JXB Advance Access originally published online on November 21, 2006
Journal of Experimental Botany 2007 58(3):391-401; doi:10.1093/jxb/erl209
RESEARCH PAPER |
Suillus variegatus causes significant changes in the content of individual polyamines and flavonoids in Scots pine seedlings during mycorrhiza formation in vitro
1Department of Applied Biology, University of Helsinki, PO Box 27, 00014 University of Helsinki, Finland
2Department of Biology, University of Joensuu, PO Box 111, 80101 Joensuu, Finland
3Department of Biology, University of Oulu, PO Box 3000, 90014 University of Oulu, Finland
4Finnish Forest Research Institute, Parkano Research Unit, 39700 Parkano, Finland
* To whom correspondence should be addressed. E-mail: Karoliina.Niemi{at}helsinki.fi
Received 29 June 2006; Revised 12 September 2006 Accepted 19 September 2006
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Abstract |
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Changes in the concentrations of individual flavonoids and polyamines (PAs) in Scots pine (Pinus sylvestris L.) cotyledonary seedlings were studied during the establishment of an ectomycorrhizal (ECM) symbiosis with two Suillus variegatus strains in vitro. Both flavonoids and PAs were analysed after 3, 7, and 14 d in dual culture, and changes in concentrations were compared with growth of the seedlings. Both S. variegatus strains caused similar responses in Scots pine seedlings. Free putrescine accumulated immediately but only transiently after inoculation. This was followed by continuous accumulation of PA conjugates in needles and stems, and free spermidine and spermine in roots, which was accompanied by mycorrhiza formation and improved growth. The fungi induced lateral root formation and main root and primary needle elongation. Inoculation caused no qualitative changes in flavonoid composition, while quantitative changes in flavonols, catechins, and condensed tannins were observed in shoots during mycorrhiza formation. These results indicate that in this in vitro system conjugated PAs and specific flavonoids, generally related to the plant's defence reactions, did not play a major role in the regulation of the establishment of the ectomycorrhizal (ECM) symbiosis in Scots pine roots. The results also clearly show that positive growth responses in shoots and roots due to S. variegatus were supported by different and highly specific changes in the synthesis of both primary and secondary metabolites in these parts of the seedling.
Key words: Ectomycorrhizas, flavonoids, Pinus sylvestris, polyamines, Suillus variegatus
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Introduction |
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Under natural conditions, Scots pine (Pinus sylvestris L.) as well as other pine species live in close association with ectomycorrhizal (ECM) fungi. These fungi cover short roots as a hyphal mantle and penetrate between epidermal and cortical cells forming a highly differentiated structure called a Hartig net. In ECM symbiosis, the fungus is dependent on the photosynthates supplied by the host plant, whereas the plant benefits from water and nutrient acquisition facilitated by the fungus. Moreover, the fungus may increase tolerance of the plant to disease and heavy metals (reviewed by Smith and Read, 1997).
Polyamines (PAs) constitute a group of basic cell components that are essential in the growth and development of all living organisms (Tabor and Tabor, 1985; Walters, 1995; Bais and Ravishankar, 2002). In plants, they have been shown to be involved in a vast variety of metabolic processes, ranging from embryogenesis and organ development (Bais and Ravishankar, 2002; Couée et al., 2004) to reactions against biotic stress (Walters, 2003). Spermidine (Spd) and spermine (Spm), and their precursor putrescine (Put), are the most common PAs that have been shown to exist in cells as free bases and perchloric acid (PCA)-soluble and -insoluble conjugated forms. Although free PAs may, in certain situations, act like inorganic cations, the major part of their function in cells arises from their interactions with DNA and RNA, different proteins, and phospholipids (reviewed by Igarashi and Kashiwagi, 2000; Bais and Ravishankar, 2002). PAs may also interact with hydroxycinnamic acids, resulting in the formation of corresponding amides (Facchini et al., 2002).
There is increasing evidence of the involvement of specific PAs not only in biotic stress reactions (reviewed by Walters, 2003) but also in symbiotic interactions with both N-fixing bacteria (Vasileva and Ignatov, 1999; Flemetakis et al., 2004; Atici et al., 2006) and mycorrhizal fungi (Kytöviita and Sarjala, 1997; Niemi et al., 2002a, 2006; Sarjala and Taulavuori, 2004). In a microcosm study with 5-month-old Scots pine seedlings, inoculation with an ECM fungus Suillus variegatus (Swartz: Fr.) O. Kunze increased Put and Spd concentrations in needles and roots (Sarjala and Taulavuori, 2004). Similarly, in a recent in vitro study (Niemi et al., 2006), inoculation with the same S. variegatus strain resulted in a transient increase in the concentrations of free Put in needles and free Put, Spd, and Spm in stems and roots of the cotyledonary seedlings of Scots pine. The increase in root PAs was followed by improved root growth. These results indicate that in ECM symbiosis, specific PAs are more related to improved growth of the host plant than to defence reactions.
