Institute of Botany, Plant Science and Biodiversity Centre, Slovak Academy of Sciences, Dúbravská cesta 9, 84523, Bratislava, Slovak Republic.
Planta. 2022 Feb 16;255(3):69. doi: 10.1007/s00425-022-03850-1.
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. 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 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, 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. Similarly, the DCDHF-DA and DHR123 oxidation was markedly reduced after the inhibition of apoplastic PRXs by NaN 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.
NBT 和 HE 可有效用于检测超氧阴离子，而 DCDHF-DA 和 DHR123 可用于检测完整大麦根尖中的过氧亚硝酸盐，前提是抑制 PRX 和氧化还原酶以避免假阳性反应。在大麦根尖中观察到强烈的过氧化物酶（PRX）和氧化还原酶活性，这些活性被NaN 明显抑制。快速且强烈的硝基蓝四唑氯化物（NBT）还原主要与根细胞的生命功能相关，而不是与超氧阴离子的形成相关。相反，NaN 强烈抑制根表面氧化还原活性，从而强烈减少甲臜的形成，但在根伸长区观察到的轻微积累是质外体超氧阴离子形成的 NADPH 氧化酶介导的结果。在细胞内检测安密霉素 A 介导的超氧阴离子形成时，需要更长的 NBT 染色时间。在 NaN 抑制 PRX 后，NBT 诱导的超氧阴离子可以被羟乙基噻唑二唑溴盐（HE）清楚地检测到，并且它局限在根过渡区。超氧阴离子清除剂 TEMPOL 在 NaN 存在下强烈抑制 NBT 还原和 HE 氧化。同样，在 NaN 抑制质外体 PRX 后，DCDHF-DA 和 DHR123 的氧化明显减少，主要在根过渡区检测到。该荧光信号不受丙酮酸盐的影响，但受过氧亚硝酸盐清除剂尿素的强烈影响。研究结果表明，如果抑制根 PRX 和氧化还原酶，NBT 和 HE 主要检测超氧阴离子，而 DCDHF-DA 和 DHR123 可有效用于检测完整大麦根尖中的过氧亚硝酸盐。抑制 PRX 和氧化还原酶对于避免在完整大麦根尖中活性氧的定位中出现假阳性反应至关重要。
