Calcium channel identification,modulation and function in the pollen tube of Pyrus pyrifolia

【摘要】：正The pollen tube has been widely usedas a model to study signal transduction in plants.The tip-high free cytosolic calcium([Ca~(2+)]_(cyt))gradient is essential for pollen tube growth.Extracellular Ca~(2+) influx mediated by plasma membrane Ca~(2+) channels is involved in maintain pollen tube[Ca~(2+)]_(cyt) gradient.To investigate the molecular mechanism of pear pollen tube plasma membrane Ca~(2+) channels,we have developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia.By using patch clamp technique,we have further identified a hyperpolarization activated calcium channel in the spheroplasts.The calcium channel displayed a strong selectivity for divalent cations,with a relative permeability sequence of Ba~(2+)≈Ca~(2+)Mg~(2+).We also showed that the channel was inhibited by the Ca~(2+) channel blockers La~(3+).Furthermore,channel activity depended on extracellular pH and pollen viability. In the next step,we tested how to modulate this channel by chemicals such as spermidine (Spd).Spd has been correlated with pollen tube growth.In our testing,Spd induces an increase in the[Ca~(2+)]_(cyt) in the pollen tube.Moreover,exogenous Spd induces the hyperpolarization activated Ca~(2+) current:the addition of Spd cannot induce the channel open probability increase in excised outside-out patches,indicating that the effect of Spd in the induction of Ca~(2+) currents is exerted via a second messenger.In our further study,we found this messenger is hydrogen peroxide(H_2O_2),and is generated during Spd oxidation,a reaction mediated by polyamine oxidase(PAO).These reactive oxygen species directly trigger the opening of the hyperpolarization activated Ca~(2+) channels in pollen.To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca~(2+) channels,two Arabidopsis mutants lacking AtPAO3 gene were isolated and characterized.Pollen from these mutants was unable to induce the opening of the Ca~(2+) channels in the presence of Spd,resulting in reduced pollen tube growth and seed number.However,a high Spd concentration triggers a Ca~(2~) influx beyond the optimal,which has a deleterious effect.These findings strongly suggest that the Spd-derived H_2O_2 signals Ca~(2+) influx,thereby regulating pollen tube growth. We are further deciphering of the downstream targets of the intracellular calcium.We identified an outward K~+ channel which sensitive to the cytosolic Ca~(2+) concentration in the pear pollen tube plasma membrane.More interestingly,the outward K~+ channel is regulated by heme/carbon monoxide controlling system.Heme of 1μmol·L~(-1) decreased outward K~+ channel opening probability,but the inhibition disappeared when heine was coapplied with 10μmmol·L~(-1) intracellular free-Ca~(2+).Conversely,exposure to heine in the presence of NADPH increased channel activity.However,with Tin protoporphyrin IX(SnPP) treatment,which inhibits hemeoxygenase activity,the inhibition of outward K~+ channel by heme occurred even in the presence of NADPH.Carbon monoxide,a product of heme catabolism by hemeoxygenase,activates outward K~+ channel in pollen tube protoplasts in a dose-dependent manner.The induced current by carbon monoxide was inhibited by the K~+ channel inhibitor tetraethylammonium (TEA~+).These data indicate a role of heine and carbon monoxide in reciprocal regulation of outward K~+ channel in pear pollen tube.