低能氩离子注入对玉米花粉萌发过程中微丝骨架和钙离子浓度分布的影响
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摘要
花粉萌发、花粉管伸长是种子植物实现有性生殖的重要环节,花粉是极有代表性的顶端生长的细胞之一。细胞顶端生长的调控是微丝骨架和Ca~(2+)等参与的十分复杂的过程,因此作为具有顶端生长特征的花粉管已成为研究Ca~(2+)信号与植物细胞骨架的典型材料。本文以玉米花粉为材料,研究了低能Ar~+注入对其萌发活性、花粉内的微丝结构、以及胞内Ca~(2+)浓度分布的影响,为进一步了解花粉极性形成,花粉萌发及花粉管生长的发育调控机理提供了新方法。主要得到如下结果:
1、利用能量为30keV,剂量为0.78×10~(15)ion/cm~2-13×10~(15)ion/cm~2的Ar~+注入玉米花粉粒,以未注入离子的花粉做为对照观察花粉体外萌发后发现:当注入剂量为5.2×10~(15)ions/cm~2离子时,玉米花粉的萌发率有了明显的提高;当注入剂量超过7.8×10~(15)ion/cm~2以后,萌发率呈现急剧下降的趋势;在注入离子剂量为9.1×10~(15)ion/cm~2时,萌发的花粉不足50%,可以视为半致死剂量。同时我们发现,一些经Ar~+注入后的花粉花粉管生长变的异常,出现了诸如萌发出两条花粉管以及花粉管出现了分枝的现象。
2、用TRITC-phalloidin对花粉粒和花粉管内的微丝骨架进行染色后,利用激光共聚焦显微镜观察了低能Ar~+注入对花粉粒及花粉管内微丝骨架的影响。用促进花粉萌发的剂量5.2×10~(15)ions/cm~2的Ar~+注入后的花粉在萌发过程中微丝骨架的动态变化明显提前,在未经过离子注入的花粉内的微丝骨架分布还处于启动初期的网络状态时,经离子注入的花粉中的微丝骨架已经完成了它的重新聚合排列,从较为随机的在萌发孔处的交叉排列状态变为浓密的平行束状或环状结构,并集中于花粉萌发孔处;而用剂量为9.1×10~(15)ion/cm~2的Ar~+注入的花粉中的微丝骨架动态变化则相对滞后。说明离子注入可能是改变了花粉内微丝骨架的动态装配,从而对花粉萌发启动产生影响的。
3、利用低温孵育法在完整的玉米花粉粒中,成功地载入酯化形式的Ca~(2+)荧光探针fluo-3 AM。结果表明,萌发的花粉内游离Ca~(2+)呈极性分布,集中分布于与萌发密切相关的萌发沟部位,以萌发孔部位的Ca~(2+)浓度最高,而在未萌发的花粉中则观察不到Ca~(2+)的极性分布,表明Ca~(2+)有可能是控制花粉萌发启动的重要因素;同样在生长的花粉管内,Ca~(2+)浓度呈现梯度分布,以花粉管尖端Ca~(2+)浓度最高,表明Ca~(2+)与花粉管伸长密切相关。用促进花粉萌发的剂量5.2×10~(15)ions/cm~2Ar~+注入后的花粉在萌发起始,细胞中Ca~(2+)浓度明显增加;而用剂量为9.1×10~(15)ion/cm~2Ar~+注入的花粉却观察到相反的现象。这一结果揭示了低能离子注入后引起了花粉内Ca~(2+)浓度的动态变化可能是微丝骨架的动态装配和花粉萌发变化的初始效应。
Pollen tube,which deliver sperm cells to the egg for fertilization in higher plants,are an established model system for examining polarized cell growth.It is well established that the actin cytoskeleton and cytosolic Ca~(2+) play a critical role in regulating cell tip growth,so the pollen tube has progressively become a model system for the study of plant cell cytoskeleton and Ca~(2+) signal transduction.The aim of this study is to investigate the effects of low energy nitrogen ion implantation on the distribution of the actin cytoskeleton and the cytosolic Ca~(2+) concentration during Maize(Zea mays L) pollen germination,which provides a new method for studying the development control mechanism of pollen polar formation,pollen germination and pollen tube growth.
Pollen grains of maize were implanted with 30 keV argon ion beams at doses ranging from 0.78×10~(15)ions/cm~2 to 13×10~(15)ions/cm~2.When compared with the control,Ar~+ implantation at the dose of 5.2×10~(15)ions/cm~2 significantly increased maize pollen germination.However,when the ion dose is larger than 7.8×10~(15)ions/cm~2,the rate of pollen germination sharply decreases.At the dose of 9.1×10~(15)ions/cm~2,less than 50%of the treated pollen germinated,can be regarded as medial lethal dose.Meanwhile,we found some abnormal germinated pollen tubes after ion implantation.For example,some pollens germinatied two tubes from one aperture,some of the pollen tubes have branched.
