Plant leaf tissue was ground into fine powder under liquid nitrogen. The frozen powder was weighed, and extraction buffer (50% methanol, 0.1% formic acid) was added immediately (1 mg, 10 μl). The plant sample was vortexed for 30 s and then centrifuged at 11,000g for 20 min at 4°C. The supernatant was transferred into a new Eppendorf tube and centrifuged once more to remove all particles.

Benzoxazinoid content was analyzed with an Acquity UPLC (Waters, USA) coupled to an ultraviolet (UV) detector and a QDa mass spectrometer (Waters, USA) using an Acquity BEH C18 column (2.1 mm by 100 mm, 1.7 μm; Waters, USA). The temperatures of the autosampler and column were set to 15° and 40°C, respectively. The mobile phase consisted of 99% water, 1% acetonitrile, and 0.1% formic acid (A), and acetonitrile and 0.1% formic acid (B). Flow rate was set to 0.4 ml min−1 with 97% A and 3% B. The injection volume was 5 μl. The elution profile was: 0 to 9.65 min, 3 to 16.4% B; 9.65 to 13 min, 16.4 to 100% B; and 13.1 to 15 min at 100% B, followed by 2-min column reconditioning at 3% B. The extracted UV trace at 275 nm was used for benzoxazinoid quantification. The following extracted ion chromatograms were used for quantification with a mass window of ±0.01 D: 164 mass-to-charge ratio (m/z) for DIMBOA [retention time (RT), 5.62 min], 372 m/z for DIMBOA-Glc (RT, 5.64 min), 432 m/z for HDMBOA-Glc (RT, 8.19 min), 402 m/z for DIM2BOA (RT, 5.81 min), 356 m/z for HMBOA-Glc (RT, 5.28 min), 342 m/z for DIBOA-Glc (RT, 4.29 min), 462 m/z HDM2BOA-Glc (RT, 8.32 min), 164 m/z for MBOA (RT, 8.17 min), and 372 m/z for MOA-Glc (RT, 5.31 min). Benzoxazinoid concentrations were determined by external calibration curves obtained from purified DIMBOA-Glc, DIMBOA, and HDMBOA-Glc standards. Five calibration points (5, 10, 50, 150, and 200 μg/ml) were used.

Quantification of phenolic acids (chlorogenic acid, p-caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid) was performed on an Acquity UPLC coupled to a Xevo G2 XS Q-TOF time-of-flight mass spectrometer (Waters, USA) equipped with an electrospray ionization (ESI) source. The compounds were separated on an Acquity BEH C18 ultra performance liquid chromatography (UPLC) column (2.1 mm by 50 mm internal diameter, 1.7 μm particle size). Water (0.1% formic acid) and acetonitrile (0.1% formic acid) were used as mobile phases A and B. The gradient profile was as follows: 0 to 1 min, 95% A in B; 1 to 5 min, 81% A in B; 5 to 5.2 min, 1% A in B; 5.2 to 6.5 min, 1% A in B; 6.6 to 8 min, 95% A. The mobile phase flow rate was 0.4 ml/min. The column temperature was maintained at 40°C, and the injection volume was 1 μl. The quadrupole orthogonal acceleration–time-of-flight (Q-TOF) was operated in ESI negative mode, and data were acquired in scan range (50 to 1200 m/z) using a cone voltage of 20 V. The elution order was as follows: 2.09 min, chlorogenic acid; 2.22 min, p-caffeic acid; 3.18 min, p-coumaric acid; 3.83 min, ferulic acid; and 4 min, sinapic acid. Chromatograms were acquired in MSE mode using a collision energy ramping of 10 to 30 V. Quantification of the compounds was performed on the basis of their exact masses: chlorogenic acid, 353.0872 m/z; p-caffeic acid, 179.0344 m/z; p-coumaric acid, 163.0395 m/z; ferulic acid, 3.83 min and 193.050 m/z; and sinapic acid, 4 min and 223.0606 m/z. Calibration solutions containing the different phenolic acids were prepared between 0 and 5 μg ml−1 to establish linear regressions.

To determine amino acid concentrations in wheat phloem, phloem samples were diluted 1:7000 with methanol:water (1:1, v/v) and spiked with 13C, 15N–labeled amino acids (algal amino acids 13C, 15N; Isotec, Miamisburg, OH, USA) at a concentration of 9 μg of the mix per milliliter. Amino acids were directly analyzed by LC-MS/MS as recently described (54). Amino acid concentrations in wheat leaves were determined using an AccQ-Tag kit (Waters, Milford, USA). Five hundred microliters of extract buffer (20 mM norleucine in MeOH) was added to 10 mg of dry and ground leaf powder. After the solution was thoroughly mixed for 15 min, 250 μl of chloroform and 500 of μl Milli-Q water were added. The mixture was centrifuged at 11,000g for 5 min at room temperature (20° to 23°C). Fifty microliters of supernatant was freeze dried into dry residue and then suspended with 50 μl of Milli-Q water. Five microliters of sample was briefly mixed with 35 μl of borate buffer and 10 μl of derivatization buffer from an AccQ-Tag Ultra Derivatization kit (Waters, USA) and then placed in a 55°C water bath for 10 min. Amino acid content was analyzed with a UPLC coupled to a UV detector and a QDa mass spectrometer (Waters, Milford, USA). Mixtures of 20 amino acids at 100, 50, 10, and 1 μg ml−1 were used as external standards for quantification. Soluble sugars, starch, and total soluble proteins were extracted and quantified as described previously (38).

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