Structural and immunocytochemical characterization of the walls of dichlobenil-habituated BY-2 tobacco cells

BY-2 tobacco cells, grown in the presence of the cellulose biosynthesis inhibitor herbicide dichlobenil (DCB), change in morphology from finely dispersed clumps and filaments to large aggregates after 6 mo. In this article, we utilize structural, cytochemical, and immunocytochemical techniques to characterize the cell wall changes that accompany the habituation of the cells to DCB. Wall structure is altered radically in the habituated cells, revealing a lamellate structure with no distinct middle lamella, even at cell junctions. Plasmodesmata traverse the cell wall by more circuitous routes than in the control cells. Habituated cells also produce unusual appositions of the wall that extend into the cytoplasm. Immunogold localization of various pectin epitopes reveals a 30-fold-greater density of gold particles detected in the walls of habituated cells compared to the controls and less density when the labeling of the middle lamellae is included. Antiextensin labeling was also found along the strands of pectic-reactive material, and the density of immunogold labeling exhibits a similar increase to that for pectin. Cellulose, detected by cellulase-gold, revealed only 2% of the reactivity of the controls, while callose detection was slightly greater compared to controls. Despite the loss of cellulose, xyloglucan is still detected, loosely associated with the strands of pectin or trapped between the pectin lamellae. Cortical microtubule arrays in habituated cells were apparently normal, despite a virtual absence of cellulose, although callose fibrils did parallel this array. These data indicate that long-term habituation to DCB results in the production of cell walls in which a pectin-extensin network has replaced cellulose as the major component of the cell wall. These changes have profound effects on wall ultrastructure and the gross morphology of the cultures.