Ms salts medium

Arabidopsis thaliana ecotype Columbia (Col-0) was used for drought experiments in this study. The double mutant of At1g79090 (PAT1) pat1 (Salk_040660) and At1g12280 (SUMM2) summ2-8 (SAIL_1152A06) pat1/summ2 tolerant to drought stress^{68 (link)} was used as positive control in this study. Surface-sterilized seeds were grown on solid Murashige–Skoog (MS) salts medium (Duchefa), with 1% sucrose and 0.8% agar, vernalized 48 h at 4 °C and then placed in the growth chamber with 150 μmol m⁻² s⁻¹ light intensity, 70% humidity, 12/12 h light/dark photoperiod, 22 °C for daytime temperature and 21 °C for night to allow germination. Then, 10-day-old germinated seedlings were transplanted in a 7 cm × 7 cm square pots with drainage holes (four plants in each pot), containing approximately 60 g of non-sterile soil (Plug og såjord, SW HORTO A/S, DK) (recipe described in Supplementary Table 2) to grow in the chamber with the same conditions.

Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used in this study. The Col-0 seeds were firstly surface-sterilized by rinsing with 1 ml of bleach for 1 min, followed by rinsing with 1 ml of 70% ethanol for 1 min. The seeds were then washed with 1 ml of sterilized water twice after removing the ethanol. The surface-sterilized Col-0 seeds were placed on the Murashige–Skoog (MS) salts medium (Duchefa) containing 1% (w/v) sucrose and 1.5% (w/v) agar, allowing germination. Seeds were firstly vernalized at −4°C for 48 h on the sterile Petri dishes filled with 30 ml MS agar medium per plate, and then cultured in a greenhouse with a photoperiod of 16-h-light/8-h-dark, 150 μmol m⁻² s⁻¹ light intensity, 60% humidity, 23°C for daytime temperature, and 18°C for night. Seven days post germination, seedlings with similar growth phenotype were selected and transferred to the new MS agar plates without addition of sucrose, and were inoculated with a bacterial suspension, and incubated under the same conditions in the greenhouse.

Young tobacco leaves were excised from axenically grown wild-type plants and cut into about 1 cm2 leaf discs. The discs were placed for 5 min in a culture of A. tumefaciens carrying the plasmid of interest, and then incubated at 25°C in 16 h of light on MS salts medium (Duchefa Biochemie), containing 3% sucrose and 0.8% phyto agar, underside down. After 2 days, the leaf discs were transferred to a shoot generation medium (1/2 MS salts supplemented with 30 g/l sucrose, 0.1 mg/l α-naphthalene acetic acid, 0.1 mg/l 6-benzylaminopurine, 50 μg/ml hygromycin, 100 μg/ml carbenicillin and 250 μg/ml cefotaxime, and 0.8% phyto agar) in order to promote callus growth, select transformed plants and to prevent further growth of Agrobacterium. Elongated shoots were excised from calli and transferred on to 1/2 MS agar supplemented with 0.1 mg/L indole-3-acetic acid, 50 mg/L hygromycin and 100 mg/L carbenicillin in order to promote roots formation. Transformed plants were grown at 25°C in 16 h of light in axenic conditions without antibiotics and propagated every 5–6 weeks.