AOBPreview originally published online on October 7, 2007
Annals of Botany 2007 100(7):1599-1603; doi:10.1093/aob/mcm243
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TECHNICAL ARTICLE |
Isolation and Characterization of Arbuscules from Roots of an Increased-arbuscule-forming Mutant of Lotus japonicus
1 Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan
2 Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan
3 National Institute of Agrobiological Resources, Kannondai, Tsukuba, Ibaraki 305-8602, Japan
4 Laboratory of Soil Science, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan
5 School of Earth and Geographical Sciences, M087, The University of Western Australia, Crawley, WA 6009, Australia
* For correspondence. E-mail solaiman{at}cyllene.uwa.edu.au
Received: 4 June 2007 Returned for revision: 23 July 2007 Accepted: 20 August 2007 Published electronically: 7 October 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Background and Aims: Previous methods for isolation of arbuscules from mycorrhizal roots are time-consuming, complex and expensive. Therefore, a simple, rapid and inexpensive method for the isolation of metabolically active arbuscules from plant root of an increased-arbuscule-forming mutant of Lotus japonicus (Ljsym78-2) is described.
Methods: Roots of the L. japonicus mutant plants Ljsym78-2 colonized by Glomus sp. were separated from soil, washed with water, immersed in CaSO4 before being cut into 5-mm pieces and homogenized with a Waring blender at 6000 rpm for 30 s. The arbuscules were purified by separation from plant tissues with a 50-µm nylon mesh, finally collecting on a 30-µm nylon mesh. Enzyme histochemical staining showed that the collected arbuscules had succinate dehydrogenase, alkaline phosphatase and acid phosphatase activities.
Key Results and Conclusions: The enzymic activity of the arbuscules was not affected after the isolation process. The establishment of this simple, rapid and inexpensive method for the isolation of metabolically active arbuscules will be useful to clarify the biochemical processes occurring in nutrient exchange at the arbuscular interface.
Key words: Arbuscular mycorrhiza, arbuscule isolation, Glomus sp., increased-arbuscule-forming mutant, Lotus japonicus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Arbuscular mycorrhizas (AM) are symbiosis between plant roots and fungi belonging to the Glomales. The mutualistic nutrient exchange in this symbiosis is characterized by the transfer of phosphorus from the mycosymbiont to the host plant and by the reverse transfer of carbon compounds derived from photosynthates (Smith and Read, 1997; Solaiman and Saito, 1997). It is now widely accepted that phosphate in the soil is taken up into the extraradical hyphae by a phosphate transporter, subsequently condensed into polyphosphate and translocated by protoplasmic streaming into the intraradical hyphae (Saito, 2000). The arbuscular hypha is probably the main site for the nutrient exchange. The presence of alkaline phosphatase (ALP) enzyme activity in the arbuscules has been used as evidence for arbuscules being the site of phosphorus exchange (Tisserant et al., 1992) with photosynthetic carbon (Solaiman and Saito, 1997; Ezawa et al., 1999). However, the carbon and phosphorus metabolism involved in the nutrient exchange at the arbuscules is largely unknown.
In order to clarify the biochemical processes occurring in nutrient exchange in the symbiotic system, it is essential to isolate the arbuscules from host tissue and to examine their biochemical activities. The separation of arbuscules from host tissue is a useful technique, especially because this endosymbiont can not be independently cultured in vitro. However, it is not easy to achieve because of the complex penetration of arbuscular hyphae into cortical cells. Intraradical hyphae were separated from their host by enzymic digestion of root tissue with cellulase and pectinase, followed by hand sorting of the hyphae under a dissecting microscope (Capaccio and Callow, 1982; Smith et al., 1985; Hepper et al., 1986). This method was laborious and expensive, and enzymic digestion of the mycorrhizal roots for >12 h reduced the metabolic activity of the hyphae as evaluated by histochemical staining for succinate dehydrogenase (SDH) activity (McGee and Smith, 1990).
