2001 Fiscal Year Final Research Report Summary
Analysis of molecular mechanisms of defense systems by ascorbate peroxidase against oxidative damages caused by excess light energy
| Project/Area Number |
12660090
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Kinki University |
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Principal Investigator |
SHIGEOKA Shigeru Kinki University, Agriculture, professor, 農学部, 教授 (80140341)
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| Project Period (FY) |
2000 – 2001
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| Keywords | ascorbate peroxidase / ascorbate / active oxygen / photooxidative stress / alternative splicing / regulation of gene expression / post-transcriptional regulation / transgenic plants |
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| Research Abstract |
Alternative splicing events in the 3'-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of SAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis-element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves, but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE.
Transgenic tobacco plants (TpTAP-12) overexpressing thylakoid membrane-bound ascorbate peroixdase (tAPX) targeted to chloroplasts were generated. The tAPX activity in TbTAP-12 plants was approximately 37-fold higher than that of the wild-type. The abilities of CO_2 fixation and PSII activity in between wild-type and TpTAP-12 plants were not changed.
The activities of thiol-modulated enzymes in the Calvin cycle, antioxidative enzymes, and levels of antioxidants were also same values in both plants. Transgenic tobacco plants -suppressing chloroplastic ascorbate peroixdase isoenzymes are generating using antisense technology.
Five lines of those plants at T_1 generation were isolated.
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