Labeling with TRITC-phalloidin and using confocal laser scanning microscope,we examined the distribution of the actin cytoskeleton in pollen grains and pollen tubes during pollen germination and pollen tube growth after argon ion implantation.The actin cytoskeleton in the pollens which implanted by 5.2×10~(15)ions/cm~2 argon ion reorganized much earlier than that in control pollens.The actin filaments formed parallel thick bundles at the aperture in ion implanted pollens while the actin cytoskeleton remained at its cross-linked thin filament network in the control pollens.On the contrary,The reorganization of actin cytoskeleton in the pollens which implanted with 9.1×10~(15) ions/cm~2 argon ion comparatively slow than that in control pollens.The results indicating that the effect of argon ion implantation on the germination of pollen might be mediated by reorganization of the actin cytoskeleton.
The calcium sensitive fluorescence indicator fluo-3 AM was loaded into intact pollen grain cells at low temperature.Results showed cytoplasmic calcium take polarized distribution in germinating pollens.The cytoplasmic calcium concentration near germinal furrow is obviously higher than others which around the aperture has highest apical concentration,but we kan not see this apical gradient[Ca~(2+)]_i in ungerminated pollens, established that Ca~(2+) plays a key role in the regulation of pollen germination.Normally growing pollen tubes possess a steep tip-focused[Ca~(2+)]_i apical gradient,and at tip region of pollen tubes have highest apical concentration of[Ca~(2+)]_i,which established that[Ca~(2+)]_i are indisputably involved in modulation of pollen tube growth.Results showed that pollen cell Ca~(2+) concentration would be increased when implanted with a dose of 5.2×10~(15)ions/cm~2 argon ion,reveal that dynamic change of Ca~(2+) might be the primary effects of the reorganization of actin cytoskeleton and pollen germination after low energy argon ion implantation.
1、利用能量为30keV,剂量为0.78×10~(15)ion/cm~2-13×10~(15)ion/cm~2的Ar~+注入玉米花粉粒,以未注入离子的花粉做为对照观察花粉体外萌发后发现:当注入剂量为5.2×10~(15)ions/cm~2离子时,玉米花粉的萌发率有了明显的提高;当注入剂量超过7.8×10~(15)ion/cm~2以后,萌发率呈现急剧下降的趋势;在注入离子剂量为9.1×10~(15)ion/cm~2时,萌发的花粉不足50%,可以视为半致死剂量。同时我们发现,一些经Ar~+注入后的花粉花粉管生长变的异常,出现了诸如萌发出两条花粉管以及花粉管出现了分枝的现象。
2、用TRITC-phalloidin对花粉粒和花粉管内的微丝骨架进行染色后,利用激光共聚焦显微镜观察了低能Ar~+注入对花粉粒及花粉管内微丝骨架的影响。用促进花粉萌发的剂量5.2×10~(15)ions/cm~2的Ar~+注入后的花粉在萌发过程中微丝骨架的动态变化明显提前,在未经过离子注入的花粉内的微丝骨架分布还处于启动初期的网络状态时,经离子注入的花粉中的微丝骨架已经完成了它的重新聚合排列,从较为随机的在萌发孔处的交叉排列状态变为浓密的平行束状或环状结构,并集中于花粉萌发孔处;而用剂量为9.1×10~(15)ion/cm~2的Ar~+注入的花粉中的微丝骨架动态变化则相对滞后。说明离子注入可能是改变了花粉内微丝骨架的动态装配,从而对花粉萌发启动产生影响的。
3、利用低温孵育法在完整的玉米花粉粒中,成功地载入酯化形式的Ca~(2+)荧光探针fluo-3 AM。结果表明,萌发的花粉内游离Ca~(2+)呈极性分布,集中分布于与萌发密切相关的萌发沟部位,以萌发孔部位的Ca~(2+)浓度最高,而在未萌发的花粉中则观察不到Ca~(2+)的极性分布,表明Ca~(2+)有可能是控制花粉萌发启动的重要因素;同样在生长的花粉管内,Ca~(2+)浓度呈现梯度分布,以花粉管尖端Ca~(2+)浓度最高,表明Ca~(2+)与花粉管伸长密切相关。用促进花粉萌发的剂量5.2×10~(15)ions/cm~2Ar~+注入后的花粉在萌发起始,细胞中Ca~(2+)浓度明显增加;而用剂量为9.1×10~(15)ion/cm~2Ar~+注入的花粉却观察到相反的现象。这一结果揭示了低能离子注入后引起了花粉内Ca~(2+)浓度的动态变化可能是微丝骨架的动态装配和花粉萌发变化的初始效应。
Pollen tube,which deliver sperm cells to the egg for fertilization in higher plants,are an established model system for examining polarized cell growth.It is well established that the actin cytoskeleton and cytosolic Ca~(2+) play a critical role in regulating cell tip growth,so the pollen tube has progressively become a model system for the study of plant cell cytoskeleton and Ca~(2+) signal transduction.The aim of this study is to investigate the effects of low energy nitrogen ion implantation on the distribution of the actin cytoskeleton and the cytosolic Ca~(2+) concentration during Maize(Zea mays L) pollen germination,which provides a new method for studying the development control mechanism of pollen polar formation,pollen germination and pollen tube growth.
