Journal of Experimental Botany Advance Access published July 25, 2006 Journal of Experimental Botany, Page 1 of 10 doi:10.1093/jxb/erl049 RESEARCH PAPER Changes in polyamine content and localization of Pinus sylvestris ADC and Suillus variegatus ODC mRNA transcripts during the formation of mycorrhizal interaction in an in vitro cultivation system Karoliina Niemi1,*, Suvi Sutela2, Hely Ha¨ggman2, Carolyn Scagel3, Jaana Vuosku2, Anne Jokela2 and Tytti Sarjala4 1 Department of Applied Biology, University of Helsinki, PO Box 27, 00014 University of Helsinki, Finland 2 Department of Biology, University of Oulu, PO Box 3000, 90014 University of Oulu, Finland 3 USDA, Agricultural Research Service, Horticultural Crops Research Laboratory, Corvallis, OR 97330, USA 4 Finnish Forest Research Institute, Parkano Research Unit, 39700 Parkano, Finland Received 24 October 2005; Accepted 27 April 2006 Abstract formation and main root elongation were greatest between the first and third week in dual culture, The involvement of polyamines (PAs) in the interaction coinciding with retarded accumulation or a decrease between Pinus sylvestris L. seedlings and an ectomy- of free PAs. These results show that accumulation of corrhizal fungus Suillus variegatus (Swatz: Fr.) O. Kunze PAs in the host plant is one of the first indicators of was studied in an in vitro cultivation system. PA the establishment of ectomycorrhizal interaction be- concentrations in seedlings were analysed after 1, 3, tween P. sylvestris and S. variegatus in the in vitro and 5 weeks in dual culture with S. variegatus, and system. changes in PA pools were compared with the growth of the seedlings. Pinus sylvestris arginine decarboxy- Key words: Arginine decarboxylase, ectomycorrhizas, lase (ADC) and S. variegatus ornithine decarboxy- ornithine decarboxylase, Pinus sylvestris, polyamines, Suillus lase (ODC) mRNA transcripts were localized during variegatus. the formation of mycorrhizas. During mycorrhiza formation, Suillus variegatus ODC transcripts were found in developing hyphal mantle and Hartig net, and Introduction P. sylvestris ADC transcripts in specific root paren- chyma cells adjacent to tracheids and in mitotic cells Mycorrhizal symbiosis is a mutualistic association between of the root apical meristem. However, no unambigu- plants and certain fungi that colonize roots. Pinus sylvestris ous difference in ADC transcript localization between L., like other Pinus species, form a symbiosis with inoculated and non-inoculated roots was observed. ectomycorrhizal (ECM) fungi that entirely cover short Regardless of the unchanged distribution of ADC roots with a hyphal mantle. Moreover, ECM fungi form transcripts, inoculation with S. variegatus increased a highly branched structure called a Hartig net between free putrescine, spermidine, and spermine concentra- epidermal and cortical cells. In ECM association, the host tions in roots within the first week in dual culture. The plant supplies simple sugars to the fungal partner, whereas concentration of free and conjugated putrescine and the fungus improves plant nutrition by increasing the conjugated spermidine also increased in the needles surface that absorbs nutrients and also by enabling the due to the fungus. The fungus-induced lateral root use of organic forms of nutrients (Smith and Read, 1997; * To whom correspondence should be addressed. E-mail: Karoliina.Niemi@helsinki.fi Abbreviations: ADC, arginine decarboxylase; ECM, ectomycorrhizal; ODC, ornithine decarboxylase; PA, polyamine; PCA, perchloric acid; Put, putrescine; Spd, spermidine; Spm, spermine. ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected] Downloaded from http://jxb.oxfordjournals.org at USDA, Eastern Regional Research Center on 11 May 2009 2of10 Niemi et al. Lindahl, et al., 2002). In addition to nutritional benefit, the et al., 2004; Acosta et al., 2005; Delis et al., 2005; host plant may gain improved tolerance against root Paschalidis et al., 2005). ADC mRNA has been found to pathogens in the presence of the mycorrhizal fungus (Smith accumulate under various stress conditions (Chattopadhyay and Read, 1997). et al., 1997; Urano et al., 2003; Hao et al., 2005), but ADC Polyamines (PAs) are low molecular weight organic has been reported to be regulated both transcriptionally cations that are essential for the development and growth and post-translationally (Malmberg and Cellino, 1994; of all living organisms (Tabor and Tabor, 1985; Walters, Watson and Malmberg 1996; J Vuosku et al., unpublished 1995; Bais and Ravishankar, 2002). PAs exist in cells as results). This indicates that different regulatory mechan- free bases or perchloric acid (PCA)-soluble or PCA-insoluble isms may be involved in ADC expression. conjugated forms. Free bases may in certain situations act In fungi, ODC seems to be the sole pathway for like inorganic cations affecting basic cell chemistry, in- production of Put, although there is also evidence of cluding pH and ion balance. However, the