Author for correspondence: Frédérique C. Guinel Tel: +1 519 884 0710 Fax: +1 519 746 0677 Email:email@example.com
• E107 is a pleiotropic mutant of Pisumsativum (pea) characterized by a decreased ability to form nodules. Its colonization by the arbuscular mycorrhizal fungus Glomusaggregatum is reported, and the mycorrhizal phenotype compared with the nodulation phenotype.
• Four cleared lateral roots from 21-d-old plants were chosen randomly, and their lengths scanned for any fungal structures. Colonization success was determined by counting the numbers of extraradical hyphae, and calculating epidermal entry and cortical entry. Graft experiments were performed to establish which organ regulates mycorrhiza-formation.
• Two blocks to infection were obvious, at the root surface, and at the interface between the epidermis and the outermost cortical cell layer. Once the fungus breached this interface, it spread within the cortex and formed normal arbuscules. The mutant phenotype was controlled by the shoot.
• The E107 mycorrhizal phenotype was designated as app+, low pen, low coi. The development of the fungal infection was similar to that of rhizobial infection and under the same shoot control. The phenotype of the mutant E107 strongly supports the hypothesis that mycorrhiza formation and nodulation are regulated by the same processes.
Pisumsativum E107 is a pea mutant characterized by its short stature and low nodulation (Kneen et al., 1990). There are necrotic spots on its basal leaves; these have been linked to an increased amount of several cations, especially iron (Welch & LaRue, 1990) and aluminium (Guinel & LaRue, 1993). As well, probably due to the accumulation of cations, wall appositions form in the leaf epidermal and lateral root endodermal cells (Guinel & LaRue, 1990). Low nodulation in E107 has been studied quantitatively; the number of rhizobial infections is decreased by a third compared with that of its parent Sparkle (Guinel & LaRue, 1992). Most of the infection threads formed are arrested in the epidermis with only 14% progressing beyond that cell layer (Guinel & LaRue, 1992). The plant hormone ethylene appears to be responsible, in part, for this epidermal block because treatment with ethylene inhibitors increases the number of successful infections (Guinel & LaRue, 1992). The mutant E107 does not fit the phenotypic codes of Caetano-Anolles & Gresshoff (1991); it is neither a nonnodulator (nod−) nor a nonfixer (nod+fix−), because the few E107 nodules that form do fix nitrogen. Thus, we call it a low nodulator. Duc et al. (1989) were the first to report that, in pea, some mutants defective in nodulation also did not form a mycorrhizal symbiosis. Recent reports have further emphasized similarities in the establishment and regulation of the two symbioses (Peterson & Guinel, 2000). These similarities have been studied at the gene and molecular levels and also at the cytological level. For example, Balestrini et al. (1999) showed that the gene PsNlec1, which encodes a lectin-like glycoprotein and is expressed in root nodules, was also detected in roots colonized by AM fungi. In both symbioses, gene expression was limited to the infected plant cells possessing fully differentiated symbiotic structures (Balestrini et al., 1999). Wegel et al. (1998), by studying the mycorrhizal phenotype of Lotusjaponicus nodulation mutants, defined genetically independent steps in the development of mycorrhizae. One step that appears important is the entrance of the fungus into the cortex; once this point in development has been crossed, the fungus is able to colonize the host and form arbuscules (Wegel et al., 1998; Bonfante et al., 2000). The parallel between the two interactions has been extended to some supernodulation mutants, for which this phenotypic characteristic is associated with an increase in mycorrhizal fungal colonization (Morandi et al., 2000).
In this report we examined the formation of arbuscular mycorrhizae in E107, a low nodulator, and compared the two symbioses in terms of development and regulation. We were especially interested in determining if the mycorrhizal phenotype of E107 would parallel its nodulation phenotype (i.e. would fungal colonization be lessened and would the mycorrhizal fungus be arrested in the epidermis). Root/shoot grafts between the mutant and its parent Sparkle allowed us to determine if the two symbioses were similarly controlled.
Materials and Methods
Development of mycorrhizae
Seeds of pea (Pisumsativum L.) from the parent Sparkle and the mutant E107 were surface-sterilized in an aqueous solution of 5% household bleach (5.25% sodium hypochlorite) for 8 min, rinsed four times with sterile water, and imbibed overnight. The seeds were germinated in the dark for 3 d in Petri plates containing filter paper moistened with water and then planted into small square pots (650 mL) containing a mixture (1 : 2, v : v) of sterile peat (Greenworld Garden Products, Pointe Sapin, NB, Canada) and Turface® (kiln-baked montmorillonite clay from Applied Industrial Materials Corp., Buffalo Grove, IL, USA). The fungal inoculum (kindly provided by R. L. Peterson, University of Guelph, Ontario, Canada) was obtained from roots of corn (Zea mays) grown in Turface® and colonized by Glomusaggregatum Schenk and Smith emend. Koske. The inoculum consisted of desiccated corn roots, cut into small pieces, mixed with the Turface® in which the corn had grown; it was added to the peat : Turface® mixture in a 1 : 10 (v : v) ratio.
