A novel application of periodic acid-Schiff (PAS) staining and fluorescence imaging for analysing tapetum and microspore development.

The precisely timed process of tapetum development and its degradation involving programmed cell death is an important molecular event during anther development. Through its degeneration, the tapetum not only provides nutritive substances to the developing microspores but also contributes to the pollen wall by way of sporopollenin, which is a complex mixture of biopolymers, containing long-chain fatty acids, phenylpropanoids, phenolics and traces of carotenoids. A number of dyes and staining methods have been used to visualize tapetal structure and its components by using light microscopy techniques, but none of these methods could differentially stain and thus distinguish tapetal cells from other cell types of anther wall. While analysing progression of tapetum development in different cell types in rice anthers, we discovered a unique property of periodic acid-Schiff (PAS) stain, which upon interaction with some specific component(s) in tapetal cells and developing microspores emits fluorescence at ~620 nm. In rice anthers, the PAS-associated fluorescence could be observed initially in tapetum and developing microspores, and subsequent to degeneration of tapetum, the fluorescence was found to emanate mainly from the pollen wall. We also show that PAS-dependent fluorescence in tapetal cells is distinct from the autofluorescence resulting from pollen wall components and is also not caused by interaction of PAS with pollen starch. Henceforth, this novel fluorescence property of PAS stain could prove to be a new tool in the toolkit of developmental biologists to analyse different aspects of tapetum development and its degeneration with little more ease and specificity.