Title: A peptide that regulates metalation and function of the Arabidopsis 1 ethylene receptor

Abstract

is localized to the ER (12), and ER localization of a proPLS::PLS:GFP fusion protein was 114 confirmed by co-localization with the ER marker dye ER-Tracker TM (Fig. 2a-c) and with an ER-  proPLS::PLS:GFP also appears to localize to the nucleus (Fig. 2). As controls, free GFP protein 117 expressed under the control of the PLS promoter is not co-localized to the ER (Fig. 2d-f) and, as 118 expected, the Golgi marker SH:GFP does not co-localize with ER Tracker (Fig. 2m-o). Trans- 119 Golgi-localized SULFOTRANSFERASE1 (ST1) mCherry (21) show proPLS::PLS:GFP does 120 not localize to the Golgi (Fig. 2j-l). To further clarify the side of the ER membrane on which PLS 121 localizes, transient expression of redox-sensitive GFP (roGFP2) fusions of PLS were carried out. 122 The different excitation properties of roGFP2 in an oxidizing (ER lumen) or reducing 123 environment (cytosol) allows discrimination of the precise location of PLS. Ratiometric analysis 124 and comparison with proteins of known localization revealed that PLS resides at the cytosolic 125 side of the ER and is not localized in the ER lumen (Fig. 2p).

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PLS interacts with the ethylene receptor protein ETR1 128 We hypothesized that PLS plays a role in receptor function and investigated whether this 129 involved direct interaction with the receptor complex. Preliminary experiments using yeast 2-130 hybrid analysis suggested that PLS interacts with ETR1 (Fig. S4). Confirmation of the physical 131 interaction between PLS and ETR1 in plants came from co-immunoprecipitation (Co-IP) analysis. 132 Agrobacterium containing a plasmid encoding PLS linked to a C-terminal GFP and ETR1 with 133 a C-terminal HA tag was infiltrated into Nicotiana benthamiana leaves for transient expression. 134 After 3 d, interaction was confirmed by western blotting after Co-IP with either anti-GFP beads 135 (showing PLS pulls down ETR1) or anti-HA beads (showing ETR1 pulls down PLS) (Fig. 3a).   (Fig. 3a, b). 143 To investigate the specificity of PLS binding, synthetic full length PLS peptide PLS(FL) was 144 introduced into the infiltrated N. benthamiana leaves 30 min before the tissue was harvested. The 145 addition of 25 nM synthetic PLS caused a ca. 80% reduction in PLS-GFP binding to ETR1-HA 146 (Fig. 3c, d), suggesting that the synthetic PLS peptide competes for ETR1 binding, and showing 147 the specificity of PLS for ETR1. Interestingly, the anti-GFP beads bound two sizes of PLS-GFP 148 protein (Fig. 3e), both of which were larger than a GFP-only control, suggesting that the PLS 149 peptide undergoes cleavage, a change in conformation, post-translational modification or 150 incomplete reduction of Cys residues on some PLS. When using ETR1-HA to pull down PLS-151 GFP, only the larger peptide was present (Fig. 3e, f), suggesting that ETR1 binds the full length 152 PLS peptide, but that PLS may be modified after ETR1 binding. 153 To pinpoint the interaction site at the receptor in more detail, binding studies were performed 154 with purified receptor variants and PLS by microscale thermophoresis (MST; Fig. 3g Cysteine residues are common metal-ligand binding residues in low molecular weight copper-162 handling peptides, and predictions of PLS structure suggests a single a-helix plus unstructured 163 region with two cysteines (CX10C arrangement where X is any amino acid), with some analogy 164 to copper-metallochaperones such as Cox17 or other CX9C twin proteins (23). In view of both 165 structural considerations and the copper-dependency of ETR1 we determined whether the two 166 cysteine residues (C6 and C17) in PLS play a functional role, such as in copper binding. A 167 mutated Arabidopsis full-length peptide in which both cysteines were replaced with serine, 168 PLS(FL C6S, C17S), was non-functional in root feeding assays (Fig. 4a), indicating the 169 importance of the cysteine residues for biological activity. To determine a possible role for PLS 170 in binding Cu(I), we first grew pls mutants and wildtype seedlings in the presence of the copper 171 chelator bathocuproine disulphonic acid (BCS), which is often used to deplete copper but notably 172 solubilises and stabilises Cu(I). Treatment of pls and wildtype seedlings with 10 μM and 50 μM 173 BCS led to an increased primary root length of both genotypes, while 100 μM BCS led to 174 significantly enhanced root growth in the pls mutant compared with wildtype (Fig. 4b); and 175 rescue of the overexpression of ethylene-responsive genes ( Fig. S5; 19). This suggests that the 176 availability of Cu(I) is growth-limiting in the pls mutant through abnormal ethylene perception.

