Analyst 1

Linear ion trap (LIT) and triple quadrupole (QQQ) scans were obtained using a triple quadrupole-linear ion trap mass spectrometer, the AB Scoex Qtrap^® 6500+ LC-MS/MS System (Sciex, Framingham, MA, USA), equipped with an ESI Turbo Ion-Spray interface. The instrument operated in positive ion mode and was controlled by Analyst 1.6.3 software (Sciex). The ESI source operated with the following parameters: Ion Source: ESI+, Source Temperature: 550 °C, Ion Spray Voltage (IS): 5500 V, Curtain Gas (CUR): Set at 35 psi. Anthocyanins were subjected to analysis using scheduled multiple reaction monitoring (MRM). Data acquisition was conducted through Analyst 1.6.3 software (Sciex). Quantification of all metabolites was performed using Multiquant 3.0.3 software (Sciex). Further optimization of declustering potentials (DP) and collision energies (CE) for individual MRM transitions was carried out. A specific set of MRM transitions was monitored for each period, corresponding to the elution of metabolites during that timeframe.

Aliquots of 10 μL plasma were prepared using derivatization as previously reported [11 ]. For internal standardization, a labeled amino acid standards set (set A, Cambridge Isotope Laboratories) was mixed with L-Asparagine (15N2, 98%, Cambridge Isotope Laboratories) and L-Tryptophan (Indole-D5, 98%, Cambridge Isotope Laboratories) and added to the precipitation reagent. AA butylester were determined by ion-pair liquid chromatography coupled to mass spectrometry detection (LC-MS/MS). 10 μL of the prepared sample were injected into the HPLC system (HPLC 1100, Agilent, Waldbronn, Germany) and chromatographic separation was performed with a XBridge C18 column (Waters GmbH, Eschborn, Germany). MS detection was performed with an API 2000 triple quadrupole instrument (Sciex, Darmstadt, Germany) with an APCI source operating in positive ion ionization mode. Data acquisition on the mass spectrometer was controlled by Analyst 1.6.2 software (AB Sciex, Darmstadt, Germany). Data handling and quantification were also performed with Analyst 1.6.2 software (AB Sciex, Darmstadt, Germany). The sums of non-essential and essential AA were computed in addition to the sum of the branched chain AA (BCAA) leucine, isoleucine and valine.

Phytohormones and sugars were analyzed from 10 mg freeze-dried, ground leaf material of the photosynthesis experiment (n = 6). Phytohormone analysis was carried out on an LC/MS/MS system as previously described (Eberl et al., 2018 (link)). Data were processed using ANALYST 1.5.2 (AB Sciex, Framingham, MA, United States) and hormones were quantified relative to the peak area of their corresponding standard (D₄-salicylic acid and D₆-abscisic acid; Santa Cruz Biotechnology, Dallas, TX, United States). For sugar analysis, extracts were diluted 1:10 with water prior to analysis on an Agilent 1200 HPLC system (Agilent, Santa Clara, CA, United States) coupled to an API 3200 tandem mass spectrometer (AB Sciex). The analytes were separated on an hydrophobic interaction liquid chromatography (HILIC)-column (apHera NH₂ Polymer; 15 × 4.6 mm, 5 μm; Supelco, Bellefonte, PA, United States) with a water/acetonitrile gradient (flow, 1.0 ml min^-1), for more details see Madsen et al. (2015) (link). The data were processed using ANALYST 1.5.2 (AB Sciex) and the compounds were quantified using an external standard curve. For this, a mixture of glucose, fructose and sucrose (Sigma-Aldrich, St. Louis, MO, United States) was analyzed at six different concentrations ranging from 20 to 1.25 μg ml^-1.

The UPLC-MS/MS system consisted of an Agilent 1290 UPLC system (Agilent Technologies, Santa Clara, CA, USA) and a QTrap 5500 tandem mass spectrometer (AB Sciex, Toronto, ON, Canada). The instrument equipped with electrospray ionization (ESI) source and operated using Analyst 1.5.2 software (AB Sciex, Toronto, ON, Canada). Multiple reaction monitoring (MRM) and positive ion mode were used for detection (Tables S4 and S5). A Phenomenex EZfaast C18 column (250 mm × 2.0 mm, 4 μm) and a Waters Atlantis T3 column (150 mm × 2.1 mm, 3 μm) were adopted to determine amino acids and tryptophan metabolites separately with mobile phase A (0.1% formic acid in water, v/v) and B (0.1% formic acid in acetonitrile, v/v). The gradient was shown in Tables S6 and S7. The sample preparation and detection method were adopted according to the previous study [45 ,46 (link)].

An LC-MS/MS system that was equipped with a 1290 HPLC instrument (Agilent Technologies, Glostrup, Denmark), a QTRAP 5500 (ABSciex, Framingham, MA, USA), and a reversed-phase column (Pursuit 5 C18 150 × 2.0 mm; Agilent Technologies, Santa Clara, USA) was employed. MS was conducted in positive ion mode with a turbo ion-spray voltage of 5500 V, while using 20 psi curtain gas, 50 psi nebulizer gas, and 50 psi drying gas at a temperature of 400 °C. The sample injection volume was 3 µL. LC separation was performed while using mobile phase A (0.1 % formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile), at a flow rate of 500 µL/min and a temperature of 40 °C. The separation gradient was as follows: 30% B at 0 min., 30 to 50% B in 30 min., 50 to 30% B in 0.1 min., and 30% B in 4.9 min. A collision energy of 15 V was used for multiple reaction monitoring (MRM), and LC-MS/MS data were analyzed by Analyst 1.5.2 software (AB Sciex). Peak area of each isotope-labeled internal standard was used to normalize that of straight or branched SCFA having the same number of carbons.