| Cytoskeleton plays important role in growth and development process of plant cells. Based on pollen and style of Pyrus Pyrifolia cultivars Hosui and Imamuraaki, effects of cytoskeleton on the growth, development and the role in stress of pear pollen tube were investigated in this study.1. The relationship between microfilament-structure of pear pollen tube and all kinds of signal molecule was investigated. Though medicament treatment, microtubule structure was observed and it was detected to take part in the process of pollen tube growth. After calcium ionophore A23187 treatment, the actin cytoskeleton was stability, but microtubule depolymerized after treating for long time; EGAT treatment depolymerized actin cytoskeleton, but showed no effect on microtubule; variations of microfilament-structure could affect the vibration and distribution of calcium ionophore in pollen tube. Variations of NO had no significant effect on pollen tube cytoskeleton while variations ROS regulated the microfilament-structure; variations of microfilament-structure influenced the vibration and distribution of NO and ROS in the pollen tube.2. The effect of the changes of cytoskeleton on the nuclear degradation of pear pollen tube and mitochondrial alteration was investigated. Nuclear degraded at 2 h after oryzalin (which inhibits microtubule polymerization) treatment, indicating that the depolymerization of microtubule is the early singal. in the way of stopping pollen tube growth; the depolymerization of cytoskeleton induced the collapse of the membrane potential and the leakage of cytochrome c, promoted the degradation of the nuclear DNA of pollen tubes and programmed cell death, while the stability of the cytoskeleton could remit the PCD during the SI of pear. In order to characterize the mitochondrial alterations in pollen tubes following the changes of cytoskeleton, transmission electron microscopy (TEM) was used to assess the change in ultrastructure. After the depolymerization of microtubules, no obvious changes in the the volume of the mitochondria was observed, but disappearance of most cristaes in the mitochondria and thickening of mitochondrial membrane were observed; cooperative process with the stabilizer of microtubules and the self-S-RNase could abate the mitochondrial alteration in the SI, and only a small part of cristaes was disappear; an increasing mitochondrial volume was observed and several cristaes were destroyed after the depolymerization of actin. The mitochondria in the pollen tube were swollen, and the cristaes were disappear after applying the stabilizer of actin, indacting that it had no obvious effects on the remission of the SI. All the results indacted that the polymerization of the microtubules could abate the breakage of SI, but the polymerization of the actin have no obvious effect on the remission of the destruction of mitochondria.3. Genes for coding microfilament-and microtubule-associated proteins were studied based on bioinformatics analysis of pear genome. Fifteen genes were speculated to be Myosi family though BLASTn and classified into 7 groups using phylogenetic analysis; furthermore, uniform distribution of these genes in pear chromosome were observed. Compared to Arabidopsis, SH3 and DIL structural domain in Myosin Ⅷ gene in pear genome were lost based on the analysis of genetic structure domain. Eleven genes were speculated to be MAP65 family though BLASTn and classified into 6 groups via phylogenetic analysis; based on homology alignment, homology of each gene in MAP65 of pear with its group gene of Arabidopsis was further observed.4. Effects of different components of pear style on pollen tube growth and cytoskeleton were investigated. After treating with taxol and S-RNase meanwhile, taxol can alleviate the inhibition of incompatibile S-RNase on tube growth, while S-RNase has no effect on germination. After treating with S-RNase, incompatibile S-RNase resulted in rapid disappearance of cortical microtubules pollen tube specifically and decrease of pollen tubes containing GC ratio significantly, while no effect was observed after treating by compatibile S-RNase. GC structure ratio was improved significantly after treating with incompatibile S-RNase plus taxol. Microfilament degradation, amplitude enhance and gradient descent of CAM, depolymerization of microfilament and microtubule, and ROS decrease influenced by incompatibile S-RNase could be stabilized by calcium ion regulator protein CAM, be relieved by microtubule stabilizer, taxol, be stabilized by scavenging agent of NO and be relieved by microtubule stabilizer, respectively. All the results indicated that microtubule plays importment role in pear pollen tube growth process, and interactions between cytoskeleton with Ca2+ or NO/ROS system in the pollen tube in self-incompatibility process of pear were also observed. In pear style, fourteen free amino acids were detected, of which glutamic acid content was the highest; after investigating the effects of different amino acids on the pollen tube growth, concentration dependent on 8 kinds of amino acids (L-, D-glutamic acid, L-, D-cysteine, L-, D-phenylalanine, D-valine and L-aspartic acid) were observed. After treating with these amino acids at high concentration, D-valine, D-phenylalanine and L-cysteine resulted in depolymerization of microfilament cytoskeleton; microfilament structure appeared and microfilament in pollen tube thicked after treating by L-phenylalanine; L-aspartic acid, D-glutamic acid and D-cysteine resulted in the disappearance of ring structure of tip microfilament cytoskeleton; no effect was observed for L-glutamic acid.5. Effects of different environmental factors (hormone, Polyamines, turgor pressure, metal ion, temperature, etc) on cytoskeleton in pollen tube were observed. Results of the effects of hormone on pollen tube growth and microfilament cytoskeleton showed that GA3 and zeatin possessed concentration-dependent on pollen tube growth regulation, with growth promotion by low concentration and growth inhibition and serious depolymerization of cytoskeleton for high concentration; low concentration of ABA promoted the pollen tube growth and resulted in the disappear of cyclic structure of microfilament cytoskeleton in the tip of pollen tube. Low concentration of spermine (0.1 mM) showed no effect on the microfilament cytoskeleton structure while high concentration of spermine and spermidine (0.5 mM) resulted in the depolymerization of the microfilament cytoskeleton, especially for spermine treatment. Osmotic pressure in pollen tube was 0.6 M, and after hypotonic treatment, pollen tube tip expanded and microfilament-structure in the tip distorted; after hypertonic treatment, microfilament distorted to cyclic structure and tip microfilament lost. Pollen tube was rather sensitive to Cu and its inhibition concentration was 5μM, while inhibition concentrations for Al3+ and Na+ were 2 mM and 50 mM, respectively. After treating with Cu for 0.5 h, fragmentation of microfilament cytoskeleton was observed, while Al3+ and Na+ need 1 h. After treating at 4℃ for 1 h, microfilament cytoskeleton depolymerized, while none depolymerization was observed and pollen tube tip just expanded when treating at 37℃ for 0.5 h. |
PDF Full Text Request