2 n morpholino ethanesulfonic acid mes

All commercially available chemicals were
used without further purification. BaseMuncher endonuclease was purchased
from AbCam. Ampicillin, dithiothreitol (DTT), and isopropyl-β-D-1-thiogalactopyranoside (IPTG) were purchased from Formedium. Escherichia coli DH5α (high efficiency) and
BL21(DE3) cells and Gibson Assembly Cloning Kit were purchased from
New England Biolabs. Ethylenediaminetetraacetic acid (EDTA)-free Complete
protease inhibitor cocktail was purchased from Roche. Deuterium oxide
(D₂O), sodium deuteroxide, 2-(N-morpholino)ethanesulfonic
acid (MES), N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic
acid (TAPS), glycerol, imidazole, lysozyme, NiCl₂, tris(hydroxymethyl)aminomethane
(Tris), polyethylene glycol 8000 (PEG-8000), and dUMP were purchased
from Merck. Agarose, dNTPs, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic
acid (HEPES), NaCl, and Phusion high-fidelity polymerase were purchased
from ThermoFisher Scientific. Tobacco etch virus protease (TEVP) was
produced as previously described.^{11 (link)}

The compounds α-lipoic acid, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ethanolamine, 2-(N-morpholino)ethanesulfonic acid (MES), saline phosphate buffer 10 mM with pH 7.4 (PBS), estradiol, Glycine, and sodium chloride were purchased from Merck-Sigma (Darmstadt, Germany). Recombinant Human Estrogen Receptor alpha protein (ERα) (ab82606 abcam) was purchased from Abcam (Cambridge, UK).

NAA membranes of 200 nm pore diameter (Whatman Anodisc Circles 13 mm, 200 nm) were purchased from Interpath services (Australia). Carboxyl-terminated MNPs (100 nm diameter, 25 mg/ml, fluidMAG-ARA) were purchased from Chemicell (Germany). Potassium ferrocyanide (K₄[Fe(CN)₆]), potassium ferricyanide (K₃[Fe(CN)₆]), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl) N′-ethylcarbodiimide hydrochloride (EDC), ethanolamine, phosphate buffered saline (PBS) tablets, 3-(triethoxysilyl) propyl isocyanate (ICN) silane, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate, and 2-(N-morpholino)-ethanesulfonic acid (MES) were purchased from Sigma-Aldrich (Australia).
Polyclonal antibodies against KLH produced in rabbit and anti-human IgG antibodies (used as control) were purchased from Sapphire Bioscience (NBP1-30443, Australia). Monoclonal antibodies raised in mouse against the N-terminus of the LRR domain of the human Flii protein (FnAb) were developed and supplied by Cowin group (Jackson et al., 2012 (link)). Custom made KLH-Flii peptide conjugate was purchased from Mimotopes (Australia).

Arabidopsis seeds (Col-0, DR5rev:GFP, DR5:GUS, WOX5:GFP, YUC2:GUS, PIN1:PIN1-GFP, coi1–40, myc2/3/4 and npr1–1) were surface sterilised. Seeds were then plated on MS medium (Sigma) supplemented with 0.05% 2-(N-morpholino) ethanesulfonic acid (MES, Sigma), 1% sucrose and 1% technical agar and amended with chitosan. Plates with no chitosan were used as controls. Seed were placed at 4 °C for 48 h in the dark to synchronise germination. They were then incubated at 21 °C and 65% relative humidity (RH) with continuous light for 21d upright to allow root elongation. Every two days plants were checked for secondary root and bolting emergence in order to identify flowering time. At harvest time, total plant weight, root weight and length and number of leaves in the rosette per plant and treatment were scored. This experiment was performed in triplicate.
Intracellular hydroxyl, peroxyl and other reactive oxygen species were detected using 2′-7′ dichlorodihydrofluorescein diacetate (H2DCFDA; Sigma, St. Louis, MO, USA). Arabidopsis roots were exposed to 1 mg ml⁻¹ chitosan for 2 h and then were dipped in H2DCFDA. Finally, we observed ROS in roots by confocal microscopy with a 490 nm excitation and a 535 nm emission filters. Untreated control roots were incorporated to evaluate the ROS levels in standard conditions.