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.

A temperature-responsive gene in sorghum encodes a glycine-rich protein that interacts with calmodulin.

Imposition of different biotic and abiotic stress conditions results in an increase in intracellular levels of Ca2+ which is sensed by various sensor proteins. Calmodulin (CaM) is one of the best studied transducers of Ca2+ signals. CaM undergoes conformational changes upon binding to Ca2+ and interacts with different types of proteins, thereby, regulating their activities. The present study reports the cloning and characterization of a sorghum cDNA encoding a protein (SbGRBP) that shows homology to glycine-rich RNA-binding proteins. The expression of SbGRBP in the sorghum seedlings is modulated by heat stress. The SbGRBP protein is localized in the nucleus as well as in cytosol, and shows interaction with CaM that requires the presence of Ca2+. SbGRBP depicts binding to single- and also double-stranded DNA. Fluorescence spectroscopic analyses suggest that interaction of SbGRBP with nucleic acids may be modulated after binding with CaM. To our knowledge, this is the first study to provide evidence for interaction of a stress regulated glycine-rich RNA-binding protein with CaM.

Characterization of Peptidyl-Prolyl Cis-Trans Isomerase- and Calmodulin-Binding Activity of a Cytosolic Arabidopsis thaliana Cyclophilin AtCyp19-3.

Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (Ki) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu(2+), which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca(2+)-dependent, and CaM-binding domain is localized to 35-70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca(2+) signaling and cyclophilin activity in Arabidopsis.