DOI: 10.5061/dryad.wpzgmsbss
title: RNA-seq data showing PLS role in gene expression control in Arabidopsis

description: 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.
description: 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 using a
 TapeStation 2200 (Agilent
 Technology) with High Sensitivity RNA ScreenTape
 (5067-5579). mRNA was
 fragmented into 120-200 bp sequences with a median size of 150 bp, and
 used as template to synthesize first strand cDNA using reverse
 transcriptase and random primers, followed by second strand cDNA synthesis
 with DNA Polymerase I and RNase H. Newly synthesized cDNA had a single
 adenine base added with ligation of adaptors, before being purified and
 amplified by PCR to make the final library. Library quality control was
 carried out again using a TapeStation with D1000 ScreenTape (catalog number
 5067-5582). RNA-seq data were processed and aligned against
 the TAIR10 (EnsemblePlants) genome using TopHat and indexed with Samtools.
 DeSeq determined differential expression. A padj-value of  ≤ 0.05 and a
 log<sub>2</sub>fold change (log<sub>2</sub>FC) of
 ≥ 0.5 were selected to identify differentially expressed genes
 (DEGs).             
 For RT-qPCR, RNA was extracted from 7 day-old seedlings (3
 biological replicates, 20 mg of tissue) as described (Thompson et al. Development
 2023). Total mRNA was extracted using Dynabeads®mRNA
 DIRECT™kit with Oligo(dT)<sub>25</sub> labelled magnetic
 beads. cDNA was prepared using a SuperScript®IV First-Strand synthesis
 system. Samples were checked for the presence of genomic DNA by PCR with
 <em>ACTIN2</em> primers ACT2 forward and reverse. Primer
 sequences were determined using Primer-BLAST
 (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).