Location and quantification of phosphorus and other

Transcription

1 Research Location and quantification of phosphorus and other Blackwell Publishing Ltd. elements in fully hydrated, soil-grown arbuscular mycorrhizas: a cryo-analytical scanning electron microscopy study M. H. Ryan 1,3, M. E. McCully 1 and C. X. Huang 1,2 1 CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia; 2 Research School of Biological Sciences, Australian National University, Canberra, ACT 0200, Australia; 3 Present address; School of Plant Biology MO81, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia Summary Author for correspondence: Megan Ryan Tel: Fax: mryan@agric.uwa.edu.au Received: 10 April 2003 Accepted: 24 June 2003 doi: /j x Concentrations of phosphorus (P), potassium (K), magnesium (Mg) and calcium (Ca) were determined in situ in fully hydrated arbuscular mycorrhizas by cryoanalytical scanning electron microscopy. The field- and glasshouse-grown plants (subterranean and white clovers, field pea and leek) were colonized by indigenous mycorrhizal fungi. The [P] in intraradical hyphae was generally mm, although up to 600 mm was recorded, and formed strong linear relationships with [K], up to 350 mm, and [Mg], up to 175 mm. Little Ca was detected. The turgid branches of young arbuscules contained mm P, up to 100 mm K and little Mg. Collapsing arbuscule branches and clumped arbuscules had greatly elevated Ca ( mm), but otherwise differed little from young arbuscule branches in elemental concentration. The [P] was low or undetectable in 86% of uncolonized cortical cell vacuoles, but was generally elevated in vacuoles surrounding an arbuscule and in the liquid surrounding hyphae in intercellular spaces. Our results suggest that both young arbuscules and intercellular hyphae are sites for P-transfer, that Mg 2+ and K + are probably balancing cations for P anions in hyphae, and that host cells may limit arbuscule lifespan through deposition of material rich in Ca. Key words: arbuscular mycorrhizal fungi (AMF), cryo-scanning electron microscopy, root intercellular spaces, root cell vacuoles, potassium, calcium, magnesium, phosphorus, host defence response, X-ray microanalysis. New Phytologist (2003) 160: Introduction Current knowledge of the structure of arbuscular mycorrhizas is strongly based on a small number of early microscopy studies conducted primarily during the 1970s and early 1980s (Cox & Sanders, 1974; Kinden & Brown, 1975a,b,c, 1976; Holley & Peterson, 1979; Walker & Powell, 1979; Gianinazzi-Pearson et al., 1981; Scannerini & Bonfante- Fasolo, 1983; Toth & Miller, 1984). Observations reported in these studies, along with those from a few more recent structural studies (Bonfante & Perotto, 1995; Dickson & Kolesik, 1999; Dickson & Smith, 2001; Vierheilig et al., 2001), underpin interpretation of all research into arbuscular mycorrhizas (see also Smith & Read, 1997). While development of methods for more direct assessment of mycorrhizal functioning has been rapid and diverse (e.g. molecular techniques, plant mutants that block hyphal development and axenic cultures), it has been difficult to mesh together the findings from morphological and functional studies. This is partly because the methods used New Phytologist (2003) 160:

2 430 Research in the structural studies, while yielding much important architectural information, have obscured essential features of the living, functioning tissues by the extraction of all water and water- and solvent-soluble materials. As a consequence, many basic aspects of mycorrhizas are still primarily subjects of speculation. For example, while the ability of arbuscular mycorrhizal fungi (AMF) to deliver phosphorus (P) to plants is well appreciated, and considered the primary benefit of the symbiosis, the actual location and method of P-transfer remains unclear. It is generally believed that the transfer of P to the plant occurs at the interface between root cortical cell cytosol and specialized, but relatively short-lived, fungal structures termed arbuscules (Smith & Smith, 1990; Gianinazzi-Pearson et al., 1991; Smith & Read, 1997; Smith et al., 2001). One of these convoluted, multibranched structures may develop in individual cortical cells after the cell wall is breached by either a large hypha growing directly from cell to cell (intracellular hypha), or by a branch from a hypha growing through intercellular spaces (intercellular hypha). Although the arbuscule may eventually fill much of the cell volume, it remains outside the cell plasma membrane, which invaginates around each small branch of the extending arbuscule. The large area of fungal/root-cell interface that is thereby formed is certainly consistent with the arbuscule being an especially efficient location for P-transfer and much evidence supports this scenario (Smith et al., 2001). However, the role of arbuscules in P- transfer remains to be proven definitively (Smith et al., 2001) and it is not possible to rule out other locations, such as intracellular hyphal coils (Dickson & Kolesik, 1999) or intercellular spaces colonized by hyphae. As pointed out by Smith & Read (1997) all fungal structures within the root lie within the root apoplast and all transfer of nutrients