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.