Flavonoids form a large and heterogenous group of secondary products having a bioactive role in, for example, cell wall rigidification, pollen tube germination, attraction of pollinators and seed dispersers, as well as defence reactions against predators, pathogens, and abiotic stress conditions (Winkel-Shirley, 2002; Dixon et al., 2005; Koes et al., 2005; Taylor and Grotewold, 2005). Recent studies have also highlighted the role of flavonoids in modulating cell signalling pathways, including polar auxin transport (Brown et al., 2001; Peer et al., 2004; Kakiuchi et al., 2006). Despite increasing evidence of the role of flavonoids and other phenolics in plant development, knowledge of their role in symbiotic ECM interactions is still scarce and contradictory. Existing reports have emphasized the role of specific flavonoids only as defence compounds, which either accumulate in the inner parts of the cortex to prevent the growth of the fungus (Weiss et al., 1997, 1999) or decrease in the lateral roots to allow the colonization of the compatible fungus (Münzenberger et al., 1990, 1995; Beyler and Heyser, 1997).
The importance of ECM fungi in the growth and morphology of roots has increased interest in using them as promoting agents in in vitro cultures. Inoculation with specific ECM fungi has induced development and growth of adventitious roots and also improved subsequent acclimatization to conditions ex vitro (reviewed by Niemi et al., 2004). However, conditions in the closed in vitro system, including nutrient and CO2 concentrations, may disturb the balance between symbiotic partners and subsequently reduce the growth of the plantlet. Recently, an in vitro cultivation system was developed to induce formation and growth of adventitious roots of Scots pine by means of inoculation with the ECM fungi Pisolithus tinctorius (Pers.) Coker and Couch and Paxillus involutus (Batsch) Fr. (Niemi et al., 2002a, b). In the present study, this in vitro system was used to investigate the effects of fungal inoculation on the contents of individual flavonoids that have been suggested to regulate the formation of ECM symbiosis. The contents of specific flavonoids in different parts of Scots pine cotyledonary seedlings were analysed 3, 7, and 14 d after inoculation with two strains of S. variegatus. Changes in flavonoid pools were compared with both growth responses and contents of PAs that in a previous study (Niemi et al., 2006) referred to the establishment of ECM symbiosis of Scots pine seedlings.
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Materials and methods |
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Biological material
The ECM fungi used in the present study were two strains of S. variegatus that were originally isolated from basidiocarps under a Scots pine stand. The strain SVT was isolated from Parkano, Valkoinenkeidas (62°0' N; 22°44' E) and SVA from Karvia, Kourajärvi (62°10' N; 22°50' E) both located in western Finland. The fungi were maintained by cultivating mycelia on Hagem agar medium (Modess, 1941). For the mycorrhiza experiments, mycelia were cultivated for 4 weeks on strips of sterile, moist filter paper overlaid on Hagem agar medium.
Seeds from the open-pollinated elite Scots pine clone K818 in the Punkaharju clone collection (61°48' N; 29°17' E) were surface-sterilized with 2% calcium hyphochlorite for 20 min, rinsed in sterile water and germinated on 0.7% water agar in glass jars. The germinating seeds were incubated for 25 d in a growth chamber at 24±2 °C under a 16 h photoperiod (140–150 µmol m–2 s–1, Osram L36W/830 and Osram L36W/77).
Growth of Scots pine seedlings in the presence of Suillus variegatus strains
Petri dishes, 14 cm in diameter, were filled with Melin Norkrans (MMN) agar medium (Marx, 1969) modified by Niemi et al. (2002b). Two 25-d-old seedlings were transferred from the germination medium and laid horizontally on sterile moist filter paper covering the agar surface. Individual seedlings were inoculated by placing two filter paper strips covered by 4-week-old mycelium of SVA or SVT on the main root. In non-inoculated cultures, mycelium-covered filter papers were substituted with sterile moist filter papers under which a small piece of fresh agar was placed. The experiment was carried out according to Niemi et al. (2006) under the same conditions as used for seed germination. One Petri dish, i.e. two seedlings, formed one replicate, and there were 15 replicates per treatment for each harvesting.