between fungus and host plant is in either direction across the apoplast. However, the apoplastic environments surrounding intercellular and intracellular fungal components termed by Smith & Read (1997) as intercellular and intracellular apoplast, respectively are likely to be quite different and these differences may be very important for nutrient transfer. The nature of the intercellular space apoplast has not been investigated in mycorrhizal roots. In the cortex of maize roots not colonized by AMF, Canny & Huang (1993) used cryoscanning electron microscopy (cryo-sem) to show that some intercellular spaces were liquid-filled, while others were gasfilled. This raises the question of whether the intercellular hyphae of AMF within roots are bathed in liquid or lie within gas-filled space. The conventional preparative methods that have been used to examine AMF within roots by high-resolution optical or electron microscopy remove all water from tissues, making it impossible to detect liquid-filled regions, or to observe fungal structures and host tissues in their fully turgid, chemically unaltered state. Plant tissues, fast frozen and cryo-planed for cryo-sem, remain fully hydrated and retain both their inherent cell shapes and the contents of liquid-filled spaces, which are then easily distinguished from gas-filled spaces. Combined with quantitative X-ray microanalysis these preparations may be used to determine accurately the elemental concentrations within cells and tissues, including liquid in intercellular space apoplast. (For further information on the use of analytical cryo-sem, see McCully et al., 2000.) In this paper we present the results of an analytical cryo- SEM study of roots, mostly from field-grown plants, colonized by indigenous AMF. As well as providing a new view of the morphology of the symbiosis, we present the first data on local P, potassium (K), calcium (Ca) and magnesium (Mg) concentrations in fully hydrated arbuscular mycorrhizas. Materials and Methods AMF-colonized roots Roots colonized by indigenous AMF were collected from subterranean clover (Trifolium subterraneum L.), white clover (Trifolium repens L.), leek (Allium porrum L.) and field pea (Pisum sativa L.). The subterranean clover was grown in a well-lit glasshouse maintained at 20 C/30 C night/day in field-collected soil from Junee, Australia (34 53 S, E). The soil contained 26 mg kg 1 of extractable P as determined following Colwell (1963), 74 mg/kg of mineral nitrogen (N) and 2.1% organic carbon, while the ph was 5.5 (CaCl 2 ). Plants were watered daily and sampled three weeks after sowing. The white clover and leek were grown in a garden in Canberra, Australia (35 15 S, E), in soil with 29 mg kg 1 extractable P, 34 mg kg 1 of mineral N, 3.2% organic carbon and a ph of 4.7. Plants were watered regularly and sampled in December when the leeks were soon to commence flowering and the clover was in flower. The field peas were grown in an agricultural field in Canberra in soil with 22 mg kg 1 extractable P, 153 mg kg 1 of mineral N, 1.7% organic carbon and a ph of 5.5. The field peas received no water other than rainfall and were sampled in April, at the commencement of flowering. Fertilizer was not applied to any of the plants. Tissue preparation, cryo-sem and X-ray microanalysis Field-grown plants were carefully excavated so that their roots remained in situ in a ball of soil and quickly brought to the nearby laboratory. Glasshouse-grown plants were brought in their pots. Individual axile roots were gently separated from the soil, shaken to remove excess soil and briefly washed. Each axile root was lifted from the water and held vertically so that fine branch roots drooped vertically and clung along the larger diameter root. Excess water was removed by gently wiping the root between thumb and forefinger, this also helped align the branch roots along the main axis. Each root with the aligned New Phytologist (2003) 160:

3 Research 431 branches was immediately frozen by plunging into liquid nitrogen (LN 2 ). Frozen roots were severed under LN 2 and either prepared immediately for observation in the cryo-sem or stored in vials in a cryostore. Segments of frozen root, 2 mm long, were cut under LN 2 and quickly affixed to stubs with low-temperature Tissue Tek (Miles Inc., Elkhart, IA, USA), transferred to a cryomicrotome under LN 2 and planed at 90 C to a smooth transverse face with glass and diamond knives. Each planed face included not only a transverse profile of the central larger-diameter root but also of the fine branch roots that were aligned along it. Planed specimens were transferred under LN 2 to a cryo-transfer unit (Oxford Instruments, Eynsham, UK) and hence to the stage of the cryo-sem (JEOL 6400, JEOL Ltd., Tokyo, Japan). Frost was removed by brief, carefully controlled gentle etching at 90 C and the specimen then cooled to 170 C and coated with evaporated, high purity aluminium. (For further details of preparation methods see Huang et al. (1994) and McCully et al. (2000)). Images were recorded at kv to a digital recorder (ImageSlave, OED Pty Ltd., Hornsby, Australia). Fungal structures and host root cortical tissue were analysed with a Link exl system (Oxford