Lithium treatment of Nicotiana tabacum microspores blocks polar nuclear migration, disrupts the partitioning of membrane-associated Ca2+, and induces symmetrical mitosis

Abstract

We have found that the normal developmental pathway of Nicotiana tabacum microspores is blocked or switched when microspores are exposed to lithium, and these effects are reversible with Ca2+ and myo-inositol. Normal development was defined by the following characteristics: changes in microspore shape from spherical to oval and then ellipsoid; two nuclear displacements, first from a central location to the cell periphery, and then from the periphery to the generative pole; a localization of membrane-associated Ca2+ at the generative pole preceding nuclear division; and, finally, an asymmetrical mitosis that results in a two-celled pollen grain with well-differentiated generative and vegetative nuclei. Lithium treatment blocked the localization of membrane-associated Ca2+ at the generative pole, and instead it was evenly distributed at both poles. Lithium treatment also blocked the asymmetrical positioning of the microspore nucleus at the generative pole and resulted in an approximately four-fold increase in the frequency of symmetrical mitosis. When Ca2+ and myo-inositol were added along with lithium, the effects were substantially decreased, and there was only a small increase in the frequency of symmetrical mitosis compared with controls. The timing of treatment was important; microspores isolated before the first nuclear displacement had a low frequency of further development, while microspores isolated immediately preceding the onset of mitosis were much less sensitive to lithium, and the result was only a small increase in the frequency of symmetrical mitosis. In microspores isolated after the first nuclear displacement, a 1-day exposure to lithium was sufficient to switch the developmental pathway from an asymmetrical to a symmetrical mitosis while still allowing limited further development. However, we have not optimized culturing conditions for embryogenesis and the furthest development observed after a 1-week culture was to four- or five-celled proembryo-like structures.