Pollen grains of maize were implanted with 30 keV argon ion beams at doses ranging from 0.78×10~(15)ions/cm~2 to 13×10~(15)ions/cm~2.When compared with the control,Ar~+ implantation at the dose of 5.2×10~(15)ions/cm~2 significantly increased maize pollen germination.However,when the ion dose is larger than 7.8×10~(15)ions/cm~2,the rate of pollen germination sharply decreases.At the dose of 9.1×10~(15)ions/cm~2,less than 50%of the treated pollen germinated,can be regarded as medial lethal dose.Meanwhile,we found some abnormal germinated pollen tubes after ion implantation.For example,some pollens germinatied two tubes from one aperture,some of the pollen tubes have branched.
Labeling with TRITC-phalloidin and using confocal laser scanning microscope,we examined the distribution of the actin cytoskeleton in pollen grains and pollen tubes during pollen germination and pollen tube growth after argon ion implantation.The actin cytoskeleton in the pollens which implanted by 5.2×10~(15)ions/cm~2 argon ion reorganized much earlier than that in control pollens.The actin filaments formed parallel thick bundles at the aperture in ion implanted pollens while the actin cytoskeleton remained at its cross-linked thin filament network in the control pollens.On the contrary,The reorganization of actin cytoskeleton in the pollens which implanted with 9.1×10~(15) ions/cm~2 argon ion comparatively slow than that in control pollens.The results indicating that the effect of argon ion implantation on the germination of pollen might be mediated by reorganization of the actin cytoskeleton.
The calcium sensitive fluorescence indicator fluo-3 AM was loaded into intact pollen grain cells at low temperature.Results showed cytoplasmic calcium take polarized distribution in germinating pollens.The cytoplasmic calcium concentration near germinal furrow is obviously higher than others which around the aperture has highest apical concentration,but we kan not see this apical gradient[Ca~(2+)]_i in ungerminated pollens, established that Ca~(2+) plays a key role in the regulation of pollen germination.Normally growing pollen tubes possess a steep tip-focused[Ca~(2+)]_i apical gradient,and at tip region of pollen tubes have highest apical concentration of[Ca~(2+)]_i,which established that[Ca~(2+)]_i are indisputably involved in modulation of pollen tube growth.Results showed that pollen cell Ca~(2+) concentration would be increased when implanted with a dose of 5.2×10~(15)ions/cm~2 argon ion,reveal that dynamic change of Ca~(2+) might be the primary effects of the reorganization of actin cytoskeleton and pollen germination after low energy argon ion implantation.
引文
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82.Malho R,Trewavas AJ.Localized apical increases of cytosolic free calcium control pollen tube orientation.Plant Cell,8:1935-1949(1996)
83.Malho R.Role of 1,4,5-inositol triphosphate induces Ca~(2+) release in pollen tube orientation.Sex Plant Report,11:231-235(1998)
84.Kohon T,Shimmen T.Ca~(2+)-induced F-actin fragmentation in pollen tubes.Prot-oplasma,141:177-179(1987)
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99.Hwang JU,Suh S,Yi HJ,Kim J,Lee Y.Actin filaments modulate both stomatal opening and inward K~+ channel activities in guard cells of Vicia faba L.Plant Physiol,115:335-342(1997)
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101.余增亮,王学栋,吴跃进等.离子注入水稻育种机理初探.安徽农业科学,1:12-16(1989)
102.余增亮等,离子束生物技术引论.安徽科技出版社,1998年12月,第一版
103.Yu ZL.Ion beam application in genetic modification.IEEE Transaction on Plasma Science,28:128-132(2000)
104.Yu ZL.Interaction between low energy ion and the complicated organism.Plasma Science&Technology.1(1):79-85(1999)
105.Yu ZL,Yang JB,Wu YJ,Cheng BJ.Transferring Gus gene into intact cells by low energy ion beam.Nuclear Instruments and Methodsin Physics Research,B 80/81:1328-1331(1993)
106.陈恒雷,吕杰,曾宪贤.离子束诱变育种研究及应用进展.生物技术通讯,2:10-13(2005)
107.虞龙,张宁.离子注入微生物诱变育种的研究与应用进展.微生物学杂志,25(2):60-83(2005)
108.袁成凌,王纪,等.离子注人花生四烯酸(AA)产生菌诱变选育及产业化研究.第三次全国离子束生物工程学大会暨第一次国际研讨会论文集,(2002)
109.胡素华,吴瑜,陈斌 单粒子微束装置的研究.核技术,25(9)742-744(2002)
110.彭士香,王绍虎,余增亮.微离子束:用预定数目的离子对细胞中指定位置进行手术的工具.真空科学与技术,18(1)38-46(1999)
111.吴李君,Tom K Hei,余增亮.微束单细胞照射的研究和发展.核科学与工程.20(1)78-83(2000)
112.Hei T.K.et al.Mutagenic effects of a single or an exact number of alpha particles in mammalian cells.Proc.Natl.Acad.Sci.(USA),94:3765-3770(1997)
113.Li-Jun Wu,Gerhand Randers-Pehrson.et al.Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells.Proc Natl Acad Sci U S A,96:4959-4964(1999)