Salicylate

Soil pH was determined using a fresh soil to water ratio of 1:5 using a pH monitor (Thermo 0rion-868, MA, USA). Soil moisture was measured gravimetrically after a 16-h desiccation at 105 °C. Soil samples for C and N analyses were air dried (2 mm mesh), handpicked to remove plant litter and fine roots, and ground. Total soil C and N content for each plot were determined by combustion (2400 II CHNS/0 Elemental l Analyzer, Perkin-Elmer, Boston, MA, USA). Dissolved organic carbon (DOC) and dissolved total nitrogen (DTN) were extracted by adding 50 ml of 0.5 MK₂SO₄ to 10 g fresh soil, shaking for 1 h, and vacuum filtering through a G4 glass fiber filter with a pore space of 1.2 μm (Fisher). DOC and DTN were determined using a total organic carbon-total nitrogen (TOC-TN) analyzer (Shimadzu, Kyoto, Japan). Ammonium (NH₄⁺) and nitrate (NO₃⁻) concentrations in extracts were assessed colorimetrically by automated segmented flow analysis (AAIII; Bran and Luebbe, Germany) using the salicylate/dichloroisocyanuric acid and cadmium column/sulfanilamide reduction methods, respectively. Through HF and HClO₄ digestion, total potassium (TK) was determined by flame photometry (FP640, INASA, China), while total phosphorus (TP) was determined using the molybdenum blue method. Available potassium (AK) was determined in 1 M ammonium acetate extracts by flame photometry (FP640, INASA, China). Soil available phosphorus (AP) was extracted by 0.5 M NaHCO₃ and determined using the molybdenum blue method. Organic carbon was determined according to potassium dichromate oxidation titration. Soil electric conductivity was determined by a conductivity monitor using a dry soil to water ratio of 1:5 (Thermo 0rion-868, MA, USA). Soil samples were air dried and homogenized by grinding in an agate mortar and then passed through a 0.149 mm sieve to analyze the elements. These samples (~ 0.4–0.5 ± 0.0001 g) were digested with nitric acid (HNO₃), hydrofluoric acid (HF), and perchloric acid (HClO₄) (5 mL: 10 mL: 5 mL) on a hot plate. Soil total Mg, Ca, K, and Fe were measured with an ICP-AES Optima 8000 (Perkin-Elmer, Waltham, MA, USA), while total Cd, chromium(Cr), Mn, copper(Cu), Zn, plumbum (Pb), and As were measured with an HPLC-ICP-MS (7700X, Agilent, USA). A certified soil reference material (GBW07408, National Research Center for Certified Reference Materaials, China) were used to ensure that the accuracy of the analytical data and the accuracy ranged from 93.9 to 107.4%. All soil variables are described in Additional file 2: Table S9.

Hyperoside (98%), astragalin (98%), avicularin (98%), azaleatin (98%), quercetin (98%), dexamethasone (DEX) and kaempferol (98%) were purchased from Shanghai Macklin Inc (Shanghai, China); paeoniflorin (98%) was purchased from Shanghai Acmec Biochemical Co. Ltd. (Shanghai, China) (+)-catechin (95%) and vanillin (95%) was purchased from Shanghai Yuanye Biochemical Co.. Ltd. (Shanghai, China); 1,1-diphenyl-2-picrylhydrazyl (DPPH), lipopolysaccharide (LPS), nitroblue tetrazolium (NBT), nicotinamide adenine dinucleotide disodium salt (NADH-2Na), and phenazine methosulfate (PMS) were purchased from Sigma-Aldrich (St. Louis, MO, United States); and ethylenediaminetetraacetic acid (EDTA), ascorbic acid (Vc), ferrous sulfate (FeSO₄), salicylate, ferrous chloride (FeCl₂), hydrogen peroxide (H₂O₂), petroleum ether, ethyl acetate, trichloroacetic acid (TCA), thiobarbituric acid (TBA) and acetic acid were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Ethanol (analytical grade) was purchased from Tianjin Guangfu Chemical Reagent Company (Tianjin, China); ketoconazole and azoxystrobin were purchased from Solarbio Co. Ltd (Beijing, China); and acetonitrile (HPLC and MS grade) was purchased from Fisher Scientific (England).

Arabidopsis stems of 5‐week‐old wild‐type and T3 homozygous C4H::qsuB lines were collected in liquid nitrogen and stored at −80 °C until further utilization. Prior the metabolite extraction, collected stems were pulverized in liquid nitrogen. For extraction of methanol‐soluble metabolites, 700–1000 mg of frozen stem powder was mixed with 2 mL of 80% (v/v) methanol–water and mixed (1400 rpm) for 15 min at 70 °C. This step was repeated four times. Pooled extracts were cleared by centrifugation (5 min, 20 000 g, at room temperature), mixed with 4 mL of analytical grade water and filtered using Amicon Ultra centrifugal filters (10 000 Da MW cut‐off regenerated cellulose membrane; EMD Millipore, Billerica, MA, USA). Filtered extracts were lyophilized and the resulting pellets dissolved in 200 μL 50% (v/v) methanol–water prior to LC‐MS analysis. An acid hydrolysis of the samples was performed for the quantification of protocatechuate and salicylate; an aliquot of the filtered extracts was dried under vacuum, resuspended with 1 N HCl and incubated at 95 °C for 3 h. The mixture was subjected to three ethyl acetate partitioning steps. Ethyl acetate fractions were pooled, dried in vacuo and resuspended in 50% (v/v) methanol–water prior to LC‐MS analysis.

Soil pH was measured in slurry with a soil-to-water ratio of 1:2 (10 g of soil in 20 ml deionized water) after shaking for 1 h at room temperature. Soil moisture was determined by calculating weight loss after drying 20 g fresh soil at 105°C for 24 h. Soil ammonium and nitrate were extracted with 2 M KCl by mixing 5 g fresh soil with 45 ml of KCl solution, followed by homogenization on a shaker at 50 rpm for 1 h and centrifuge at 3,000 g for 10 min. The supernatant was filtered through a 0.45 μm Nylon filter and the ammonium and nitrate concentrations were then determined colorimetrically following salicylate method (Bower et al., 1980 (link)) and VCl₃-Griess method (García-Robledo et al., 2014 (link)), respectively. Total phosphorus (TP) and available phosphorus (AP) concentrations were measured using a continuous segmented flow analyzer (Auto Analyzer 3, Seal Analytical Inc., Mequon, WI, United States) following the EPA method 365.1 (Determination of Phosphorus by Semi-Automated Colorimetry, Revision 2.0). Soil organic matter (OM) content was measured by calculating loss on ignition and dissolved organic carbon (DOC) content was measured using a TOC analyzer (Shimadzu, Kyoto, Japan).