Inhibition of peroxidases and oxidoreductases is crucial for avoiding false-positive reactions in the localization of reactive oxygen species in intact barley root tips.

Abstract

Main conclusion NBT and HE may be efficiently used for the detection of superoxide, while DCDHF-DA and DHR123 for the detection of peroxynitrite in intact barley root tips, only if PRXs and oxidoreductases are inhibited to avoid false-positive reactions. Strong peroxidase (PRX) and oxidoreductase activities were observed in the barley root tips that were markedly inhibited by NaN 3 . Rapid and strong nitro-blue tetrazolium chloride (NBT) reduction is associated mainly with the vital functions of root cells but not with superoxide formation. In turn, the inhibition of root surface redox activity by NaN 3 strongly reduced the formation of formazan, but its slight accumulation, observed in the root elongation zone, was a result of NADPH oxidase-mediated apoplastic superoxide formation. A longer staining time period with NBT was required for the detection of antimycin A-mediated superoxide formation inside the cells. This antimycin A-induced superoxide was clearly detectable by hydroethidine (HE) after the inhibition of PRXs by NaN 3 , and it was restricted into the root transition zone. TEMPOL, a superoxide scavenger, strongly inhibited both NBT reduction and HE oxidation in the presence of NaN 3 . Similarly, the DCDHF-DA and DHR123 oxidation was markedly reduced after the inhibition of apoplastic PRXs by NaN 3 and was detectable mainly in the root transition zone. This fluorescence signal was not influenced by the application of pyruvate but was strongly reduced by urea, a peroxynitrite scavenger. The presented results suggest that if the root PRXs and oxidoreductases are inhibited, both NBT and HE detect mainly superoxide, whereas both DCDHF-DA and DHR123 may be efficiently used for the detection of peroxynitrite in intact barley root tips. The inhibition of PRXs and oxidoreductases is crucial for avoiding false-positive reactions in the localization of reactive oxygen species in the intact barley root tip.

Plant phenotyping relevance

オオムギ根端における活性酸素種の局在検出法を比較・検証し、ペルオキシダーゼ等による偽陽性を抑制する条件を評価しているため、植物状態の取得法が中心である。

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