Study On The Characteristics And Pathway Of Incompatible Pollen Tube Death Mediated By Style S-RNase In Pyrus Pyrifolia

Using Pyrus pyrifolia cultivars ’Kosui’(S4S5) and’Imamuraaki’(S1S6) as materials, and a new method of isolation mitochondria or a method of mitochondrial membrane potential stain in intact pollen tube as procedure, the features of incompatible pollen tube death and the signal transduction mechanisms of self-incompatibility were studied in vitro, and some experimental results were validated in vivo. The main results as follows:1. A new method was developed for isolation mitochondria from a few amounts of pollen tubes. The method of homogenization method usually represents the critical step affecting the yield of mitochondria isolated from plant cells. The problem of balancing total disruption of cell wall structures with need to maximize preservation of intact mitochondria places crucial emphasis on the optimization of both the intensity and the duration of homogenization. We developed a method for cell homogenization, which homogenize the pollen tube on cell sieve and purify the mitochondria by differential density centrifugation. The isolated mitochondria were tested with transmission electron microscope, flow cytometry. The results showed that the isolated mitochondria were intact and with physiological activity. What’s more important, the isolated mitochondria are of high purity. This new method was easy, fast, reliable and efficient.2. The influence of SI in incompatible pollen tubes growing was evaluated. The MTT stain and Evarisblue stain were used to evaluate the time of incompatible pollen tube losing viability and death under S-RNase challenge in vitro, respectively. The results showed that the frequencies of viable pollen tube and live pollen tube were less changed at 30 min; at 60 min, the frequencies decreased obviously, and at 120 min, the values were less than half of control. Furthermre, the pollen tubes were stained with anilin blue, the result showed that incompatible pollen tubes were arrested in style at 9 h after pollination. Those data were helpful for the following experiments.3. It was demonstrated that death of incompatible pollen tubes shared some feature of programmed cell death (PCD). To investigate whether the death of incompatible pollen tube belongs to PCD, the fluorescence stain, transmission electron microscope, western blotting were used to observe the effect of S-RNase on mitochondria and nuclear DNA, and the caspase-like activity was tested in incompatible pollen tube at the same time. The results showed that the mitochondrial membrane potential (ΔΨmit) of incompatible pollen tube was specifically collapsed by S-RNase at 30 min after SI challenge in vitro. Moreover, it was also confirmed by western blotting at 2 h after SI challenge that ΔΨmit collapse induced cytochrome c leakage into the cytosol. Swollen mitochondria had been detected by transmission electron microscopy as early as 1 h after SI challenge, the swollen mitochondria accompany with reduction of crista, reducing of electron density and large bleb. The most important, the nuclear DNA degradation was detecteded both in vitro and in vivo, and the vegetative nucleus was degraded in advance of the generative nucleus. Otherwise, the data showed that the caspase-like activity was not triggered in incompatible pollen tube under S-RNase challenge. These results suggested that the death of incompatible pollen tube might belong to PCD and caspase-like activity was not involved in nuclear DNA degradation.4. The effect of tip-localized reactive oxygen species (ROS) on SI was evaluated, and a cascade signal pathway of nuclear DNA degradation was uncovered. The results showed that S-RNase specifically disrupted tip-localized ROS of incompatible pollen tubes via arrest the ROS formation in mitochondria and cell walls. The mitochondria ROS disruption was related to the mitochondria alteration, while cell walls ROS disruption was related to quantity of NADPH decrease. Tip-localized ROS disruption not only decreased the Ca2+ current, depolymerized the actin cytoskeleton, but it also induced nuclear DNA degradation. This was the first time to report a cascade signal pathway of self-incompatibility in P. pyrifolia L.