Saito (1995) developed a method to isolate metabolically active intraradical hyphae from AM onion roots with only 1 h of enzymic digestion. The metabolic activity of the isolated hyphae, based upon the evaluation of SDH staining, was not affected by this enzymic separation procedure. In addition, a mass of intraradical hyphae almost free of plant debris can be collected after Percoll gradient centrifugation. Hyphae separated by this protocol were used for a radiorespirometric assay (Solaiman and Saito, 1997) to study the carbon metabolism therein. But this method is still laborious and expensive, and 2–3 h are needed to complete the isolation of the intraradical hyphae or arbuscules. No suitable method is available at present for isolation of arbuscules. There is a need for highly colonized plant roots from which arbuscules can be isolated without intensive labour.
Highly arbuscule-forming mutants of Lotus japonicus, Ljsym78-1 and 78-2, which showed increased colonization by arbuscules on their roots compared with the wild-type ‘Gifu’, have been reported previously (Solaiman et al., 2000). The majority of the arbuscules on the mutant roots were SDH active and were well-developed and physically tough. These arbuscules were easily released from the plant root cells when the plant root was torn with needles. These findings encouraged the establishment of a rapid and simple method for isolating arbuscules from the mycorrhizal colonized roots of the mutant. The method may lead to advances in the understanding of biochemical processes of nutrient exchange between arbuscules and plant cortical cells. The procedure for isolating arbuscules from the mutant roots, and the quantity and quality of the isolated arbuscule fraction are described in this paper.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Plant material
The Lotus japonicus mutant, Ljsym78-2, generated by EMS (ethylmethane sulfonate) treatment of L. japonicus B-129 ‘Gifu’, was used in this study. This mutant has a hypernodulating (Kawaguchi et al., 2002) and increased arbuscule forming (Arb++) phenotype (Solaiman et al., 2000). The mutated loci were confirmed to be monogenic and recessive. Ljsym78-2 was confirmed to be allelic to Ljsym16 and har1 (Ljsym34) (Schauser et al., 1998; Wopereis et al., 2000; Nishimura et al., 2002). The mutant has a nitrate tolerant, shoot-controlled hypernodulating phenotype with increased lateral root formation. When inoculated by AM fungus, Glomus sp. R-10, the mutant, showed increased arbuscular colonization and the majority of the arbuscules had SDH activity and were well-developed and physically tough (Solaiman et al., 2000). As a control, L. japonicus wild-type ‘Gifu’ was used in this examination.
Potting medium
Washed sand was mixed with Akadama soil (subsoil of a volcanic ash soil; Kodaira Engei Shizai Co., Ltd, Japan) in equal volumes, sterilized by autoclaving at 121 °C for 1 h and supplemented with NH4NO3, KH2PO4 and KCl at 0·53 g, 0·027 g and 0·107 g L–1 mix, respectively. AM fungal inoculum containing spores of Glomus sp. R-10 (Idemitsu Kosan Co. Ltd, Japan; R-10 is the commercial name) was mixed well with the potting mixture (1 : 10, v/v). Glomus sp. R-10 gave the highest colonization rate among several Glomus species in tests with various crops including legumes (A. Narutaki, pers. comm.; Idemitsu Cosan Co. Ltd., Japan).
Mycorrhizal colonization
Seeds of Ljsym78-2 and wild-type ‘Gifu’ were taken in Eppendorf tubes and dipped in concentrated H2SO4 for 10 min with gentle shaking. After removal of H2SO4, seeds were rinsed in distilled water and then germinated on sterilized moist filter paper in Petri dishes at 25 °C in the dark. Upon germination, the seedlings were transplanted individually to nursery trays (six plants per tray) containing the sand–soil–inoculum mixture (300 g of mix per six plants). Seedlings of ‘Gifu’ were grown without inoculum to check for contamination with pathogens in the sterilized mix. Plants were grown in a growth chamber (day: 20 h, 25 °C, photosynthetic photon flux density 70 µmol m–2 s–1; night: 4 h, 22 °C). Sampling was done at 9 and 15 weeks after transplanting seedlings for the isolation of arbuscules.