Once a week, the seedlings received 20 mL of low-phosphate nutrient solution made of 0.4 mM KH2PO4, 2.5 mM Ca(NO3)2 4H2O, 2 mM K2SO4, 1 mM MgSO4 7H2O, 40 mM FeIII EDTA with 1 mL L−1 micronutrient solution (50 mM KCl, 25 mM H3BO3, 2 mM MnSO4 H2O, 2 mM ZnSO4 7H2O, 0.5 mM CuSO4 5H2O and 0.5 mM Na2MoO4 2H2O), pH = 6.8. Seedlings were placed in a growth-room under both sodium halide and cool white fluorescent lamps with a total intensity of 250 µmol m−2 s−1 PPF, with a 22°C/18°C, 16 h/8 h, day/night cycle. Plants were harvested 18 dap (days after planting), that is when they were 21 d old.
Processing of mycorrhizal roots
Complete root systems were excised just below the cotyledons and processed as outlined in Brundrett et al. (1994) with slight modifications. Roots were fixed overnight in 50% ethanol, transferred to 5% KOH (w/v), autoclaved 15 min at 120°C to clear, rinsed in water and finally placed in the stain at 50°C for 1 h. The stain consisted of 0.1% Chlorazol Black E, 85% lactic acid and glycerol (1 : 1 : 1, v : v : v). The roots were destained and kept in 100% glycerol until observed.
Four secondary roots from four different root systems were selected randomly, mounted on slides in glycerol and scanned along their entire lengths from the point of attachment to the primary root to the tips. Roots were observed with a Reichert Microstar IV compound light microscope equipped with bright-field optics (objectives Plan Achro ×10/0.25 and ×40/0.66). Micrographs were taken on Pan F (50ASA) Kodak film. Plates were prepared using Adobe® Photoshop® 5.0 software (Adobe Systems Inc., Seattle, WA, USA).
The numbers of hyphae external to the root (referred to as extraradical hyphae or ERH), arrested in the epidermis, within the outer and inner cortical cell layers, and differentiated into arbuscules were recorded. The epidermal entry (i.e. the total number of hyphae having penetrated the epidermis divided by the total number of ERH) and the cortical entry (i.e. the total number of hyphae penetrating the cortex divided by the total number of ERH) were calculated. In addition, the success of colonization (i.e. sites with arbuscules) was determined by dividing the number of colonization sites with arbuscules by the total number of ERH. The ability to form arbuscules was estimated by scoring the number of cortical hyphae with arbuscules and dividing this number by the total number of cortical hyphae. For ease of comparison, all values were converted to percentages. However, to compare with nodulation data (Guinel & LaRue, 1992), fungal entry was also calculated on a per cm of lateral basis (total entry points divided by total root length observed).
Regulation of mycorrhizae-formation and nodulation
For the mycorrhiza studies, the soil was a mixture of peat : Turface® (1 : 3, v : v) in which the fungal inoculum (same fungus as above but cultivated on leeks (Allium porrum), supplied by J. N. Klironomos, University of Guelph, Canada) had been mixed in a 1 : 10, v : v (inoculum : substrate) ratio. Individual seedlings were first transferred into the pots containing the inoculum, then were grafted. Three grafting experiments were performed.
For the nodulation studies, the substrate consisted of peat : Turface® (1 : 2, v : v) mixed with the inoculum, Rhizobiumleguminosarum bv. viciae 128C53K (Nitragin® Inoculants, Liphatech Inc., Milwaukee, WI, USA), which was grown in yeast-mannitol broth. Once the bacterial culture reached an absorbance of 0.550 at 600 nm wavelength, it was diluted into sterile water (2%, v/v) and 5 ml was added at the crown of each seedling just after the grafting procedure. The nutrient solution used was low in nitrogen (i.e. as above but with 2 mM KH2PO4 and only 0.5 mM Ca(NO3)2 4H2O). Two grafting experiments were performed; some inoculated nongrafted controls were also planted.
