Breĭgina M A, Smirnova A V, Matveeva N P, Ermakov I P
Tsitologiia. 2009;51(10):815-23.
We applied quantitative fluorescent microscopy to study membrane potential alterations during pollen germination and in growing pollen tube. Two voltage-sensitive dyes were applied: DiBAC4(3) was used to detect average membrane potential values in pollen grains and isolated protoplasts; Di-4-ANEPPS gave an option of membrane potential mapping on pollen protoplast and pollen tube surfaces. We have found out that tobacco pollen grain activation is accompanied by hyperpolarization of the vegetative cell plasma membrane by about 8 mV. Lily pollen protoplasts were significantly hyperpolarized (-108 mV) with respect to the pollen grains (-23 mV) from which they were isolated. We found polar distribution of the membrane potential along the protoplast surface, and longitudinal potential gradient along the pollen tube. In the presence of plasma membrane H(+)-ATPase inhibitor, sodium orthovanadate (1 mM) or its activator fusicoccin (1 microM), the longitudinal voltage gradient altered but did not disappear. Anion channel blocker, NPPB (40 microM), fully discarded the gradient in pollen tubes. Obtained results give evidence of the plasma membrane hyperpolarization during pollen germination and uneven potential distribution on pollen grain and tube surfaces. Inhibitory analysis showed involvement of the plasma membrane H(+)-ATPase and anion channels in membrane potential regulation.
我们应用定量荧光显微镜来研究花粉萌发过程中和生长中的花粉管内的膜电位变化。使用了两种电压敏感染料：DiBAC4(3)用于检测花粉粒和分离原生质体中的平均膜电位值；Di-4-ANEPPS可用于绘制花粉原生质体和花粉管表面的膜电位图。我们发现，烟草花粉粒的激活伴随着营养细胞质膜超极化约8 mV。百合花粉原生质体相对于分离它们的花粉粒（-23 mV）显著超极化（-108 mV）。我们发现膜电位沿原生质体表面呈极性分布，沿花粉管呈纵向电位梯度。在存在质膜H(+)-ATP酶抑制剂原钒酸钠（1 mM）或其激活剂藤霉素（1 microM）的情况下，纵向电压梯度发生改变但并未消失。阴离子通道阻滞剂NPPB（40 microM）完全消除了花粉管中的梯度。获得的结果证明了花粉萌发过程中质膜的超极化以及花粉粒和花粉管表面电位分布不均。抑制分析表明质膜H(+)-ATP酶和阴离子通道参与膜电位调节。
