Evaluation Of UV Damage At DNA Level In Several Plant Protoplasts Using SCGE And UV Resistant Physiological Indicators Detecting

Comet assay as also called the single cell gel electrophoresis(SCGE)is a sensitive,reliable,and rapid method which can detect double or single DNA breaks and alkal-labile sites in eukaryotic individual cells.In this study,this method was applied to measure DNA damage of several plant protoplasts,which induced by UV. An improvement method that is used for detecting different plant DNA damages.In our previous asymmetric somatic hybridization of plants,some asymmetry hybrids were obtained by UV irridiated protoplasts of Bupleurm scorzonerifolium (380μW/cm²,5min)and Thellungiella salsuginea(380μW/cm²,2min)before asymmetric somatic hybridization between Arabidopsis thaliana/B,scorzonerifolium and A.thaliana/T,salsuginea.The chromotins of receptor eliminated and introgressed into genomes of donors were investgated in these somatic hybrids.On the contrary., many results showed that the chromosomes of donor fragmented,translocted or introgressed into the genomes of receptor in the somatic hybridization in which Triticum aestivum as one partners and heterologous genus grasses were UV-irradiated before fusion.Since ionization and excitation could be induced by primary UV-radiation led to the production of new chemical species in donor and hybrid cell, such as hydroxyl radicals,hydrogen radicals,and solvated electrons produced have a finite probability of interacting with DNA sites.To investigate the relationship between the dosages of UV-radiation on the protoplasts of donor and the chromosomes elimination/introgression of receptors or donors in the somatic hybrids. we use SCGE to evaluate UV damage at DNA level on six plant protopasts(B. scorzonerifolium,T.salsuginea,T.aestivum L.,Hasypyrum villosum and Thinopyrum intermedium(Hest)Nevski.)and using CASP(Comet assay software project) software to accurately analyze the images.The related physiological indicators produced by UV- radiation on plant materials were also detected and analyzed.The following experiments were operated in this research:The values of OTM(value of damage on cells)could reflect the response for different dosages of UV-irradiation on six plant protoplasts.We found that the value of OTM of B.scorzonerifolium,A.thaliana,T.salsuginea,T.aestivum and S,villosum don't have obvious images of DNA smearing(DNA migration length)at the low doses of UV-irradiation.While the value of OTM of H.villosum is the highest in these plant materials.The images of SCGE had the longest DNA smearing after UV irradiated its protoplasts for 1 min.The OTM values of the other five protoplastes varied greatly during the medium doses of UV(380μW/cm²)irradiatied for 2-5min. The OTM values of B.scorzonerifolium and T.salsuginea are lower than that A.thaliana,T.aestivum and T.intermedium,accordingly,the images of SCGE have longer DNA smearing in A.thaliana,7.aestivum and 7.intermedium as compared to B.scorzonerifolium and T.salsuginea with increase in UV exposure time.The DNA damage level of 7.salsuginea rises sharply with increase in UV exposure time (380μW/cm² for 5-10min)using SCGE analysis;while the OTM values of B. scorzonerifolium rises gently.Just about 20%of DNA damages can be detected in the cells of B.scorzonerifloium,the other DNA samples all diffused in the gel to be detected with UV exposure time rised to(380μW/cm²)12.5min.According to above data,the OTM value of B.scorzoneriflium is the lowest and H,villosum,is the highest, respectively.UV-resistant order of six plant materials arranged descending roughly:B. scorzonerifolium＞T.salsuginea＞T.aestivum＞A.thaliana＞T.intermedium＞H. villosum.1)The concentration of flavonoids in all six kinds of plant cells was measured using AI(NO3)3-NaNO2-NaOH method before and after UV-irradiation.Before and after UV irradiation,the concentration of flavonoids in B.scorzonerifolium was the highest. and the concentration of flavonoids in A.thaliana and T.salsuginea are the lowestin all other plant materials mensurated by ultraviolet spectroscopy.Accompaned by the rise of UV dosage,the content of flavonoids in all six kinds of plant materials are all gradually increased and then decreased after reaching a peak.The concentration of flavonoids in B.scorzonerifolium cells reached the maximum at UV-5min,while in T. salsuginea cells reached the maximum at UV-15min.The concentration of flavonoids in A.thaliana,T.intermedium and H,villosum reached the peak at UV-10min.2)The concentration of phenolic compounds in B.scorzonerifolium,T.salsuginea, A.thaliana,T.intermedium,H.villosum and T.aestivum were measured after UV-irradiation using Prussian Blue(PB)method as compared to untreat sample.The concentration of polyphenols in B.scorzonerifolium is the highest in six plant materials.A sharp increase in the level of polyphenols was observed in six kinds of plant materials with increase in the time of exposure of samples to UV-radiation.A significant change in the content of polyphenols was observed in calli of B.scorzonerifolium till it reached the maximum level when added the UV irradiation for 5 min,and the production of polyphenols enhanced with increase the UV exposure for 10 min in all other five samples.Above results lead us to believe that UV radiation induced secondary metabolites accumulation have an important role in protection B.scorzonerifolium cells against UV radiation promoted flavonoids paths processes.3)The activities of POD in B.scorzonerifolium,T.salsuginea,Arabidopsis thaliana, H.villosum,T.intermediumand T.aestivum were detected before and after UV-irradiation.No significant regularity of POD content was observed in all six samples.Compared to other samples,the activity of POD in B.scorzonerifolium calli was the lowest in the control.Calli of B.scorzonerifolium had higher potential to accumulate POD in response to UV irradiation for 5 min,and then decreased sharply to the lowest level.Almost the same level of POD activity of B.scorzonerifolium. T.aestivum,T.salsugmea,and H.villosum was detected in the time of exposure of samples to UV-radiation for 20 min.The results suggested that UV could induce the activity of POD in plants. 4)The concentration of Pro in B.scorzonerifolium,T.salsuginea,A.thaliana,H. villosum,T.intermedium and T.aestivum were measured before and after UV exposure by acidic-ninhydrin method.The content of Pro was the highest in A. thaliana,and was the lowest in T.salsuginea in control samples.Calli of six kinds of sample exposed to UV radiation showed a time dependent increase in the level of Proline.No significant Pro accumulation was show in the calli of B.scorzonerifolium with increase in duration of UV exposure in its all five samples.Calli of T.salsuginea had higher potential to accumulate Pro in response to UV irradiation more than 10 min.Besides Proline accumulation in T.salsuginea during UV-exposure might be one of the modes to counteract UV radiation promoted ROS generation.5)The activities of O₂^?in B.scorzonerifolium,T.salsuginea,A.thaliana,H. villosum,T.intermedium and T.aestivum were measured before and after UV-irradiation by hydroxylammoniumchloride autoxidation method.The activities of O₂^?in the calli of B.scorzonerifolium and T.aestivum were the lowest in the six plants in controls.A sharp increase in the level of O₂^?was observed in T.salsuginea upon exposure to UV-radiation,and no significant change was observed in calli of B.scorzonerifolium.O₂^?generation is one of the initial cytochemical responses of plants exposed to UV radiation.To sum up,Secondary metabolite,such as flavonoids and polyphenols accumulation is the most frequent metabolic responses exhibited by B.scorzonerifolium exposed to UV stresses.POD and Pro accumulation in other plant samples during UV-exposure might be one of the modes to counteract UV radiation promoted ROS generation.