D9754

Where specified, any bleached visual pigment was regenerated with the artificial chromophore analog 9-cis-Retinal. Stock solutions of 9-cis-Retinal (R5754; Sigma–Aldrich) in ethanol (100 mM) were prepared in darkness and stored at −80°C. On the day of the experiment, an aliquot was thawed and diluted to a final concentration of 100 µM in Ames' medium integrated with 1% wt/vol fatty acid-free bovine serum albumin (A8806; Sigma–Aldrich), an effective solubilizing and protective agent (Li et al., 1999 (link)). This solution was delivered to the preparation directly in the recording chamber, without modifying flow rate or temperature, for 20–25 min followed by washout. A pharmacological blockade of gap junctions was attempted with meclofenamic acid (MFA; M4531, Sigma–Aldrich) and 2-aminoethyl diphenylborinate (2-APB; D9754, Sigma–Aldrich). The I_h current was blocked with 4-Ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD7288; cat. no. 1000, Tocris, United Kingdom).

In situ detection of flavonoids, including flavonols, was done using fluorescence microscopy. Stem samples were taken 28 dpi from the bottom, middle and top parts of the tested potato plants. Free hand cross-sections were immersed in NEU reagent (1% methanolic 2-aminoethyl diphenylborinate, Sigma-Aldrich D9754) before observation under a fluorescence microscope (Vanguard Microscopes, Model 1486FL, VEE GEE Scientific Inc., Kirkland, WA USA) with filter set Exciter, Dichroic and Emitter wavelengths of 365, 400, and 535 nm, respectively. The NEU reagent reveals the presence of many flavonoids in bright yellow or orange under UV-366 nm.

All compounds for treating embryos were dissolved in DMSO, diluted in 0.5× E2 Embryo Medium and embryos treated by immersion. The compounds, and concentrations used, with catalogue numbers were diphenyleneiodonium chloride (DPI), 40 µM (D2926, Sigma); thapsigargin, 6.25 µM (T9033, Sigma); bisindolylmaleimide I (GF109203X), 85 µM (S7208, Selleckchem); YM-254890, 32 µM (10-1590-0100, Focus Biomolecules); 2-aminoethyl diphenylborinate (2-APB), 2.5 µM (D9754, Sigma), BI-D1870, 1.2 µM (Axon-1528, Axon Medchem); dimethyl fumarate, 9 µM (242926, Sigma); phorbol 12-myristate 13-acetate (PMA), 37.5 or 125 ng/ml (P8139, Sigma); U0126, 100 µM (9903, Cell Signaling Technology); PD184352 (CI-1040), 1.3 µM (S1020, Selleckchem). Unless otherwise stated, controls for all experiments were exposed to 0.5% DMSO carrier in 0.5× E2 Embryo Medium.