Author for correspondence: Hans Schnyder Tel: +49 8161 713242 Fax: +49 8161 713243 Email: firstname.lastname@example.org
• The aim of this work was to disentangle phosphorus status-dependent and -independent effects of arbuscular mycorrhizal fungus (AMF) on leaf morphology and carbon allocation in perennial ryegrass (Lolium perenne).
• To this end, we assessed the P-response function of morphological components in mycorrhizal and nonmycorrhizal plants of similar size.
• AMF (Glomus hoi) stimulated relative P-uptake rate, decreased leaf mass per area (LMA), and increased shoot mass ratio at low P supply. Lower LMA was caused by both decreased tissue density and thickness. Variation in tissue density was almost entirely caused by variations in soluble C, while that in thickness involved structural changes.
• All effects of AMF were indistinguishable from those mediated by increases in relative P-uptake rate through higher P-supply rates. Thus the relationships between relative P-uptake rate, leaf morphology and C allocation were identical in mycorrhizal and nonmycorrhizal plants. No evidence was found for AMF effects not mediated by changes in plant P status.
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Soil P availability has been recognized as a key factor limiting crop growth and grassland production on many soils. Symbiosis with arbuscular mycorrhizal fungus (AMF) is known to increase the nutritional status of grassland plants, especially with respect to P (Smith & Read, 1997). The production of hyphae seems to incur a smaller C cost per unit absorbing area than that of roots, and it allows the exploration of soil volumes that are not accessed by roots (Koide, 1991). However, as obligate symbionts, the AMF relies on the plant host for the supply of C assimilates required for its growth, maintenance and functioning. As the AMF draws C from the host, it has been proposed that the overall effect of AMF on the growth of the host is dependent on the cost–benefit relationship of the symbiosis (Smith & Smith, 1996; Johnson et al., 1997). However, mechanistic understanding of the terms of exchange of this symbiosis is far from complete (Fitter, 2005). A still unsolved aspect is whether AMF influence on growth is entirely mediated by changes in plant P status, or whether there are P status-independent AMF effects. It is known that both P addition and AMF colonization could promote plant growth by increasing the leaf area per unit of plant biomass (leaf area ratio, LAR) (Baas & Lambers, 1988), which contributes to an increase in the C assimilation on a whole-plant basis (Lambers & Poorter, 1992). But are AMF and P supply effects on growth and its morphological determinants quantitatively equal? This manuscript is an attempt to elucidate this question.
Efficient resource uptake and optimal biomass allocation are important determinants of competitive ability and stress tolerance. Plants have a great capacity to control the expansion of their organs, and optimize light interception by increasing the proportion of leaf area and shoot biomass if soil nutrient availability is improved (Lambers & Poorter, 1992). A recent meta-analysis of the published literature concluded that plant morphological components were major determinants of growth responses to nutrient deficiencies (Poorter & Nagel, 2000). Differences in LAR can be caused by variation in the leaf mass per area (LMA) and in the fraction of plant mass present in leaves (leaf mass ratio, LMR). There is a close association between plant growth rate and the components of LMA (Lambers & Poorter, 1992), but still little is known about the effects of nutrient availability on leaf morphology (Meziane & Shipley, 1999). LMA often shows a considerable plasticity in response to environmental factors, and this can be attributed to either the density or thickness of the leaf tissue (Witkowski & Lamont, 1991; Lambers & Poorter, 1992). Phosphorus deficiency in plants is generally associated with accumulation of nonstructural compounds, as more photosynthates are produced than can be consumed in growth (Rao et al., 1989). Therefore it is possible that the increases in LMA often found in response to P deficiency do not reflect structural changes in leaf architecture, but result merely from passive accumulation of water-soluble compounds (de Groot et al., 2001; Miller et al., 2002). Clearly, a functional interpretation of changes in LMA must distinguish between effects related to changes in the soluble fraction, and those associated with a change in the proportion of structural C per unit of leaf area (Witkowski & Lamont, 1991; Miller et al., 2002).
