Abstract:

Fluorescent probe acetoxymethyl ester of Fluo-3 (Fluo-3/AM) was loaded into the cells of Liriodendron tulipifera × L. chinense anther under low temperature at 4 ℃ to observe the distribution of free Ca²⁺ during the pollen development using confocal laser scanning microscopy(CLSM). The dynamic distribution of free Ca²⁺ was measured with the relative changes of Ca²⁺ fluorescence intensity. We found that the Ca²⁺ 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,Ca²⁺ fluorescence intensity of cytoplasm was stronger than the tetrad wall. Actually,there was no Ca²⁺ 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 Ca²⁺ distribution of microspore was not symmetrical. There was stronger Ca²⁺ 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 Ca²⁺ fluorescence intensity was well-proportioned in different cell layer of anther wall. In succession,the middle layer of anther wall had the strongest Ca²⁺ 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 Ca²⁺ fluorescence intensity from microspore to pollen maturity until the tapetum was disappeared completely. The dynamic distribution of Ca²⁺ in anther wall shows the evidence that the Ca²⁺ flux flows from outer layers to inner layers. Moreover,the characteristic distribution of free Ca²⁺ is closely related to the important conversion taches in the development of pollen.

Key words: Liriodendron tulipifera ×, L. chinense;pollen development;free Ca²⁺;low temperature loading