The Regulation Of Hyperpolarization- Activated Calcium Channel In The Pollen Tube Of Pyrus Pyrifolia

Ion channels play important roles in regulation pollen tube growth. In this study, Ca2+ channels of Pyrus Pyrifolia cultivars’Housui’ pollen have been studied by multiple techniques. The main results as follows:1. Firstly, we studied the relation between the Ca2+ channels and pollen development. We have identified a similar Ca2+ channel in the pollen as which in the pollen tube. The whole current intense of the channel in the pollen was small than which in the pollen tube. However, neither open probability nor channel conductance of the single channel was changed. Thus, we speculate that the expression amount of the channels in the pollen was lower than which in the pollen tube. Furthermore, the Ca2+ channels expression were associated with the flowers age, pollen pre-hydration and the pollen tube length.2. Many signal-transduction processes in higher plant cells have been suggested to be triggered by signal-induced opening of Ca2+ channels in the plasma membrane. Cyclic nucleotides increase cytosolic free Ca2+ concentration in the pollen tube has been identified previously. However, direct evidence for activation of plasma-membrane Ca2+ channels by cAMP in pollen has not yet been obtained. Here, we report that cAMP activated a hyperpolarization activated Ca2+ channel in the Pyrus pyrifolia pollen tube. Outside-out single channel configuration identified that cAMP directly increased the Ca2+ channel open probability without affecting channel conductance. Interestingly, cAMP-induced currents show that these currents are permitting permeation of both Ca2+ and K+. However, the cAMP-evoked Ca2+ currents could not be substituted by cGMP. Cytosolic free Ca2+ concentration elevated to more than 1 μM significantly decreased the cAMP-induced currents. These results provide direct evidence for cAMP activation of hyperpolarization-activated Ca2+-permeable channels in the plasma membrane of pollen tube, which, in turn, can modulate cellular responses regulating pollen tube growth.3. Spermidine (Spd) has been correlated to various physiological and developmental processes in plants, including pollen tube growth. In this work, we show that Spd induces an increase in the cytosolic Ca+ concentration that accompanies pollen tube growth. Using the whole-cell patch clamp and outside-out single-channel patch clamp configurations, we show that exogenous Spd induces a hyperpolarization-activated Ca+ 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 Ca2+currents is exerted via a second messenger. This messenger is the H2O2, and it is generated during Spd oxidation; a reaction mediated by polyamine oxidase (PAO). These reactive oxygen species trigger opening of the hyperpolarization-activated Ca2+-permeable channels in pollen. To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca2+-permeable channels, two Arabidopsis mutants lacking expression of the peroxisomal-encoding AtPAO3 gene, were isolated and characterized. Pollen from these mutants was unable to induce the opening of the Ca2+-permeable channels in the presence of Spd, resulting in reduced pollen tube growth and seed number. However, a high Spd concentration triggers a Ca2+influx beyond the optimal, which results in being deleterious. These findings strongly suggest that the Spd-derived H2O2 signals Ca2+ influx, thereby regulating pollen tube growth.4. Sphingosine-1-phosphate is derived from sphingosine under the treatment of sphingosine kinase; it serves as an important signaling molecule involved in cell differentiation, proliferation and death process. In plants, studies have shown that its participation in the ABA regulated guard cells opening and closing. We have tested the effect of sphingosine-1-phosphate on pollen tube growth. The results showed that sphingosine-1-phosphate through the heterotrimeric G protein activation of the pollen plasma membrane hyperpolarization activated calcium channels, making the increase in calcium influx, regulation of pollen tube growth.5. The effect of heme on the outward K+(K+out) channels of Pyrus pyrifolia pollen tube protoplasts were studied using patch clamp technique. Heme directly decreased K+ out channels opening probability without affecting the unitary conductance, but the inhibition disappeared when heme was coapplied with increasing intracellular free-Ca2+. Conversely, exposure to heme in the presence of NADPH increased channel activity. However, with SnPP treatment, which inhibits hemeoxygenase activity, the inhibition of K+ out channels by heme occurred even in the presence of NADPH. Carbon monoxide (CO), a product of heme catabolism by hemeoxygenase, directly activated pollen tube protoplasts K+ out channels in a dose-dependent manner, but the induced currents was inhibited by the traditional K+ channel inhibitor Tetraethylammonium (TEA+). These data indicate a role of heme and CO in reciprocal regulation of the K+ out channels in pear pollen tube.6. Low temperature has significantly impact on pollen germination and pollen tube elongation. We have tested the effect of low temperature on pollen development. The results showed that pollen [Ca2+]cyt increased significantly upon the treatment of low temperature (10℃). Both of the influx of extracellular Ca2+ and Ca2+ release from intracellular store was involved in this response. The influx of extracellular Ca2+ in this response was mediated by membrane Ca2+-permeable cation channels. Outward K+ channel also participate in this response. Thus, we speculate that low temperature induced pollen tube growth inhibition is a Ca2+ elicited signal transduction cascade.