Differentiation and degeneration of cells that play a major role in tobacco anther dehiscence

Dehiscence is the terminal step in anther development that releases pollen grains from the wall of each theca at a specific site between the two locules. In tobacco, two groups of cells—the circular cell cluster and the stomium—are required for anther dehiscence and define the position at which pollen is released. The processes responsible for the differentiation of the circular cell cluster and the stomium from cells in specific anther regions are unknown. Nor is it understood what initiates the programmed degeneration of these cell types that ultimately is responsible for pollen release from the anther. We characterized stomium and circular cell cluster differentiation and degeneration using both light and transmission electron microscopy throughout anther development, from the emergence of stamen primordia to anther dehiscence at flower opening. We observed that histological changes within primordium L1 and L2 cells destined to become the stomium and circular cell cluster occur at the same time after the differentiation of surrounding locule regions. Sub-epidermal cells that differentiate into the circular cell cluster divide, enlarge, and generate vacuoles with calcium oxalate crystals prior to any detectable changes in pre-stomium epidermal cells. Differentiation and division of cells that generate the stomium occur after cell degeneration initiates in the circular cell cluster. Prior to dehiscence, the stomium consists of a small set of cytoplasmically dense cells that are easily distinguished from their larger, highly vacuolate epidermal neighbors. Plasmodesmata connections within and between cells of the stomium and circular cell cluster were observed at different developmental stages, suggesting that these cells communicate with each other. Circular cell cluster and stomium cell death is programmed developmentally and occurs at different times. Degeneration of the circular cell cluster occurs first, contributes to the formation of a bilocular anther, and generates the site of anther wall breakage. The stomium cell death process is complete at flower opening and provides an opening for pollen release from each theca. We used laser capture microdissection and real-time quantitative reverse-transcription polymerase chain reactions to demonstrate that stomium cells can be isolated from developing anthers and studied for the presence of specific mRNAs. Our data suggest that a cascade of unique gene expression events throughout anther development is required for the dehiscence program, and that the differentiation of the stomium and circular cell cluster in the interlocular region of the anther probably involves cell signaling processes.