The Study Of The Effect Of Extracellular Calmodulin On Cytosolic Calcium In Pollen And Its Signal Transduction Pathway

In this paper, lilium daviddi pollen as material, the calcium sensitive fluorescence indicator fluo-3 AM was loaded into intact pollen grain cells, confocal laser scanning microscopy was used as main method, the effect of extracellular calmodulin on cytosolic calcium concentration and its signal transduction pathway were investigated. The result is as below: When cytosolic calcium is being measured with acetyl methyl ester of calcium sensitive fluorescence indicator, eliminating the effect of extracellular lipases is the critical step for successful dye loading. Loading indicator at room temperature was unsuccessful, but low temperature may inhibit the extracellular lipases, indicators can be loaded successfully. Exogenous purified calmodulin can elevate intracellular calcium ion concentration, the optimum concentration of CaM is 10-7mol/L. The effect of higher or lower concentration CaM all reduced. The result show that as a signal , purified exogenous CaM can stimulate cytosolic Ca²⁺, then Ca²⁺ may modulate metabolism in cytoplasm as a secondary messager. Cell membrane nonpermeable inhibitor of calmodulin –W7-agarose–and the anti-serum of calmodulin can decrease the cytosolic calcium level, the results show that the endogeneous extracellular calmodulin may play an important role in maintaining and accelerating the cytosolic calcium level in pollen grain cell. The activity of heterotrimeric G protein in cell membrane affect the cytosolic calcium. After treated by CTX ,activator of heterotrimeric G protein, the cytosolic calcium elevated markedly. In pollen cells treated with PTX, the inhibitor of heterotrimeric G protein, cytosolic calcium decreased. When the pollen cells which pre-treated with PTX were treated by exogenous calmodulin, calmodulin can not effect cytosolic calcium level. In pollen cells which had been treated by anti-serum of CaM, CTX can elevate the cytosolic Ca²⁺ markedly. The result indicate that heterotrimeric G protein play an important role in the effect of exogenous calmodulin on cytosolic calcium, in this signal transduction chain, heterotrimeric G protein locate at the downstream of extracellular calmodulin. The activity of calcium channels in plasma membrane affect the cytosolic calcium, verapamil-an inhibitor of voltage-dependent calcium channels in plasma membrane-treatment can decrease the cytosolic calcium, but nifedipine had no effect. The result indicate that the calcium channel which sensitive to verapamil but insensitive to niedipine participate in maintaining calcium influx into cytoplasm of pollen cells under normal condition. The pollen cells pre-treated with verapamil were treated with CTX or CaM consequently, the cytosolic calcium elevated. The elevation amplitude in pollen cells treated by CTX is just the same as singly CTX treatment, but in pollen cells which treated by CaM, the elevation amplitude is lower than cells which treated with CaM singly. It is deduced that the calcium channel may be as the downstream effector of heterotrimeric G protein which modulated by extracellular calmodulin, play an important role in the effect of extracellular calmodulin on cytosolic calcium in pollen cells. At same time, the effect of CaM or CTX were not totally eliminated by verapamil , there may be other calcium influx pathway participate in the effect of extracellular calmodulin and CTX on cytosolic calcium in pollen cells. When pollen cells were treated with heparin, no obvious change of cytosolic calcium was found, it is deduced that in pollen cells without treatment, the activity of lipositol signal transduction pathway is low and cannot be detected. CTX had no effect on cytosolic calcium concentration in pollen cells pretreaterd with heparin, the result show that lipositol signal transduction pathway participate in the effect of CTX on cytosolic calcium in pollen cells, maybe a main pathway. Pollen cells pre-treated by heparin were treated by CaM, cytosolic calcium elevated, but the elevation amplitude was lower than single CaM treatment. The lipositol signal transduction system maybe participate in the effect of extracellular calmodulin on cytosolic calcium in pollen cells. But the effect of CaM was not totally eliminated by heparin, there may be other calcium influx pathway participate in the effect of extracellular calmodulin on cytosolic calcium in pollen cells.