Development of Symbiotic Engineering for Phytoremediation

• Project/Area Number: 15208032
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Boundary agriculture
• Research Institution: Osaka University
• Principal Investigator: YAMASHITA Mitsuo (2004) Osaka Univ., Eng., Assoc.Prof., 大学院・工学研究科, 助教授 (40220347); 室岡 義勝 (2003) 大阪大学, 大学院・情報科学研究科, 教授 (60029882)
• Co-Investigator(Kenkyū-buntansha): ONO Hisao Osaka Univ., Eng., Assis.Prof., 遺伝情報実験センター, 助手 (40224274) ; HAYASHI Makoto Osaka Univ., Eng., Assis.Prof., 大学院・工学研究科, 助手 (30291933) ; 山下 光雄 大阪大学, 大学院・工学研究科, 助教授 (40220347)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥36,920,000 (Direct Cost: ¥28,400,000、Indirect Cost: ¥8,520,000); Fiscal Year 2004: ¥7,540,000 (Direct Cost: ¥5,800,000、Indirect Cost: ¥1,740,000); Fiscal Year 2003: ¥29,380,000 (Direct Cost: ¥22,600,000、Indirect Cost: ¥6,780,000)
• Keywords: phytoremediation / symbiotic engineering / Cd / metallothionein / Phytochelatin / heavy metal binding protein / ファイトレメディエーション

Research Abstract

In this research project, we have been developed a new bioremediation technology utilizing the symbiotic relationships of a leguminous plant and its rhizobia to clean up soils contaminated with heavy metals. In the rhizosphere, plant roots secrete various nutrients and attractants stimulating microbes to swarm plant roots. We believe that microbes have the ability to gather and carry metal ions to plant roots. The metal ions released by these microorganisms are taken in the plants. The phenomena gave me an idea for bioremediation using plants and their microbes.
Our major findings for this 2 year project included :
a)We constructed transgenic renge-soh (Astragalus sinicus) with tetrameric metallothionein (MTL4) gene for increased uptake of heavy metals by plants. To express a foreign gene in bacteroids in nodules, we chose the two promoters, PnifH and PnolB. The MTL4 gene expressed in nodules, and nodules accumulated 2 to 7 times more cadmium than nodules without MTL4.
b)To further improv e plant uptake of heavy metals, we introduced the phytochelatin synthetase(PCS) gene. Another naturally occurring metal-binding peptide, PCs are an attractive alternative to MTs because they offer the potential for enhanced affinity for, and selectivity of, heavy metals. We constructed vectors with Arabidopsis PCS gene under the control of nifH promoter. The gene expressed and catalyzed the synthesis of PCn, with values of n from 2 to 7.
c)We found expression of the AtPCS gene in M.huakuii subsp.rengei B3 along with the increased ability of bacterial cells to bind Cd 10to20 fold. The B3 strain carrying the AtPCS gene was used to infect A.sinicus roots, and nodules formed. The AtPCS gene expressed in nodules, and we found 1.5 times the increased accumulation of Cd in these engineered nodules.
d)Although recombinant free-living bacteria accumulated 20 times more Cd than wild type bacteria, bacteroids in plant root nodules increased Cd accumulation less than 2 times. We expected that there might be a limitation of Cd transportation into nodules. Thus, we isolated the AsIRT1 gene fro Arabidopsis and transferred it together with AtPCS to the B3 strain. The AsIRY1 and AtPCS genes expressed in the bacteroid in plant nodules. Our results show bacterial cells having the IRT1 gene stimulated the incorporation of Cd. However, no increased accumulation of Cd in nodules was observed. Interestingly, we found that Cd accumulated more in roots infected with B3 cells carrying the AtPCS and AtIRT1 genes.
e)We cultivated A.sinicus carrying the MT and AtPCS genes in rice field soil containing cadmium. Cadmium ions accumulated significantly in roots and nodules. Our results showed about 9 % Cd accumulation for this engineered plant.