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Metal binding in biology is challenging to predict because the formation of metal-protein 187 complexes is a combined function of metal affinity for a given protein and metal availability, 188 which would need to be known for Cu(I) in the Arabidopsis cytosol in the case of PLS (Fig. 2p).

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Cu(I) occupancy of the cytosolic copper chaperone ANTIOXIDANT PROTEIN 1 (ATX1) tracks 190 with fluctuations in available cytosolic Cu(I) such that its affinity approximates to the mid-point 191 of the range of Cu(I) availabilities within this eukaryotic compartment (25,26). Arabidopsis 192 ATX1 was therefore expressed and purified to determine a 1:1 ATX1:Cu(I) stoichiometry and 193 affinity KD Cu(I) of 5.47 (±0.6) x10 -18 M (Fig. 4f inset, Fig. S10). Figure 4f     were performed for 10 minutes at 75°C at 25W power. Cys and His residues were coupled at low 277 temperature (10 minutes at room temperature followed by 10 minutes at 50°C, 25W). Arg   (19).

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For RT-qPCR, RNA was extracted from 7 day-old seedlings (3 biological replicates, 20 mg of 336 tissue) as described (32). Total mRNA was extracted using Dynabeads®mRNA DIRECT™kit 337 with Oligo(dT)25 labelled magnetic beads. cDNA was prepared using a SuperScript®IV First-338 Strand synthesis system. Samples were checked for the presence of genomic DNA by PCR with 339 ACTIN2 primers ACT2 forward and reverse. Primer sequences were determined using Primer-340 BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Primers are listed in Table S2.

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Protein-protein interaction studies 343 Yeast 2-hybrid 344 The GAL4 two-hybrid phagemid vector system was used to detect protein-protein interactions 345 in vivo in yeast, using the reporter genes β-galactosidase (lacZ) and histidine (HIS3) in the YRG-346 2 yeast strain, essentially as described (34). DNA that encodes the target (ETR1) and bait (PLS)  382 BiFC was carried out essentially as described previously (35). Full-length Arabidopsis ETR1 and 383 PLS cDNA sequences were cloned respectively into the vectors pDH51-GWYFPn (AM779183, 384 to form ETR1-YFPn) and pDH51-GW-YFPc (AM779184, to form PLS-YFPc), and the CTR1 385 cDNA was cloned into pDH51-GW-YFPc (CTR1c), as a control for ETR1 interactions. Intact 386 YFP plasmid was also used as a positive control and YFPc alone was used as a negative control 387 as described previously (34). Plasmids were kindly provided by Prof. Don Grierson, University   Additionally, liquid cultures of GV3101 containing the p19 protein that is encoded by the tomato 411 bushy plant virus were also prepared in order to suppress post-transcriptional gene silencing (36).

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The overnight cultures were grown until an OD600 of approximately 0.6 was reached, and then   with 50 μl GFP beads or HA beads and incubated for 30 minutes at 4°C, mixing every 2 minutes.

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The mixture was centrifuged at 2500 x g for 2 min at 4°C, washed twice with 500 μl ice-cold 445 dilution buffer, and the beads were transferred to a new microcentrifuge tube. The target protein 446 was eluted with the addition of 100 μl 2x SDS sample buffer (120 mM Tris pH 6.8, 50 mM 4% 447 (w/v) SDS, 20% (v/v) glycerol) and the sample was boiled for 10 minutes at 95°C to dissociate 448 immunocomplexes from the beads. The mixture was centrifuged for at 2500 x g for 2 minutes at 449 4°C to separate the beads, and the supernatant was transferred to a new microcentrifuge tube.

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The supernatant was used in SDS-PAGE analysis.      Table S1, expression data 572 on Dryad (19). For protein-protein interaction and protein localization studies, assays were 573 carried out independently between 2 and 10 times (Figs. 2, 3 legends). At least three biological 574 replicates were used for plant growth assays, RNA-seq and gene expression (RT-qPCR) 575 experiments, metal binding assays (Fig. 4). Normalised values from at least three biological