For each study (mycorrhizae-formation and nodulation), at least 15 plants were analysed for each type of graft (reciprocal and control for both lines of pea). All grafted plants were harvested 28 d after inoculation. The number of nodules was recorded for each plant. For the mycorrhiza study, root systems were excised just below the cotyledons and fixed in 50% ethanol. The primary root was then removed and the laterals cut into 1 cm fragments and processed as described previously. A random sample of each root system was taken; 15 root fragments were mounted per slide. At least 2 slides were examined per root system of each grafted plant. Each fragment was scanned for mycorrhizal fungal structures (extraradical hyphae, appressoria, hyphae in the epidermis and cortex, and arbuscules) with a Zeiss Axiostar light microscope equipped with bright-field optics (objectives Apo-Plan ×20/0.45 and CP-Achromat ×40/0.65). Colonization was quantified in the same manner as above.
The mean value (± SE) for each parameter calculated in the mycorrhiza study was compared between Sparkle and E107 using student t-tests (P ≤ 0.05). For grafted plants, the mean value (± SE) for each parameter calculated was compared between all combinations of control and reciprocal grafts using student t-tests (P ≤ 0.05). Statistics were performed using SigmaPlot® 2000 software (SPSS Inc., Chicago, IL, USA).
Development of mycorrhizae in Sparkle and E107
In general, secondary roots were more colonized than primary roots but less colonized than tertiary roots. Because the tertiary roots at 21 dap were short, we examined only the secondary roots.
The parent Sparkle
The parent Sparkle was colonized in a normal manner, with symbiotic structures similar to those of Gallaud’s Arum-type (Smith & Read, 1997). Numerous extraradical hyphae grew along the root surface and several appressoria, variable in shape and in size, formed per hypha (Fig. 1a). Often, once a hypha had entered the epidermis, a typical ‘fanning’ pattern (Fig. 1b) of colonization was observed; the main hypha generally divided in two and continued branching as it progressed in the cortex. Upon reaching the inner cortex, fungal hyphae penetrated cortical cells and differentiated into trunks and fine branches forming arbuscules (Fig. 1c). By 3 wk, the colonization units could be consisting of 100 or more cells containing arbuscules. At that age, only a few vesicles (Fig. 1d) were formed in the root cortex.
In E107 colonization took place following a sequence of events (Figs 1e–g) similar to that described above. Appressoria (Fig. 1e) were similar in morphology to those observed on Sparkle. Once the fungus contacted the root, obvious structural modifications such as wall appositions were never seen in the E107 epidermis. Fungal hyphae were able to penetrate the epidermis and colonize large areas of the cortex (Fig. 1f). Hyphae that reached the cortex spread laterally in the root, entered inner cortical cells (Fig. 1f) and differentiated into arbuscules (Fig. 1g) and vesicles that were similar to those observed in Sparkle. However, most of the times, hyphae were unable to progress into the cortex; they grew parallel to the root surface within the epidermal layer (Fig. 1h). The hyphae often swelled in an attempt to penetrate the outer periclinal wall of the outermost cortical cell layer (inset, Fig. 1h).
Fungal hyphae commonly entered through root hairs of both types of pea (Fig. 2a–d). Extraradical hyphae were wrapped around individual root hairs in a helicoidal manner (Fig. 2a) and penetrated just proximal to the tip (Fig. 2b). Although appressoria-like structures were occasionally observed on the hair surface, they did not appear to be required for penetration. Root hair entry, as the one directly through the epidermal cell layer, could lead to successful (Fig. 2c) or to unsuccessful colonization with a block in the epidermis (Fig. 2d).
In E107, colonization was rare, that is only 35% of the extraradical hyphae (ERH) were able to penetrate the root surface, half as many as in Sparkle (Fig. 3a). This significant large decrease (P = 7.1 × 10−6) in penetration indicates a block at the root surface. In the mutant E107, of the ERH, 10% progressed to the cortex (Fig. 3a) and 1.9% differentiated into arbuscules. These values contrasted significantly with those of the normal parent Sparkle, where 39% entered the cortex (Fig. 3a) and 7.7% formed arbuscules (P = 5.9 × 10−6 and 0.0321, respectively). These results indicate the existence of an additional block at the boundary between the epidermis and the outermost layer of the cortex.
When considering only hyphae that penetrated the roots, on a per cm of lateral root basis, whereas Sparkle had 3.89 ± 0.57 (± SE) fungal entry points, E107 had only 1.88 ± 0.33. As a percentage, 75% in E107 were unable to progress further than the epidermis, whereas only 49% were arrested in Sparkle (not significant with a P-value of 0.1205). However, for those that penetrated the cortex, no significant difference (P = 0.4050) was found between the two pea lines in the number of hyphae differentiating into arbuscules.
