL2 in ET-induced root EIN3 and that ET promotes root hair elongation through tran- hair growth, we hypothesized that EIN3/EIL1 might also positively scriptional activation of RSL4 by EIN3/EIL1. regulate RSL class II genes in parallel with RHD6/RSL1. EIN3 and RHD6 Exhibit Protein–Protein Interaction. RHD6 was repor- RSL4 Is a Direct Target of EIN3. To determine whether EIN3/ tedtodirectlyactivateRSL4 transcription (12), although evidence EIL1 regulate RSL genes, we measured RSL transcript levels in of RHD6 binding to the RSL4 promoter was lacking. We in- ein3 eil1 and in transgenic plants with inducible EIN3 over- vestigated whether EIN3 and RHD6 coordinately regulate RSL4 expression. Exogenous ET treatment increased RSL4 and RSL5 transcription. A yeast two-hybrid assay showed that EIN3 and transcript levels, but only the increase of RSL4 was completely RHD6 interacted with each other (Fig. S3B). Consistently, an dependent on EIN3/EIL1 (Fig. 3A). In plants with inducible EIN3 in vitro pull-down assay demonstrated a direct interaction between overexpression, RSL4 mRNA levels increased as the induction HA-tagged EIN3 protein and His-tagged RHD6 protein (Fig. 4A). time increased (Fig. 3B). RSL5 levels were very low in untreated A luciferase complementation imaging (LCI) assay was also per- plants but dramatically increased after ET treatment and induction formed in tobacco leaves and Arabidopsis protoplasts, and verified of EIN3 overexpression (Fig. 3 A and B). RSL4 was previously the EIN3–RHD6 interaction in planta (Fig. S3 C–E). EIN3-RFP reported to positively regulate RSL5 (11); thus, the effect of ET protein driven by EIN3 promoter colocalized with RHD6-GFP and EIN3 induction on RSL4 transcription likely promoted RSL5 driven by RHD6 promoter in H cell files of Arabidopsis roots (Fig. expression. In contrast, RSL2 and RSL3 transcript levels were either 4B). A coimmunoprecipitation assay provided further in vivo evi- slightly reduced or largely unchanged upon exogenous ET applica- dence of the association between EIN3 and RHD6 (Fig. 4C). tion or EIN3 induction (Fig. 3 A and B). To determine whether EIN3 and RHD6 act together to regulate The changes in RSL4 mRNA abundance indicated that RSL4, the quadruple mutant ein3 eil1 rhd6 rsl1 was generated by RSL4 maybeadirecttargetofEIN3.Analysisofthepro- crossing ein3 eil1 and rhd6 rsl1. Notably, due to the absence of two moter region of RSL4 uncovered a putative EIN3-binding site distinct positive regulators, RSL4 was barely expressed in ein3 (EBS, 5′-ATGTAT-3′, starting at -853 upstream of the RSL4 gene). eil1 rhd6 rsl1 and did not respond to ET treatment (Fig. 4D). We In vitro electrophoretic mobility shift assays (EMSAs) confirmed the conducted a dual-luciferase reporter experiment (29) in Arabidopsis

Feng et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 rhd6 rsl1,andein3 eil1 rhd6 rsl1 roots were obtained by RNA sequencing. A total of 956 differentially expressed genes (DEGs) were identified in ein3 eil1 rhd6 rsl1 vs.Col-0.Biologicalprocessesin- volved in root hair development were statistically overrepresented among the 956 DEGs, including cell wall organization, cell tip growth, response to stimulus, and root epidermis differentiation (Fig. S4A). Moreover, the majority of the 956 genes had a greater fold change in ein3 eil1 rhd6 rsl1 vs. Col-0 than in either double mutant versus Col-0 (Fig. S4B), suggesting that gene expression coregulation by EIN3/EIL1 and RHD6/RSL1 occurs at loci throughout the genome and not only at RSL4. Next, we identified 187 genes induced by ET in an EIN3/EIL1- dependent manner in rhd6 rsl1 (ET-promoted genes). Compared with other published genes related to root epidermis morphology, strikingly, 43 of 154 core H genes (10) were included (Fig. 5C), but none of the 54 N genes was found, strongly indicating that EIN3/EIL1 and RHD6/RSL1 positively regulate hair formation. In rhd6 rsl1, the expression level of 43 H genes induced by ET was still lower than that in Col-0 without ET. Such an expression trend was consistent with the finding that ET only partially restored the hair growth defect in rhd6 rsl1 (Figs. 2 and 5 A and B), suggesting that the coordinated activity of EIN3/ EIL1 and RHD6/RSL1 is needed to fully activate root hair initiation. In light of the coactivation of the hair elongation factor RSL4 Fig. 4. EIN3 physically interacts with RHD6. (A) Pull-down analysis shows direct by EIN3/EIL1 and RHD6/RSL1, we found 25 of the 43 H genes to interaction between EIN3 and RHD6 in vitro. EIN3-HA and His-TF-RHD6 (TF- be RSL4-regulated genes (12, 32) (Fig. 5C). The remaining 18 genes RHD6) were incubated for 4 h before precipitation by nickel-nitrilotriacetic acid not influenced by RSL4 (Fig. 5C) were considered to be potential agarose. TF, trigger factor. (B) Colocalization of EIN3 and RHD6 in H cells (white initiation stage factors. Furthermore, coexpression analysis of the arrows). Six-day-old F1 progeny of pRHD6::GFP:RHD6 × pEIN3::EIN3:RFP were treatedwithETfor2h.(C) Coimmunoprecipitation of EIN3 and RHD6. Whole- proteinextractionfromroottipsofpRHD6::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.