The basic aim of this study was to disentangle P status-dependent and -independent effects of AMF on leaf morphology and C allocation in perennial ryegrass (Lolium perenne). To this end, responses of plant growth, leaf morphology and C allocation of mycorrhizal and nonmycorrhizal perennial ryegrass plants of similar size were compared as a function of relative P-uptake rate. Awareness of size-dependent effects is a prerequisite for plant growth studies, as many morphological ratios change in conjunction with plant size (Coleman et al., 1994). In order to assess unbiased treatment effects, it is therefore essential to take into account ontogenic drifts by, for example, comparing plants of common size (Eissenstat et al., 1993; Wright et al., 1998; de Groot et al., 2001). Specifically, we addressed the following questions: (i) is there a consistent quantitative relationship between P availability, plant growth, leaf morphology and C allocation in perennial ryegrass? (ii) If so, is this relationship modified by P status-independent effects caused by the presence of AMF colonization? Further, (iii) which are the specific morphological components explaining plant response to changes in P availability? To our knowledge, this is the first comparative study of AMF effects on the P response of leaf morphology and biomass allocation in a grass.
Materials and Methods
Plant material, AMF inoculation and growth conditions
Seeds of perennial ryegrass (Lolium perenne L. cv. Condesa) were washed for 20 min in NaOCl (6% active chlorine) for surface sterilization, and sown into tubes (diameter 5 cm, height 35 cm) filled with quartz sand (0.3–0.8 mm). Previously all tubes had been fertilized with fine powdered Hyperphos (0.564 g kg−1 sand, 30.5% P2O5) as a P source of low plant availability. In half the tubes, plants were inoculated with the AMF Glomus hoi BEG 104 (provided by Dr A. Heinemeyer, University of York, UK), which had been propagated in glasshouse conditions for 3 months on Plantago lanceolata L. plants as a host. The inoculum, consisting of roots and soil material (15 ml per tube), was mixed thoroughly with the quartz sand used as substrate. Tubes with and without AMF inoculum were placed in separate plastic boxes (56 × 76 × 37 cm) to prevent contamination of the control plants with AMF.
Plants were grown in a growth chamber (VKZPH 005-120-S, Heraeus Vötsch GmbH, Balingen-Frommern, Germany) with relative humidity kept between 65 and 75%, temperature 20°C during the 16 h light period and 15°C in the dark, and 425 µmol m−2 s−1 photosynthetic photon flux density at plant level. Plants were watered by automatic irrigation systems supplying 25 ml nutrient solution to every plant four times a day. During the first 5 wk, plants were irrigated with modified half-strength P-free Hoagland's solution (2.5 mm KNO3, 2.5 mm Ca (NO3)2, 1 mm MgSO4, 0.5 mm KCl, 0.5 mm NaCl, 0.125 mm Fe-EDTA, 23 µm H3BO3, 4.5 µm MnSO4, 0.38 µm ZnSO4, 0.16 µm CuSO4, 0.05 µm Na2MoO4). Thereafter, until the end of the experiment, four soluble P treatments were applied: nil (0 mm); low (0.02 mm); intermediate (0.1 mm); and high (0.5 mm) P supply, delivered as KH2PO4 in the nutrient solution described above. Potassium supply was maintained constant between treatments by reducing the KCl supply proportionally. Once a week tubes were flushed with 125 ml distilled water to prevent salt accumulation.
Five plants per treatment combination were sampled at the end of the dark period 68, 74 and 83 d after sowing. Roots were freed from the soil substrate by washing with tap water. A sample of the fresh root material was weighed and used for detection of AMF colonization. Shoots were separated into mature tillers (at least one fully developed leaf) and daughter tillers, and counted. Mature tillers were dissected into four compartments: (i) expanding leaf; (ii) youngest fully expanded leaf (defined as the youngest one having its ligule exposed); (iii) all other mature leaves; and (iv) dead material. Each leaf was then dissected into lamina and sheath. The area of the laminas was measured with an Li-3100 leaf area meter (Li-Cor Inc., Lincoln, NE, USA). Samples were then fresh-weighed, frozen in liquid nitrogen, freeze-dried, dry-weighed, ground and stored at −25°C.
