Effects Of Low Energy Argon Ion Implantation On The Distribution Of The Actin Cytoskeleton And The Cytosolic Ca²⁺ Concentration During Maize Pollen Germination

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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 0.78×10¹⁵ions/cm² to 13×10¹⁵ions/cm².When compared with the control,Ar⁺ implantation at the dose of 5.2×10¹⁵ions/cm² significantly increased maize pollen germination.However,when the ion dose is larger than 7.8×10¹⁵ions/cm²,the rate of pollen germination sharply decreases.At the dose of 9.1×10¹⁵ions/cm²,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 5.2×10¹⁵ions/cm² 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 9.1×10¹⁵ ions/cm² 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[Ca²⁺]_i in ungerminated pollens, established that Ca²⁺ plays a key role in the regulation of pollen germination.Normally growing pollen tubes possess a steep tip-focused[Ca²⁺]_i apical gradient,and at tip region of pollen tubes have highest apical concentration of[Ca²⁺]_i,which established that[Ca²⁺]_i are indisputably involved in modulation of pollen tube growth.Results showed that pollen cell Ca²⁺ concentration would be increased when implanted with a dose of 5.2×10¹⁵ions/cm² argon ion,reveal that dynamic change of Ca²⁺ might be the primary effects of the reorganization of actin cytoskeleton and pollen germination after low energy argon ion implantation.