【作者】 沈捷； 徐进； 施季森；

【Author】 Shen Jie Xu Jin Shi Jisen Key Laboratory of Forest Genetics & Biotechnology (Nanjing Forestry University),Ministry of Education,China,210037

【机构】 南京林业大学林木遗传与生物技术省部共建教育部重点实验室；

【摘要】 Ca2+作为偶联胞外信号与胞内生理生化反应的第二信使,其动态变化在启动花粉萌发、调节花粉管伸长等环节起着重要作用。目前,对花粉发育过程中Ca2+的分布研究主要集中在花粉的萌发和花粉管的生长方面,对小孢子发生和花药中Ca2+的分布研究较少,裸子植物小孢子母细胞的形成和发育过程以及花粉管的结构与被子植物存在差异,同时由于体外萌发困难等因素,研究较为缓慢。我们以杉木不同无性系花粉为材料,运用低温装载法在完整花粉粒中成功地载入酯化形式的Ca2+荧光探针Fluo-3-AM,利用激光共聚焦显微技术对花粉细胞质中游离Ca2+的荧光强度和分布变化特点进行观测,以研究杉木花粉对环境的响应以及在自身的应答反应过程中Ca2+分布的特征,为进一步探讨杉木花粉萌发过程中钙信号参与各种生命活动提供证据。结果表明,杉木花粉萌发时,萌发孔或萌发沟附近区域含较多的游离Ca2+,呈现出一种不均匀分布的趋势;随着花粉的水合,萌发孔或萌发沟部位的质膜扩张,激活了该部位质膜上的Ca2+通道,导致外源Ca2+从该部位进入胞质;而外源Ca2+的进入又造成一个含高浓度Ca2+的萌发孔区,这一梯度控制着花粉管伸长生长的速度和方向;随着花粉管的突起和生长,在花粉细胞的萌发沟和新生的花粉管内分布着大量的Ca2+,主要存在于花粉管的尖端。通过进一步的观察我们发现,大量分布Ca2+的花粉细胞的萌发沟和新生的花粉管尖端,往往也有内质网的分泌小泡大量聚集。内质网分布于整个花粉管内,而不是集中于一个明显区域或者仅仅局限于位于花粉管顶端的质膜附近,因此我们认为在杉木花粉萌发过程中,内质网系统并不是维持花粉管Ca2+梯度的唯一内源钙库,分泌小泡可能也是细胞的内源钙库之一。更多还原

【Abstract】 Calcium as a universal second messenger regulates a range of cellular function and physiological processes in plants and animals.The changes of the cytosolic free Ca2+ could be the cause for changes in cell physiology and the generation of an appropriate response.Ca2+ has being widely studied during pollen germination and pollen tube elongation in many plants.In the present study we have reinvestigated the issue of free calcium gradients in pollen tubes.We used Chinese fir (Cunninghamia lanceolata (Lamb.)Hook.) pollen as materials,loaded the calcium sensitive probe Fluo-3/AM into intact pollen cells at low temperature.The dynamic distribution of free Ca2+ was measured with the relative changes of Ca2+ fluorescence intensity by confocal laser scanning microscopy (CLSM).Results showed cytoplasmic calcium take polarized distribution in germinating pollens.The cytoplasmic calcium concentration near germinal furrow is obviously higher than others,and the highest is around germinal pore and tip of the pollen tube,the lowest is in pollens nuclear.All the places where at high Ca2+ lever are abundant of secretory vesicles.The endoplasmic reticulum(ER) encloses a continuous space in the whole pollen cell instead of located at the tip of the pollen tube.Experimental analysis suggested that ER and Golgi complex are not the only major mobilizable intracellular Ca2+ stores in pollen tubes,the dense core secretory vesicles may also be a dynamic Ca2+ store.更多还原