Two distinct cell sources of H2O2 in the lignifying Zinnia elegans cell culture system

The use of transdifferentiating Zinnia elegans mesophyll cells has proved useful in investigations of the process of xylem differentiation from cambial derivatives. Cultured mesophyll cells can be induced by external stimuli to proceed through temporally controlled developmental programs which conclude in the formation of single-cell-derived dead vascular tracheids and parenchyma-like elements. However, there is a gap in our knowledge concerning the role played by reactive oxygen species (O-2(-) and H2O2) in the development of these vascular elements. In this study, we show by the following four independent and highly selective methods that transdifferentiating Z. elegans mesophyll cells are capable of producing reactive oxygen species: the 2,3-bis(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, which monitors O-2(-) production, and the xylenol orange, 2,7-dichlorofluorescein diacetate, and CeCl3 assays, which monitor H2O2 production and localization. The joint use of these biochemical (XTT and xylenol orange) assays and cytochemical (2,7-dichlorofluorescein diacetate and CeCl3) probes revealed that transdifferentiating Z elegans mesophyll cells do not show an oxidative burst but live in a strongly oxidative state during the entire culture period. In this state, H2O2 is produced by both tracheary and parenchyma-like elements, the nonlignifying parenchyma-like cells acting quantitatively as the main source. The existence of these two sources of H2O2 in this in vitro cell culture system may be especially relevant during the later stages of tracheary cell wall lignification, in which lignifying tracheary elements become hollow. In the case of differentiating tracheary elements, H2O2 was located in the same place and at the same time as the onset of tracheary element lignification, i.e., at the primary cell wall during secondary thickening, supporting the view that the H2O2 produced by this in vitro culture system is destined for use during lignin biosynthesis.