Instruments) using the Be window. Spectral data for P, K, Ca and Mg were converted to elemental concentrations using frozen standards prepared exactly as the specimens (Fig. 1). The analysis does not distinguish between ions and elements in insoluble form and we express concentrations of elements in all structures as a concentration in mm. (See McCully et al. (2000) for details of the analysis and quantification methods, and hints for maximizing the reliability of the results.) The lower limit of reliable detection of elements by the X-ray microanalysis was considered to be 10 mm and data that fell from 0 to 10.0 mm were ascribed a notional value of 5 mm. Fig. 1 Calibration graph for [P] used to convert standard ratios obtained by X-ray analyses to concentrations (see details in McCully et al., 2000). Measurements from frozen standard solutions (ordinate) of K 3 PO 4 were planed, etched, coated and analysed under identical conditions to those used for the tissue samples. The abscissa shows measured values of the standard ratio (% ratio 1000/live time) derived from means of two replicate measurements of 10 spectra from each of the standard solutions. The line is a linear regression of standard ratio on concentration (y = x ; r 2 = ). Similar calibrations used for quantifying [K], [Mg] and [Ca] were also strong with r 2 = for [K], r 2 = for [Mg] and r 2 = for [Ca]. Experimental design and data analysis One transverse root face from each of seven subterranean clover roots, seven white clover roots, two leek roots and two field pea roots, in each case taken from at least two plants, were examined. In each root face, numerous measurements were made of elemental concentrations in a number of structures: (1) Hyphae present in intercellular spaces (intercellular hyphae) and/or hyphae present inside cells (intracellular hyphae). (2) Arbuscule trunk hyphae. These were distinguished from intracellular hyphae by being present in the midst of smaller arbuscule branches. (3) Arbuscules. When branches were well defined, and large enough, the analysis was conducted inside individual branches. In other arbuscules numerous hyphae, and presumably any immediately surrounding host cell material, were analysed together. Arbuscules were divided into three development stages, consistent with the lifecycle and morphology described by Kinden & Brown (1975b,c, 1976) and Toth & Miller (1984). These were termed young, collapsing and clumped. Young arbuscules had numerous well-defined, turgid branches. In collapsing arbuscules some fungal walls appeared to be breaking down, although this was not necessarily apparent throughout the arbuscule. In clumped arbuscules, no individual branches remained and the arbuscule consisted of a clump of relatively smooth material. (4) The vacuole of uncolonized cortical cells. The remainder of the cytoplasm and the nucleus in the highly vacuolated cortical cells were too narrow in the transverse profiles to be accurately analysed. (5) The vacuole of cortical cells containing an arbuscule. In some cases arbuscules filled cells so completely that this measurement was not possible. (6) The liquid, or other substances, in uncolonized intercellular spaces. (7) The liquid, or other substances, surrounding intercellular hyphae. Intercellular spaces generally presented a relatively small surface area in the transverse tissue faces, depending on where the planed surface intersected the space. Analyses were only done where the area of the liquid was large enough to ensure the analysis did not include input from hyphae in the space or surrounding cells. Such areas were relatively rare. Unless otherwise noted, results are only presented from metabolically active cortical tissues, arbitrarily defined as those with cortical cell vacuole [K] > 20 mm. New Phytologist (2003) 160:

4 432 Research Fig. 2 Cryoplaned transverse face of a white clover (Trifolium repens) root, viewed by cryo-scanning electron microscopy, showing arbuscular mycorrhizal fungal structures in some cells and intercellular spaces of the mid and inner cortex (e.g. cells marked with an asterisk). The fine white lines or dots in the cortical cell vacuoles are solutes sequestered during freezing. It is from preparations like these that Figs 3 5 have been taken. Bar, 50 µm. Statistical examination of the elemental concentration data was complicated by the variable number of analyses available in the seven categories described, and by the small number of analyses available in some instances, especially for the leek and field pea. Hence, data for some structures are presented only for the two clovers, or are combined across all hosts. Data are generally presented as mean ± SD. All data analysis was conducted using the SPSS software SIGMAPLOT version (SPSS, Chicago, IL, USA). No data were excluded from the data sets presented unless specifically mentioned. Results Structural features Within the planed, transverse faces of uncolonized root cortical cells, only the frozen cell vacuole contents, with lines or dots of sequestered solutes, could always be distinguished. The profiles of the cytoplasm and the nucleus were too narrow to be confidently identified. Colonization by AMF was most commonly found in the mid and