Biochemical assays for phenylpropanoid pathway activity
Tomato roots, stems and leaves were harvested seven dai with 400M. incognita J2s (three weeks after transplantation). Stem samples consisted of the stem section between roots and cotyledons, leaf samples of the first, second and third leaves and root samples of the whole root system. Samples were ground in a liquid nitrogen-chilled mortar. Lignin and phenolic compound quantification were repeated three times, using five biological replicates per treatment (consisting of 2-3 pooled seedlings).
Lignin was quantified using the acetyl bromide assay. Cell walls were prepared as in Van Acker et al. (2013), and the resulting extract was dried in a fume hood, weighed and suspended in 300 µl 25% acetyl bromide (Sigma-Aldrich). After two hours incubation (50°C, 800 rpm), samples were placed on ice and diluted with 1.5 ml acetic acid. After centrifugation (10 minutes, 19 330g, RT), 300 µl supernatant was mixed with equal volumes 2M NaOH and 0.5M hydroxylamine (Sigma-Aldrich) and centrifuged (10 minutes, 19 330g, RT). Absorbance at 280 nm was measured in a microplate reader (Tecan Infinite F200 Pro) and lignin content was calculated according to Barnes & Anderson (2017). Because no extinction coefficient for tomato lignin was available, that of A. thalianareported by Xue et al. (2008) was used.
Free and cell-wall bound phenolic compounds were quantified using the Folin-Ciocalteu assay (Ainsworth & Gillespie 2007). Free phenolics were extracted from ±100 mg tissue with 20 µl mg-1 80% (v/v) ethanol (30 minutes, RT). After centrifugation (5 minutes, 19 330g, RT), 125 µl supernatant was mixed with 675 µl distilled water, 37.5 µl Folin-Ciocalteu reagent (Sigma-Aldrich) and 375 µl 20% (w/v) Na2CO3. After 15 minutes, absorbance at 765 nm was measured in a spectrophotometer (VWR UV1600PC) and phenolic compound concentration was calculated using a gallic acid standard curve.
The residue left after ethanol extraction was washed with 80% ethanol and extracted overnight with 20 µl mg-1 1M NaOH to release cell wall-bound phenolic compounds. After centrifugation (5 minutes, 19 330g, RT), 125 µl NaOH extract was mixed with 1.05 ml distilled water and 37.5 µl Folin-Ciocalteu reagent and absorbance was measured as for free phenolic compounds.