Image-Analysis-Of-Sucrose-Treatment.md

Plant growth and image analysis of sucrose treatment assay

Arabidopsis thaliana Col-0 wild-type seeds were surface sterilized with the chlorine gas method (see the Fluorescein section of Material and methods). The seedlings were grown in sterile RoPod v5 chambers containing 3 ml of solid growth medium (0.5 × MS (Serva, ref. 47,515); MES 5 mM (Sigma, ref. M8250); pH 5.7 adjusted with KOH; 0.8% Plant agar (Duchefa, ref. M1002)), as described in Supplementary Methods. The injection hole of the RoPod v5 was closed with micropore tape. The chambers were placed into a 12 × 12 cm dish, and positioned with an angle of 45° into a plant growth cabinet with side illumination, 16 h light, 20 °C for six days. The recording was made using a 90°-flipped microscope for vertical specimen mounting (Zeiss AxioVert 200M, ITK MMS-100 linear stage and Hydra controller, Hamatsu OrcaC11440-36U camera, Zeiss 10 × PlanNeoFluar objective). LED bulb compatible for plant culture (LED power 150W, Chip SMD2835, 215 pcs Warm, 40pcs Red, 5pcs blue) was set to illumination intensity of 100 µmol m−2.s−1 measured at the proximity of the RoPod). Tubing connected to a syringe was inserted into the injection hole and taped on the microscope prior to imaging start in order to limit the vibration during injection. Two RoPod v5 were mounted at the same time on the microscope. A basal line was recorded for 4 h for both control and treated samples. Then, 8 ml of 0.5 × MS liquid supplemented with 1% sucrose were added in one of the RoPods (final concentration: 0.8%) and control specimen were treated with 8 ml of 0.5 × MS without sucrose. Root hair growth was recorded for an additional 8 h. For each root, two consecutive XY fields of view were recorded to obtain images of the root hairs at different stages of development throughout the recording time (pixel dimensions 0.92 µm.px−1, image size 1200 × 1920 px, 16-bit). In addition, a Z-stack encompassing 240 µm with optical sections of 30 µm was carried out to obtain a clear view of the entire root hairs. Finally, an image was recorded every 8 min. The images were analyzed with Fiji software. The detailed method illustrated with figures, the macros used for the analysis, as well as a tutorial video to use those macros are available in the Supplementary Methods, Figure S3, File S5–8 and Movie S4. To test these macros, an example image is provided in the Supplementary File S9. The data produced by Fiji were analyzed using R studio software. The scripts used for the analysis are available in the Supplementary File S10.