In situ detection of Esr proteins secretion during maize microspore embryogenesis and their secretion blockage show effects on the culture progression

In vitro plant cells in culture release proteins and carbohydrates, but the active molecules responsible for sustaining the switch in embryogenic development and progression have not yet been identified. In maize, the Esr genes encode for small hydrophilic proteins and are expressed in the restricted region of maize endosperm surrounding the embryo, the embryo surrounding region (ESR). In the present work, the possible influence of secreted molecules in the liquid medium during microspore-derived embryo development, and specifically the presence of Esr proteins has been analysed in maize microspore cultures. The study has been carried out by in situ monitoring of the structural and cellu lar organization of developing embryos and the subcellular localization of the Esr proteins by immunofluorescence and immunogold labelling. The results obtained using confocal and electron microscopy revealed that Esrproteins were localized in elements of the secretory pathway and cell walls in microspore-derived embryo cells during early embryogenesis. Esr proteins were also detected in the liquid medium of maize microspore cultures and accumulated at 20 days of culture. Tunicamycin treatment to block protein glycosilation and therefore secretion inhibited microspore-derived embryo development, which was subsequently recovered by supplementation with medium containing all the secreted factors from a well-developed microspore culture. Esr labelling was not present in non-developing microspore embryos of cultures treated with tunicamycin, whereas labelling was present again in the Golgi elements and secretory vesicles of embryo cells when development was restored. The results indicate that Esr proteins are part of the secreted proteins, which show a nursing or signalling role during in vitro embryo development in maize microspore embryogenesis cultures, and provide new evidence for an endosperm-like function of microspore-derived embryo structures during the early stages.