Hydrogen peroxide and expression of hipI-superoxide dismutase are associated with the development of secondary cell walls in Zinnia elegans

Abstract

A special form of a CuZn-superoxide dismutase with a high isoelectric point (hipI-SOD; EC 1.15.1.1) and hydrogen peroxide (H2O2) production were studied during the secondary cell wall formation of the inducible tracheary element cell-culture system of Zinnia elegans L. Confocal microscopy after labelling with 2',7'-dichlorofluorescin diacetate showed H2O2 to be located largely in the secondary cell walls in developing tracheary elements. Fluorescence-activated cell sorting analysis showed there were lower levels of H2O2 in the population containing tracheary elements when H2O2 scavengers such as ascorbate, catalase, and reduced glutathione were applied to the cell culture. Inhibitors of NADPH oxidase and SOD also reduced the amount of H2O2 in the tracheary elements. Furthermore, addition of these compounds to cell cultures at the time of tracheary element initiation reduced the amount of lignin and the development of the secondary cell walls. Analysis of UV excitation under a confocal laser scanning microscope confirmed these results. The expression of hipI-SOD increased as the number of tracheary elements in the cell culture increased and developed. Additionally, immunolocalization of a hipI-SOD isoform during the tracheary element differentiation showed a developmental build-up of the protein in the Golgi apparatus and the secondary cell wall. These findings suggest a novel hipI-SOD could be involved in the regulation of H2O2 required for the development of the secondary cell walls of tracheary elements.

Alternative Splicing Studies of the Reactive Oxygen Species Gene Network inPopulusReveal Two Isoforms of High-Isoelectric-Point Superoxide Dismutase

“…Isoforms of Cu/Zn-SOD named high-isoelectric-point superoxide dismutases (hipI-SODs) have been detected in pine (Pinus spp. ), Zinnia, and Populus in our laboratory and shown to be involved in the regulation of ROS and plant development (Schinkel et al, 1998Karpinska et al, 2001;Karlsson et al, 2005;Srivastava et al, 2007). These enzymes have substantially higher pI values than other known SODs (.9.5) and monomeric masses of about 16 kD.…”

“…One of the AS enzymes identified in the ROS network was a recently described hipI-SOD with suggested roles in the development and lignifications of the secondary cell wall (Karpinska et al, 2001;Karlsson et al, 2005;Srivastava et al, 2007). BLAST searches of the Populus genome database for hipI-SOD sequences by Srivastava et al (2007) showed that the Populus genome contains two hipI-SOD genes, designated PthipI-SODC1 and PthipI-SODC2.…”

A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism

“…Other candidates include plasma membrane-located RBOH enzymes (Ros Barceló, 1998;Suzuki et al, 2011;Kärkönen and Kuchitsu, 2015). RBOHs generate apoplastic superoxide that dismutates to H 2 O 2 either nonenzymatically in the acidic pH present in the cell wall or enzymatically via a superoxide dismutase (SOD)-catalyzed reaction (Karlsson et al, 2005). Surprisingly, none of the putative RBOH genes were induced in lignin-forming conditions, but the expression of several RBOH genes was high in all non-lignin-forming conditions (also at time 0; Fig.…”

“…Several plant cell wall-and plasma membranelocated sources for apoplastic reactive oxygen species (ROS; superoxide, H 2 O 2 , and hydroxyl radical) exist (Kärkönen and Kuchitsu, 2015), including apoplastic peroxidases, various oxidases, and plasma membrane-located enzymes such as respiratory burst oxidase homologs (RBOHs; also called NADPH oxidases) and quinone reductases. There are indications that RBOHs produce ROS in zinnia (Zinnia elegans) during vascular xylem lignification (Ros Barceló, 1998) and in in vitro tracheary element (TE) formation (Karlsson et al, 2005;Pesquet et al, 2013), with some participation of peroxidases (Karlsson et al, 2005). In addition, in Arabidopsis, ROS produced by AtRBOHF are essential for the formation of lignin-composed Casparian strips in the root endodermis (Lee et al, 2013).…”

Non-Cell-Autonomous Postmortem Lignification of Tracheary Elements inZinnia elegans

“…Reactive oxygen species (ROS) have been shown to be required for full TE lignification (Karlsson et al, 2005;Ros Barceló, 2005). To define whether the ROS are produced by non-TEs, experiments were again conducted with the 144-h-old, PA-treated TE cell cultures that consisted of dead and non-lignified TEs and living non-TEs.…”