Epson1680

For the analysis of root morphology, the whole root system of the same plants used for the collection of exudates was scanned using the WinRHIZOTM system (WinRhizo software, EPSON 1680, WinRHIZO Pro2003b, Regent Instruments Inc., Quebec, QC, Canada). The dry weight of shoots and roots was recorded after drying at 65 °C until constant mass.

At harvest, root morphology parameters (total root length, number of tips, root volume, and average root diameter) were determined using the Winrhizo software (EPSON1680, WinRHIZO Pro2003b, Regent Instruments Inc., Quebec, Canada). Afterward, the roots and leaves were separated and dried at 65°C until constant weight was reached, a portion of the root apparatus was frozen in liquid nitrogen and kept at −80°C for gene expression analysis. Roots and leaves tissues were acid digested (HNO₃ 65% v/v) in a single digestion chamber (SRC) microwave digestion system (UltraWAVE, Milestone, Shelton, CT, United States) and the Cu concentration was determined by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES, Arcos Ametek Spectro, Germany).

The phytotoxicity potential of the three metabolites, gramine, hordenine and N-methyltyramine was investigated by measuring the main growth root parameters of lettuce (Lactuca sativa L.) once treated with the three alkaloids. Seeds of lettuce were placed in petri dishes laid out with filter paper (Whatman N°41, Whatman, Maidstone, UK) and soaked with 1 mL of solution of each alkaloid. The solutions were previously prepared from 5 mM MES buffer in distilled water adjusted to pH 6.15 by adding NaOH as described in [49 (link)], in which gramine, hordenine and N-methyltyramine were added in order to reach concentrations of 0.5 mM and 1 mM, obtaining different treatments. Controls were also prepared using the buffer alone. Petri dishes were sealed with Parafilm and incubated in the dark in the climate chamber for 48 hours. Thereafter, the germinated seeds were counted, and the main growth parameters were measured to calculate the inhibition percentage of the three metabolites investigated on Lactuca sativa L.. Root parameters were assessed by scanning the seedlings with WinRHIZO^TM system (WinRhizo software, EPSON1680, WinRHIZO Pro2003b, Regent Instruments Inc., Quebec, Canada).