Mycorrhizal colonization of the roots was determined by histological detection of mycorrhizal structures after root staining. A sample of the fresh root material was cleared in KOH (10% w/v) for 10 min at 105°C, acidified in HCl (1% v/v) for 5 min, then stained with Trypan Blue (0.05% w/v; Sigma-Aldrich, Steinheim, Germany) in acid glycerol for 10 min at 105°C. Stained roots were briefly submerged in distilled water, arranged lengthwise on microscope slides, then mounted in glycerol-gelatine. The percentage of root length with AMF colonization was determined in the mounted roots by evaluating 100 random intersections for each plant, using a line graticule in the eyepiece of a compound microscope at ×125 magnification (Giovanneti & Mosse, 1980).
One representative shoot sample for each plant was made by pooling proportional fractions of dry biomass of each organ. The C concentration was then determined in roots, shoot and separate lamina samples on 0.7 mg aliquots of dry, ground material using an elemental analyser (NA1500, Carbo Erba Instruments, Milan, Italy). Phosphorus concentration was determined on samples of roots and shoot (25 mg) which were ashed in a muffle furnace (4 h at 500°C). The resulting ash was digested in HNO3/HCl, and quantities of P were measured by phosphovanado-molybdate colorimetry (Hanson, 1950). Reference material of ground grass leaves was included with every 10 samples to check digestion and analytical procedures. All data are presented as P and C masses.
For analyses of different C fractions, 20 mg dry mass of the youngest fully expanded leaf were extracted with 2 ml distilled water for 10 min at 93°C and 45 min at room temperature (Schnyder & de Visser, 1999). After centrifugation (10 000 g for 15 min) the supernatant was used for analyses of water-soluble fractions, while the residual pellet was used for starch determination. The content of water-soluble N and of water-soluble C in 100 µl of the supernatant was analysed with the elemental analyser, using sulphanilamide (Merck, Darmstadt, Germany) as a standard. Water-soluble amino-C content was estimated as soluble N content × 2.6 (Schnyder & de Visser, 1999). Water-soluble carbohydrates were analysed on an aliquot of the extract. For this, samples were hydrolysed in 0.1 m H2SO4 for 20 min at 93°C, and the reducing power of the hydrolysed carbohydrates was detected photometrically at 425 nm after reduction of a potassium ferricyanide solution (Schnyder & de Visser, 1999). Fructose and glucose (Merck) were used as standards. Finally the residual pellets were hydrolysed in a mixture of 5 ml dimethylsulfoxide (DMSO) and 1.25 ml HCl (8 m) for 30 min at 60°C. Starch was determined colorimetrically after neutralization with 1.25 ml NaOH (8 m) and equilibration with citric buffer (0.112 m; pH = 4) by an enzymatic test combination (catalogue no. 207748, Boehringer, Mannheim, Germany). The content of C in water-soluble carbohydrates and starch was estimated as total hexose units × 0.4. Structural C fraction was then estimated as: Cstructural = Ctotal − (Csoluble + Cstarch).