Seedlings were cultivated in the presence of the fungus for 0, 3, 7, and 14 d. At harvest, shoot and root fresh weights, the length of the main root, and the number of lateral roots were determined. The number of root tips covered with a hyphal mantle was evaluated using a dissecting microscope.
Six more seedlings per treatment were harvested for examination of mycorrhizal structures by light microscopy according to the method described by Niemi and Häggman (2002). After fixation, the root samples were infiltrated and embedded in Spurr resin kit (ASL). The sections were cut in a LKB IV ultratome and stained with toluidine blue (Merck).
Analysis of polyamines from Scots pine seedlings
After the growth parameters were determined, needles, stems, and roots of non-inoculated and inoculated seedlings were analysed for PAs. For each harvest, 10 seedlings were pooled to form one sample for analyses. PAs were determined from three samples of needles, stems, and roots of non-inoculated and inoculated seedlings. Free, PCA-soluble, and PCA-insoluble conjugated PAs in the needles, stems, and roots were extracted in 5% (w/v) PCA according to Sarjala and Kaunisto (1993) and Fornalé et al. (1999). PAs in the crude and hydrolysed extracts were dansylated and then separated by high-performance liquid chromatography (HPLC; Merck Hitachi) as described by Sarjala and Kaunisto (1993). Concentrations of PAs are expressed as nmol g–1 fresh weight of plant material.
Analysis of flavonoids from Scots pine seedlings
Non-inoculated and inoculated (with both SVA and SVT) seedlings of 25-d-old Scots pine were grown as in the PA experiment. The seedlings were harvested 0, 3, 7, and 14 d after inoculation and there were 15 replicates (two seedlings in a Petri dish represented one replicate) per treatment for each harvesting. After the growth parameters were determined, needles, stems, and roots of non-inoculated and inoculated seedlings were analysed for flavonoids. For each harvest, seedlings were pooled to form one sample of 30–40 mg fresh weight for analyses. Flavonoids were determined from three samples of needles, stems, and roots of non-inoculated and inoculated seedlings. The samples were freeze-dried and stored at –80 °C until analysis.
Freeze-dried needle (6–9 mg), stem (4–6 mg), and root (3–5 mg) samples were first homogenized with a roughened glass rod and then extracted with an Ultra-Turrax homogenizer in 600 µl of cold methanol for 1 min. The samples were left to stand in an ice bath for 15 min, and then centrifuged at 11 000 g for 3 min. Extraction was repeated three more times while standing for 3 min. Supernatants were combined, and methanol evaporated. The dried samples were dissolved in 400 µl of water:methanol (1:1, v/v) and analysed by HPLC as described by Julkunen-Tiitto and Sorsa (2001). The compounds were identified and quantified based on their retention time, spectral characteristics, and HPLC–mass spectrometry (API-ES, positive ions) (Julkunen-Tiitto and Sorsa, 2001). The glycosides and diacylated derivatives of kaempferol were quantified against the standard astragalin (kaempferol-3-glucoside), and glycosides of quercetin and myricetin against the standards hyperin (quercetin-3-galactoside) and myricitrin (myricetin-3-rhamnoside), respectively. The standard (+)-catechin was used for quantification of catechin and gallocatechin.
Soluble condensed tannins were analysed from the HPLC samples and insoluble (bound) condensed tannins from the dried extract residue by acid butanol assay, as reported by Porter et al. (1986).
Statistical analyses
Growth, PA, and flavonoid results between non-inoculated seedlings and those inoculated with either SVA or SVT were compared using either analysis of variance (ANOVA) combined with Tukey's honestly significant difference test or non-parametric Kruskall–Wallis test combined with Mann–Whitney U-test with Bonferroni correction (Zar, 1984; Altman, 1991). The non-parametric test was used when the conditions for ANOVA, i.e. normal distribution and homogeneity of variances, were not met. Growth parameters between PA and flavonoid experiments were also compared, and no significant (P >0.05) difference was observed. Therefore, only the growth data of the first experiment are shown and discussed. All statistical analyses were conducted with SPSS/PC statistical version 12.0 (SPSS Inc., Chicago, IL, USA).
