Fluorescent staining and microscopy
Plant root tips from all shoot phenotype groups were cut into 0.5 cm segments starting from the distal tip. Root tip segments were embedded in 5% (W/V) agarose in deionized water and sectioned to 100 µm thickness using vibratome (Vibratome 1000 Plus Sectioning System) as previously described (Pradhan Mitra and Loqué, 2014). The sections were transferred into CoroNa Green incubation buffer (20 mM 3-(N-morpholino)propanesulfonic acid, 0.5 mM calcium sulfate, 200 mM sorbitol). For CoroNa Green staining, 50 µg of CoroNa Green reagent (Thermo Fischer, C36676) was resuspended in 100 µL dimethyl sulfoxide, and this solution was further diluted in incubation buffer to make 0.1 mM staining solution. Sections were either incubated in staining solution or incubation buffer (as controls) for 16 h in the dark at room temperature before imaging. SNARF-1 staining (Thermo Fisher, C1270) was performed using a protocol adapted from Rosquete et al, 2019. Briefly, 1 µL of 10 mM SNARF-1 was diluted in 1 mL of CoroNa Green incubation buffer, and sections are incubated in this solution in the dark for 3 h at room temperature before imaging (Rosquete et al., 2019). Suberin staining was performed using a protocol adapted from Naseer et al (Naseer et al., 2012). Briefly, sections were incubated in 0.1 mg/mL Fluorol Yellow 088 (Santa Cruz Biotechnology CAS-81-37-8) in lactic acid for 1 h at 70 °C in the dark, rinsed in double deionized water, and imaged while mounted in 50% glycerol. FDA staining (Sigma, #F7378) was performed using protocol adapted from Jones et al (Jones et al., 2016). Briefly, live root sections were incubated in 4 µg/mL of FDA in incubation buffer for 5 min in the dark and imaged within 30 min.