Relative growth rate (RGR, mg C g−1 C d−1) and relative phosphorus-uptake rate (RPUR, mg P g−1 P d−1) during the sampling period (68–83 d after sowing) were calculated as the slope of the least squares regression lines of the loge-transformed values of C and P masses against time, respectively. The LAR (cm2 g−1 C) was determined as total plant leaf area divided by plant mass. LAR is associated with the amount of lamina mass invested to construct a unit of leaf area (LMA) and with the fraction of plant mass allocated to the leaf laminas (LMR). The LMA (g C cm−2) was estimated for different C fractions (total, structural and nonstructural), and calculated as lamina C mass divided by leaf area. In addition, LMA was factorized into lamina tissue density (g C g−1 f. wt) and thickness (mm). Lamina tissue density was estimated as the ratio of lamina mass to leaf fresh weight, and tissue thickness as the ratio of leaf fresh weight to leaf area under the assumption that leaf volume is approximately equal to leaf fresh weight (Garnier & Laurent, 1994). LMR (g C g−1 C) was calculated as total lamina mass divided by plant mass, and factorized into shoot mass ratio (g C g−1 C) and lamina : shoot ratio (g C g−1 C). Shoot P : C ratio and all morphological components (plant and leaf) were analysed on plants of similar size (0.5–0.9 g C per plant) selected from the three harvests, instead of chronological analyses, to distinguish treatment effects from ontogenetic drifts (Coleman et al., 1994). All harvested plants that fitted within this range were used. Leaf morphology and C fraction analyses were performed on laminas of the youngest fully expanded leaf from mature tillers of the size-selected plants.
All data were previously checked for normality and homogeneity of variances. Root mycorrhizal colonization was analysed by one-way anova and a posteriori mean comparisons were performed with Tukey's test (Steel & Torrie, 1988). Differences in RGR and RPUR within P treatments were analysed by two-way anova with time and mycorrhizal inoculation as independent variables (skipping the intermediate harvest), and loge-transformed values of C or P mass as dependent variables. A significant time × treatment interaction denotes a significant difference in RGR or RPUR, respectively (Poorter & Lewis, 1986). The relationship between RGR and RPUR was examined by simple correlation analysis, and an F test for differences between mycorrhizal and nonmycorrhizal plants was performed (Steel & Torrie, 1988). All other variables were analysed by two-way anova, with P and AMF inoculation as main factors, on plants of similar size, and contrasts between specific treatments were performed by a priori LSD mean tests (Steel & Torrie, 1988). Variables that involve percentages were arcsine square-root transformed before analysis (Steel & Torrie, 1988). Quadratic-plateau functions and all statistical analyses were performed by the statistical package sas version 8.2 (SAS Inc., Cary, NC, USA). Results are shown as mean and standard error of the means.
Phosphorus uptake, AMF colonization and plant growth
Both P supply and AMF inoculation affected the P status of the plants, expressed as shoot P : C ratios (w/w; Fig. 1a). Planned comparison tests revealed that all P treatments differed significantly in this parameter (dfe = 41; P < 0.05). Also the P : C ratio was higher (P = 0.008) in mycorrhizal plants under low P supply (0.02 mm), but was not affected by mycorrhiza in the other P treatments (P > 0.05; Fig. 1a). Phosphorus treatment had a significant effect (dfe = 16; P < 0.001) on the percentage of AMF colonization (Fig. 1b). None of the control plants observed was contaminated with AMF.
The RGR and RPUR were positive and approximately constant during the sampling period (Fig. 2). Overall, RGR was linearly related to RPUR, and the relationship was not different between AMF treatments (P > 0.05), not far from the 1 : 1 line in both treatments (Fig. 3). This suggests that the P : C ratio of accumulated biomass was rather similar to that of the standing biomass, although somewhat lower in intermediate and high P supply. In consequence, plant P : C ratios remained relatively stable over the harvesting period. Hence plants within each treatment can be considered to be close to a nutritional steady state.