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Results |
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Growth and mycorrhiza formation of Scots pine seedlings
Inoculation with both S. variegatus strains drastically increased the growth of seedlings (Fig. 1A–C). Seven days after inoculation, main roots (Fig. 1A) and primary needles (Fig. 1B) were significantly (P <0.05) longer in inoculated than non-inoculated seedlings. Inoculation also resulted in a significant (P <0.05) increase in the number of lateral roots (Fig. 1C). The root/shoot ratios of the inoculated seedlings started to increase soon after inoculation, whereas in non-inoculated seedlings it slightly decreased during the experiment (Fig. 1D). Mycorrhiza formation started immediately after the first lateral roots were formed and, 7 d after inoculation, hyphae of SVA and SVT covered 46% and 49% of the lateral root tips, respectively. At the end of the experiment, the corresponding percentages were 52 and 54. Both fungi formed a Hartig net between epidermal and cortical cells, and no difference in the intensity of Hartig net formation was observed between two strains.
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Polyamine concentrations in Scots pine seedlings
In non-inoculated seedlings, the concentration of free Put doubled in needles, stems, and roots during the first 3 d of the experiment. After that, the concentration stayed relatively stable (Fig. 2A–C). Concentrations of free Spd (Fig. 2D–F) and Spm (Fig. 2G–I) varied relatively little during the experiment. Dual culture for 3 d with both SVA and SVT caused a significant (P <0.05) increase in the concentration of free Put in needles (Fig. 2A), after which the concentration started to decrease slowly, approaching the level of the non-inoculated seedlings at the end of the experiment. In contrast, free Spd and Spm concentrations decreased in the needles and, 7 d after inoculation, they were significantly (P <0.05) lower in inoculated than non-inoculated seedlings (Fig. 2D, G). In stems, inoculation increased the concentration of free Put and also of free Spd already within the first 3 d in dual culture (Fig. 2B, E). In roots, fungus-induced accumulation of free Put occurred more slowly, and the concentration started to decrease later than in needles and stems (Fig. 2C). In contrast, free Spd and Spm continued to accumulate in the inoculated roots until the end of the experiment (Fig. 2F, I).
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In non-inoculated seedlings, the concentrations of PCA-soluble conjugated PAs were low and relatively constant throughout the 2 week experiment (Fig. 3A–I). Inoculation with S. variegatus significantly (P <0.05) increased the concentration of PCA-soluble conjugated Put and Spd in the needles after 7 d in dual culture (Fig. 3A, D), whereas a slight decrease was observed in Spm concentration 3 d after inoculation (Fig. 3G). Inoculation also increased the concentrations of soluble conjugated Put, Spd, and Spm in stems starting from the third day in dual culture (Fig. 3B, E, H). The effects of the inoculation on the contents of PCA-soluble conjugated PAs in roots were highly variable, and no significant difference between non-inoculated and inoculated seedlings was observed (Fig. 3C, F, I). Small amounts of PCA-insoluble Spd were found only in single samples, and no Put or Spm was detected as PCA-insoluble conjugated forms (data not shown).
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Flavonoid concentrations in Scots pine seedlings
The needles of Scots pine seedlings contained non-acylated and acylated flavonols (Fig. 4), as well as catechins and condensed tannins (Fig. 5). A novel methyl flavonol in Scots pine was extracted and tentatively identified as methyl-myricetin 3-glucoside [571 M+23.495 (M+1)] (Fig. 4H). The concentrations of non-acylated kaempferol and quercetin derivatives decreased towards the end of the experiment (Fig. 4A–F), whereas myricetin derivatives remained more stable (Fig, 4G, H). Inoculation with both S. variegatus strains enhanced the decrease of all non-acylated flavonols, although not always significantly (P <0.05) (Fig. 4A–H). In contrast, inoculation increased the amount of diacylated flavonols, especially dicoumaryl isorhamnetin in needles (Fig. 4I, J). At the end of the experiment, the needles of the seedlings inoculated with SVA and SVT contained 44 and 25 times more dicoumaryl-isorhamnetin, respectively, than the needles of the non-inoculated seedlings, whereas the concentration of isorhamnetin was about twice as high in non-inoculated than in inoculated seedlings. In the needles of the non-inoculated seedlings, the concentration of catechins and condensed tannins stayed constant throughout the experiment (Fig. 5A, D, G, J). In contrast, in the needles of the inoculated seedlings, the concentrations of catechin, gallocatechin, and soluble tannins started to increase already within the first 3 d in dual culture (Fig. 5A, D, G) and the concentration of cell wall-bound tannins a few days later (Fig. 5J).