Regulation of mycorrhizae-formation and nodulation
The reciprocal Sparkle/Sparkle (S/S) grafts had significantly higher proportions of epidermal and cortical entry than E107/E107 (E/E) grafts (Fig. 3b), as in nongrafted plants (compare Fig. 3a,b). In general, grafts with Sparkle scions had higher values for both epidermal and cortical entry than those with E107 scions, regardless of the stock. Therefore, the mycorrhizal phenotype of E107 is shoot-controlled.
In grafts with E107 scions, fungal entry into the epidermis was decreased compared with grafts with Sparkle scions, indicating a block at the root surface. Of the hyphae that penetrated the root, about 70% were blocked in the epidermis. By contrast, about 40% of hyphae were blocked in the epidermis of grafts with Sparkle scions. These values reinforce the existence of an epidermal block in the development of mycorrhizae in E107.
There were more nodules on nongrafted Sparkle and on grafted plants with Sparkle scions (Fig. 4). Grafts with E107 scions had significantly fewer numbers of nodules than grafts with Sparkle scions; however, these numbers were similar to those of nongrafted E107. Thus, the nodulation phenotype of E107 is also shoot-controlled.
The mycorrhizal phenotype of E107, a low nodulator
This is the first time that a mutant forming few but functional nodules is reported also to be low in forming mycorrhizae; E107 is able to form successful associations with G. aggregatum but only at a low frequency. The lessened ability to form mycorrhizae was a result of two major blocks: one at the root surface, the other at the boundary between the epidermis and the outer cortex.
These results match the nodulation phenotype described by Guinel & LaRue (1992), that is E107 is able to form nodules, but at a lower frequency than Sparkle. The number of infection threads in the epidermal layer is about 1/3 of the amount in Sparkle, suggesting a block at the root surface. Furthermore, the infection thread is often arrested in the epidermis (about 85% vs 45% in Sparkle, Guinel & LaRue, 1992). Those infections that do extend into the cortex form functional nodules.
Comparison of E107 to other mycorrhizal mutants
Several mycorrhizal phenotypes have been described which assist in the categorization of mycorrhizal mutants (Peterson & Guinel, 2000). In the pea and fababean (Vicia faba) mutants described by Duc et al. (1989), appressoria form but the infections are aborted, often limited to one or very few cells just around the point of entry. Myc−1 mutants, correlated with the strictly nod− phenotype, are characterized by infections that are aborted at a very early stage of development; appressoria are formed, but the fungus is unable to penetrate the root (Gianinazzi-Pearson et al., 1991). In myc−2 mutants, linked to the nod+ fix− phenotype, arbuscular mycorrhizal fungi are able to form appressoria and intercellular hyphae; however, they do not form arbuscules, but rather only stumpy branches within the inner cortical cells (Gianinazzi-Pearson et al., 1991). Appressoria form on the roots of the alfalfa myc− mutants described by Bradbury et al. (1991), but the fungus generally fails to enter the roots. Recently, Wegel et al. (1998) studied four nodulation mutants of Lotus japonicus, in which the mycorrhizal fungus entered the epidermis but was often prevented from entering the cortex. However, hyphae that were able to penetrate developed normal mycorrhizal structures. This mutant phenotype was named Coi− (cortex invasion). Further characterizing these L. japonicus mutants, Bonfante et al. (2000) focused on two LjSym4 gene mutations. LjSym4–2 exhibits a strong block to fungal colonization in that the hyphae never proceed past the epidermal layer (Bonfante et al., 2000). In this mutant, the extraradical hyphae differentiated into abnormal appressoria upon contact with the host root; colonization of the epidermis occurred with fungal entry followed by hyphal swelling and eventual death of the epidermal cell (Bonfante et al., 2000). Similarly, LjSym4–1 exhibits an epidermal block with numerous aborted colonization attempts; however, the fungus is able occasionally to cross the epidermal layer and to invade the root cortex resulting in the formation of arbuscules (Bonfante et al., 2000). When the epidermis–cortex interface is breached, colonization units spread laterally in a manner similar to that observed in the wild-type (Bonfante et al., 2000).
Although the mycorrhizal phenotype of E107 is very similar to that of LjSym4–1, it is unique in displaying two blocks, one at the root surface and one at the epidermis. According to the recent terminology of Smith & Read (1997), which attempts to provide a framework for mutant comparison, E107 can be described as Apr+ (appressorium) and low Pen (penetration). However, this terminology does not take into account the passage from the epidermis to the outer cortex, a transition that has been shown by Wegel et al. (1998), Bonfante et al. (2000), and this paper to be an important check-point in mycorrhizal development. To emphasize the partial block that the fungus encounters in E107 at this check-point, we include the term Coi (Wegel et al., 1998) in its description. Thus, E107 can be classified as Apr+, low Pen and low Coi.
