1923===Lygodium japonicum

RESEARCH PAPER Lygodium japonicum fern accumulates copper in the cell wall pectin Haruyoshi Konno 1, *, Takako Nakato 1 , Susumu Nakashima 1 and Kenji Katoh 2 1 Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan 2 Shionogi Research Laboratories, Shionogi & Co., Ltd., Osaka 553-0002, Japan Received 19 November 2004; Accepted 7 April 2005 Abstract The present work reports the results of a study on the growt h kinetics and chara cteri zation of matrix poly- saccharides in the cell walls of Lygodium japonicum protha llium grown in the presence of copper (Cu). When the prothallium was cultured in the media con- taining 0.2 mM or 0.4 mM CuSO 4 , it showed a rapid accumulati on of Cu wi th a maximum uptake of Cu measured in the ce ll s up to 20 d of cu l tu r e. The maximum rate of Cu uptake into the prothallium was greater for 0.4 mM Cu-treated cells (17.2 lmol g 21 DW) tha n for 0.2 mM Cu- treate d cel ls (3. 2 lmol g 21 DW). Cel l wal ls wer e iso lat ed fro m bot h untrea ted control and Cu-tr eate d cell s and then extract ed seque ntial ly with cyclohex ane- trans -1,2-di aminetetr a-aceta te (CDTA), Na 2 CO 3 , 1 M KOH, and 4 M KOH. The amount of pectin solubi li zed fr om 0. 4 mM Cu- tr eated cell walls de- cr eased to 53% of it s level in the control, wher eas the amount of hemicellulose solubilized from the Cu- treated cell walls represented 82% of that from control cell walls. Whe n the pol ysa cch arides wer e fra cti on- at ed by ani on- exc hange chromatography int o four carbohydra te components, consi derable incre ases in fractions PI-3 and PII-3 elut ed wit h 0.5 M NaCl were observed in CDTA-soluble (PI) and Na 2 CO 3 -soluble (PII) pectic polymers from Cu-treated cell walls. Frac- tions PI-3 and PII-3 were composed predominantly of uronic acid (more than 71% of total sugars). Approxi- mately 66% of Cu within the cell walls was released f ro m th e 0. 4 m M C u- tr ea te d ce ll s wi th th e endo - pecta te-l yase treatme nt, sugge sting that most of the Cu that accumulated into the Lygodium prothallium is ti ghtl y bound to the homogalact uronan of the cell wall pectin. Key word s: Copp er accumulat ion, endo -pec tate lyas e, fern (pteridoph yte), homogalactu ronan, Lygodium japonicum , pectin structure, phytoremediation, prothallium. Introduction Heavy metals such as copper (Cu) and zinc are required by biological systems as catalyt ic and structural components of pro tei ns and as cof act ors essential to pla nt gro wth and development. However, since many heavy metals can bind easily to sulphydryl groups in the active sites of enzymes and structural proteins, a high heavy metal concentration int erf ere s wit h the met abo lis m for nor mal gro wth , cel l proliferation, and differentiation of most plant cells. The pollution of soil and water with Cu, iron, zinc, and lead is wid esp rea d; the ref ore, hig h lev els of tol erance to these met als are req uir ed by pla nts gro wing in the se contam- inated sites. It has been reported to date that a small number of wild plants are capable of the extraction of heavy metals fro m con tamina ted soi l and wat er by con cen tra tin g the metals in the ir roo ts and shoot s (Kumar et al., 199 5). Currently, environmental remediation using the ability of the endemic plants to accumulate heavy metals has been proposed as ‘phytoremediation’ (Reeves and Baker, 2000). Various mechanisms have been suggeste d for toler ance and det oxi ﬁca tion of pot ent ial ly tox ic hea vy met als in pla nts (Hall, 2002). It is generally well demonstrated that small cyst eine- cont ainin g pepti des capa ble of bindi ng heav y metal s, terme d phyt ochel atin s and metal lothi onei ns, are rapidly synthesized within several plant cells in response to toxic levels of heavy metals (Steffens, 1990). The roots of some plant species also excrete speciﬁc organic materials, suc h as cit rat e, mal ate , and histi din e, tha t can bind to Cu, nickel, and zinc, and, consequently, the formation of * To whom correspondence should be addressed. Fax: +81 86 434 1249. E-mail: [email protected] Journal of Experimental Botany, Vol. 56, No. 417, pp. 1923–1931, July 2005 doi:1 0.10 93/jx b/eri 187 Adva nce Access pub lication 31 May, 2005 ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjo urnals.org

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1923===Lygodium japonicum