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Myricetin derivatives and hyperin found in needles were not detected in stems, and the concentrations of most non-acylated flavonols found were clearly lower in stems than in needles (Fig. 6A–E). As in needles, the concentrations of most non-acylated flavonols decreased in stems towards the end of the experiment, and the presence of both fungi enhanced the decrease, although not always significantly (P <0.05) (Fig. 6A–E). Inoculation caused a transient increase in the concentration of dicoumaryl-astragalin in stems (Fig. 6F). However, at the end of the experiment, stems of the inoculated seedlings contained less diacylated flavonols than stems of the non-inoculated seedlings (Fig. 6F, G). As in needles, the fungus increased the amount of catechin, gallocatechin, and soluble tannins in stems (Fig. 5B, E, H). Cell wall-bound tannins did not differ between non-inoculated and inoculated seedlings (Fig. 5K).
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Of the flavonoids found in shoots (needles and stems), only catechin, gallocatechin, and condensed tannins were detected in roots (Fig. 5C, F, I, L). Roots contained remarkably more catechin and condensed tannins at the beginning of the experiment than needles and stems (Fig. 5C, I, L). A decrease in catechin and condensed tannins was observed towards the end of the experiment but, in contrast to shoots, no significant difference between non-inoculated and inoculated seedlings was observed in roots. However, the concentration of gallocatechin was increased in the presence of SVT (Fig. 5F).
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Discussion |
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In the present in vitro study, changes in the concentrations of individual flavonoids and PAs in Scots pine seedlings were followed and compared with growth responses during the establishment of the ECM symbiosis with two S. variegatus strains. Free Put accumulated immediately but only transiently after inoculation, and was followed by continuous accumulation of PA conjugates in shoots and free Spd and Spm in roots, as well as improved growth. Inoculation caused quantitative changes in individual flavonoids in shoots, whereas in roots no clear changes were observed. These results show that regardless of the improved growth of both shoots and roots in the presence of the fungus in vitro, changes in the contents of individual free and conjugated PAs and flavonoids due to the fungi were highly specific in different parts of the seedling.
PAs are known to play an essential role in rhizogenesis and root growth (Couée et al., 2004). Recent reports have also implicated PAs in ECM interactions (Kytöviita and Sarjala, 1997; Sarjala and Taulavuori, 2004; Niemi et al., 2002a, 2006). In the present study, inoculation with two S. variegatus strains caused highly similar changes in PA contents in the roots of Scots pine cotyledonary seedlings, as did in vitro inoculation with SVT in the roots of Scots pine cotyledonary seedlings representing another seed origin (Niemi et al., 2006). The fungus-induced changes in root PAs also support the results obtained from the microcosm study using 5-month-old seedlings (Sarjala and Taulavuori, 2004). In the present study, inoculation caused a transient increase in free Put in roots, whereas free Spd and Spm accumulated throughout the 2 week experiment coinciding with lateral root formation and main root elongation. This agrees with earlier studies on the importance of free Spd and Spm in lateral root development and primary root growth (Hummel et al., 2002; reviewed by Couée et al., 2004) and, therefore, in this study, high Put production in roots immediately after inoculation might be due to the need for the precursor for Spd and Spm synthesis. In this study, the need for free Spd and Spm for root growth is supported by the fact that PCA-soluble conjugated PAs were not accumulated in the roots as a result of inoculation. This also indicates that soluble conjugated PAs that have been implicated in defence against pathogens and wounding (Pearce, et al., 1998; Cowley and Walters, 2002a, b) did not play an important role in the regulation of ECM fungal growth within the roots.
Fluctuation of free and conjugated PAs in shoots differed from that in roots of the inoculated seedlings. In the shoots, the concentrations of free Put and Spd increased immediately but only transiently after inoculation, except for Spd in needles. The decrease of both free Spd and free Spm in needles coincided with accumulation of Put and Spd conjugates. Conjugated forms have not been shown to function as a source of stored amines (Faccini et al., 2002) and, therefore, conjugated Put and Spd seemed to have a specific role in the regulation of the growth and development of the shoots in vitro. However, Spm conjugates were not accumulated in needles regardless of the decrease of free Spm. These results show that the overall balance between free and conjugated forms of single PAs is important in cellular metabolism. Moreover, needles and roots seem to differ in their needs for PAs during intensive growth.