Isolation of arbuscules
Isolation of arbuscules was carried out according to the method for the isolation of internal hyphae (Saito, 1995) with several modifications. Fresh roots were separated from soil by washing with water, immersed in cold 0·5 mM CaSO4 for a few minutes for homeostasis, before being cut into 5-mm pieces. The root pieces (5 g) were collected on nylon mesh (50 µm) and washed with washing buffer (WB) (0·3 M mannitol, 1 mM DTT, 0·01 M Tris–HCl; pH 7·4). The washed root pieces were transferred to the 100-ml container of a homogenizer of a Waring Blender (Cell Master CM-100, Iuchi Seieido Co. Ltd, Japan) with 40 mL of cold WB, and homogenized for 30 s at 6000 rpm. Buffer was used in these steps to minimize the degradation of fungal structures. The homogenate was filtered through two layers of cheesecloth and the residue again homogenized with 40 mL of cold WB. This homogenization/filtration process was repeated twice more. The combined filtrates were further filtrated through a 50-µm and then collected onto a 30-µm nylon mesh. The residue on the 30-µm nylon mesh was gently collected with a Pasteur pipette as the arbuscule fraction. All the procedures were carried out at 0–4 °C.
Composition of the isolated arbuscule fraction and enzyme activities of the arbuscules
The isolated arbuscule fraction was subjected to an assessment of its components, and histochemical observation of SDH, ALP and acid phosphatase (ACP) according to the method described by Saito (1995) and McDonald and Lewis (1978). The isolated arbuscules were incubated at 35 °C for 2 h in the following solutions: SDH, 0·25 M sodium succinate, 0·05 M Tris–HCl buffer (pH 7·6), 0·5 mM MgCl2, 1 mg mL–1 Nitroblue tetrazolium (McDonald and Lewis, 1978); ALP, 4 mM 
-naphtyl acid phosphate, 0·6 mg ml–1 Fast Blue RR, 0·1 M Tris–HCl buffer (pH 8·5); ACP, same as ALP but with 0·1 M sodium acetate buffer (pH 4·0) instead of Tris buffer. After incubation the arbuscules were washed with deionized water and collected on a 30-µm nylon mesh to remove excess substrate.
After the washing, the arbuscule fractions treated for SDH, ALP and ACP staining were counterstained with 0·5 mg mL–1 acid fuchsin (Saito et al., 1993), transferred to lactoglycerol and mounted on glass slides. Under the light microscope, the number of particles belonging to the following five categories was counted separately; (1) arbuscules, (2) fragmented internal hyphae, (3) vesicles, (4) root hairs, and (5) broken cell wall fragments. This counting also was performed for approx.100 particles on five different glass slides (in total approx. 500 particles).
The arbuscules with or without SDH, ALP or ACP activity were enumerated separately. To evaluate the loss in the SDH, ACP and ALP activity of arbuscules during the isolation process, the percentages of SDH, ACP and ALP active arbuscules in the roots of Ljsym78-2 were compared with those in the isolated arbuscules. The fresh roots of Ljsym78-2 were cut into 2- to 3-mm pieces, stained for SDH, ACP or ALP and counterstained with acid fuchsin as described above. The stained root pieces were squashed on a slide glass and mounted with lactoglycerol. The percentage of arbuscules showing SDH, ACP or ALP activity was examined.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Mycorrhizal colonization
The percentage of root length colonized by Glomus sp. R-10 was 90 % in Ljsym78-2 (60 % arbuscular colonization) and 75 % in wild-type ‘Gifu’ (40 % arbuscular colonization) at 9 weeks after sowing which was very similar to previous reports (Solaiman et al., 2000).
Isolation of arbuscules
The dry weights of the arbuscule fraction obtained from 1 g of fresh root of Ljsym78-2 and wild-type ‘Gifu’ were 3·1 mg and 0·7 mg, respectively. The above-mentioned homogenization speed (6000 rpm) and duration (30 s) gave the largest amount of intact arbuscules among several combinations tested (data not shown). Filtration of cheesecloth filtrate through 50-µm nylon mesh efficiently removed root hairs and large broken plant cell wall fragments. In fact, root hairs and broken plant cell wall fragments accounted for approx. 70 % of particles trapped on the 50-µm nylon mesh and arbuscules approx. 29 %. To collect intact arbuscules, the filtrate was further passed through 30-µm nylon mesh, and the residue on the mesh was recovered. This mesh was chosen from the size of the arbuscules formed in the roots of Ljsym78-2 (Solaiman et al., 2000).