root cell protoplasts and found that transient expression of EIN3 together with RHD6 activated RSL4 transcription more effectively than expression of either EIN3 or RHD6 alone (Fig. 4E). Based on these results, we conclude that EIN3 and RHD6 associate with each other and coactivate RSL4 transcription.

ET Promotes Root Hair Initiation Through EIN3/EIL1 and RHD6/RSL1. Besides promoting root hair elongation, RHD6/RSL1 are known regulators of root hair initiation (8, 9). ET also promotes root hair initiation under hairless conditions (10, 30, 31). We carefully observed the surface changes of rhd6 rsl1 and rsl4 rsl2 in response to ACC and found that ACC led to bulge formation in both mutants, a marker event of successful hair initiation (Fig. 5 A and B). When higher ACC concentrations were used to treat rhd6 rsl1,the number of root hairs and hair length both increased (Fig. 2). Nevertheless, large portions of the root epidermal regions of Fig. 5. ET promotes root hair initiation through EIN3/EIL1 and RHD6/RSL1. rhd6 rsl1 remained hairless (Fig. 5 A and B), suggesting that the (A) Representative roots show hair bulge formation induced by 2 μMACCin effect of ET on root hair initiation partially depends on the pres- 6-d-old hairless mutants. (Scale bar: 200 μm.) (B) Root hair/bulge percentage in ence of RHD6/RSL1. In rsl4 rsl2, all H positions formed bulges H cells. The percentage was calculated based on bulge and hair numbers per upon ACC treatment (Fig. 5 A and B). However, the lack of RSL4/ H cell. Two hundred cells per sample were counted. ee, ein3 eil1; ee42, RSL2 ledtotip-growthfailureofthehairbulges,andnolength- 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 bi- measurable hair was observed (Figs. 2 and 5). We further examined ’ hair initiation in ein3 eil1 rhd6 rsl1 and ein3 eil1 rsl4 rsl2. Neither ological replicates. Statistical significance was determined by a Student s t test (***P < 0.001). (C) Summary diagram for the identification of root hair initia- bulge formation nor hair growth was found even in the presence of tion genes. RNA sequencing and differential expression analysis were per- ACC, illustrating the importance of EIN3/EIL1 and RHD6/RSL1 formed using roots of Col-0, ee, 61,andee61 after air or ET treatment. Among in ET-induced root hair initiation. 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 Identification of Genes Coregulated by EIN3/EIL1 and RHD6/RSL1 in hair genes based on coexpression analysis (Fig. S5).Theseninegenesplus Root Hair Initiation. For genome-wide analysis of EIN3/EIL1 and 18 non–RSL4-regulated H genes comprise the group of 27 hair initiation can- RHD6/RSL1 interaction, transcriptome profiles of Col-0, ein3 eil1, didate genes. EPGs, ET-promoted genes.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1711723115 Feng et al. Downloaded by guest on September 28, 2021 187 genes revealed compact clustering of previously published root hair genes, including core root epidermal genes (10), RSL4- dependent genes (12, 32), and H cell-enriched genes (33) (Fig. S5). Although nine genes within the compact cluster were not found in previous studies, they may nevertheless participate in root hair development considering the similarity of their expression patterns to those of other root hair genes. These nine genes and the 18 non–RSL4-regulated H genes comprised a candidate pool of 27 putative downstream target genes of RHD6/RSL1 and EIN3/ EIL1 involved in root hair initiation (Table S1). More analysis details are provided in SI Identification of Genes Coregulated by EIN3/EIL1 and RHD6/RSL1 in Root Hair Initiation.

