L 1 agar

The batch reactors and agar plates contained Luria–Bertani (LB) broth consisting of 1 g L ${}^{-1}$ NaCl (Glassworld, South Africa), 20 g L ${}^{-1}$ tryptone (Oxoid, UK) and 10 g L ${}^{-1}$ yeast extract (Oxoid, UK), with 5 g L ${}^{-1}$ agar (Sigma Life Science, Spain) added to the agar plates solution only. The stock solution of 10,000 mg L ${}^{-1}$ Pb(NO ${}_3$ ) ${}_2$ consisted of 1.6 mg Pb(NO ${}_3$ ) ${}_2$ (Glassworld, South Africa) in 100 mL distilled water. Metabolic activity was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma, Aldrich, MO, USA). The nitrate concentration was determined using a nitrate test (Supelco, Germany). Ethylenedinitrilotetraacetic acid disodium salt (EDTA) (Supelco, Germany) was used in the determination of the extracellular and intracellular Pb(II) concentration.

Nutrient broth (NB) consisting of 3 g L^-1 meat extract, 5 g L^-1 peptone and salts was used as a medium for bacterial cultures. Nutrient agar petri-dishes were prepared with composition 3 g L^-1 meat extract, 5 g L^-1 peptone, and 15 g L^-1 agar. Sabouraud medium was used for maintenance of Candida spp. and it contained 40 g L^-1 glucose, 10 g L^-1 peptone, 20 g L^-1 agar, pH 5.6.
1,1-Diphenyl-2-picryl-hydrazyl (DPPH) was purchased from Sigma (St. Louis, MO, United States). Methanol used was of HPLC grade. Polyamide knitted fabric with density 23 columns cm^-1 and 15 rows cm^-1, weight 12 g m^-2, 154.7 Denier and average thickness 0.309 mm was supplied by Colora S.A. (Thessaloniki, Greece). The PA fabric was washed with detergent Felosan NFG before use for the removal of impurities.

Both wild-type (Col-0) and mutant plants were grown in vitro on 14 cm diameter Petri dishes at 21 °C under a 16 h day (110 µmol m⁻² s⁻¹) and 8 h night regime. The mutant line was grown together with the appropriate control on one plate to correct for plate effects. For each condition (MS or mannitol), 4–6 plates with eight wild-type and eight mutant seeds per plate were sown. Half of the plants were grown on solid (9 g l⁻¹ agar, Sigma) 1/2 MS control medium and the other half on solid 1/2 MS medium with the addition of 25 mM d-mannitol (Sigma). The plates were photographed at 16 or 22 DAS, and all images were analysed using ImageJ (Schindelin et al., 2015 (link)) to measure the projected rosette area. In total, four independent experiments were performed for aha2-4 and crrsp38-1 at 22 DAS and two independent experiments for aha2-4 at 16 DAS. To determine statistical significance of the raw data, an ANOVA test followed by a Tukey’s post-hoc test was performed (Supplementary Tables S4–S9).