Studies On The Cytological Damage Effects Of Low Energy Ion Implantation On Pollen Grains And Pollen Tubes

In 1986, low-energy ion beams were first explored by Chinese researchers as anew agent for mutation induction in rice. Subsequently, the technology ofion-beam-induced gene transfer into either plant or bacterial cells was successfullyestablished. Through 20 years of development, the ion beam biotechnology hasaccomplished outstanding results in both fundamental research and applied research.However, detailed descriptions on the mechanisms of biological effects caused by ionimplantation are insufficient. Especially there is little understanding of the underlyingmechanisms responsible for its cytological damage effects at cellular or subcellularlevel. Most of the ion beam implantation work have been accomplished by usingseeds as model plant material. Their findings were about the biological effects of ionimplantation expressed on the development stage of seedlings or mature individuals.On other hand, a lot of work used biomacromolecule, such as plasmid DNA, asimplanted material to study the direct interaction of low energy ions and biomolecule.However, theses simulation experiment findings could not account for the realcytological effects of low energy ions in living cells. Cells are the basic units ofstructure and function in an organism. And cells are the valid targets of ionimplantation. So, there is a need for a better understanding of the direct interaction oflow energy ions and living cells.An ideal model system is a prerequisite for the studying on the basic cytologicalmechanisms of low energy ion implantation in living individual cell. The pollen grainis a monoploid and is rather simple in structure. The ease of culture and handling inthe lab make it a suitable and relatively simple, yet reliable, tool to investigate thebioeffects of low-energy ion implantation on plant cell. Gymnosperms pollen hassome other characteristics, including ease of storage from harvest until use, great survivability, and high yield quantity. In this study, pollen grains of gymnospermsPinus thunbergii and Cedrus deodara were implanted with 30 keV nitrogen ionbeams at doses ranging from 1×10¹⁵ ions/cm² to 15×10¹⁵ ions/cm². The cytologicaldamage effects of N⁺ implantation on the pollen cell wall, cell membrane,cytoskeleton framework, cell nucleus and proteome structure were examined. Themajor findings were described as follows:1. The effects of N⁺ implantation on pollen germination and pollen tube growthof Pinus thunbergii and Cedrus deodara were observed using light microscope andlaser scanning confocal microscope(LSCM). The threshold dose for a significanteffectiveness in pollen germination, tube length and tube width was 3×10¹⁵ ions/cm².N⁺ implantation within dose range of 3×10¹⁵ ions/cm² to5×10¹⁵ ions/cm² increased therate of pollen germination in Cedrus deodara. The dose response has a shoulderappearance—it first increased at small doses then decreased with increasing dose.However, the dose-response curve in P. thunbergi was different from that seen in C.deodara. Generally N⁺ implantation did inhibit pollen germination in P. thunbergi.The dose-response curve presented a particular pattern of saddle-like. An apparentpeak of germination rate was observed at the dose of 7×10¹⁵ ions/cm²(saddle-point).In addition, N⁺ implantation with same dosage range produced similarcytomorphological changes in pollen tubes of Pinus thunbergii and Cedrus deodara,namely the diameter of the tube increased and the tube length decreased. Bycomparison, the biological effects of nitrogen ion beams on pollen germination andpollen tube growth in P. thunbergi were different from those induced by UV andgamma-ray radiation, indicating that low energy ion implantation might work atdifferent way when compared to low-LET radiation.2. By using atomic force microscope (AFM), scanning electronmicroscope(SEM) and light microscope, the effects of N⁺ implantation on Cedrusdeodara pollen exine substructure were investigated. Results of light microscopyshowed that N⁺ implantation with doses bigger than 5×10¹⁵ ions/cm² significantlychanged the pollen color from pale yellow to deep rust. Atomic force microscopyand scanning electron microscopy distinctly demonstrated the erosion of the pollen exine caused by N⁺ implantation in the micrometer to nanometer range. Typicalresults showed that the erosion degree was linearly proportional to the ion dose. Thethreshold dose beginning to etch pollen wall was 3×10¹⁵ ions/cm². Generally, ionimplantation reduced exine strata and made pollen wall thin to some extent. With thedose increasing, ion implantation severely etched the pollen surface and resulted inthe formation of scattered 'holes', "crater", or even crack-like structures, which wouldbe the ion-beam-generated pathways for exogenous macromolecule transfer.3. The accumulation of superoxide free anion radical and malondialdehyde(MDA), the relative membrane permeability, as well as activities of some protectiveenzymes