杂种鹅掌楸花粉发育过程中细胞游离Ca^(2+)的动态变化

Dynamic Change of Free Ca^(2+) during Pollen Development of Liriodendron tulipifera×L. chinense

利用钙离子荧光探针Fluo-3/AM低温装载技术,在激光扫描共聚焦显微镜下观察杂种鹅掌楸花粉发育不同阶段细胞内游离Ca2+的分布,以其相对荧光强度的强弱来衡量细胞内游离Ca2+水平的动态变化。研究发现在初生造孢细胞中Ca2+荧光较弱,次生造孢细胞和小孢子母细胞时期Ca2+荧光增强。早期四分体小孢子细胞质中Ca2+荧光强度较强,胼胝质壁无荧光;后期细胞质中的Ca2+荧光减弱,而胼胝质壁处Ca2+荧光增强。小孢子内Ca2+分布不均匀,细胞质中的Ca2+荧光较弱,细胞壁处Ca2+荧光较强,二细胞花粉时期,细胞呈现较强的Ca2+荧光。在花药壁组织内Ca2+分布也呈现规律性的变化:造孢组织时期,花药壁组织Ca2+荧光强度在不同壁层组织中分布均匀;小孢子母细胞时期,药壁中层细胞Ca2+荧光最强,绒毡层细胞次之;单核小孢子时期,绒毡层细胞呈解体状态,Ca2+荧光最强,并保持到二核花粉时期直至绒毡层完全消失,但此时花药纤维层发育形成,表现出较强的Ca2+荧光。花药组织中Ca2+分布动态显示出Ca2+由外而内流动的迹象,而且细胞内游离Ca2+分布特征与花粉发育过程的重要转变环节密切相关。

Fluorescent probe acetoxymethyl ester of Fluo-3 （Fluo-3/AM） was loaded into the cells of Liriodendron tulipifera x L. chinense anther under low temperature at 4 ℃ to observe the distribution of free Ca^2＋ during the pollen development using confocal laser scanning microscopy （ CLSM）. The dynamic distribution of free Ca^2＋ was measured with the relative changes of Ca^2＋ fluorescence intensity. We found that the Ca^2＋ fluorescence intensity of primary sporogenous tissue was lower than secondary sporogenous tissue. The fluorescence intensity became stronger gradually from the stage of sporogenous cell to microspore mother cell. In the earlier stage of microspore tetrad, Ca^2＋ fluorescence intensity of cytoplasm was stronger than the tetrad wall. Actually, there was no Ca^2＋ fluorescence intensity in the callose wall of the tetrad. Later on, fluorescence intensity of cytoplasm in microspore tetrad was subdued, and callose wall of microspore tetrad was enhanced. Similarly, the Ca^2＋ distribution of microspore was not symmetrical. There was stronger Ca^2＋ fluorescence intensity in the microspore wall, and weaker in the cytoplasm by contraries. At the same time, the regular changes were presented in anther wall tissue during the development of pollen. In the stage of sporogenous tissue, the Ca^2＋ fluorescence intensity was well-proportioned in different cell layer of anther wall. In succession, the middle layer of anther wall had the strongest Ca^2＋ fluorescence intensity, and the tapetum took second place during the meiosis of microspore mother cell. Subsequently, the tapetum cell began to disorganize, and it kept the strongest Ca^2＋ fluorescence intensity from microspore to pollen maturity until the tapetum was disappeared completely. The dynamic distribution of Ca^2＋ in anther wall shows the evidence that the Ca^2＋ flux flows from outer layers to inner layers. Moreover,the characteristic distribution of free Ca^2＋ is closely related to the important conversion taches in the development of pollen.