Movement of generative cell and vegetative nucleus in tobacco pollen tubes is dependent on microtubule cytoskeleton but independent of the synthesis of callose plugs

Abstract

The role of microtubules (MTs) in vegetative nucleus (VN) and generative cell (GC) transport was investigated by comparing VN and GC distribution with callose plug formation in tobacco pollen grains germinated and grown for 12 h with the plant-specific anti-MT drug oryzalin. The VN-GC complex or VN alone was located close to the tube tip in 100% of controls, but in only 5% of oryzalin-treated tubes. Instead, in 38% of oryzalin tubes, the complex or VN occurred close to the last-formed callose plug; in 40% between or in the middle of plugs; and in 17%, in or near the grain. An aberrant microfilament (MF) cytoskeleton was revealed by expression of a green fluorescent protein-talin fusion protein in living oryzalin-treated tubes. The abnormal MF structures probably resulted from the absence of MTs and impaired – or were a consequence of – VN and GC movement into the tube tip. In oryzalin tubes with several callose plugs, the VN and GC could be in or near the grain, indicating that callose plug synthesis is not dependent on the movement of VN and GC into the tube. VN and GC movement and callose plug formation are apparently independent events, in which the transport of the VN-GC complex must precede callose plug synthesis. Maintenance of the correct developmental program requires an intact MT cytoskeleton, otherwise no fertile pollen tubes are formed.