Kong Lingan, Wang Mao, Wang Qinli, Wang Xiaohua, Lin Jinxing
Key Laboratory of Photosynthesis and Molecular Environment Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China.
Tree Physiol. 2006 Aug;26(8):1001-12. doi: 10.1093/treephys/26.8.1001.
To investigate the roles of protein phosphatases 1 and 2A in the development of pollen tubes of Picea wilsonii Mast., pollen grains were cultured in standard medium in the presence and absence of the protein phosphatase inhibitors okadaic acid and calyculin A. At nanomolar concentrations, these compounds blocked pollen tube growth, causing abnormal morphologies of the pollen tubes. Studies with Fluo-3 revealed that the inhibitors reduced the pollen-tube tip-to-base cytoplasmic calcium (Ca(2+)) gradient and arrested extracellular Ca(2+) uptake. The transmission electron microscope observations indicated that the fusion of paramural bodies with plasma membranes occurred frequently in the tip and sub-tip regions of control pollen tubes, but fusion rarely occurred in inhibitor-treated pollen tubes. Staining with aniline blue showed that callose accumulated in the tip regions of inhibitor-treated pollen tubes. Immunolabeling of pollen tubes revealed that acidic pectin epitopes recognized by the monoclonal antibody JIM5 were present in the tip region and on the flanks of the sub-tip in normal pollen tubes. In inhibitor-treated pollen tubes, these epitopes existed only in the extreme tip region and at higher concentrations than in control pollen tubes. The esterified pectin recognized by JIM7 was located preferentially at the extreme tip region in normal pollen tubes, but at basal sites in inhibitor-treated tubes. Fourier transform infrared (FTIR) analysis further confirmed the changes in acidic and esterified pectin distributions and their relative contents. These results suggest that protein phosphatase 1 or 2A, or both, are involved in the regulation of Ca(2+) uptake across the plasma membrane, in exocytotic activity and in the biosynthesis of cell wall components, all processes that occur in the tip region of pollen tubes and that control pollen tube development.
为了研究蛋白磷酸酶1和2A在青杄花粉管发育中的作用，在有和没有蛋白磷酸酶抑制剂冈田酸和花萼海绵诱癌素A的情况下，将花粉粒培养在标准培养基中。在纳摩尔浓度下，这些化合物会阻止花粉管生长，导致花粉管形态异常。用Fluo-3进行的研究表明，抑制剂降低了花粉管尖端到基部的细胞质钙（Ca(2+)）梯度，并阻止了细胞外Ca(2+)的吸收。透射电子显微镜观察表明，壁旁体与质膜的融合在对照花粉管的尖端和亚尖端区域频繁发生，但在抑制剂处理的花粉管中很少发生融合。用苯胺蓝染色表明，胼胝质在抑制剂处理的花粉管尖端区域积累。花粉管的免疫标记显示，单克隆抗体JIM5识别的酸性果胶表位存在于正常花粉管的尖端区域和亚尖端的侧面。在抑制剂处理的花粉管中，这些表位仅存在于极端尖端区域，且浓度高于对照花粉管。JIM7识别的酯化果胶在正常花粉管中优先位于极端尖端区域，但在抑制剂处理的花粉管中位于基部。傅里叶变换红外（FTIR）分析进一步证实了酸性和酯化果胶分布及其相对含量的变化。这些结果表明，蛋白磷酸酶1或2A，或两者都参与了跨质膜的Ca(2+)吸收、胞吐活性以及细胞壁成分生物合成的调节，所有这些过程都发生在花粉管的尖端区域，并控制着花粉管的发育。
