Sub-cellular location of H2O2, peroxidases and pectin epitopes in control and hyperhydric shoots of carnation

In carnation shoots (Dianthus caryophyllus cv. Killer), hyperhydricity was induced in in vitro culture using a low agar concentration. Using transmission electron microscopy, cytochemical techniques and immunolocation of JIM5 and JIM7 pectin epitopes, we followed the sub-cellular modifications of cell walls in relation to peroxidase activity and hydrogen peroxide accumulation during hyperhydricity induction. Peroxidase activity revealed a significant induction of the stomatal and epidermal cells as well as of the intercellular spaces of hyperhydric leaves. Similarly, hydrogen peroxide accumulated in the epidermal cell walls and the intercellular spaces of hyperhydric leaves. Immunolocation of an epitope recognised by the JIM5 antibody revealed the main unesterified nature of the cell walls. Such an epitope was located in the epidermal cell walls as well as in the corners of cell junctions in control leaves. However, hyperhydric leaves showed a total reduction of JIM5 labelling in the corners of cell junctions and a significant reduction of the intercellular spaces and the middle lamella. Highly-methylsterified pectin, recognised by the JIM7 antibody, was present to a slight extent in cell walls in control and hyperhydric leaves. We propose that the altered anatomy observed in hyperhydric carnation leaves could be regulated by the concomitant actions of pectin methyl esterases and free radicals, modifying the structure of the pectin and polysaccharides of the cell walls.