EIN3/EIL1 and RHD6/RSL1 Mediate Diverse Root Hair Stimuli. Tran- scriptome profiling revealed a more general role of EIN3/EIL1 and RHD6/RSL1 as gene expression coregulators during root hair formation. RSL4, the target gene coactivated by EIN3 and RHD6, is required for several root hair-inducing signals, including nutrient deficiency and hormone application (12, 14, 15). We therefore assessed whether EIN3/EIL1 and RHD6/RSL1 mediate the effects of these stimuli. Four root hair stimuli, application of auxin or cytokinin and depletion of phosphorus or nitrogen, were used to assess the response in rhd6 rsl1, ein3 eil1,andein3 eil1 rhd6 rsl1. The rhd6 rsl1 was responsive to both hormone treatments but not to nutrient deficiency (Fig. 6 A–D and Fig. S7), suggesting that RHD6/RSL1 are essential for mediating the effects of nutrient depletion but not hormone application. The ein3 eil1 was responsive to all four stimuli, although root hair length in the mutant was relatively shorter than that of wild type under all conditions PLANT BIOLOGY (Fig. 6 A–D). The ein3 eil1 rhd6 rsl1 quadruple mutant was insensitive to all four stimuli and had no visible root hairs (Fig. 6 A–D and Fig. S7), highlighting the importance of EIN3/EIL1 and RHD6/RSL1 coordination in response to these treatments. Discussion Research over the past few decades has clearly established the importance of ET in regulating plant root hair development. However, the underlying molecular mechanisms were poorly understood. In this study, we found that ET promotes root hair formation at both the initiation and elongation stages. Moreover, the Fig. 6. Relative root hair length of Col-0, ein3 eil1(ee), rhd6 rsl1(61),and master regulators EIN3/EIL1 mediate the effects of ET at both ein3 eil1 rhd6 rsl1(ee61) under the following stimuli: auxin [indole-3-acetic acid stages. EIN3 directly binds the promoter region of the hair length- (IAA)] (A), cytokinin [6-benzylaminopurine (6-BA)] (B), nitrogen (N) deficiency determining gene RSL4 and activates its transcription to promote (C), and phosphorus (P) deficiency (D). The length of Col-0 root hair was cali- root hair elongation. EIN3 also physically interacts with RHD6, brated as 1.0. Data are means ± SD (n = 10 roots). The values were compared between treated and untreated samples within the same genotype. Statistical another essential regulator of root hair development upstream of ’ < RSL4. EIN3/EIL1 and RHD6/RSL1 function in parallel and syn- significance was determined by a Student s t test (***P 0.001). ND, not de- ergistically as positive regulators of root hair initiation and elon- tectable. (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/ gation. Based on these findings, we propose the following model for EIL1 interact with RHD6/RSL1 (R6; yellow hexagons) in H cells to directly activate ET-induced root hair growth. In wild-type roots where EIN3/ the transcription of RSL4, an essential positive regulator of root hair tip growth, EIL1 levels are low, RHD6/RSL1 are mainly responsible for the as well as a set of genes involved in hair initiation. (Top Left)Intheabsenceof induction of RSL4 and root hair initiation genes to maintain normal ET, root hair initiation and elongation are mainly regulated by R6, with root hair growth. Upon ET treatment, EIN3/EIL1 accumulate and E3 playing a minor role. (Bottom Right)Intherhd6 rsl1 mutant, ET-induced and complex with RHD6/RSL1 to synergistically activate the expression stabilized E3 independently promotes hair initiation and elongation to partially of hair initiation genes as well as RSL4 to potently increase hair restore hair growth of the hairless mutant. (Bottom Left)Intheabsenceofboth growth. In rhd6 rsl1, ET-induced EIN3/EIL1 act independently to ET-induced E3 and functional R6, the hairless phenotype is observed. partially rescue the hair initiation and elongation defects (Fig. 6E). ET, a widely documented stress hormone, helps plants adapt to various environmental challenges. In the proposed model, the initiation (34–38). The ein3 eil1 rhd6 rsl1 remains hairless in the ET signal is integrated with the internal root hair development presence of auxin applications (Fig. 6A and Fig. S7 A and B), re- pathway, with EIN3/EIL1 conferring stress responsiveness to the vealing that ET does not promote root hair initiation and elongation EIN3–RHD6 transcription