Composition of the arbuscule fraction
Light microscopic observation of the SDH-stained arbuscule fractions counterstained with acid fuchsin revealed that the arbuscule fraction contained arbuscules, fragmented internal hyphae, vesicles, root hairs and broken cell wall fragments (Fig. 1). Vesicles and cell wall fragments were observed infrequently. Both intact and partially broken arbuscules were observed.
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The proportion of each component in the arbuscule fraction (the number of particles included in each component/total number of particles) was visually assessed under a light microscope using acid fuchsin-counterstained arbuscule fractions prepared from Ljsym78-2 roots and ‘Gifu’ roots (Table 1). In the arbuscule fraction prepared from roots of Ljsym78-2 grown for 9 weeks, 56 % of the visible particles were arbuscules, a value higher than that for the fraction from ‘Gifu’ roots (33 %). The percentage of fragmented internal hyphae in the arbuscule fractions was 23 % in both Ljsym78-2 and ‘Gifu’. Root hairs were present in both fractions, but at lower frequency in the fraction from roots of Ljsym78-2 (18 %). Root hairs present in the arbuscule fraction might be reduced by the enzyme digestion (cellulase and pectinase) of root tissue and/or the Percoll gradient centrifugation of the arbuscule fraction, but neither process was included in the proposed isolation method to achieve the rapid arbuscule isolation.
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As a result, the proportion of fungal structures other than vesicles (arbuscules and internal hyphae) was as much as 80 % in the arbuscule fraction obtained from roots of Ljsym78-2 by the present method.
The mutant roots grown for 9 weeks under the above-mentioned conditions seemed well suited to the isolation of arbuscules. Roots grown for a shorter period had a lower biomass and lower percentage of root length colonization, which resulted in a lower recovery of the arbuscule fraction (data not shown). The roots grown for longer period (15 weeks) were not suitable because the arbuscules occupied only 16 % of the isolated fraction (Table 1). This might be caused by the presence of senescent arbuscules in old roots.
Enzyme activities of the isolated arbuscules
Detailed assessment of the arbuscules in the fraction from roots of Ljsym78-2 revealed the percentage of SDH-, ACP- and ALP-active arbuscules (active arbuscules/total arbuscules) to be 84, 69 and 82 %, respectively (Table 2). The proportion of SDH-active arbuscules was significantly higher in the mutant roots than ‘Gifu’ roots.
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The SDH, ACP and ALP activities of arbuscules in root tissue of Ljsym78-2 were 87, 91 and 91 %, respectively (Table 2). There was only a small reduction in the SDH, ACP and ALP activity of the arbuscules due to the isolation process.
Root of the increased-arbuscule-forming mutant of Lotus japonicus (Ljsym78-2) colonized by Glomus sp. R-10 is good material for isolation of intact arbuscules. From the mycorrhizal colonized root, the arbuscule-enriched fraction can be isolated simply and quickly. The majority of the particles in the fraction were arbuscules (56 %) and internal hyphae (23 %). Loss of the enzymic activity of the arbuscules during the isolation process was not significant. The establishment of a simple and quick method of isolating enzyme-active arbuscules should help to clarify the biochemical processes occurring in nutrient exchange at the arbuscules. It is worth noting that due to its simplicity and quickness, the present modified method may enable the isolation of arbuscules with intact RNA, which could lead to elucidation of the molecular mechanisms governing the nutrient exchange at arbuscules. This isolation method can also be used in other arbuscular mycorrhizal fungi and host plant systems as we tested this not only in mutant but also in wild-type ‘Gifu’ as well as in other host plants such as onion.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The authors thank Dr Motoshi Suzuki of Idemitsu Kosan Co., Ltd, Tokyo, Japan for supplying spores of Glomus sp. R-10. We extend our thanks to Dr Masanori Saito of the National Grassland Research Institute for invaluable suggestions. We thank Dr Paul Blackwell, Department of Agriculture and Food, Western Australia for his critical reading of this manuscript. We thank both anonymous reviewers for their valuable comments and suggestions on the manuscript.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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