Qtrap 5500

The LC-MS/MS system was composed of Agilent 1200 HPLC (Agilent Technologies, Waldbronn, Germany) and a QTRAP5500 tandem mass spectrometer. The compounds were separated in a Zorbax Eclipse XDB-C₁₈ column (4.6 × 150 mm, 5 μm) (Agilent Technologies, Santa Clara, CA, USA). Gradient elution (0.6 mL·min^–1) was performed with the same mobile phases as in the preparative analysis. The turbo ion spray operated at 550 °C; voltage, 5.5 kV; nebuliser gas pressure, 60 psi; curtain gas pressure, 20 psi. To determine the content of the samples, an IDA (information-dependent acquisition) mode was used, and ions within the m/z range 500–1250 and intensity higher than 5 × 10⁵ cps were fragmented. The assessment of the relative content of APs in the extract was performed in multiple reaction monitoring mode (MRM). The following transitions were monitored: 807→402, 231, 120 for anabaenopeptin AP806Ne (m/z 807); 821→448, 248, 120 for AP820Ne (m/z 821); 835→448, 248, 120 for AP SA6 (m/z 835); and 837→448, 248, 120 for AP836Ne (m/z 837). The collision energy was 60 eV, and the dwell time was 100 msek.

Physicochemical (nutrient contents and pH) and biological (enzymatic activities and concentrations of IAA and Trp) variables were measured in the dissolved phase of the soil samples. After three generations of Arabidopsis cultures (Fig. 1a), soil samples were then collected randomly from each pot, air-dried, homogenized, and sieved to obtain particles < 1 mm. The activity of soil enzymes (β-glucosidase, chitinase, urease, and nitrate reductase) and soil N content (NH₄⁺ and NO₃⁻ contents) were subsequently measured using 0.5 g of soil (n = 4) following the manufacturer’s instructions of corresponding commercial reagent kits (COMIN, Suzhou, China). The concentrations of two key metabolites (IAA and Trp) were measured in the soil dissolved phase. Five grams of soil was mixed with 5 g of NaCl and 20 mL of acetonitrile for 5 min, followed by centrifugation at 1000g for 10 min. The concentrations of IAA and Trp in the supernatants were measured by Liquid Chromatography-Electrospray Ionization-Mass/Mass Spectroscopy (LC-ESI-MS/MS, Q-Trap 5500, Agilent Technologies, USA).

Quantifications of phytohormone levels were conducted according to a method described previously (Wu et al., 2007 (link)). In brief, at 2 d and 4 d post infiltration, approximately 500 mg infiltrated leaves from 10 individual plants with the same treatment were harvested and pooled to generate one biological replicate. Samples were homogenized in 1 mL acetonitrile (1% famic acid). Four nanogram of D4-SA, D6-JA, 1 ng JA-Ile-D6 to were added to the aqueous extraction solvent as internal standards. All samples were then vortexed for 2 min and centrifuged at 14,000 rpm for 10 min at 4°C. Eight hundred microlitres of supernatants was collected and evaporated to dryness using a vacuum concentrator to dry. Residues were resuspended in 200 µL of acetonitrile: H₂O (1:1, v/v) and centrifuged at 14,000 g for 10 min. The supernatants were then collected and analyzed with a high-performance liquid chromatography-tandem mass spectrometry system (Agilent 1290 Infinity LC, QTRAP 5500, AB SCIES). Wheat leaves incubated with the pEDV6 empty vector were used as control groups. There were five biological replicates for each treatment and time point.

As in our previous studies^{7 (link),108 (link)–112 (link)}, fecal SCFAs were measured using LC–MS/MS at the University of Illinois-Chicago Mass Spectrometry Core using previously published methods^{113 (link),114 (link)}. The LC–MS/MS analysis was completed on an AB Sciex Qtrap 5500 coupled to the Agilent UPLC/HPLC system. All samples were analyzed by Agilent poroshell 120 EC-C18 Column, 100 Å, 2.7 µm, 2.1 mm × 100 mm coupled to an Agilent UPLC system, which was operated at a flow rate of 400 µl/min. A gradient of buffer A (H₂0, 0.1% Formic acid) and buffer B (Acetonitrile, 0.1% Formic acid) was applied as 0 min, 30% of buffer B; increase buffer B to 100% in 4 min; maintain B at 100% for 5 min. The column was then equilibrated for 3 min at 30% B between the injections with the MS detection in negative mode. The MRM transitions of all targeted compounds include the precursor ions and the signature production ion. Unit resolution is used for both analyzers Q1 and Q3. The MS parameters, such as declustering potential, collision energy, and collision cell exit potential, are optimized in order to achieve optimal sensitivity. SCFAs are presented as individual SCFAs (μg/g), including butyric acid, propionic acid, acetic acid, and valeric acid, as well as total SCFAs (sum of 4).