such as superoxide dismutase (SOD), peroxidase(POD) and catalase(CAT)were measured in P. thunbergi pollen following N⁺ implantation, with an aim toinvestigate the physiological mechanism of cell damage caused by ion implantation.The results showed that the superoxide free anion radical generation rate, the contentof MDA and the electrolyte leakage of the cell membrane gradually raised with thedose increasing, indicating the peroxidation of cell membrane lipid was induced andthe membrane system was damaged. The SOD activity was enhanced at the dose of1×10¹⁵ ions/cm², and decreased rapidly with the dose increasing, while the activitiesof POD and CAT sharply decreased. It was suggested the SOD was the majorprotective enzyme that involved in activated oxygen metabolism in P. thunbergipollen.4. The effects of N⁺ implantation on total soluble protein composition and SODisozyme, POD isozyme, as well as CAT isozyme in P. thunbergi pollen tubes werestudied by means of SDS-PAGE gel electrophoresis and PAGE gel electrophoresis.The data showed that N⁺ implantation induced the obvious differences of the solubleprotein bands and isozyme spectrums when compared to that of control sample,suggesting that low energy ion implantation was effective on producing DNAdamage.5. Like every other eukaryotic cell, the pollen tube contains an elaboratecytoskeletal apparatus, which mainly consists of microtubules and actin filaments. Awell-characterized cytoskeletal apparatus plays important roles in pollen tube growth. Elongation in gymnosperms pollen tubes is fundamentally distinct from angiosperms,CytoskeletaI organization in pollen tubes of gymnosperms differs from that seen inangiosperm pollen tubes. This study investigated the effects of low energy nitrogenion implantation on the cytoskeleton organization in pollen tube of Pinus thunbergiiby using immunolabeling for microtubules and rhodamine-phalloidin fluorescencelabeling for actin filaments. The results of confocal microscopy showed that N⁺implantation caused the disorganization of microtubules throughout pollen tubes,especially in swollen tips. The cytological consequences of ion implantation treatmentwere coincident with that of colchicine treatment. These results indicated thatdisruption of microtubules induced by N⁺ implantation was responsible formorphological abnormalities in the pollen tubes. Furthermore, confocal microscopyalso showed that ion implantation disrupted actin filament cytoskeleton organizationin pollen tube. There was a distinct correlation between the inhibition of pollen tubegrowth and the disruption of actin cytoskeleton organization, indicating that an intactactin cytoskeleton was essential for continued pollen tube elongation in Pinusthunbergii.6. The single-cell gel electrophoresis test or comet assay is a microgelelectrophoresis technique that measures DNA damage at the level of single cells.Alkaline single-cell gel electrophoresis enables sensitive detection of DNAsingle-strand breaks in eukaryotic cells. The study employed single cell gelelectrophoresis to analyze the DNA damage in nuclei isolated from pollen exposed tolow energy nitrogen ion beams and used confocal microscopy to assess the nucleidamage induced directly by ion implantation. The results of confocal microscopyshowed that ion implantation caused dose-dependent increases in nuclei damage.Some kinds of fragmentation of pollen nuclei were detected. The alkaline cometassays revealed that ion implantation induced increased DNA damage, whenevaluated by tail moment and tail length in migrated DNA. To the best of myknowledge, this was the first observation that DNA single-strand breaks induced bylow energy ion implantation had been detected using the alkaline comet assay. Due toits sensitivity and rapidity, the alkaline comet assay would prove to be a useful tool in assessing DNA damage caused by low energy ion implantation.The present data demonstrated that 30 keV nitrogen ion beam implantation coulddamage pollen cell constituents, including pollen wall, cell membrane, cytoskeletonand cell nucleus, etc. The structure damages might be either the direct consequence ofimplanted ion action or the indirect events initiated by the accumulation of superoxidefree anion radical. The present study assumed that the cytoskeleton system in pollengrains, namely, the microtubule and the actin filament, might provide a key target inresponses to ion beams implantation. The ion implantation could directly or indirectlyinduce the disruption of cytoskeleton organization in pollen cells and subsequentlycaused cytological changes in elongating pollen tubes. With regard to the mechanismsof cytological damage effects induced by low energy ion implantation, manyproblems remained unsolved. To test our assumption, further evidence would beexpected from the genetic and molecular analysis. The present study might provide aclue for further research onto the interaction between low-energy ion beams andpollen cellular organization and its practical application.