The distribution of and relationship between F-actin and G-actin were investigated in pollen grains and pollen tubes of Lilium davidii Duch. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. Circular F-actin bundles were found to be the main form of microfilament cytoskeleton in pollen grains and pollen tubes. Consistent with cytoplasmic streaming in pollen tubes, there were no obvious F-actin bundles in the 10- to 20-microm tip region of long pollen tubes, only a few short F-actin fragments. Labeling with fluorescein isothiocyanate (FITC)-DNase I at first established the presence of a tip-focused gradient of intracellular G-actin concentration at the extreme apex of the tube, the concentration of G-actin being about twice as high in the 10- to 20-microm region of the tip as in other regions of the pollen tube. We also found that the distribution of G-actin was related negatively to that of the F-actin in pollen tubes of L. davidii. Caffeine treatment caused the G-actin tip-focused gradient to disappear, and F-actin to extend into the pollen tube tip. Based on these results, we speculate that the circular F-actin bundles may be the track for bidirectional cytoplasmic streaming in pollen tubes, and that in the pollen tube tip most of the F-actin is depolymerized into G-actin, leading to the absence of F-actin bundles in this region.

MATERIALS AND METHODSWe demonstrate here the presence of actin and myosin in pea (Pisum sativum L.) tendrils.

Pollen is an excellent source of actin for biochemical and physiological studies of the actomyosin system in higher plants. We have developed an efficient method to prepare relatively high levels of actin from the pollen of maize (Zea mays L.). The procedures of purification include acetone powder preparation, saturated ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, a cycle of polymerization-depolymerization, and Sephacryl S-200 gel filtration. The average yield of actin is 19 milligrams per 100 grams of pollen grains extracted. This is comparable with those of Acanthamoeba castellanii and human platelets. The purified pollen actin is electrophoretically homogeneous and its molecular mass is 42 kilodaltons. The amino acid composition and circular dichroism spectrum of pollen actin are identical to those of muscle actin. The actin purified from pollen is able to polymerize to F-actin. The pollen F-actin activated the activity of the muscle myosin ATPase sevenfold. amino acid composition, circular dichroism spectrum, and activation of myosin ATPase activities. MATERIALS AND METHODS ChemicalsDEAE-cellulose and Sephacryl S-200 were obtained from Pharmacia. ATP-Na2 salt, imidazole, SDS, acrylamide, bismethylene-acrylamide, Tris, and monoclonal antibodies against smooth-muscle a-actin were from Sigma. All other reagents used were of analytical grade. Maize PollenMaize (Zea mays L.) plants were grown in the Experimental Station of Beijing Agricultural University in Beijing.The cultivar was Nongda No. 14. Pollen were collected in the middle of July when the plants were in pollination stage. About 1 kg fresh weight of maize pollen was collected and stored in a desiccator placed in a freezer (-700C) for use. It has been well established that the actomyosin system is universally distributed in higher plants (9). The actomyosin system is an important component of cytoskeleton, which plays a pivotal role in the maintenance of cell shape, cell motility, mitosis, and cytokinesis. The presence of actin and myosin in higher plants has been identified in Amaryllis, Haemanthus, tomato, conifer root, pollen of Luffa, pea tendrils, and pollen tube of lily (3,5,13,17,20,21,23) by biochemical and immunological methods. But the concentrations of actin in plant are lower than they are in animal cytoplasmic tissues (14). So the biochemical and physiological studies of plant actomyosin are limited, unlike the cases in other nonmuscle cells such as slime molds and Acanthamoeba castellanii. However, we found that pollen is a rich source of actin for purification. We have developed an efficient method to prepare sufficient quantities of pure actin for biochemical and physiological studies such as the conformational changes during the polymerization of actin and the reassembly of microfilaments in vitro. Here we describe this method, which includes acetone powder preparation, ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephacryl S-200 gel filtration. Furthermore, the pollen actin w...

Tubulin was purified by a combination of acetone powder preparation, DEAE Sephadex A-50 chromatography, Sephacryl S-300 gel filtration, and Mono Q anion exchange chromatography from the pollen of ginkgo (Ginkgo biloba L.), a typical gymnosperm. The average yield of tubulin is 2 mg per 100 g of pollen grain. The purified tubulin is electrophoretically homogeneous. It seems to be composed of two subunits on SDS-PAGE and is resolved as two major spots on two-dimensional electrophoresis, preliminarily indicating that there are no obvious tubulin isotypes in ginkgo pollen. The apparent molecular weights of the two subunits are about 54 kDa and 52 kDa respectively, estimated from the SDS-PAGE. It was also demonstrated that tubulin from ginkgo pollen is immunochemically related to animal brain tubulin, and the purified tubulin was polymerized to microtubular aggregates in the presence of taxol and GTP in vitro.

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