RNA-seq data showing PLS role in gene expression control in Arabidopsis

The vital plant ethylene receptor (ETR1) contains an essential Cu(I) ion, and as for other vesicular cupro-proteins, the final step of Cu(I) maturation at the endoplasmic reticulum (ER) is undefined. We previously discovered that mutants in the Arabidopsisgene POLARIS (PLS), encoding a 36 amino acid peptide, exhibit constitutive ethylene signalling responses. Here we report a 1:2 thiol-dependent Cu(I):PLS2 complex, with an affinity of 3.79 (±1.5) x1019 M-2. We demonstrate interactions with the transmembrane domain of ETR1, the Cu(I) chaperones ATX1 and CCH, and Cu(I)-transporting P1B-type ATPase RAN1 at the ER. Formation of Cu(I)-dependent PLS-cuproprotein adducts at the ER provides a mechanism to modulate the metalation of ETR1, thereby regulating its activity and representing a distinct mechanism for plant hormone receptor regulation., RNA was extracted from 7 day-old seedlings grown on half strength MS10 medium using the Sigma-Aldrich Plant Total RNA Kit (STRN50) and the On-Column DNase I Digestion Set (DNASE10-1SET), essentially as described previously (Thompson et al. Development 2023). Tissue was ground in liquid nitrogen before incubation in lysis solution containing 2-mercaptoethanol at 65°C for 3 min. Debris was removed by centrifugation and column filtration and RNA was captured onto a binding column using the supplied binding solution. DNA was removed by wash solutions  and DNase treatment on the column. Purified RNA was eluted using RNAase free water.
       The Illumina HiSeq 2500 System was used for RNA sequencing of three biological replicate samples, with libraries prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Plant Sample Preparation kit (RS-122-2401), essentially as described (Thompson et al. Development 2023). Ribosomal RNA (rRNA) was removed and purified RNA was quality checked...,

植物关键的乙烯受体（ethylene receptor, ETR1）含有必需的一价铜离子（Cu(I)）。与其他囊泡铜蛋白（vesicular cupro-proteins）一样，其在内质网（endoplasmic reticulum, ER）中完成一价铜离子成熟的最后步骤仍未明确。我们此前发现，拟南芥（Arabidopsis）POLARIS（PLS）基因——编码一条36个氨基酸的肽段——的突变体表现出组成型乙烯信号响应。本文报道了一种1:2的巯基依赖型Cu(I):PLS2复合物，其结合亲和力为3.79(±1.5)×10¹⁹ M⁻²。我们证实该复合物可与ETR1的跨膜结构域（transmembrane domain）、一价铜离子伴侣蛋白（Cu(I) chaperones）ATX1和CCH，以及内质网中负责一价铜离子转运的P1B型ATP酶（P1B-type ATPase）RAN1发生相互作用。在内质网中形成依赖Cu(I)的PLS铜蛋白加合物，为调控ETR1的金属化修饰提供了一种机制，进而调节其活性，这代表了一种独特的植物激素受体调控模式。

本研究使用Sigma-Aldrich植物总RNA提取试剂盒（STRN50）与柱上DNase I消化试剂盒（DNASE10-1SET），从培养于1/2 MS10培养基的7日龄幼苗中提取RNA，操作流程基本参照此前发表的方法（Thompson等，Development 2023）。将组织在液氮中研磨后，置于含2-巯基乙醇的裂解液中65℃孵育3分钟。通过离心与柱过滤去除残渣，利用配套结合液将RNA捕获至结合柱中。通过洗涤液及柱上DNase处理去除DNA，最后用无RNA酶水洗脱得到纯化RNA。

本研究采用Illumina HiSeq 2500系统对三份生物学重复样本进行RNA测序，文库构建使用Illumina TruSeq Stranded Total RNA with Ribo-Zero Plant样本制备试剂盒（RS-122-2401），操作流程基本参照此前发表的方法（Thompson等，Development 2023）。实验移除了核糖体RNA（rRNA），并对纯化后的RNA进行质量检测……