complex. Meanwhile, due to its strict simply through increasing auxin biosynthesis. Other effects on pro- expression pattern in H cells, RHD6 confers spatiotemporal speci- moting hair formation by ET–auxin interplay, including trans- ficity to the complex. In this way, different internal and external portation and signaling, still need further investigation. Similar to signals converge at key nodes through associated transcription fac- EIN3, ARF5 is also a direct regulator of RSL4 in mediating auxin- tors that provide flexibility and adaptability to ever-changing envi- promoted polar growth (14). Combining findings in this study, RSL4 ronments. Consistent with this hypothesis, the simultaneous loss promoter is suggested to be a direct, central point of convergence for of both EIN3/EIL1 and RHD6/RSL1 led to virtual insensitivity to auxin signaling via ARF5 and for ET signaling via EIN3, and that it various root hair-inducing signals, underscoring the central role of is regulated by the major hair cell differentiation regulator RHD6. the EIN3/EIL1–RHD6/RSL1 transcription complex in signaling in- In addition to RSL4, EIN3/EIL1 and RHD6/RSL1 coregulate a tegration. Notably, ET signaling is known to act upstream of auxin subset of genes that likely contribute to root hair initiation and biosynthesis in the root tip on cell elongation and polar root hair elongation. Of the 27 candidate root hair initiation genes identified

Feng et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 in this study, some have been reported to participate in root hair enhance their respective DNA-binding ability. Third, RHD6 may formation. For example, overexpression of ROOT HAIR SPECIFIC adopt a de-repression mechanism by competing with EIN3-associ- 3 (RSH3) leads to spiral, bent, and branched hair morphologies ating repressors in H cells, and RHD6 interaction could release (39). LEUCINE-RICH REPEAT/EXTENSIN 1 (LRX1) encodes EIN3 from an otherwise repressed state. Further investigation of a chimeric leucine-rich repeat/extensin protein, and the lrx1 mutant these alternative mechanisms is needed to fully understand the ac- exhibits aberrant root hair formation, including aborted, swollen, tion and importance of the EIN3–RHD6 complex. and branched hairs (40). Lotus japonicus (Lj)RHL1-LIKE 3 (LRL3), encoding a bHLH subfamily XI protein, rescues the Materials and Methods hairless defect of Ljrhl1 mutants (41). Plant Materials. The A. thaliana mutant ein3 eil1 and transgenic plant As master regulators, EIN3/EIL1 associate with a group of tran- iEIN3 ein3 eil1 ebf1 ebf2 and iEIN3 ein3 eil1 were described previously (43, 44). scriptional regulators in hormone cross-talk. All EIN3-associated The rhd6 rsl1 and rsl4 rsl2 double mutants and transgenic pRHD6::GFP:RHD6 proteins reported thus far repress its biological function (27, 42, 43). and RSL4ox plants were gifts from Liam Dolan, University of Oxford, Oxford, Intriguingly, this study uncovered a class of EIN3-associated tran- United Kingdom. Details about plant growth conditions and treatments are scription regulators that enhance its activity, although how this en- described in SI Materials and Methods. Plant transformation, root hair length hancement is achieved is unclear. Several possibilities can be measurement, gene expression, ChIP-qPCR, EMSA, yeast two-hybrid assay, pull- considered. First, RHD6 may act as a positive regulator that down analysis, LCI, dual-luciferase reporter assay, and sequencing analysis were enhances EIN3 transcription activity. Although RHD6 directly carried out according to protocols described in SI Materials and Methods. regulates RSL4 expression, no DNA-binding ability has been demonstrated. RHD6 may be recruited by other DNA-binding ACKNOWLEDGMENTS. We thank Liam Dolan (University of Oxford) for factors, such as EIN3, to the RSL4 promoter. Second, RHD6 may sharing several plant materials. This work was supported by the National directly bind to a specific DNA sequence in the RSL4 promoter, Natural Science Foundation of China (Grant 91017010), the Peking-Tsinghua while EIN3 binds to the EBS motif. In turn, the association between Center for Life Sciences, and start-up funding from the Southern University of the two classes of transcription factors could mutually and greatly Science and Technology (to H.G.).

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