To estimate the phenolic acid contents of root samples, the tissue was ground, extracted in 5 mL of 1 M NaOH per g of root tissue, and passed through a 0.22 µm filter. The pH of the filtrate was adjusted to 2.5 with HCl, and the filtrate was extracted five times in an equal volume of ethyl acetate. The pooled extracts were lyophilized, and the residue was dissolved in 5 mL of methanol for the measurement of FA CA and CHA content via HPLC analysis. The extraction of the root exudates followed the same procedure used for the measurement of these phenolic acids.
For HPLC analysis of each example, CA, FA and CHA were quantified using a 1260 HPLC device (Agilent Technologies, Santa Clara, CA, USA) equipped with a ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, 5 μm). The mobile phases for CA, FA and CHA were a 1:1 mixture of 100% methanol and 0.2 M phosphoric acid, a 1:1 mixture of 100% methanol and 0.1 M phosphoric acid, and a 1:1 mixture of 100% acetonitrile and 0.05 M phosphoric acid, respectively, provided at a constant flow of 11 mL min−1. The detection of CA, FA and CHA was performed at 323, 300 and 327 nm, respectively, and the column temperature was maintained at 30 °C. All experiments were performed in triplicate.
For HPLC analysis of each example, CA, FA and CHA were quantified using a 1260 HPLC device (Agilent Technologies, Santa Clara, CA, USA) equipped with a ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, 5 μm). The mobile phases for CA, FA and CHA were a 1:1 mixture of 100% methanol and 0.2 M phosphoric acid, a 1:1 mixture of 100% methanol and 0.1 M phosphoric acid, and a 1:1 mixture of 100% acetonitrile and 0.05 M phosphoric acid, respectively, provided at a constant flow of 11 mL min−1. The detection of CA, FA and CHA was performed at 323, 300 and 327 nm, respectively, and the column temperature was maintained at 30 °C. All experiments were performed in triplicate.