研究報告/第80号

農業・園芸総合研究所研究報告

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第80号：2012年3月発行

第80号抄録

高等植物の葉緑体、ミトコンドリアにおける

紫外線誘発DNA損傷（CPD）に対する光修復機構に関する研究

高橋　正明

The photoreactivation of ultraviolet-B-induced CPDs in

chloroplasts and mitochondria in higher plants.

Masaaki Takahashi

太陽光に含まれる紫外線B（280-320 nm; UVB）は、植物細胞内の核、葉緑体、ミトコンドリアゲノム上にDNA損傷の一つであるシクロブタン型ピリミジン二量体(CPD)を誘発する。CPDの蓄積はUVBによる植物の生育抑制の主要因であり、これまでの研究から、核でのみ青色光を利用して修復するCPD光回復酵素による光修復機構の存在が確認されている。本研究ではオルガネラでのCPD光回復量を解析することで、イネとアラビドプシスの葉緑体、ミトコンドリアでCPD光修復能が存在することを明らかにした。また、細胞組織学的な解析で、イネの各オルガネラにCPD光回復酵素が局在していることを示した。これらの結果は、高等植物が進化の過程で、CPD光回復酵素を核、葉緑体、ミトコンドリアに移行させて、効率的にCPDの修復を行う生存戦略機構を獲得したことを示唆している。

〔キーワード〕紫外線、オルガネラ、ミトコンドリア、葉緑体、CPD、光修復、光回復酵素、DNA損傷、

Summary

Introduction

Plants use sunlight as an energy source for photosynthesis; however, plants DNA is exposed to the harmful effects of ultraviolet-B (UVB) radiation (280 to 320 nm) in the process. UVB radiation damages nuclear, chloroplast and mitochondrial DNA. Cyclobutane pyrimidine dimers (CPDs) are major UVB-induced DNA lesions, and are one of the principal causes of UVB-induced growth inhibition in plants. Repair of CPDs is therefore essential for plant survival under UVB-containing sunlight. Nuclear repair of the UVB-induced CPDs involves a photoreversal of CPDs, called photoreactivation, mediated by CPD photolyase, which monomerizes the CPDs in DNA by using the energy of near-UV and visible light (300-500 nm), and this is the major mechanism for repairing CPDs in plants.

In addition to the nuclear genome, the cells of higher plants contain two additional genomes: one in chloroplasts and another in mitochondria. The chloroplast and mitochondrial genomes contain genes that encode many proteins important for photosynthesis and respiration, respectively. UVB radiation also induces the formation of CPDs in these organellar DNAs. Thus, chloroplasts and mitochondria must also utilize a pathway(s) that repairs CPDs and efficiently removes the DNA lesions prior to replication and transcription.To date, several studies examining CPD repair activity under conditions of visible light or darkness in chloroplasts and mitochondria in higher plants have been reported. But, it is therefore unclear whether the light-dependent removal of CPDs in cpDNA and mtDNA is mediated by CPD photolyase, and the potential existence of a repair pathway, including one targeting photoreactivation, for repairing CPDs in chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) in higher plants remains unclear.

To ascertain the existence of pathways that repair UVB-induced CPDs in cpDNA and mtDNA in rice, we used a site-specific assay for detecting CPDs based on T4 endonuclease V (T4 endo V) digestion and Southern blot analysis. Moreover, we did same experience in Arabidopsis, which possessed lower photorepair activity than that of rice, and we discuss the relationship between the existences of photorepair system in organelles and UVB sensitivity in plants.,

Results

1　Mechanism of CPD photorepair of organelles in rice.

We analysed the existence of photoreactivation in nuclei, chloroplast and mitochondria, with a site-specific assay for detecting CPDs based on T4 endonuclease V (T4 endo V) digestion and Southern blot analysis. Exposure to UVB radiation induce CPDs in organelles, and CPDs were diminished in chloroplasts and mitochondria as well as in nuclei.There were no significant change in response to dark conditions, so these results strongly suggest that, in addition to nuclei, rice chloroplasts and mitochondria are able to efficiently photoreactivate CPDs.

To determine whether the photoreactivation activity in nuclei, chloroplasts and mitochondria was mediated by CPD photolyase, we measured CPD photoreactivation activity in all DNA-containing organelles of rice strains with different levels of CPD photolyase activity. The Surjamkhi strain which is deficient in CPD photorepair when compared with the Sasanishiki strain, the CPD photoreactivation activity in all three organelles was higher in Sasanishiki than in Surjamkhi leaves. Furthermore, CPD photoreactivation was undetectable in any organelle in transgenic rice (AS-D) engineered to express antisense RNA targeting CPD photolyase and in which there was a very low level of CPD photolyase activity. We also examined CPD photoreactivation activity in transgenic rice (S-C) that was constructed by transformation with the cDNA sequence of rice CPD photolyase and that therefore had higher CPD photolyase activity than that of UV-resistant Sasanishiki plants. CPD photoreactivation activity in chloroplasts and mitochondria was markedly higher in S-C plants than in Sasanishiki plants. These results indicate that the photoreactivation activity in chloroplasts and mitochondria is mediated by CPD photolyase which is encoded by a single gene and not a splice variant,.