inner cortex (Fig. 2). Intercellular hyphae were often present, and in cells colonized by AMF, intracellular hyphae, arbuscules and fungal vesicles could be identified, and appeared surrounded by the host cell vacuole (Figs 2 5). The contents of hyphae showed a more structured appearance than host cell vacuoles (Fig. 3, insets i and ii) and the contents of some hyphae appeared denser than others (Fig. 3). Intercellular hyphae were generally 2 7 µm in diameter, usually with one hypha in each intercellular space, although occasionally two or three were observed (Fig. 3). In subterranean clover, leek and field pea the transverse faces of the intercellular hyphae were usually circular, leaving some liquid-filled space surrounding them in the usually triangular profiles of the intercellular spaces (Fig. 3, inset i). In all four species some intercellular spaces without hyphae were also liquid-filled, while others were gas-filled. In some white clover roots, intercellular hyphae appeared to be surrounded by expanded gel (Fig. 3). In white clover the large-diameter, often irregularly shaped, intercellular hyphae were frequently closely adpressed to all surrounding cell walls and sometimes appeared to have pushed these walls inwards (Fig. 2, and Fig. 3, main figure and inset ii). Intracellular hyphae were observed commonly only in white clover and leek and were µm diameter and always circular in transverse profile (Fig. 3). Arbuscules at the three development stages defined earlier were present in the roots. Young arbuscules had numerous, turgid, distinct branchlets ranging from 0.6 µm to 2.9 µm diameter and usually filled a large volume of the colonized cortical cell (Figs 2 4). Within young arbuscules there were often one or two much larger diameter hyphae that were assumed to be trunk-hyphae. Collapsing arbuscules ranged from those that had turgid, distinct branches together with areas where some branches had coalesced, to those in which coalesced branches had formed into loosely connected, frequently rather ropey, strands (Figs 3 and 4). In places these strands were quite homogeneous in appearance while in other places they showed a meshwork of aggregated, collapsed branches (Fig. 3). Where the clumped branches had been planed to expose a suitable section, they often appeared to be encased in a clearly defined outer shell (short thick arrows Fig. 3). Collapsing arbuscules occupied a decreasing volume of the root cell as the degree of collapse increased. Clumped arbuscules had no distinct branches remaining and consisted of a single relatively smooth clump that appeared homogeneous, except for very thin remains of the original fungal walls and the distinct, thin outer shell (Fig. 3, cell labelled C). Clumped arbuscules were centrally located in the host cell and occupied relatively little of the cell volume. The transverse faces of white clover roots often contained all three stages of arbuscule development (Fig. 3). In subterranean clover, while young and collapsing arbuscules were observed in metabolically active roots (Fig. 4), clumped arbuscules were observed only in metabolically inactive roots. The fungal vesicles observed all had a smooth, homogeneous appearance in section except for a well-defined outer rim (Fig. 5). Because of the low numbers of vesicles in the material examined we are unable to describe their development definitively. The evidence we have, however, suggests that they are New Phytologist (2003) 160:

5 Research 433 Fig. 3 Detail of fungal structures in roots of white clover (Trifolium repens; main figure and inset ii) and subterranean clover (Trifolium subterraneum; inset i). Cells labelled A, B, and C contain arbuscules classified as young, collapsing, and clumped, respectively. A distinct surface layer surrounds clumping regions of arbuscules (short thick arrows). Cells labelled D contain transversely planed profiles of intracellular hyphae, while many intercellular spaces also contain hyphae (H). Intercellular hyphae in white clover often appear to be pushing inwards the walls of surrounding host cells (long arrows). They are also sometimes of irregular shape, as in inset (ii) where a portion of a large intercellular hyphae is seen in tangential view. Intercellular spaces (S) contain liquid contents that are either electron nonemissive (dark) or lighter, (the latter suggesting expanded mucilage). Such content can be observed in spaces where planed profiles of hyphae are also present (indicated with asterisk). Cells labelled E contain no fungal structures in the planed profile. Preparation as in Fig. 2. Bar, 10 µm. New Phytologist (2003) 160:

