Apoptosis in developing anthers and the role of ABA in this process duringandrogenesis in Hordeum vulgare L.

Intra-nucleosomal cleavage of DNA into fragments of about 200 bp was demons trated to occur in developing anthers, in which microspores had developed i rate the mid-late to late uni-nucleate stage in situ, i.e. at the verge of mitosis. The same was observed, but to a much larger extent, if these anthe rs were pre-treated by a hyper-osmotic shock. Pretreatment of anthers befor e the actual culture of microspores was required for optimal androgenesis o f microspores. The use of the TUNEL reaction, which specifically labels 3' ends of DNA breaks, after intranucleosomal cleavage of DNA, revealed that D NA fragmentation mainly occurred in the loculus wall cells, tapetum cells a nd filament cells. TUNEL staining was absent or infrequently observed in th e microspores of developing anthers in situ. Electron microscopy studies sh owed condensed chromatin in nuclei of loculus wall cells in the developing anthers. These observations at the chromatin and DNA level are known charac teristics of programmed cell death, also known as apoptosis. Features of ap optosis were infrequently found in microspores from freshly isolated mature anthers. However, most tapetum cells had disappeared in these anthers and the remaining cell structures showed loss of cellular content. The viabilit y of microspores in pre-treated anthers was comparable to those in freshly isolated anthers and almost four times higher than in anthers from control experiments. This observation was correlated with three to four times less microspores showing TUNEL staining and a two times higher level of ABA in t he anther plus medium samples than in controls. Addition of ABA to the cont rols enhanced the viability and lowered the occurrence of apoptosis linked characteristics in the microspores. These data suggest that pre-treatment i s effective in stimulating androgenesis because it leads to an increase in ABA levels which protects microspores from dying by apoptosis.