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 inh ibitor herbicide dichlobenil (DCB), change in morphology from finely disper sed clumps and filaments to large aggregates after 6 mo. In this article, w e utilize structural, cytochemical, and immunocytochemical techniques to ch aracterize the cell wall changes that accompany the habituation of the cell s to DCB. Wall structure is altered radically in the habituated cells, reve aling a lamellate structure with no distinct middle lamella, even at cell j unctions. Plasmodesmata traverse the cell wall by more circuitous routes th an in the control cells. Habituated cells also produce unusual appositions of the wall that extend into the cytoplasm. Immunogold localization of vari ous pectin epitopes reveals a 30-fold-greater density of gold particles det ected in the walls of habituated cells compared to the controls and less de nsity when the labeling of the middle lamellae is included. Antiextensin la beling was also found along the strands of pectic-reactive material, and th e density of immunogold labeling exhibits a similar increase to that for pe ctin. Cellulose, detected by cellulase-gold, revealed only 2% of the reacti vity 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 di d parallel this array. These data indicate that long-term habituation to DC B results in the production of cell walls in which a pectin-extensin networ k has replaced cellulose as the major component of the cell wall. These cha nges have profound effects on wall ultrastructure and the gross morphology of the cultures.