6 434 Research Fig. 4 Detail and elemental concentrations of arbuscules in a subterranean clover (Trifolium subterraneum) root. The arrows point to regions that were analysed: A, transverse profile of a small branch of a young arbuscule (34 mm P, 120 mm K, 26 mm Ca, < 10 mm Mg); B, arbuscule branches just beginning to coalesce (45 mm P, 96 mm K, 65 mm Ca, < 10 mm Mg); C, vacuole in cell containing a young arbuscule (< 10 mm P, Mg & Ca, 76 mm K); D, intercellular hypha (204 mm P, 188 mm K, 29 mm Ca, 43 mm Mg); and E, vacuole of cell with no fungal structures present in the planed face (< 10 mm P and Ca, 98 mm K, 14 mm Mg). The lower limit of reliable detection of the elements was considered to be 10 mm. Preparation as in Fig. 2. Bar, 10 µm. Fig. 5 A fungal vesicle (V) in a subterranean clover (Trifolium subterraneum) root. The contents of the vesicle appear homogeneous, it is surrounded by a clearly defined rim, and it appears to have expanded to fill much of the cell in which it is contained. Preparation as in Fig. 2. Bar, 10 µm. initiated within a cortical cell no earlier than when arbuscules in surrounding cells begin to collapse, and then expand to fill the cell and ultimately kill it so that the vesicle becomes essentially extracellular in the root tissue. Elemental analyses Intraradical hyphae had the highest [P], averaging mm (Figs 4 and 6), and reaching 600 mm in one white clover root where all hyphae had > 350 mm. Hyphal [P] was much higher in the two clovers than the leek and field pea. Two hyphae in one field pea root contained no P but high [K] and were excluded from data analyses. Arbuscules averaged mm P in all four hosts (Fig. 6). The [P] in cortical cell vacuoles was much lower than in fungal tissues (generally < 25 mm) and tended to be slightly higher in vacuoles surrounding arbuscules than in vacuoles of cells that appeared uncolonized (Fig. 6). In subterranean clover, 58% of cells containing an arbuscule in the planed face had a detectable concentration of P (> 10 mm) in the vacuole (n = 19) compared with 14% of cells where no fungal structures were present in the planed face (n = 14). The [K] ranged from 20 to 350 mm and was higher in intraradical hyphae than in arbuscules or cortical cell vacuoles (Fig. 6). The [Ca] tended to be highest in arbuscules, while [Mg] was generally highest in intraradical hyphae (Fig. 6). Hyphal [P] showed a strong linear correlation with both [K] and [Mg], but not [Ca] (Fig. 7). If P is assumed to be present primarily as a monovalent anion, it is balanced in a near 1 : 1 relationship with K + and Mg 2+ combined (e.g. the analysis at D in Fig. 4, Fig. 8). In subterranean clover arbuscules (Fig. 9), trunk hyphae had similar profiles of [P], [K], [Ca] and [Mg] to neighbouring intercellular hyphae, but slightly higher [K] and much higher [P] and [Mg] than finer arbuscule branches. Indeed, Mg was rarely present in arbuscule fine branches (e.g. the analysis at A in Fig. 4, Fig. 9). The data on elemental concentrations in arbuscules and surrounding cortical cell vacuoles presented in Fig. 6 were further investigated by division according to arbuscule development stage (Fig. 10). An adequate number of analyses were available only for the two clovers and for subterranean clover, New Phytologist (2003) 160:

7 Research 435 Fig. 6 Concentrations of P, K, Ca and Mg in intraradical hyphae (IRH), arbuscules of any development stage (Arb), the vacuole of arbuscule-containing root cortex cells (VacC) and the vacuole of root cortex cells where no fungal structures were present in the planed face (VacU): (a) subterranean clover (Trifolium subterraneum) (IRH n = 28, Arb n = 24, VacC n = 19, VacU n = 14); (b) white clover (Trifolium repens) (IRH n = 29, Arb n = 22, VacC n = 17, VacU n = 7); (c) leek (Allium porrum) (IRH n = 7, Arb n = 5, VacC n = 5, VacU n = 3); and (d) field pea (Pisum sativum) (IRH n = 9, Arb n = 5, VacC n = 4, VacU n = 4) (mean ± SD). As the lower limit of reliable detection of the elements was considered to be 10 mm, lower measurements were ascribed a value of 5 mm. Results from intercellular and intracellular hyphae did not differ and were combined. analyses of clumped arbuscules were available only from metabolically inactive roots. The [P] in arbuscules remained relatively stable as they senesced, especially in white clover. While arbuscule [K] was 50% lower in clumped arbuscules, host vacuolar [K] remained relatively stable as arbuscules senesced. [Ca] was greatly elevated in the coalescing branches of collapsing arbuscules, even when branches were only slightly collapsed (e.g. Figure 4B), but was not elevated in turgid branches in the same arbuscule (Fig. 10). [Ca] remained high in clumped arbuscules. [Ca] was particularly high in white clover, where up to 250 mm was recorded. There was no relationship between [Ca] and [P] in collapsing or clumped arbuscules (r 2 = 0.006). One white clover clumped arbuscule registered no Ca, but was included in analyses. Host cell vacuoles did not exhibit elevated [Ca] (Fig. 10); also elevated in the coalescing branches of collapsing and clumped white clover arbuscules was [Mg] (Fig. 10). In field pea, two clumped arbuscules were analysed and contained 11 mm and 20 mm of P, with 36 and 58 mm of Ca. In liquid-filled intercellular spaces not colonized by AMF, P was generally not detectable (Fig. 11). However when an intercellular space contained a hypha, [P] in surrounding liquid, or gel-like substance, was often mm and reached up to 100 mm. There was no relationship between [P] in individual hyphae and [P] in fluid in the surrounding intercellular space (r 2 = 0.001). Since [K] did not vary between colonized and uncolonized intercellular spaces it is unlikely that the enhanced P in colonized spaces resulted from inclusion of a portion of undetected underlying