Research Products

• [Journal Article] Root hair abundant genes LjRH101 and LjRH102 encode peroxidase and xyloglucan endotransglycosylase in Lotus japonicus (2005)
  • Author(s): T.Maekawa
  • Journal Title: Journal of Bioscience and Bioengineering 99(1) Pages: 84-86
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots. (2005)
  • Author(s): H.Imaizumi-Anraku
  • Journal Title: Nature 433 Pages: 527-531
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein. (2005)
  • Author(s): D.Hamada
  • Journal Title: FEBS Journal 272 Pages: 756-768
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Expression of a Major House Dust Mite Allergen Gene from Dermatophagoides farinae in Lotus japonicus accession Miyakojima MG-20 (2005)
  • Author(s): T.Kato
  • Journal Title: Journal of Bioscience and Bioengineering 99(2) Pages: 165-168
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Lotus burttii takes a position of the third corner in the Lotus molecular genetics triangle (2005)
  • Author(s): Kawaguchi, M
  • Journal Title: DNA Reseach 99(2)(in press)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots. (2005)
  • Author(s): H.Imaizumi-Anraku^*, N.Takeda^*, M.Charpentier, J.Perry, H.Miwa, Y.Umehara, H.Kouchi, Y Murakami, L.Mulder, K.Vickers, J.Pike, A.Downie, T.Wang, S.Sato, E.Asamizu, S.Tabata, M.Yoshikawa, Y.Murooka, G.-J.Wu, M.Kawaguchi, S.Kawasaki, M.Parniske, M.Hayashi (^*These authors contributed equally to this work)
  • Journal Title: Nature 433 Pages: 527-531
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Root hair abundant genes LjRH101 and LjRH102 encode peroxidase and xyloglucan endglycosylase in Lotus japonicus. (2005)
  • Author(s): Maekawa T, Hayashi M, Murooka Y.
  • Journal Title: J.Biosci.Bioeng. 99(1) Pages: 84-86
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein. (2005)
  • Author(s): D.Hamada, T.Kato, T.Ikegami, K.N.Suzuki, M.Hayashi, Y.Murooka, T.Honda, I.Yanagihara.
  • Journal Title: FEBS Journal 272 Pages: 756-768
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Lotus burttii takes a position of the third corner in the Lotus molecular genetics triangle. (2005)
  • Author(s): Kawaguchi, M.^*, Pedrosa-Harand, A.^*, Yano, K.^*, Hayashi, M., Murooka, Y, Saito, K., Namai, K., Nishida, H., Shibata, D., Sato, S., Tabata, S., Sandal, N, Stougaard, J., Bachmair, A., Grant, WF.(^*These authors contributed equally to this work)
  • Journal Title: DNA Res. (in press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Expression of a Major House Dust Mite Allergen Gene from Dermatophagoides farinae in Lotus japonicus accession Miyakojima MG-20. (2005)
  • Author(s): T.Kato, Y.Goto, M.Hayashi, K.Ono, Y.Murooka
  • Journal Title: J.Biosci.Bioeng. (in press)
  • NAID: 110002695643
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Root hair abundant genes LjRH1Ol and LjRH102 encode peroxidase and xyloglucan endglycosylase in Lotus japonicus. (2005)
  • Author(s): T.Maekawa
  • Journal Title: Journal Bioscience and Bioengineering 99(1) Pages: 84-86
• [Journal Article] Lotus burttii takes a position of the third corner in the Lotus molecular genetics triangle (2005)
  • Author(s): Kawaguchi, M
  • Journal Title: DNA Research (in press)
• [Journal Article] Expression of a Major House Dust Mite Allergen Gene from Dermatophagoides farinae in Lotus japonicus accession Miyakojima MG-20. (2005)
  • Author(s): Tomoaki Kato
  • Journal Title: Journal Bioscience and Bioengineering (in press)
• [Journal Article] Pollen development and tube growth are affected in the symbiotic mutant of Lotus japonicus, crinkle. (2004)
  • Author(s): M.Tansengco
  • Journal Title: Plant and Cell Physiology 45(5) Pages: 511-520
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Molecular analysis of promoter elements from Propionibacterium freudenreichii. (2004)