To confirm the location of CPD photolyase within cells, transgenic S-C plants were examined by immunoelectron microscopy (IEM) using an anti-rice CPD photolyase antibody. Gold particles were detected in the chloroplasts, mitochondria and nuclei. We also prepared organelle-enriched fractions from the leaves or calli of S-C plants and analyzed crude organellar extracts by western blot using anti-rice CPD photolyase antiserum. CPD photolyase was detected in all organelle-enriched fractions, demonstrating that CPD photolyase translocates to the chloroplasts, mitochondria and nuclei of rice.

2　The analysis of CPD photoreactivation of organelles in Arabidopsis and UVB tolerance of transgenic

Arabidopsis that was constructed by transformation with the rice CPD photolyase.

To date, several studies were demonstrated, but it is unclear that the existence of CPD photorepair activity in chloroplasts and mitochondria in Arabidopsis. Pilot study demonstrated that Arabidopsis is deficient in CPD photorepair and sensitive to UVB when compared with rice. There are several possibilities on these results. (1)The existence of CPD photoreactivity in organelles is involved in tolerance to UVB in plants. (2)The sensitivity to UVB is owe to the lower activity of CPD photolyase. (3)Mechanism of tolerance to UVB in Arabidopsis is differ from that of rice. To elucidate the difference the torelance to UVB between rice and Arabidopsis, we analyzed the CPD photoreactivation of organelles in Arabidopsis and the relationship between the existences of photorepair system in organelles and UVB sensitivity in plants. CPD photolyase functions in all organelle in rice plants.

First, we analyzed the existence of photoreactivation in each organelle of Landsberg erecta (Ler) with quantitative PCR (qPCR) with genome-specific primers and Southern blot analysis with T4 endo V. We revealed that there are CPD photoreactivation in chloroplast and mitochondria. But, the activity in each organelle is lower than those of rice. Futhermore, we prepared the transgenic lines overexpressing rice CPD photolyase in uvr2-1 which is deficient in CPD photoreactivation (Osphr lines), and they were grown under enhanced UVB in order to test whether the overexpression CPD photolyase has a positive effect. As a result, the Osphr lines have high CPD photoreactivity ten times more than those of Ler in chloroplast, mitochondria and nuclei, and Osphr lines acquired the tolerance to UVB. For the wild type, daily UVB doses of 25.9 kJ m-2 day-1 given over 14 days led to reduction of the fresh mass to 20%. The over expression lines also showed a reduction of fresh mass under UVB corresponding to 31, 40% at 25.9 kJ m-2 day-1 for the lines Os3 and Os10 respectively. However, these data also indicated that Os3 and Os10 couldn’t get same torelance level for UVB when compared with Osphr overexpressed line in rice (S-C).

Discussion

In this study, we clearly demonstrated that rice and Arabidopsis CPD photolyase, which is encoded by a single-copy gene in the nuclear genome, translocates to chloroplasts, mitochondria, and nuclei and repairs UVB-induced CPDs in all three genomes.CPD photolyase is a simple and efficient repair system andis widely distributed among species ranging from bacteria to higher organisms. Thus, these results indicate that plants have evolved a CPD photolyase that functions in all DNA-containing organelles to protect cells from the harmful effects of UVB radiation.

There are photoreactivation in chloroplasts, mitochondria and nuclei in Arabidopsis, it is unprobable that whether the photorepair in organelle is there or not are cause of the sensitivity to UVB in Arabidopsis. But, the activity of CPD photolyase in Arabidopsis are low level compared with that of rice, so that it might be the the one of the cause of the untorelance to UVB in Arabidopsis. Osphr transgenic lines in Arabidopsis couldn’t acquire the same torelance level to UVB when compared with Osphr overexpressed line in rice. These results indicated that UVB-induced growth inhibition in Arabidopsis is caused by CPD, but also other factors may effect strongly.

A single DNA repair enzyme, CPD photolyase, functions in chloroplasts, mitochondria, and nuclei and is subjected to “triple targeting”, which is, to our knowledge, the first demonstration. In the future, we will elucidate the molecular mechanism of translocation to organelles, the significance of the CPD photoreactivation in organelles, and the resistance mechanism to UVB in plants.