Basal salt mixture

For sugar measurements, seedlings of Arabidopsis thaliana (ecotype Columbia; N60000 NASC Nottingham, UK) were used as a wildtype plant. A T-DNA insertion mutant (GabiKat_489_D10) in the galactokinase gene (At3g06580; galK) was obtained from NASC and verified by PCR. This mutant was previously characterized by Egert et al. (2012) (link) and shown to have no galactokinase activity. Seeds were surface sterilized by ethanol and incubated on 0.5× MS plates (Basal Salt Mixture, Duchefa #M0245, Haarlem, Netherlands), pH 5.7 (KOH) containing 0.8% plant agar (Duchefa) and 5 mM sucrose. After 7 days they were transferred to 0.5× MS plates, pH 5.7 (KOH) with 0.8% plant agar and 5 mM galactose. Plants were grown in growth chamber under short day conditions at 23°C with 8 h light (approximately 150 μE·m⁻²·s⁻¹) and 18°C in the dark. The roots of the seedlings were harvested after 14 days growth on galactose containing medium, frozen in liquid nitrogen and stored at −80°C.

Arabidopsis seeds were stratified by immersion in water and incubation at 4°C for 3 days. Surface-sterilization was initiated by soaking seeds in 1 ml of 70% (v/v) ethanol for 5 min. Ethanol was replaced by 1 ml of bleach solution [20% (v/v) commercial bleach with 3.5% (w/v) effective chloride; 0.1% (v/v) Tween-20] and seeds were further incubated for 10 min. After being rinsed five times with sterile water, seeds were resuspended in a sterile 0.08% (w/v) agarose solution. When required, seeds were subjected to heat shock. Sterilized seeds were dispersed onto plates containing MS-based medium [41 (link)] composed of 1x basal salt mixture (Duchefa), 1.5% (w/v) sucrose, 0.5 g l⁻¹ MES and 0.8% (w/v) agar at pH 5.7. Plates were sealed with parafilm to prevent dehydration and placed horizontally in a growth room, under the following conditions: photoperiod of 16 h light/8 h darkness; light intensity of 100 μmol photon m⁻² s⁻¹ and 23°C.

In this work Arabidopsis thaliana (ecotype Columbia 0) was used as a WT plant. Previously described T‐DNA insertion mutants glcak1‐1 (SALK_076931) and glcak1‐2 (SALK_127949c) were obtained from the Arabidopsis Biological Resource Center and another two mutant lines glcak1‐3 (contains CAS9 nuclease) and glcak1‐4 were created by CRISPR/Cas9 technology. Plants were grown in a growth chamber in pots on standard soil (type ED73) under long‐day conditions with 16‐h light at 150 μmol·m⁻²·s⁻¹. Temperature during the light phase was 23 °C and 18 °C in the dark. For RT‐PCR and labelling experiments seedlings were grown in liquid medium. Seeds were surface sterilised in ethanol and grown in 0.5 × MS (Basal Salt Mixture, Duchefa #M0245; Duchefa, Haarlem, the Netherlands), pH 5.7 (KOH), with 1 g·l⁻¹ sucrose for 10 days. Then the medium was changed to 0.5 × MS (or 0.5 × MS with 0.37 MBq myo‐[2‐³H]‐inositol; final concentration in medium is 0.625 μm) for the next 3 days. For sugar measurements sterile seeds were incubated on 0.5 × MS (without micronutrients; Duchefa #M0221), pH 5.7 (KOH), plates with 0.8% plant agar. Harvested samples were immediately used for experiments or frozen in liquid nitrogen and stored at −80 °C until used.