In nonmycorrhizal plants RGR increased 2.3-fold between the lowest and highest P-supply rates (Fig. 3). Two-way anova analyses within P treatments indicated that the RGR of mycorrhizal plants was significantly higher than nonmycorrhizal plants only when RPUR was improved (Table 1), which occurred both under low (0.02 mm) and high (0.5 mm) P-supply rates (by +40 and +52%, respectively; Fig. 3). Similarly, at intermediate P supply (0.1 mm), C and P masses of mycorrhizal plants were significantly higher than those of nonmycorrhizal ones (Fig. 2; Table 1), although differences in RGR and RPUR were not apparent. Similarly, RGR and RPUR of mycorrhizal plants in the 0 mm soluble P treatment tended to be higher than those of nonmycorrhizal plants (+23 and +9%, respectively). However, the resolution of the data was not good enough to detect these effects as statistically significant (Fig. 3).
Table 1. Results of two-way anova for effects of arbuscular mycorrhizal fungus (AMF) and time on carbon and phosphorus plant masses
Source of variation
Plant C mass
Plant P mass
Associated mean squares (MS) and number of degrees of freedom (df) for each source of variation are given. Significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05) within soluble P treatments. Significant AMF × time interaction denotes differences in relative growth rate (RGR) and relative phosphorus-uptake rate (RPUR) within soluble P treatments.
Effects of P nutrition on leaf morphology and C allocation
Leaf and plant morphology were closely related to RPUR. Increasing RPUR significantly increased LAR up to a plateau at intermediate P supply (Fig. 4; dfe = 41; P = 0.034). This plateau coincided with the attainment of minimum values of LMA (Fig. 5a) and maximum values of LMR (Fig. 6a). LMA decreased by 200% with increasing RPUR, and was the main factor affecting LAR. This response was associated with changes in both density and thickness of the lamina tissues (Fig. 5b,c; Table 2). In nonmycorrhizal plants the concentration of soluble C decreased 3.5-fold between the lowest and highest P-supply rates (Table 3), explaining the sharp decrease in total tissue density (Fig. 5b). Yet a slight decrease (compared with the variation in total tissue density) in the structural tissue density was also detected at highest P supplies (Fig. 5b; Table 2). The decrease in soluble C at high RPUR was mainly caused by decreases in water-soluble carbohydrates (WSC) and soluble amino-C contents. Conversely, treatments had no effect on starch content, which was very low in all treatments (Table 3). Lamina tissue thickness decreased with increasing RPUR, and was the main determinant of changes in structural LMA (Fig. 5a,c). The increase in LMR between the lowest and highest value of RPUR was nearly 25% (Fig. 6a), and was caused by an increase in the shoot mass ratio (Fig. 6b). RPUR had virtually no effect on the lamina : shoot ratio (Fig. 6c; Table 2).
Table 2. Results of two-way anova for the effects of phosphorus (P) supply rate and arbuscular mycorrhizal fungus (AMF) on different variables for perennial ryegrass (Lolium perenne) plants of similar size
(LAR) leaf area ratio (LMR) leaf mass ratio (SMR) shoot mass ratio (LMA) leaf mass per area (DEN) lamina tissue density. Associated mean squares (MS) and number of degrees of freedom (df) for each source of variation are given. Significant differences: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05.
Table 3. Effects of phosphorus (P) supply and arbuscular mycorrhizal fungus (AMF) on fractions of nonstructural carbon (C) in leaf laminas: soluble C, water-soluble carbohydrates (WSC-C), water soluble amino-C (Amino-C) and starch-C, in perennial ryegrass (Lolium perenne) plants grown for 12 wk with different P-supply rates
Leaf lamina content (mg C g−1 f. wt)
54.7 ± 4.7
23.4 ± 2.6
16.6 ± 2.3
0.3 ± 0.1
68.7 ± 4.1
35.5 ± 2.3
12.7 ± 1.6
0.8 ± 0.1
42.0 ± 3.5
17.8 ± 1.9
16.3 ± 2.7
0.5 ± 0.2
27.1 ± 3.1
11.8 ± 1.2
11.8 ± 1.1
0.2 ± 0.1
18.1 ± 2.3
7.2 ± 0.9
10.1 ± 0.6
0.4 ± 0.1
13.1 ± 1.9
5.1 ± 1.2
8.7 ± 0.7
0.5 ± 0.2
15.6 ± 2.4
6.4 ± 1.6
9.8 ± 1.1
0.4 ± 0.2
18.4 ± 2.8
8.4 ± 1.5
10.8 ± 1.6
0.5 ± 0.3
Source of variation
Values are means ± SE for plants of similar size (n = 4–8). Associated mean squares (MS) and number of degrees of freedom (df) for each source of variation are given. Significant differences: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05.