hypha within the volume analysed. Discussion Fungal morphology The morphology of mycorrhizas observed in our study is generally consistent with other reports. Arbuscules are known to collapse first in their finest branches, eventually forming smooth clumps that are finally absorbed by the cell, which itself remains viable and may even be recolonized (Kinden & Brown, 1975a,b,c, 1976; Dickson & Smith, 2001). The smooth texture of the frozen-planed vesicle contents is suggestive of lipids, consistent with the histochemical results of Nemec (1981) that indicated a high content of neutral lipid. However, the diameter of arbuscule branches is generally reported as 1 µm (Kinden & Brown, 1975c; Toth & Miller, 1984), which is lower than in the present study, probably because the tissues observed in the earlier studies were completely dehydrated during preparation. Our values are more likely to provide a realistic measure, as freezing would be expected to increase the volume of watery liquid up to about 9%, which, when translated to linear measurements, New Phytologist (2003) 160:

8 436 Research Fig. 8 Charge balance of P with Mg and K in the intraradical hyphae of subterranean clover (Trifolium subterraneum), white clover (Trifolium repens), leek (Allium porrum) and field pea (Pisum sativum), assuming that P was a monovalent anion and Mg and K were present as Mg 2+ and K + (r 2 = 0.85). The dashed line represents the 1 : 1 ratio. Fig. 7 The [P] in intraradical hyphae of subterranean clover (Trifolium subterraneum, filled circles), white clover (Trifolium repens, open circles), leek (Allium porrum, filled triangles) and field pea (Pisum sativum, open triangles); relationship with (a) [K] (r 2 = 0.73 all data, r 2 = 0.88 white clover only); (b) [Ca] (r 2 = all data, r 2 = white clover only); and (c) [Mg] (r 2 = 0.81 all data, r 2 = 0.89 white clover only). As the lower limit of reliable detection of the elements was considered to be 10 mm, lower measurements were ascribed a value of 5 mm. means a maximum increase of only around 2%. Thus, while dimensions of fungal structures preserved by rapid freezing should be close to those of their native state, conventional preparative methods can produce marked alterations through loss of turgor, total dehydration and chemical changes that affect cell wall integrity. Phosphorus concentrations in arbuscules and hyphae Our results confirm and extend those of earlier X-ray microanalytical studies that showed significant amounts of P in intraradical hyphae and/or arbuscules of arbuscular mycorrhizas (Schoknecht & Hattingh, 1976; Walker & Powell, 1979; White & Brown, 1979; Cox et al., 1980). To our knowledge, our results provide the first in situ quantitative measurements of P in these mycorrhizas. All earlier studies used preparative methods that would not reliably retain ions or soluble compounds and most did not use standards for quantification. Cox et al. (1980) using preparations from which soluble P would have been lost, determined that the P in insoluble polyphosphate granules would result in a P concentration about g cm 3 (i.e. 8 mm) in fungal cytoplasm in intercellular hyphae, approximately an order of magnitude lower than found in the present study. The few available values determined using other techniques are mm in extraradical and intraradical hyphae (calculated by Smith et al., 2001 from Solaiman et al., 1999) and 338 mm in extraradical hyphae (Nielsen et al., 2002). Phosphorus transfer in arbuscules A significant role for young arbuscules in P transfer is consistent with the lower [P] in arbuscule fine branches compared with trunks and intercellular hyphae, and the greater proportion of cortical cell vacuoles which contained a detectable [P] when an arbuscule was present. Root cell vacuoles are known to be a P reservoir to allow maintenance of cytosol P; however, P in both the cytosol and vacuoles may generally be maintained well below 10 mm (Lee & Ratcliffe, 1993). Thus, the relatively small increase in [P] in cortical cell New Phytologist (2003) 160:

9 Research 437 vacuoles when an arbuscule was present (Fig. 6) probably reflects changes in vacuolar P being below the level reliably detected by the X-ray microanalysis and, presumably, the rapid movement of P through the root to the vascular tissue. A technique capable of resolving concentrations < 10 mm is required to gain further insights into P-transfer in arbuscules. The maintenance of P in clumped arbuscules (Fig. 10) indicates the fungi did not, as has been hypothesized elsewhere (Kinden & Brown, 1976; Walker & Powell, 1979; Dickson & Smith, 2001), withdraw a significant amount of P from the senescing arbuscule. The host cell could presumably use the P remaining in clumped arbuscules if they were gradually digested (Kinden & Brown, 1976). Fig. 9 Concentrations of P, K, Ca and Mg in intercellular hyphae, and the trunk hyphae and fine branches of young arbuscules in subterranean clover (Trifolium subterraneum) roots (n = 5, mean ± SD). Arbuscules were paired with the closest intercellular hypha. As the lower limit of reliable detection of the elements was considered to be 10 mm, lower measurements were ascribed a value of 5 mm. Phosphorus and carbon transfer in