LITHIUM TREATMENT OF NICOTIANA-TABACUM MICROSPORES BLOCKS POLAR NUCLEAR MIGRATION, DISRUPTS THE PARTITIONING OF MEMBRANE-ASSOCIATED CA2+, AND INDUCES SYMMETRICAL MITOSIS

We have found that the normal developmental pathway of Nicotiana tabac um 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: chang es in microspore shape from spherical to oval and then ellipsoid; two nuclear displacements, first from a central location to the cell perip hery, and then from the periphery to the generative pole; a localizati on of membrane-associated Ca2+ at the generative pole preceding nuclea r division; and, finally, an asymmetrical mitosis that results in a tw o-celled pollen grain with well-differentiated generative and vegetati ve nuclei. Lithium treatment blocked the localization of membrane-asso ciated Ca2+ at the generative pole, and instead it was evenly distribu ted at both poles. Lithium treatment also blocked the asymmetrical pos itioning of the microspore nucleus at the generative pole and resulted in an approximately four-fold increase in the frequency of symmetrica l mitosis. When Ca2+ and myoinositol were added along with lithium, th e effects were substantially decreased, and there was only a small inc rease 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 develop men t, while microspores isolated immediately preceding the onset of mitos is were much less sensitive to lithium, and the result was only a smal l increase in the frequency of symmetrical mitosis. In microspores iso lated after the first nuclear displacement, a 1-day exposure to lithiu m was sufficient to switch the developmental pathway from an asymmetri cal to a symmetrical mitosis while still allowing limited further deve lopment. However, we have not optimized culturing conditions for embry ogenesis and the furthest development observed after a 1-week culture was to four- or five-celled proembryo-like structures