A consistent feature of the relationships presented above was that they were virtually the same in mycorrhizal and nonmycorrhizal plants (Figs 4–6). For instance, higher RPUR associated with presence of mycorrhiza at low P supply (0.02 mm) resulted in an increase in LAR and their components (P < 0.01) in the same way as in nonmycorrhizal plants (Figs 4–6; Table 3).
AMF affected plant growth, leaf morphology and C allocation solely through enhancement of P uptake
All effects of mycorrhizal symbiosis on plant growth, leaf morphology and C allocation resulted from the effect of AMF on P capture. Thus we observed no P status-independent effects of AMF on perennial ryegrass plants. The RGR of mycorrhizal plants was significantly higher as a result of improved RPUR. Interestingly, this occurred at both low and high P-supply rates. Perennial grasses are generally considered to be less dependent on mycorrhiza than legumes and other grassland species (Schweiger et al., 1995; Hartnett & Wilson, 2002) because they usually possess a highly branched root architecture with very long root hairs, which appears to render benefits from increases of P-uptake rate by mycorrhizal symbiosis less likely (Jakobsen et al., 2005). However, there is evidence of a high degree of diversity in the functional effect of mycorrhiza in relation to environmental conditions and the identity of both partners involved in the relationship (Munkvold et al., 2004; Smith et al., 2004; Jakobsen et al., 2005). Enhancement of growth by AMF when P availability is high, although a counterintuitive result, has already been reported in perennial ryegrass growing in association with Glomus species (Powell & Daniel, 1978; Hall et al., 1984). In our experiment, AMF colonization of the roots was lower at high P supply. However, the presence of mycorrhiza continued to play a positive role in P uptake and plant growth. This suggests that the degree of AMF colonization was a poor indicator of AMF activity and its effect on the host plant (Son & Smith, 1988; Jongen et al., 1996; Smith et al., 2004). More importantly, this indicates that no parasitism-like effect was apparent over the applied range of P-supply rates. We can not exclude the possibility that higher P supplies might lead to mycorrhiza-induced growth depressions, but this threshold was not achieved under our experimental conditions. Parasitic mycorrhizal associations may occur when environmental factors cause net costs to exceed net benefits; a effect that is most commonly reported at low light intensity or when defoliation exacerbates C stress (Smith & Smith, 1996; Johnson et al., 1997).
Decreases in LMA and WSC concentration and increases in shoot mass ratio have often been reported in mycorrhizal plants (Buwalda & Goh, 1982; Freeden & Terry, 1988; Son & Smith, 1988; Jongen et al., 1996; Wright et al., 1998; Müller et al., 1999). Our results are in agreement with these observations. These responses have commonly been attributed to C stress and growth depression caused by the presence of AMF in the roots. Interestingly, our results indicate that such responses were indirect effects mediated by the improvement in P nutrition of the plants. The AMF is widely thought to consume 4–20% of total photosynthates (Douds et al., 1988; Jakobsen & Rosendahl, 1990). Although the present study did not attempt to construct a C balance of the symbiosis, it nonetheless indicates that, for a given P-capture level (RPUR), a determined C gain (RGR) was made irrespective of the presence of AMF. Therefore any additional C cost of the AMF appears to be counterbalanced, for example by reductions in metabolic costs associated with P uptake and utilization and/or enhancements of C assimilation rates (Douds et al., 1988; Wright et al., 1998; Black et al., 2000), so that an overall neutral effect on whole-plant C accumulation was observed. How these effects are physiologically controlled, and whether the cost–benefit relationship of the mycorrhizal symbiosis could be affected by severe C stress (for example caused by defoliation through herbivory or mowing), merits further experimental investigation.
