EGTA对‘秦冠’苹果花粉管Ca^(2+)、微丝分布以及囊泡运输的影响

Effects of EGTA treatment on calcium,actin distribution and vesicle trafficking of apple (Malus pumila Mill.) pollen tubes

【目的】研究Ca^(2+)螯合剂EGTA对花粉萌发和生长过程中花粉管形态、Ca^(2+)分布、细胞骨架和胞内运输的影响。【方法】以Ca^(2+)螯合剂EGTA处理‘秦冠’苹果成熟花粉,显微镜观察其对花粉萌发、花粉管生长的影响。运用免疫荧光标记技术结合荧光显微镜及激光共聚焦扫描显微镜观察花粉管内Ca^(2+)分布、微丝分布和囊泡运输模式。【结果】发现低浓度的EGTA对花粉萌发和花粉管生长影响很小,甚至有促进作用。用较高浓度的EGTA处理花粉后,花粉萌发和花粉管生长均受到明显抑制,花粉粒甚至不萌发,花粉管有扭曲膨大短小等现象。用400μmol/L EGTA处理花粉后,花粉管顶端钙离子内流稍有增加,用2mmol/L EGTA处理后,花粉管顶端钙离子交换被抑制。用400μmol/L EGTA处理后,花粉管内游离Ca^(2+)在花粉管顶端聚集并呈现浓度梯度分布,与对照的分布模式一致,而用2mmol/L EGTA处理后顶端聚集模式消失。对照和400μmol/L EGTA处理后花粉管内微丝与花粉管生长方向平行排列,而用2 mmol/L EGTA处理后花粉管内微丝断裂、丝状结构不明显,无序分布在花粉管内。对照花粉管内囊泡运输在顶端以及近顶端区域聚集,用400μmol/L EGTA处理后分布模式变化不大,而用2mmol/L EGTA处理后,囊泡运输增加,布满整个花粉管内。【结论】钙可能作为一种信号因子,通过影响细胞骨架的构成和胞内囊泡的运输模式,从而调节苹果花粉的萌发及花粉管生长。

[Objective] It is well established that the calcium is essential for pollen germination and tube growth. In this study we investigated the effects of EGTA,which is Ca2＋ chelator, on apple （Malus pumila Mill.） pollen tube growth. [Methods]Cultured pollens were fluorescent-labeled and immune-labeled in vitro and observed under fluorescent microscope combined with confocal laser scanning microscope. [Results]Our results showed that EGTA has no or even promotive effects on pollen tube growth at the low concentration,while high concentration of EGTA inhibited pollen germination and tube growth and led to the morphological abnormality of pollen tubes. After treatment with 400 tzmol/L of EGTA, the flow of calcium at the top of the pollen tube increased slightly. After treatment with 2 mmol/L of EGTA, the calcium ion exchange at the top of the pollen tube was inhibited. Fluo-3/AM labeling indicated that the gradient of cytosolic calcium （[Ca2＋ ]c） in the pollen tube tip was abolished by exposure to high EGTA. High concentration of EGTA affected actin microfilaments. The actin filaments in the control pollen tubes constituted a contiguous network of bundles throughout the tube, and that the aetin cables were parallel to the growth axis. After treatment with high concentration of EGTA the actin filaments broke down. Furthermore, vesicle traffic- king, as indicated by FM4-64 fluorescence,was perturbed due to [Ca^2＋] Be disruption when pollen were treated with high EGTA concentration. In control pollen tubes,FM4-64 fluorescence was found in the cytoplasm of the apex and sub-apex, while high EGTA concentration treatment led to FM4-64 fluorescence throughout the entire tube. [Conclusion]Our results suggested that calcium mediates the processes of pollen tube growth in Malus pumila, and its disruption resulted in abnormal growth by disturbing the [Ca^2＋ ]c gradient and vesicle trafficking .