To characterize ANR initiation and primordium, the ClearSee protocol was applied as described previously (52). Briefly, seedlings exposed to various treatments were fixed using 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS) for 30 min. Fixed seedlings were washed twice with PBS and then immersed in ClearSee solution [10% (w/v) xylitol, 15% (w/v) sodium deoxycholate, and 25% (w/v) urea in water]. After incubating the seedlings in ClearSee solution in the dark at room temperature for 2 to 3 days, laser scanning confocal microscopy (Zeiss LSM 510 microscope) was used to visualize the roots. To determine pDR5Rev::GFP fluorescence at the collet region, live seedlings were directly used for laser scanning confocal microscopy (Zeiss LSM 510 microscope) examination.

For fluorescence intensity quantification of pDR5Rev::GFP and pPIN3::PIN3-GFP marker lines, ImageJ software (http://rsbweb.nih.gov/ij/) was used after taking confocal microscopy photos. All calculations are background subtracted.

To monitor ANR emergence, 3 to 4 dps pDR5::nlsYFP and pLAX3::LAX3-YFP Arabidopsis seedlings were embedded in 8% agarose and sectioned with a vibratome (Leica VT1000S vibratome). Sections were immediately stained with SCRI Renaissance 2200 (53) for 5 min to visualize cell walls. Images were acquired using a Zeiss LSM 880 with Airyscan using an Objective C-Apochromat 40×/1.2 DIC M27 water immersion. YFP fluorescence was excited with a 514 nm laser and detected with 519- to 620-nm wavelength, and the emission was recorded at 570 nm. For SCRI Renaissance, fluorescence was excited with a 405 nm laser and detected at 410- to 507-nm, and the emission was recorded at 459 nm. Images were processed with Zeiss ZEN software and Adobe Photoshop.

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