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

Root hairs are an extensive structure of root epidermal cells and are critical for nutrient acquisition, soil anchorage, and environ- mental 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 en- hance root hair initiation by selectively regulating a subset of core root hair (H) 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 in- trinsic regulators in modulating plant organ development. Overall design: Col-0, ein3 eil1, rhd6 rsl1 and ein3 eil1 rhd6 rsl1 were grown vertically on MS medium for 6 d and then treated with or without ET at 10 ppm for 4 h. Two biological replicates were prepared for each condition. Library preparation and high-throughput sequencing were conducted in accordance with the manufacturers’ instructions. Root total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen). Sequencing libraries were prepared using the TruSeq mRNA preparation kit (Illumina). RNA quality and library quality were examined by a Bioanalyzer 2100 instrument (Agilent), and paired-end, 100-bp deep sequencing was per-formed on an Illumina HiSeq 2000 platform.

根毛是固着生长植物根表皮细胞的延伸结构，对养分吸收、土壤锚定与环境互作至关重要。植物激素乙烯（ethylene, ET）可促进根毛生长，同时介导多种刺激根毛细胞发育的信号通路的调控效应。然而，乙烯诱导根毛生长的分子基础仍未被充分阐明。

本研究发现，乙烯激活的转录因子乙烯不敏感3（ETHYLENE-INSENSITIVE 3, EIN3）可与已被广泛验证的根毛细胞正向调控因子根毛缺陷6（ROOT HAIR DEFECTIVE 6, RHD6）发生物理互作，且二者可直接协同激活根毛长度决定基因RHD6同源类似物4（RHD6-LIKE 4, RSL4），从而促进根毛伸长。转录组分析进一步揭示了调控因子组合EIN3/EIL1（EIN3-LIKE 1, EIN3类似物1）与RHD6/RSL1（RHD6-LIKE 1, RHD6同源类似物1）的平行调控功能。EIN3/EIL1与RHD6/RSL1可通过选择性调控一组核心根毛（H）基因，协同增强根毛起始过程。综上，本研究揭示了由EIN3/EIL1与RHD6/RSL1组成的关键转录复合物，其可调控根毛的起始与伸长过程，并为整合环境信号与内源调控因子以调控植物器官发育提供了分子框架。

总体实验设计：将野生型Col-0、ein3 eil1双突变体、rhd6 rsl1双突变体以及ein3 eil1 rhd6 rsl1四突变体在MS培养基上垂直培养6天，随后用10 ppm的乙烯处理或不做处理，处理时长为4小时。每个实验条件设置两个生物学重复。文库构建与高通量测序严格按照厂商说明书操作。采用RNeasy Plant Mini Kit (Qiagen)提取根组织总RNA。使用TruSeq mRNA建库试剂盒（Illumina）构建测序文库。通过Agilent 2100生物分析仪检测RNA与文库质量，随后在Illumina HiSeq 2000测序平台上进行100 bp双端深度测序。