  • Author(s): Y-Z.Piao, N.Kawaraichi, R.Asegawa, P.Kiatpapan, H.Ono, M.Yamashita, Y.Murooka.
  • Journal Title: J.Biosci.Bioeng. 97(5) Pages: 310-316
  • NAID: 110002665304
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Production of vitamin B_<12> in genetically engineered Propionibacterium freudenreichii. (2004)
  • Author(s): Y.-Z.Piao, M.Yamashita, N.Kawaraichi, R.Asegawa, H.Ono, Y.Murooka.
  • Journal Title: J.Biosci.Bioeng. 98(3) Pages: 167-173
  • NAID: 110002665354
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Effect of expression of hemA and hemB genes on production of porphyrin in Propionibacterium freudenreichii. (2004)
  • Author(s): Y-Z.Piao, P.Kiatpapan, M.Yamashita, Y.Murooka.
  • Journal Title: Appl.Enviroment.Microbiol. 70(12) Pages: 7561-7566
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Expression islands clustered on the symbiosis island of the Mesorhizobium loti genome. (2004)
  • Author(s): Uchiumi, T., Ohwada, T.Itakura, M., Mitsui, H., Nukui, N., Dawadi, P., Kaneko, T.Tabata, S., Yokoyama, T., Tejima, T., Saeki, K., Omori, H., Hayashi, M., Maekawa, T., Sriprang, R., Murooka, Y., Tajima, S., Simomura, K., Nomura, M., Suzuki, A., Shimoda, Y., Sioya, K., Abe, M., Minamisawa, K.
  • Journal Title: J.Bacteriol. 186(8) Pages: 2439-2448
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Pollen development and tube growth are affected in the symbiotic mutant of Lotus japonicus, crinkle. (2004)
  • Author(s): M.Tansengco, H.Imaizumi-Anraku, M.Yoshikawa, S.Takagi, M.Kawaguchi, M.Hayashi, Y.Murooka.
  • Journal Title: Plant Cell Physiol. 45(5) Pages: 511-520
  • NAID: 10012938459
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Pollen development and tube growth are affected in the symbiotic mutant of Lotus japonicus, crinkle. (2004)
  • Author(s): M.Tansengco
  • Journal Title: Plant Cell Physiology 45(5) Pages: 511-520
• [Journal Article] Enhanced accumulation of Cd^<2+> by a Mesorhizobium sp.transformed with a gene from Arabidopsis thaliana coding for phytochelatin synthase. (2003)
  • Author(s): R.Sriprang, M.Hayashi, H.Ono, M.Takagi, K.Hirata, Y.Murooka.
  • Journal Title: Appl.Environ.Microbiol. 69(3) Pages: 1791-1796
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] crinkle, a novel symbiotic mutant that affects the infection thread growth and alters the root hair, trichome, and seed development in Lotus japonicus. (2003)
  • Author(s): M.Tansengco, M.Hayashi, M.Kawaguchi, H.Imaizumi, Y.Murooka.
  • Journal Title: Plant Physiol. 131 Pages: 1054-1063
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] Biotechnology in Agriculture and Forestry, Vol.52 (eds. Nagata/Tabata) (2003)
  • Author(s): K.Harada (共著)
  • Publisher: Springer-Verlag, Berlin, Heidelberg
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] R.Sriprang: "Enhanced accumulation of Cd^<2+> by a Mesorhizobium sp. transformed with a gene from Arabidopsis thaliana coding for phytochelatin synthase"Applied and Environmental Microbiology. 69(3). 1791-1796 (2003)
• [Publications] M.Tansengco: "Crinkle, a novel symbiotic mutant that affects the infection thread growth and alters the root hair, trichome, and seed development in Lotus japonicus"Plant Physiology. 131. 1054-1063 (2003)
• [Publications] 室岡義勝: "共生工学による環境修復"日本農芸化学会誌. 77(2). 146-149 (2003)
• [Publications] Uchiumi T: "Expression islands clustered on island of Mesorhizobium loti genome"Journal of Bacteriology. (in press). (2004)
• [Publications] 室岡義勝: "高機能プロバイオティクスとしての乳酸菌L137株"日本乳酸菌学会誌. 14(2)(in Press). (2004)
• [Publications] P.Kiatpapan(共著): "ACS Symposium Series 862 Fermentation Biotechnology"American Chemical Society, Washington,DC (ed.B.C.Saha). 221-232 (2003)
• [Publications] 平山令明(翻訳・共訳): "タンパク質ハンドブック"丸善(株). 540 (2003)