Functional Analysis Of Poly(ADP-ribose) Glycohydrolase Gene PARG1 In Regulation Of Defense And Stress Responses In Arabidopsis Thaliana

Poly(ADP-ribosyl)ation is a post-translational protein modification that plays important roles in many cellular processes in mammal systems. Emerging evidence indicates that poly(ADP-ribosyl)ation is also involved in regulation of growth/development, stress and defense response in plants. In this study, I studied the possible function of Arabidopsis poly(ADP-ribose) glycohydrolase 1 (PARG1) in disease resistance and abiotic stress responses using T-DNA insertional mutant and overexpression transgenic lines.Bioinformatics analysis reveals that there are two genes encoding for poly(ADP-ribose) glycohydrolases. RT-PCR analysis showed that expression of PARG1 was up-regulated in Arabidopsis plants after inoculation with Botrytis cinerea(B. cinerea), a necrotrophic fungal pathogen. Similarly, expression of PARG1 was also significantly induced in Arabidopsis plants after treatment with salicylic acid (SA) or light. By contrast, the expression of PARG1 was reduced when Arabidopsis plants was treated with SA under the light. Expression of PARG1 in Arabidopsis was not be induced after treatment with jasmonic acid (JA) and 1-aminocyclopropane-l-carboxylic acid (ACC). PPARG1::GUS transgenic lines was generated and GUS staning showed that the PARG1 gene was expressed in root tips, leaves and anthers. Subcellular localization analysis using 35S-GFP::PARG1 showed that the PARG1 protein was localized in the nucleus and cytoplasm of Arabidopsis cells.To study the function of PARG1 in disease resistance and abiotic stress responses, two T-DNA insertion line, parg1-3 in Ws-0 background and parg1-4 in Col-0 background were identified from genotyping screening. Meanwhile, transgenic lines overexpressing the PARG1 gene, driven by CaMV 35S promoter, in pargl-3 mutant and wild type Col-0 background were also generated. Homozygous lines with single copy of the PARG1 transgene were obtained through antibiotic resistance segregation screenings. Two independent transgenic lines overexpressing PARG1 gene in parg1-3 mutant background (PARG1-OE1/parg1-3 and PARG1-OE2/parg1-3) or wild type Col-0 background (PARG1-OE1/Col-0 and PARG1-OE2/Col-0) were selected for further studies. Disease phenotypes of the wild-type, pargl and PARG1-OE plants were compared after inoculation with B. cinerea or Pseudomonas syringae pv. tomato DC3000(Pst DC3000). As compared with the wild-type plants, the pargl plants showed increased resistance to B. cinerea and Pst DC3000. However, the transgenic PARG1-OE plants showed significant increased susceptibility to the above-mentioned pathogens. Furthermore, growth of B. cinerea and Pst DC3000 in leaves of the pargl plants was much less than in the wild-type plants; whereas growth of B. cinerea and Pst DC3000 in leaves of the PARG1-OE plants was significantly increased. After inoculation with B. cinerea, expression of Pathogenesis-related protein gene(PR) was similar in the wild-type, pargl and PARG1-OE plants. These results indicate that PARG1-mediated defense response against B. cinerea may be independent on PR genes. These data suggest that PARG1 plays an important role in Arabidopsis defense responses against both necrotrophic and biotrophic pathogens.To further study the function of PARG1 in disease resistance response, we examined accumulation of reactive oxygen species and levels of apoptosis in Arabidopsis after inoculation with B. cinerea. It was observed that, after B. cinerea infection, PARG1-OE plants accumulated much more reactive oxygen species in leaves but the pargl plants accumulated less reactive oxygen species in leaves, when comopared with those in the wild-type plants. As compared with the wild-type plants, the pargl plants showed reduced level of apoptosis after B. cinerea infection, whereas the PARG1-OE plants showed accelerated cell death after inoculation with B. cinerea, Pst DC3000 (avrRPM1) or Pst DC3000. these results suggest that PARG1 may be also involved in regulation of cell death. Poly(ADP-ribose)(PAR) induces apoptosis in pargl plants, but did not induce cell necrosis. Ac-DEVD-CHO, a caspase3 inhibitor, inhibited partially PAR- induced apoptosis. These results demonstrate that PAR, which regulated by PARGl, can induce apoptosis, and this process is partially dependent on caspase-apoptosis signaling pathways.I also examined the possible function of PARG1 in Arabidopsis abiotic stress tolerance using genetic mutant parg1-3 and transgenic PARG1-overexpressing plants. Expression of PARG1 in wild type plants was upregulated in response to drought and oxidative stress treatments. Germination rates of the parg1-3 seeds were reduced under mannitol or methyl viologen (MV) treatment as compared with the wild type seeds, but the parg1-3 seeds showed similar germination rate to the wild type seeds under ABA treatment. The parg1-3 plants showed reduced tolerance to drought, osmotic and oxidative stresses, with increased levels of cell damage under osmotic and oxidative stress and reduced survival rate under water-withholding drought stress, as compared with the wild type plants. Stomatal apertures of parg1-3 plants failed to close under water-withholding drought stress, but showed similar closure patterns to the wild type plants after ABA treatment. As compared with those in wild type plants, drought stress/ABA-responsive genes in parg1-3 plants showed comparable expression levels under drought stress condition, whereas expression level of some of oxidative stress-related genes in parg1-3 plants was reduced after MV treatment. Overexpression of PARG1 in parg1-3 mutant and wild type Col-0 background did not cause any phenotypical changes in response to drought, osmotic and oxidative stresses and ABA treatment. These results suggest that PARG1 plays important roles in abiotic stress tolerance.Taken together, data presented in this thesis suggest that PARG1 plays important roles in disease resistance and abiotic stress responses.