| Studies on the effects of UV-B radiation on plants have been going on for a long time.According to the size of radiation dose,the research directions mainly focus on two aspects.On the one hand,the appropriate dose of UV-B radiation,as a regulatory signal,can initiate the photomorphogenesis of plants with UVR8 as the recognition site,and make plants undergo rigorous and orderly changes in morphological structure,metabolism and other aspects,so as to adapt to the inevitable UV-B radiation in the environment.On the other hand,the high dose of UV-B radiation exceeds the threshold of plant self-regulation,which makes the plant unable to heal itself,causing irreversible changes,and even affecting the genetic material of the plant,resulting in the death of the plant.Chromatin assembly factor 1(CAF-1),a conserved molecular chaperone of histones,has been extensively studied in yeast,mice,Arabidopsis thaliana and humans.Based on previous studies,the CAF-1 complex plays an important role in several biological processes,including gene transcription,DNA replication and repair.However,it remains to be explored whether CAF-1 complex is involved in enhancing the ROS damage and DNA damage repair of Arabidopsis thaliana after UV-B stress.The large subunit FAS1 mutant Atfas1-4 of the CAF-1 complex was purchased from the Arabidopsis resource center in our lab in the early stage,and the pure and mutant were obtained after identification.Combined with the characteristics of enhanced UV-B radiation damage to Arabidopsis thaliana and the function of CAF-1 complex,this study used the existing mutant strain Atfas1-4 under enhanced UV-B radiation to study,which laid a foundation for further revealing the mechanism of enhanced UV-B radiation damage repair.The main findings are as follows:(1)Atfas1-4 was identified as homozygous mutant by triple primer method.The expression of FAS1 in Atfas1-4 mutant was measured by semi-quantitative method,and the content of FAS1 was significantly reduced,indicating the loss of function of FAS1 in the mutant.(2)Compared with Wild type(WT),Atfas1-4 had shorter root length,shorter plants height,smaller leaves area,more trichomes,shorter pods and lower seeds setting rate;(3)The expression of FAS1 was measured by semi quantitative method at 0,1,2 and 3hours after UV-B irradiation.The expression of FAS1 changed with the dose.Compared with the control group,the expression of FAS1 decreased at 1 hour,increased at 2 hours,and decreased more than 1 hour at 3 hours When processing.The results suggest that FAS1 in Arabidopsis responds to enhanced UV-B radiation.As FAS1 is a component of CAF-1complex,it is inferred that CAF-1 complex is related to enhanced UV-B radiation response.(4)After treatment with enhanced UV-B radiation at a dose of 3.6 KJ·m-2·d-1,root length was significantly shorter in the treatment group compared with the control group,and Atfas1-4UV-B root length was more severely inhibited than the WT UV-B root length,both of which were extremely different(P<0.01).The analysis speculated that FAS1 deletion rendered the CAF-1 complex Hypofunctional,resulting in enhanced sensitivity of Atfas1-4 to enhanced UV-B radiation and aggravated root growth inhibition.(5)Studies in which changes in reactive oxygen species were measured by DAB and NBT staining found that Atfas1-4 mutants stained more than the wild type in the control group and the treated group had increased levels of staining relative to the control group,and Atfas1-4 UV-B stained more than the WT UV-B.The results showed that Atfas1-4 significantly increased the amount of H2O2,02-·after the enhanced UV-B treatment.Transfer of Atfas1-4UV-B to GSH containing MS medium restored growth with partial restoration of root length,presumably after enhanced UV-B radiation,the Atfas1-4 mutant produced more ROS and was severely subjected to oxidative stress,and the enhanced root length inhibition of Atfas1-4under UV-B radiation was partly affected by the accumulation of more ROS.(6)Superoxide dismutase(SOD)and Catalase(CAT)were measured to reflect the changes in antioxidant enzymes in Arabidopsis roots.After testing,the activities of SOD and CAT in the control group were upregulated compared with the WT;however,the elevation of SOD and CAT in the Atfas1-4 UV-B treated group was much behind that of the WT UV-B compared with the control group.The results showed that the reduced viability of antioxidant enzymes produced by the Atfas1-4 mutant after enhanced UV-B irradiation,resulting in increased ROS accumulation,affected root development,leading to short roots.(7)Observation of flavonol content in the mutant Atfas1-4 by staining with2-Aminoethyl diphenylborinate(DPBA)revealed that,in the control group,there was little difference between the WT and Atfas1-4 fluorescence intensities,whereas in the treated group,the fluorescence intensities were significantly enhanced compared with the control group;The fluorescence intensities of Atfas1-4 UV-B were obviously enhanced compared with WT UV-B;the results illustrated that enhancing UV-B radiation enhanced the flavonol content,and Atfas1-4 resisted excessive reactive oxygen species by producing more flavonols.(8)In the control group,Atfas1-4 mutants had undergone DNA damage with slight tailing,as observed by comet assay.Compared with the control group,all the treated groups showed tailing,which was more severe in Atfas1-4 UV-B,and its Olive tail movement(OTM)was more than twice that of WT UV-B,indicating that Atfas1-4 mutants exacerbated DNA damage after enhanced UV-B treatment.Infer that the mutants are less resilient to enhanced UV-B radiation due to a loss of function mutation in FAS1,and when stressed,damage repair is greatly reduced.In conclusion,the phenotypic changes of Atfas1-4 mutant before and after enhanced UV-B radiation were observed.It was found that under enhanced UV-B radiation,Atfas1-4mutant appeared the phenotype of root length inhibition and aggravation.In order to explore the reasons,the expression of FAS1,the change of reactive oxygen species,the change of antioxidant enzymes and DNA damage were measured.It was speculated that the root length inhibition of Atfas1-4 mutant was aggravated by the insufficiency of CAF-1 complex function due to the lack of FAS1,which affected the activity of antioxidant enzymes,and led to the accumulation of active oxygen;meanwhile,the insufficiency of CAF-1 function would increase the effect on the increase of antioxidant enzyme activity The sensitivity of strong UV-B radiation can damage and enhance DNA damage repair after UV-B radiation. |
PDF Full Text Request