Actin filament organization and function in pollen development and induction of microspore embryogenesis of Brassica napus L. cv. Topas

Actin filament (AF) organisation during microspore/pollen development of Brassica napus L. cv. Topas was investigated using non-fixed cells. Actin filaments radiated from one side of acentric nucleus into the cytoplasm of microspores and surrounded the spindle apparatus during pollen mitosis I. Cytochalasin D treatment resulted in nuclear and spindle displacement. The vegetative cell of the mid-bicellular pollen grain contained cortical parallel AF bundles aligned transversely to the future axis of pollen grain elongation. Cytochalasin D treatment before AF bundle formation inhibited elongation. Pollen grains at anthesis showed AFs subjacent to the furrows of the vegetative cell and cytochalasin D treatment at this stage inhibited pollen tube germination.; Parallel cortical microtubules co-aligned with the AF bundles in mid-bicellular pollen grains. Disruption of AF bundle formation resulted in disorganised cortical microtubules. Microtubule disruption inhibited pollen grain elongation but did not affect the AF arrays. Thus it appears that AFs oriented cortical microtubules which in turn determined pollen grain shape.; Embryogenesis is induced by heat-treating microspores at 32.5°C for 24 h which were isolated near the first pollen mitosis. Heat treatment was accompanied by reorganisation of the AF cytoskeleton. Perinuclear AFs radiated into the cytoplasm from the central nucleus, AFs were present in the cytoplasm and surrounded a displaced spindle. Cytochalasin D treatment, which also resulted in nuclear and spindle relocation and induced embryogenesis, indicated that AFs participate in nuclear tethering and division plane determination.; Heat- and cytochalasin D-treated microspores contained either discontinuous walls that disappeared after 48 h of culture, or continuous cross-walls that appeared stable. Cytochalasin D-treated microspores dividing within 8 h of treatment showed microtubule phragnioplast disorganization that resulted in abnormal cell walls. This confirms the AF role in maintaining the integrity of the leading edge of the phraginoplast. The data support the hypotheses proposed for this project: actin filaments were present at all stages of microspore/pollen grain development, disruption of the actin cytoskeleton produced an embryogenic response, abnormal cell walls formed following cytochalasin D treatment were unstable and AF organisation influenced pollen grain shape.