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 P_1B-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 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 log₂fold change (log₂FC) 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)₂₅ 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 ACTIN2 primers ACT2 forward and reverse. Primer sequences were determined using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).