Molecular Clone And Functional Analysis Of PLC In Pollen

Our previous work verified that heterotrimeric G protein was involved in regulation of pollen germination, tube growth and in the transmembrane mechanism of extracellular CaM signal. In the present study, we provide further evidence that PLC signaling pathway was presented in lily pollen and involved in extracellular CaM signal transduction mechanism in pollen by molecular, biochemical and physiological approaches. These evidence included:The PIP₂-dependent PLC activity was also detected in purified lily pollen protoplasts. It was found that PLC activity was stimulated by both exogenous purified calmodulin and heterotrimeric G protein agonist cholera toxin. While, the stimulated effect on PLC activity caused by exogenous calmodulin was inhibited by heterotrimeric G protein antagonist pertussis toxin, and PLC antagonist U-73122 inhibited the effect caused by cholera toxin. We also measure the free Ca²⁺ level in germinating pollen grains, and found that exogenous purified calmodulin was able to increase the free Ca²⁺ level, and their effects on Ca²⁺ level were inhibited by U-73122.Two kinds of full length of PLC cDNAs (LdPLCl and LdPLC2) were cloned from Lily pollen. Sequence analysis showed that these two PLC genes shared high similarity with PLCs from other plant species. Northern blot and RT-PCR analysis verified that the expression level of LdPLCl was induced in germinating pollen, whereas LdPLC2 expression level did not change during pollen germinating process.The interaction between heterotrimeric G proteins and PLC was detected in a yeast two-hybrid system. The result showed that both LdPLCl and LdPLC2 can physical interacted with Gp, but neither Ga nor GIn addition, Arabidopsis PLC knock-out and overexpression lines were also characterized, and the result proved that lose of function of AtPLC(At3g55940) induced AtPLC(At3g08510) gene expression and increase pollen germination rate, whereas gain of function of AtPLC(At3g55940) repressed AtPLC(At3g08510) expression, and decrease pollen germination.All the data above suggested that PLC signaling pathway was present in lily and Arabidopsis pollen and involved in signal transduction of extracelluar calmodulin to regulate the pollen tube growth.