Cell death and 14-3-3 proteins during the induction of barley microspore androgenesis

In the barley (Hordeum vulgare L.) anther, tapetum and loculus wall cells undergo programmed cell death (PCD) at the time around the first pollen mitosis, at the uninucleate stage of microspore development. This is the stage where androgenesis is most efficiently induced in barley microspores. Induction of androgenesis is characterized by a switch of the normal pollen developmental pathway towards an embryogenic route via a stress pre-treatment of anthers for 4 days in mannitol solution. We were interested in studying the involvement of members of the 14-3-3 family of regulatory proteins during barley androgenesis induction. With the use of isoform-specific antibodies against the three 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C, we have studied their immunolocalization and expression level in anthers. All isoforms were localized in the microspores and in anther wall cells at the induction stage. At this period, 14-3-3A processing was found to take place in tapetum and loculus wall cells, where in situ DNA fragmentation was detected by TUNEL assay. After 4 days pre-treatment to induce androgenesis, anther wall cells degenerated and two types of morphologically distinct microspores were observed, enlarged and non-enlarged cells. At this stage, 14-3-3 isoforms were mainly localized in the microspores. 14-3-3A processing was found to be induced by stress and it could only be detected in non-enlarged cells with decreased viability after pre-treatment. Viable enlarged cells and pollen under normal in vivo development showed no visible 14-3-3A processing. The identification of 14-3-3A processing in anther wall cells and in microspores with decreased viability represents the first link between the processing of a specific 14-3-3 isoform in cells undergoing death pathway. The implications of this post-translational event in barley anthers are discussed.