Two different S. variegatus strains also caused similar changes among flavonoids in the shoots of Scots pine seedlings. Both fungal strains enhanced the decrease in non-acylated flavonols. Initially, the concentrations of most non-acylated flavonols in needles and stems were relatively high compared with the needles of the older Scots pine seedlings (Lavola et al., 2003; Roitto et al., 2005), which might indicate that the fungus-induced decrease of non-acylated flavonols was related to the developmental stage and/or improved growth of the shoots in vitro. On the other hand, inoculation increased dicoumaroyl-astragalin/astragalin and dicoumaroyl-isorhamnetin/isorhamnetin ratios in needles but decreased them in stems. Acylated forms have been suggested to protect needles of Scots pine against abiotic stress (Lavola et al., 2003) and, therefore, the increase in acylated flavonols in needles instead of stems might be due to fungus-induced tolerance against abiotic stress. However, the production of carbon-consuming acylatated flavonols concomitantly with intensive elongation of the needles of the inoculated seedlings may also be related to a certain developmental stage of the seedling.
In the presence of both fungal strains, a large amount of catechins and finally condensed tannins were accumulated in the shoots. Studies performed both in the growth chamber (Lavola and Julkunen-Tiitto, 1994) and in the field (Booker and Maier, 2001; Mattson et al., 2005) have shown that synthesis of catechins and condensed tannins in needles follows an increase in the CO2 concentration. On the other hand, increased availability of nutrients has been shown to decrease catechin and condensed tannins in the needles concomitantly with increasing growth of Scots pine seedlings (Lavola et al., 2003). In the closed in vitro system, the situation might be different, however, and in the present study, accumulation of catechins and condensed tannins in needles and stems started immediately after inoculation and continued throughout the intensive growth period. This all occurred at the same time as acylation of flavonols in needles and the formation of PA conjugates in the whole shoots, showing that the growth of the symbiotic ECM fungus in the roots causes a large shift in carbon allocation within the upper parts of the seedling. Certain flavonols and PA conjugates have been implicated in stress reactions of plants and it remains to be elucidated whether the ECM fungus-induced accumulation of different flavonols as well as PA conjugates, which are also related to stress reactions of plants, in the shoots of in vitro plants might improve the acclimatization of the plants to conditions ex vitro.
In contrast to shoots, the concentrations of catechin and condensed tannins showed a tendency to decrease in the roots of both non-inoculated and inoculated seedlings and, regardless of high mycorrhiza frequencies, the fungi caused hardly any changes in the flavonoid concentrations of the roots. Earlier reports have focused on the role of catechins as regulators of ECM colonization and have shown contradictory results. Weiss et al. (1997, 1999) suggested that catechin and epicatechin accumulated in the inner part of the cortex to prevent the growth of the ECM fungus into the inner cortex, whereas Beyler and Heyser (1997) and Münzenberger et al. (1995) reported that reduction of catechin and epicatechin in the root tips is a prerequisite for rapid mycorrhization. Similarly, Schützendübel and Polle (2002) showed that Scots pine short root tips covered by the mycelium of P. involutus (Batsch) Fr. contained less catechin than non-mycorrhizal root tips. In the present study, catechins and tannins were not localized at the tissue level, but the results clearly show that the closed cultivation system did not cause accumulation of individual flavonoids at concentrations that might prevent the formation of ECMs and the growth of the roots of Scots pine seedlings.
To summarize, in the closed in vitro culture system, inoculation with two S. variegatus strains resulted in drastic changes in the contents of PAs and flavonoids during the formation of mycorrhizal symbiosis. The fungi improved the growth of both shoots and roots, but the changes in ratios between free and soluble conjugated forms of single PAs were very different depending on the part of the seedling. Quantitative changes in flavonols, catechins, and condensed tannins were observed in shoots but not in roots during mycorrhiza formation, indicating that in in vitro, specific flavonoids together with conjugated PAs, generally implicated in the plant's defence reactions, did not have a major role in the regulation of the establishment of the ECM symbiosis in Scots pine roots. The results of the present study show that positive growth responses in shoots and roots due to the compatible fungus S. variegatus were supported by different and highly specific changes in the synthesis of both primary and secondary metabolites in these parts of the seedling.
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Acknowledgements |
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We are grateful to Ms Anneli Käenmäki, Ms Eeva Pihlajaviita, and Ms Tarja Salminen from the Finnish Forest Research Institute, Ms Pirkko Leikas-Lazànyi from the Institute of Biotechnology, Ms Outi Nousiainen from the University of Joensuu, and Ms Aira Vainiola from the University of Helsinki for their technical assistance. This work was supported by the Academy of Finland (projects 53440 to TS, 64308 to RJ-T, and 202415 to KN), and the Finnish Cultural Foundation (a grant to KN).
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Abbreviations |
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ECM, ectomycorrhizal; PA, polyamine; PCA, perchloric acid; Put, putrescine; Spd, spermidine; Spm, spermine.
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