Control of mutant phenotypes
Grafting has been used to study the control of mutant nodulation phenotypes. The increased number of nodules in supernodulating and hypernodulating legumes is generally controlled by the shoot (Table 1). By contrast, nonnodulating and nonfixing legumes are usually characterized by root-control of the mutant phenotype (Table 1). However, exceptions to these generalizations exist, emphasizing that the regulation of these two symbiotic associations is quite complex. For example, Hamaguchi et al. (1992) found that root factors modified the shoot-control of supernodulation in a soybean mutant. In another instance, Postma et al. (1988) reported that supernodulation in a pea mutant was root-controlled whereas nonnodulation in another mutant was influenced by both root and shoot. Furthermore, Markwei & LaRue (1997) reported that low nodulation in the Sparkle-derived mutant E132 (sym21) was shoot-controlled. E107 is another example that low incidence of nodules can be controlled by the shoot.
Table 1. Root or shoot control of nodulation in mutants
Similar grafting studies with mycorrhizal plants are relatively limited. Gianinazzi-Pearson et al. (1991) and Vierheilig & Piché (1996) conducted grafting experiments with pea mutants unable to form nodules or mycorrhizae; these workers found that the lack of rhizobial and fungal symbiotic structures was root-controlled. E107 differs from these mutants in two ways; firstly, it is able to form both of these associations although at a low frequency, and secondly, both characteristics are controlled by the shoot. An interspecific grafting experiment between lupin (Lupinus albus), a legume species unable to form mycorrhizae, and pea showed that shoot factors are involved in the inhibition of mycorrhizae formation (Gianinazzi-Pearson & Gianinazzi, 1989), results substantiated by the present study.
Correlation between nodulation and mycorrhizae-formation
There is growing evidence that the processes that lead to the initiation of nodules and mycorrhizae are similarly regulated. Many nodulation legume mutants have been used to study the relationship between these two associations; several examples of these mutants are shown in Table 2. Many nod− mutants are also myc−, although this is not always the case. Mutants that are able to form nodules but are unable to fix nitrogen can be associated with either the myc+ or the myc− phenotypes, whereas supernodulating mutants tend to be colonized normally or even at higher than normal frequencies (Morandi et al., 2000).
Table 2. Comparison of nodulation and mycorrhizal phenotypes in legume mutants
Landeweert R, Peterson RL & Guinel FC (pers. comm.).
E107 serves as an excellent tool to study the similarities between nodulation and mycorrhizae-formation. The blocks are comparable in their location (root surface and epidermal/cortical boundary). The similarity goes even further when quantitative data are considered. For the nodulation symbiosis, whereas Sparkle displays 6.2 rhizobial entry points per cm of lateral root, of which 64% enter the cortex, E107 has only 2.2; of these, 14% enter the cortex (Guinel & LaRue, 1992). For the mycorrhizal symbiosis, in Sparkle, there are 3.89 fungal entry points per cm of lateral root, of which 54% penetrate the cortex, whereas for E107, there are only 1.88; of these 24% penetrate the cortex (this paper). Also, the two processes in this mutant are similarly controlled by the shoot.
Duc et al. (1989) were the first to describe strong similarity in the genetic systems controlling the infection process of the two microsymbionts. Several authors have expanded on that observation (Gianinazzi-Pearson et al., 1994; Hirsch and Kapulnik, 1998; Albrecht et al., 1999). E107 is a good advocate for this hypothesis because quantification of its mycorrhizal phenotype mirrors that of its nodulation phenotype: It is a low mycorrhiza-former and a low nodulator with a strong block to both microsymbionts at the root surface and at the interface between the epidermis and the cortex (this paper, Guinel & LaRue, 1992). The correlation between the two symbioses is further emphasized by the fact that both are limited by the shoot.
We designate the E107 symbiotic phenotype as low nod, Apr+, low Pen, low Coi. The difficulty of placing this mutant among other mycorrhizal mutants reinforces the need for not only detailed observations but also a quantitative assessment of fungal colonization which would reflect the exact location of the defect.
This work was supported by a Natural Science and Engineering Research Council (NSERC) operating grant to FCG and an NSERC Undergraduate Student Research Assistantship to CMR. Additional funding was provided by Wilfrid Laurier University. We would like to thank Cameron Bell, a grade 12 co-op student, for initiating this study, Laurie Sloetjes for the nodulation study in grafted plants and Drs RL Peterson and TA LaRue for helpful discussion.