Study On The Configuration Of Actin Cytoskeleton And The Relationship Between Actin Cytoskeleton And [Ca～(2+)]_i In Pyrus Pyrifolia Pollen (Tubes) During Self-incompatible Response

Studies on the self-incompatibility （SI） have been mostly focused on the area of pistil S and pollen F-box gene, and their products. Many progresses on clone, expression, separation, purification and function of gene have been achieved. But there is still a long way for us to understand the mechanisms of SI throughout. Although it’s believed that the interactivity of pistil and pollen results in the SI reaction, the mechanism of signal transduction during the pistil and pollen recognition is still less reported.Using Pyrus pyrifolia cultivars, ’Hosui’ （S₃S₅） and ’Imamuraaki’ （S₁S₆） as meterials, the effects of S-RNase, the product of pistil S gene in Pyrus pyrifolia, and dynamic of actin cytoskeleton on the pollen germination, tube growth, the dynamic of cytosolic free calcium concentration （[Ca2+]i）, and stylar S-RNase on the ultrastructure of pollen tubes in vitro were studied. The signal transduction of stylar S-RNase and dynamic of actin cytoskeleton mediating the pollen tube growth were discussed. The main results are listed as follows:1. Optical microscopy and transmission electron microscopy were used to in vitro study the effects of the stylar S-RNase of different Pyrus pyrifolia varieties on the germinations, and pollen-tube growths and ultrastructures of autogamous （incompatible） and xenogamous （compatible） pollens. The results showed that the stylar S-RNase inhibited the pollen germination and pollen-tube growth of the incompatible pollens, but hardly exerted any influence on the germination and pollen-tube growth of the compatible pollens. The incompatible and compatible pollens had similar ultrastructures at their growth; after being cultured for 24 hours, the compatible pollen tubes were full of cytoplasm and organelles but the incompatible pollen tubes only contained a small amount of cytoplasm at the front tip, and thickened cell wall between which and cytoplasm there existed a layer of callose and a electronically transparent partition.2. The effects of actin cytoskeleton depolymerized reagent cytochalasin B and stabile reagent phalloidin on pollen germination and tube growth have been studied in vivo and in vitro. The results showed: 1) lower concentration of phalloidin could promote pollen germination and tube growth, but high concentration inhibited germination and growth in vitro; CB inhibited pollen germination and tube growth, and the higher was the concentration, the stronger was inhibitory effect. 2) Phalloidin also promoted self-pollen germination and tube growth after self-pollination in vitro; while CB inhibited pollen germination and tube after cross-pollination. Results showed that actin cytoskeleton was involved in Pyrus pyrifolia pollen germination and tube growth, and might play a role in Pyrus pyrifolia self-incompatibility response.3. The distribution of actin cytoskeleton in Pyrus pyrifolia pollen and tube was observed by the method of chemical fixation and phalloidin-fluorescence labeling, with either fluorescence or confocal microscopy. The results showed: 1) using MBS treatment before chemical fixation and DMSO instead of regular fixation, the image of actin cytoskeleton could be conserved intactly and distinctly. 2) FITC-ph labeling could avoid disturbance of spontaneous fluoresce of pollen ektexine.4. Configurations of actin cytoskeleton in Pyrus pyrifolia pollen and effects of stylar S-RNases and CB on the pollen tubes growth rate and the dynamics of actin cytoskeleton have been investigated with optical microscope, fluorescence microscope and confocal microscope. Results showed that: 1) the growth rates of pollen tubes which treated by self-stylar S-RNase and CB were decreased at 50 min and 30 min, respectively, after treatment; While non-self-stylar S-RNase has no effect on pollen tube growth, just as control. 2) Actin filaments in normal pollen grain exist as fusiform or circular structure. When pollen germinates, actin filaments assemble around one of germination pores, and then actin bundles orient axially throughout the shank of growing tube. There is devoid of actin filaments 5-15μm to the tube tip. While self-stylar S-RNase is added in basal medium, pollen germination and tube growth were inhibited. There were different configurations of actin cytoskeleton followed by the culturing time: within 20 rain, actin configurations in self-pollen and tube were similar as normal; but after 20 min of treatment, actin filaments in the pollen tube translated gradually into network from the shank to the tip; finally, there was punctate actin in the whole tube. Although actin filaments of self-pollen grain also disintegrated into punctate foci, the change was slower than that of tube. Furthermore, the alteration of actin cytoskeleton was prior to the arrest of pollen tube growth. These results suggested that Pyrus pyrifolia stylar S-RNase induced alterations of actin cytoskeleton in self-pollen grains and tubes.5. The effects of Ca2+ on Pyrus pyrifolia pollen germination, tube growth and the distribution of actin cytoskeleton in pollen and tube were studied with optical microscopy and confocal microscopy. The results showed that: 1) High concentration (10^-1 mol/L) and very low concentration (＜10^-4 mol/L) of Ca²⁺ in the medium caused severe inhibition of pollen germination and tube growth, but Ca2+ of 10-3 mol/L was necessary for pollen germination and tube growth. However, Ca²⁺ specific chelating agent EGTA and Ca²⁺ channel blockers verapamil led to the inhibition of pollen germination and tube growth. In addition, high concentration of Ca²⁺ ionophore A23187 （＞5μmol/L） impeded pollen germination and tube growth, but lower concentration （2.5μmol/L） promoted pollen germination and tube growth. 2) High or lower Ca²⁺ concentration induced disintegration of actin cytoskeleton in pollen and tube; Ca²⁺ concentration increased and actin cytoskeleton disintegrated after slef-pollination. These results indicated that appropriate Ca²⁺ concentration is necessary for pollen germination and tube growth. High Ca²⁺ concentration inhibited pollen germination and tube growth, and this process carded out though regulation of actin configuration and distribution. Self-stylar S-RNase might induce alterations of actin cytoskeleton by [Ca²⁺]_i.6、The effects of CB and phalloidin on [Ca²⁺]_i and Ca²⁺ channel has been studied with confocal microscopy and patch clamp. The results showed that CB could promoted [Ca²⁺]_i to increase and activated Ca²⁺ channel in plasma membrane; while phalloidin had no effect on [Ca²⁺]_i and activated Ca²⁺ channel. So, disruption of actin cytoskeleton might inhibit pollen germination and tube growth by activing Ca²⁺ channel in plasma membrane and increasing the concentration of [Ca²⁺]_i.