Histochemical Demonstration and Localization of H2O2 in Organs of Higher Plants by Tissue Printing on Nitrocellulose Paper

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Abstract
A sensitive tissue-print assay for the detection and histological localization of H2O2 in freshly cut organ sections was developed by impregnating nitrocellulose paper with a mixture of Kl and soluble starch. H2O2 transferred from the cut surface of the section to the dried paper forms I2, which can be visualized by the intensely colored I2-starch complex. The detection limit of the assay is in the range of 0.1 to 0.2 mmol L-1 H2O2. Due to the rapid immobilization of H2O2 in the paper, very clear prints of the tissue distribution of H2O2 can be obtained with a spatial resolution on the level of single cells. The application of this rapid and simple assay is explored in five experimental examples demonstrating that the in vivo level of H2O2 varies strikingly in different tissues and can be regulated by developmental factors such as hormones, light, and wounding. The results show that: (a) In the hypocotyl of soybean (Glycine max L.) seedlings the apoplastic H2O2 level increases strongly from top to base, accompanied by characteristic changes in its histological distribution. (b) In the epicotyl of pea (Pisum sativum L.) seedlings the induction of lateral expansion by ethylene is correlated with a depletion of H2O2 in the cell walls of the expanding tissues. (c) In the hypocotyl of bean (Phaseolus vulgaris L.) seedlings H2O2 is primarily localized in a ring of parenchymatic tissue between xylem and cortex next to lignifying cells but not in the lignifying cells themselves. (d) In the hypocotyl of sunflower (Helianthus annuus L.) and cucumber (Cucumis sativus L.) seedlings the light-mediated inhibition of elongation growth is correlated with a strong increase in H2O2 in the epidermis and in the vascular bundles. (e) Potato (Solanum tuberosum L.) tubers show high levels of H2O2 only in the outer cell layers but are able to accumulate H2O2 in the inner tissue upon wounding.

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