Plant materials, secretion of substances and mucilage staining

Seeds of various wild crucifers and leguminous species were collected in the field in Israel or obtained from the Israel Plant Gene Bank (S1 Table). No specific permissions were required for all locations from which seeds were collected. These locations include Road sides in central and northern Negev or the campus area at Midreshet Ben Gurion. Seeds of Arabidopsis thaliana (Col and Ler) wild type and mucilage mutant plants gl2 and mum4 (kindly provided by T. Western and S. Harpaz-Saad) were collected from plants grown in growth room at 22°C±2°C under long day photoperiod.

Analysis of substances released from seeds of various plant species was standardized either based on seed weight or on seed surface area. Briefly, 10 mg of seeds were incubated in 100 μl of phosphate-buffered saline (PBS) at 4°C for 8–12 h after which the aqueous phase was collected by centrifugation (4°C, 11,000 rpm, 5 min) and the supernatant was used immediately or stored at -20°C until used. Because, the seeds examined in this study have various volume, shape and weight, in some experiments, standardization was based on surface area of seeds equivalent to 10 mg of Arabidopsis seed surface area. Seeds examined in this study were either ellipsoid in shape (e.g., Arabidopsis thaliana) or round (e.g., Sinapis alba), and therefore we calculated the surface area of ellipsoidal seeds by the equation: SA = 4π/(a^pb^p+a^pc^p+b^pc^p)/3]^1/p where SA is the surface area, a, b and c are the semi-principal axes of the ellipsoidal seed, π = 3.14 and p = 1.6, while the surface area of round seeds by the equation SA = 4πr², where SA is the surface area, π = 3.14 and r, is the radius of the seed. All calculations were performed using on line calculators (https://planetcalc.com/149/). Accordingly, the surface area of 10 mg Arabidopsis seeds is calculated by the surface area of one seed (~0.4 mm²) multiply by the number of seeds in 10 mg (~400 seeds). Thus, the number of seeds attaining the same surface area of other Brassicaceae species was calculated by dividing the surface area of 10 mg Arabidopsis seed (~160 mm²) by the surface area of one seed of a given Brassicaceae species. Finally, in experiments where comparison were performed between seeds, embryos and seed coats we use a given number of seeds from which embryos and seed coats were dissected.

Mucilage was stained with Ruthenium red essentially as described [32]. Seeds were hydrated with either distilled water or 50 mM EDTA for 90 min, washed with distilled water and incubated with 0.01% Ruthenium Red (Sigma-Aldrich) 90 min. The seeds were washed with distilled water to remove access dye and observed under a binocular microscope (Zeiss).