非损伤微侧技术在植物单细胞及亚细胞水平的应用

世界知名的花粉管研究专家Dr.Peter K. Hepler以前的研究已经证明在Papaver rhoeas的花粉管中细胞内自由Ca²⁺的增高是由杂交不亲合引起的，但关键的问题是没有证实细胞外的Ca²⁺是否参与了此过程。为了证明这个问题，他们利用了非损伤微测技术，检测罂粟花粉管细胞外的Ca²⁺的变化情况。通过检测，最后证实在自交不亲合过程中有细胞外的自由Ca²⁺的参与。这篇文章体现非损伤微测技术在植物生理学研究中的独特作用：该技术可以对震荡信号进行检测，并且可以得到真实、稳定的信号^{[The Plant Journal]}。

Figure 1. Oscillations of Ca²⁺ influx at the pollen tube tip.

Measurements of oscillating net influx of Ca²⁺ at the apex of a growing pollen tube of P. rhoeas were measured using a Ca²⁺±selective vibrating probe and are displayed graphically here. The plot shows net Ca²⁺ flux (pmol cm±2 sec±1), with net influx as a negative reading, and net efflux as a positive reading. The latter part of the plot shows a short background measurement 10 mm away from the pollen tube, in order to demonstrate that the fluxes measured are attributed to the pollen tube, and not noise.

同时Dr.Peter K. Hepler通过多年的研究还提出花粉管伸长部位应该有一段固有碱化带的存在，为了证实这一假说，Xu Yue等应用H⁺微电极和O₂极谱电极同时测定了百合花粉管生长过程中H⁺和O₂进出的变化。结果表明，在花粉管碱化区域的H⁺表现为外流，而O₂表现为内流，因此，推测花粉管碱化区域有丰富的活跃的线粒体^{[Journal of Integrative Plant Biology]}。

Figure 2. H⁺ efflux and O₂ influx in the z-axis plane over the alkaline band region.

Pollen tubes, 800–2 500 µm in length, were selected for measurements. The H⁺/O₂ fluxes, in terms of ∆µVz and ∆µAz, at the surface of a pollen tube were measured by moving the electrode 2 µm away to the tube surface. Background references were taken at least 500 µm away from any pollen grains or tubes and that value was subtracted from the surface measurements. The inset image shows a screen shot of the H⁺/O₂ probes and a growing pollen tube with a growth rate of 14.634 µm/min. This figure is a typical plot of results from five independent experiments.

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