major part of ADC activity in certain fungal species. In the mycelium the physiological functions of PAs has been attributed to of an ECM fungus Paxillus involutus (Batsch) Fr., Put their ability to bind to important anionic cell constituents, synthesis proceeded predominantly via ODC, but ADC including nucleic acids, phospholipids, proteins, and phe- was also present in the mycelium (Fornale´ et al., 1999). nolic compounds (Del Duca and Serafini-Fracassini, 1993; Furthermore, Sannazzaro et al. (2004) observed both Bagni and Tassoni, 2001; Facchini et al., 2002). ODC and ADC activity in the spores of an arbuscular my- Putrescine (Put), spermidine (Spd), and spermine (Spm) corrhizal fungus Gigaspora rosea. However, to date, only are the most common PAs in plants and they have been genes encoding ODC have been identified in fungi reported to be involved in cell proliferation, embryo, floral, (Blasco et al., 2002; Ninˇo-Vega et al. 2004; Morel and fruit development (Bais and Ravishankar, 2002), as et al., 2005) and, depending on the species, ODC has well as rhizogenesis (Coue´e et al., 2004) and responses been regulated at the transcriptional (Blasco et al., 2002) to biotic stress (Walters, 2003). There is also increasing or post-transcriptional level (Ninˇo-Vega et al., 2004). evidence of the role of PAs in mycorrhizal interactions. An in vitro method has been developed to induce ECM fungi have been observed to produce and release formation and growth of P. sylvestris roots by means of in- different PAs in vitro depending on the species and strain oculation with specific ECM fungi (Niemi et al., 2002a, b). (Zarb and Walters, 1994; Fornale´ et al., 1999; Sarjala, 1999; A previous study using this in vitro cultivation system Niemi et al., 2002a, 2003) and, in studies on mycorrhiza showed that ECM fungi and specific PAs have a synergistic formation, ECM roots of P. sylvestris were found to contain effect on adventitious root formation of P. sylvestris (Niemi higher concentrations of PAs than non-inoculated roots et al., 2002a). In the present work, this in vitro method was (Kyto¨viita and Sarjala, 1997; Sarjala and Taulavuori, 2004). used to study changes in growth and PA metabolism in In plants, Put can be synthesized directly from ornithine P. sylvestris seedlings as they form mycorrhizal interaction in a reaction catalysed by ornithine decarboxylase (ODC) with an ECM fungus Suillus variegatus (Swatz: Fr.) (EC 4.1.1.17) or from arginine after decarboxylation to O. Kunze. Changes in concentrations of free and PCA- agmatine by arginine decarboxylase (ADC) (EC 4.1.1.19) soluble and -insoluble PAs in different parts of the seed- and subsequent conversion of agmatine into Put by lings were analysed, and P.sylvestris ADC and S. variegatus agmatine iminohydrolase (EC 3.5.3.12) and N-carbamoyl- ODC mRNA transcripts were localized in developing putrescine aminohydrolase (EC 3.5.1.53). Put is converted mycorrhizal root system. into Spd and subsequently into Spm by the addition of aminopropyl moieties from decarboxylated S-adenosyl- methionine (SAM) in reactions catalysed by Spd synthase Materials and methods (EC 2.5.1.16) and Spm synthase (EC 2.5.1.22), respectively Biological material (Bais and Ravishankar, 2002). The ECM fungus, S. variegatus was originally isolated from a Genes encoding ADC have been successfully cloned basidiocarp under a P. sylvestris stand in western Finland and was from several plant species (Bell and Malmberg, 1990; maintained by cultivating on modified Hagem agar medium (Modess, Watson and Malmberg, 1996; Nam et al., 1997; Mo and 1941). For inoculations, the mycelia were cultivated for 3 weeks on Pua, 2002; Hao et al., 2005). In developing zygotic strips of moist filter paper lying on modified Melin–Norkrans (MMN) agar medium (Marx, 1969) containing 3.7 mM KH PO , 1.9 mM embryos of P. sylvestris, Put biosynthesis has also been 2 4 (NH4)2HPO4, 0.45 mM CaCl2, 0.43 mM NaCl, 0.61 mM found to occur preferentially through an ADC pathway and MgSO4.7H2O, 0.2 lM thiamine-HCl, 30.8 lM FeCl3.6H2O, and to be involved in mitosis (J Vuosku et al., unpublished 55.5 mM glucose, pH 5.8. For RNA isolations, the mycelia grew for results). However, Arabidopsis is currently the only plant 4 weeks on the moist cellophane membrane (P 400, Visella Oy, species studied that does not have an ODC gene (Hanfrey Valkeakoski, Finland) lying on modified Hagem agar medium. Seeds from the open-pollinated elite P. sylvestris clone 884, et al., 2001; Hummel et al., 2004). In plants possessing originating from the Punkaharju clone collection in Finland both ADC and ODC systems, the enzymes have been (618489 N; 298179 E), were surface-sterilized with 2% calcium proposed to have different physiological roles (Flemetakis hypochlorite for 20 min, rinsed in sterile water and germinated on Downloaded from http://jxb.oxfordjournals.org at USDA, Eastern Regional Research Center on 11 May 2009 Polyamines and ectomycorrhizal interaction 3of10 0.7% water agar in glass jars.