Dmaca

The histochemical staining of condensed tannin (CT) in leaves was carried out by staining tissues in a solution of ethanol: 6 M HCl (1:1) containing 0.1% (w/v) dimethylamminocinnamaldehyde (DMACA) (Sigma-Aldrich) for 3 to 6 min57 .

Retinyl acetate, β-carotene, α-carotene, vitamin D₃ (> 95% pure), deuterated vitamin D₃, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), phytic acid, DMACA, vanillin, iron III, pancreatin, ammonium thiocyanate, catechins, α-amylase, pepsin, bile, ferric chloride and sodium carbonate were purchased from Sigma Aldrich (Saint-Quentin-Fallavier, France). Lutein, lycopene, phytoene and phytofluene were purchased from Extrasynthèse (Genay, France). Soyasaponin I was generously supplied by Stéphane Georgé, CTCPA Avignon. Canned tomato pulp, red bell peppers and Isio4 oil (Lesieur, Asnières, France) were purchased from a local supermarket (Marseille, France). Methanol, acetonitrile, hexane, dichloromethane, methyl-tert-butylether, acetone, ethanol absolute anhydrous, sulfuric acid, hydrochloric acid, nitric acid, formic acid, hydrogen peroxide were purchased from CarloErba Reagents (Peypin, France).

For DMACA staining of developing grains, transverse portions of the central region of grains at 15 days post‐anthesis (dpa) were frozen to a stub in OCT (Tissue‐Tek: Sakura Finetek USA) in liquid N₂. Sections, 30 μm, were cut on a Leica CM1850 cryostat and pressed on to polylysine slides before being stained in DMACA (1% DMACA [4‐dimethylaminocinnamaldehyde, Sigma] in 0.7 M HCl) for 1–2 min (Li et al., 1996 (link)). All images were taken using a Zeiss Axiophot microscope and MetaMorph imaging software (Molecular Devices LLC, San Jose, CA, USA).

The Arabidopsis tt2 mutant lacking PA biosynthesis in the seed coat was transformed with A. tumefaciens strain LBA4404 containing the binary construct pCAMBIA2301G-SsMYB3 by floral dipping [31 (link)]. Harvested seeds were selected on MS medium containing 3% (v/v) sucrose and with 100 mg/L kanamycin. For phenotypic analysis of CT accumulation in the seed coat, T₁ seeds were stained with dimethylaminocinnaldehyde reagent (DMACA, Sigma) according to a previous report [32 (link)].

The total flavanols content was determined according to the Feucht and Polster [82 (link)] method, using the DMACA (p-dimethylaminocinnamaldehyde) reagent (Sigma-Aldrich, Poznań, Poland). The absorbance was measured using a spectrophotometer (UV–VIS UV-6100A, Metash Instruments Co., Ltd., Shanghai, China) at the wave-length of λ = 640 nm. (+)-Catechin (Sigma-Aldrich, Poznań, Poland) was used as a standard. The results were expressed as the µg catechin equivalent, per 1 g of dry matter (µg CE/g d.m.). The determinations were performed in six independent replications.

Phytochemical screening was performed with thin layer chromatography using pre-coated silica gel plates (Alugram Xtra Sil G/UV254, Machery-Nagel). The solvent system was EtOAc/Acetic acid/Formic acid/H₂O (100:11:11:20 v/v). For each extract, 10 µL (5 mg/ml) were spotted on the TLC plate. The same revealers as in Santos et al. (Santos et al., 2018 (link)) were used, except for total and condensed tannins, for which ferric chloride (Oliveira et al., 2015 (link)) and DMACA (Lea, 1978 (link)) (Sigma-Aldrich) were used. Folin-Ciocalteu reagent (VWR chemicals) was also used to reveal all the polyphenols. The following standards were used as controls on TLC plates: quercetin, (+)-catechin, gallic acid, aloin, stigmasterol, glycyrrhetic acid and quinin.