Pan Yan-Yun, Wang Xin, Ma Li-Geng, Sun Da-Ye
Institute of Molecular Cell Biology, Hebei Normal University, Shijiazhuang, Hebei, PR China.
Plant Cell Physiol. 2005 Oct;46(10):1657-65. doi: 10.1093/pcp/pci181. Epub 2005 Aug 5.
The phosphatidylinositol-specific phospholipase C (PI-PLC) activity is detected in purified Lilium pollen protoplasts. Two PI-PLC full length cDNAs, LdPLC1 and LdPLC2, were isolated from pollen of Lilium daviddi. The amino acid sequences for the two PI-PLCs deduced from the two cDNA sequences contain X, Y catalytic motifs and C2 domains. Blast analysis shows that LdPLCs have 60-65% identities to the PI-PLCs from other plant species. Both recombinant PI-PLCs proteins expressed in E. coli cells show the PIP(2)-hydrolyzing activity. The RT-PCR analysis shows that both of them are expressed in pollen grains, whereas expression level of LdPLC2 is induced in germinating pollen. The exogenous purified calmodulin (CaM) is able to stimulate the activity of the PI-PLC when it is added into the pollen protoplast medium, while anti-CaM antibody suppresses the stimulation effect caused by exogenous CaM. PI-PLC activity is enhanced by G protein agonist cholera toxin and decreased by G protein antagonist pertussis toxin. Increasing in PI-PLC activity caused by exogenous purified CaM is also inhibited by pertussis toxin. A PI-PLC inhibitor, U-73122, inhibited the stimulation of PI-PLC activity caused by cholera toxin and it also leads to the decrease of Ca(2+) in pollen grains. Those results suggest that the PPI-PLC signaling pathway is present in Lilium daviddi pollen, and PI-PLC activity might be regulated by a heterotrimeric G protein and extracellular CaM.
在纯化的百合花粉原生质体中检测到磷脂酰肌醇特异性磷脂酶C（PI-PLC）活性。从大卫百合的花粉中分离出两个PI-PLC全长cDNA，即LdPLC1和LdPLC2。从这两个cDNA序列推导的两个PI-PLC的氨基酸序列包含X、Y催化基序和C2结构域。Blast分析表明，LdPLC与其他植物物种的PI-PLC具有60-65%的同一性。在大肠杆菌细胞中表达的两种重组PI-PLC蛋白均显示出PIP(2)水解活性。RT-PCR分析表明，它们在花粉粒中均有表达，而LdPLC2的表达水平在萌发的花粉中被诱导。当将外源纯化的钙调蛋白（CaM）添加到花粉原生质体培养基中时，它能够刺激PI-PLC的活性，而抗CaM抗体则抑制外源CaM引起的刺激作用。PI-PLC活性被G蛋白激动剂霍乱毒素增强，被G蛋白拮抗剂百日咳毒素降低。百日咳毒素也抑制由外源纯化CaM引起的PI-PLC活性增加。一种PI-PLC抑制剂U-73122抑制了霍乱毒素对PI-PLC活性的刺激，并且它还导致花粉粒中Ca(2+)的降低。这些结果表明，PPI-PLC信号通路存在于大卫百合花粉中，并且PI-PLC活性可能受异源三聚体G蛋白和细胞外CaM的调节。