intercellular spaces This study provides the first evidence that the intercellular hyphae of AMF are generally surrounded by liquid and/or gel and that [P] in this medium can be enhanced adjacent to the hyphae. The presence of gel possibly accounts for the particulate material occasionally seen in conventional electron micrographs of intercellular spaces containing hyphae (e.g. Figure 17 in Holley & Peterson, 1979). It seems most likely that the P we detected in the intercellular spaces containing hyphae was released locally from the hyphae and not from adjacent root cells because where profiles of liquid-filled intercellular spaces did not include a hypha (e.g. Figure 3, inset i) P content was always low (Fig. 11), even when surrounding cells contained arbuscules. Whether root cells bordering colonized intercellular spaces can readily take up intercellular P (Gianinazzi-Pearson et al., 1991) and whether P transfer is possible, or perhaps enhanced, when intercellular hyphae and cell walls are tightly adpressed to each other, are unknown. Arbuscular mycorrhizas with Paris-type morphology do not form intercellular hyphae, instead consisting of only intracellular structures. Since P-uptake and growth of the host plant may be enhanced by Paris-type mycorrhizas (Cavagnaro et al., 2003), it appears that P-transfer in arbuscular mycorrhizas is by no means confined to intercellular spaces and there is a role, and perhaps a dominant one, for intracellular structures such as arbuscules or hyphal coils, if present. The presence of liquid in intercellular spaces also raises the possibility of carbon transfer in these spaces. Intercellular spaces in plant tissues are preferred sites for colonization by endophytic bacteria (Hecht-Buchholz, 1998; McCully, 2001). Except in sugarcane, where these spaces contain sucrose solution, little is known about the presence of carbon compounds in intercellular space liquid, but there are indications that such compounds are present and thus available to colonizing microbes (see review by McCully, 2001). The existence of mutant plants that block the formation of arbuscules, but allow spread of intercellular hyphae strongly suggests that mycorrhizal hyphae can access some carbon New Phytologist (2003) 160:

10 438 Research Fig. 10 Concentrations of P, K, Ca and Mg in arbuscules and surrounding root cortical cell vacuoles in cells with young, collapsing or clumped arbuscules in: (a) subterranean clover (Trifolium subterraneum) (n = 3 9); and (b) white clover (Trifolium repens) (n = 4 12) (mean ± SD). In collapsing arbuscules, data are presented from both hyphae that appeared turgid and coalescing hyphae of relatively smooth appearance. As the lower limit of reliable detection of the elements was considered to be 10 mm, lower measurements were ascribed a value of 5 mm. No clumped arbuscules were observed in roots of subterranean clover that contained young arbuscules, however, clumped arbuscules were frequently found in roots where the majority of cells appeared metabolically inactive and analyses of these are included and marked with asterisks. compounds from intercellular spaces (Smith & Read, 1997). For example, when Gigaspora margarita colonized the Ljsym4-1 mutant of Lotus japonicus, the fungus was only very infrequently able to penetrate cell walls to form arbuscules (9% of colonization contained arbuscules), but 8% of root length was still colonized by intercellular hyphae (Novero et al., 2002). In the wild type, 31.4% of root length was colonized, presumably reflecting greater carbon transfer due to a greater proportion of arbuscules (73% of colonization contained arbuscules). Thus, while it appears transfer of P and carbon compounds can occur via intercellular hyphae, for carbon compounds at least, transfer may be slower than via arbuscules, with their far greater ratio of surface area to volume. Arbuscules do represent a large energy investment by the fungus and in some situations it may be more efficient for host and fungus to rely on transfer via intra- or inter-cellular hyphae. For example, in the long-lived, slow growing roots of many plant species in natural ecosystems, AMF may persist as intercellular hyphae for a number of years following the deterioration of arbuscules formed when roots were young (e.g. Smilacina racemosa; Brundrett & Kendrick, 1990a,b). Interestingly, however, a rapid and substantial P benefit in presence New Phytologist (2003) 160:

11 Research 439 Fig. 11 [P] and [K] in intercellular hyphae (n = 22), surrounding intercellular spaces (n = 22) and uncolonized intercellular spaces (n = 11) (mean ± SD). As colonized intercellular spaces with adequate area to analyse were rare, results from subterranean clover (Trifolium subterraneum), white clover (Trifolium repens) and leek (Allium porrum) are combined. Most colonized intercellular spaces were liquid-filled, although a few in white clover contained a substance of denser appearance (Fig. 3). As the lower limit of reliable detection of the elements was considered to be 10 mm, lower measurements were ascribed a value of 5 mm. Overall, 66% of colonized intercellular spaces and 18% of uncolonized intercellular spaces contained a detectable concentration of P. of only