Cell viability assays

The viability of cells at the root tips was measured using 2,3,5-triphenyltetrazolium chloride (TTC; Tokyo Chemical Industry Co., Ltd.) as described previously (Yamauchi et al., 2014b). TTC is a vital stain that is reduced and turns red in living cells. The assays were carried out on the same batch of roots that were used for ROL measurements. The 0–10-mm apical parts of the marked roots were cut, weighed, and immediately transferred to tubes containing 100 μl TTC solution. They were then incubated for 30 min at 40 °C, rinsed with deionized water, and destained in tubes containing 150 μl of 95% (v/v) ethanol overnight at 25 °C in darkness. Cell viability was expressed as the absorbance of the extracted solution at 520 nm as determined using a DU800 spectrophotometer (Beckman Coulter Inc., Brea, CA, USA).