intraradical hyphae (unspecified whether intra- and inter-cellular) has been reported for Salix repens with less than 5% of root length colonized (van der Heijden, 2001). Potassium and magnesium concentrations in fungal hyphae One of the most consistent findings of this study was the very strong linear relationship of [P] with [K] and [Mg] in the intraradical hyphae (Figs 7 9). Clearly, K and Mg are the balancing cations for the predominant P forms in the fungal hyphae. However, we can only speculate about the exact form, and even the solubility, of these elements. While some soluble P in hyphae of AMF may be in the form of polyphosphate, a linear polymer of orthophosphate, polyphosphate may only represent a small proportion of the P present (Solaiman et al., 1999). Polyphosphate has a lower osmotic impact than equivalent amounts of orthophosphate and may bind strongly to cations including Mg 2+ (Cramer & Davis, 1984). Significantly, Mg has not been detected in the hyphae of arbuscular mycorrhizal fungi in earlier studies using X-ray analysis. Instead, relatively large Ca peaks suggested that this element formed the major balancing cation (Schoknecht & Hattingh, 1976; Walker & Powell, 1979; White & Brown, 1979; Cox et al., 1980). Similar evidence for high Ca concentrations that would appear to balance P in ectomycorrhizas is common in the earlier literature, but this Ca accumulation has been shown definitively by Orlovich & Ashford (1993) and Bücking & Heyser (1999) to be an artefact resulting from the use of preparative methods that allow migration of soluble components before analysis. Our finding of high [Mg] but low [Ca] in the fungal hyphae is similar to the results of Bücking & Heyser (1999) in an ectomycorrhiza, suggesting that the failure of earlier studies of ectomycorrhizas and arbuscular mycorrhizas to detect Mg rather than Ca, is because mobile Ca was immobilized by the formation of insoluble P compounds and the Mg was lost. The high [K] in intraradical hyphae and arbuscule trunk hyphae is indicative of high turgor pressure. High turgor pressure would aid hyphae to push through cell walls and invaginate the host cell plasma membrane. In white clover, high turgor may enable intercellular hyphae to enlarge intercellular spaces. Calcium and arbuscule senescence High [Ca] in collapsing and clumped arbuscules has not been previously reported. As stores of Ca were not detected in host cell vacuoles or fungal hyphae, the origin of the Ca remains to be elucidated. Arbuscule collapse is thought to coincide with loss of metabolic activity in arbuscule branches (Vierheilig et al., 2001), an increase in host metabolic activity (Kinden & Brown, 1976) and, possibly, the recognition by the host cell of the fungus as an invader (Salzer et al., 1999; Vierheilig et al., 2001). It is possible that the Ca may be contained in, or infiltrated from, the host-derived encasement material observed in this study (Fig. 3) and by Kinden & Brown (1976) and Cox & Sanders (1974) to be deposited as fungal walls fragment and collapse. Scannerini & Bonfante-Fasolo (1979) showed that this encasement material was heavily stained with ruthenium red, strongly suggesting a high acidic polysaccharide component. Calcium would be expected to bind to such material. Such a scenario is suggestive of a host defence response. Calcium is well known to mediate plant responses to stress, including fungal pathogens through changes in cytosol [Ca], although these fluctuations are in the nanomolar range (Xu & Heath, 1998). High [Ca] is also present in cell wall appositions (papillae) which are deposited to provide a physical barrier to fungal pathogens (Bonello et al., 1991). New Phytologist (2003) 160:

12 440 Research Deposition of Ca would presumably seriously disrupt functioning of the specialized host plasma membrane that surrounds arbuscules. Overall, our results are consistent with the arbuscule lifecycle being strongly delimited by the host cell, a conclusion supported by the finding of hydrogen peroxide synthesis in host cells containing senescing arbuscules (Salzer et al., 1999). However, the trigger for such control is unknown. The length of arbuscule lifecycle has been reported to range from around 8 d in fast-growing annual crops (onion, bean, tomato; Alexander et al., 1989) to several months in plants in natural ecosystems where fine arbuscule branches collapse very slowly (Brundrett & Kendrick, 1990a). Conclusions Our results indicate the considerable potential of cryomicroscopy and quantitative X-ray microanalysis of fully hydrated tissues to provide unique new insights into the functioning of arbuscular mycorrhizas by revealing for the first time the concentrations and precise locations of specific elements within the root and fungal structures. The striking finding that the distribution patterns of P, Mg, K and Ca were similar in mycorrhizal associations of four different host plants, plants grown in three soil types and under widely different conditions, and, presumably, colonized by three different populations of AMF, now suggests that such distribution may play a basic role in the functioning of the mycorrhizal association. 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