Effects of P nutrition status – as a function of P supply or AMF – on leaf morphology and C allocation
The improvement of P nutrition, either by the addition of P fertilizer or by AMF inoculation, affected both components of LAR: LMA and LMR. Remarkably, increasing RPUR significantly affected LAR and its components up to a plateau, and from this point leaf characters and C-allocation patterns appeared to attain morphogenetic limits. From this point, further changes in RGR are no longer related to morphological adjustments, and might reflect increases in net assimilation rate. There is increasing evidence that C assimilation is influenced by the demands of sinks (Farrar, 1992; Paul & Foyer, 2001; Pieters et al., 2001). If this is the case, the improvement in P capture could have altered the number of active meristems and thus increased the sink strength (Pieters et al., 2001). In our study the increase in LMR was completely caused by an increase in shoot mass ratio, representing a common response associated with increasing P-supply rates (Baas & Lambers, 1988; Wright et al., 1998; de Groot et al., 2001), as the lamina : shoot ratio is a quite conserved quantity in grass species. However, LMA was decreased to a much greater extent than the positive change in LMR. Therefore our results strongly suggest that the adjustment of leaf morphology (LMA) rather than plant C allocation (LMR) was the main factor explaining differences in LAR as affected by P supply in perennial ryegrass.
This study revealed strong effects of P nutrition status on both components of LMA: tissue density and thickness. LMA was twofold lower under high P supply in comparison with P-deficient conditions. High LMA is often associated with P deficiency because of high concentrations of nonstructural compounds (Rao et al., 1989; Ryser et al., 1997; de Groot et al., 2001). Variation in leaf thickness has been reported in response to changes in light intensity and nutrient availability (Witkowski & Lamont, 1991; Meziane & Shipley, 1999), but its functional determination as related to changes in environmental conditions is still unclear. In our experimental conditions, both LMA components varied in the same direction with increasing P, thus resulting in leaves with lower tissue density and thickness. However, there was a major difference in the response of these two variables. On the one hand, more than 90% of the change in tissue density was caused by variations in the content of water-soluble C compounds. Thus, apparently, P deficiency had a greater effect on C use than on C assimilation (cf. Radin & Eidenbock, 1986; Rao et al., 1989), leading to the reported increase in lamina density by accumulation of WSC (probably in the form of fructans). In agreement, we found that P deficiency markedly reduced relative cell expansion rates (M.K. and co-workers, unpublished results). Additionally, the slight increment in structural density could be related to decreased cell size (Radin & Eidenbock, 1986), which might increase the proportion of cell walls per unit of cell volume.
On the other hand, changes in tissue thickness are inherently associated with responses in structural tissues. Therefore, while the increase in tissue density in response to P deficiency was a consequence of a passive accumulation of soluble C, changes in thickness must have been entirely associated with active responses of the growing tissue. In the present study, thickness was measured as the amount of water per unit leaf area (tissue thickness, as defined here). The increase in water per unit area observed under P deficiency could be associated with a decrease in the amount of mesophyll intercellular spaces. Restricted cell expansion under low P availability thus might produce a more compact tissue, with more water per unit area. Decreases in LMA, through having thinner leaves or leaves with lower tissue density, enhance light interception and photosynthetic capacity per unit of leaf mass (Lambers & Poorter, 1992; Poorter & Evans, 1998; Reich et al., 1998). Leaf morphological changes affect nutrient distribution per unit of leaf area, which, in turn, affects the productivity of nutrients allocated to leaves (Poorter & Evans, 1998). Ryser et al. (1997) reported a close relationship between productivity of leaf P and the ability to distribute it over a large leaf area. Our results indicate that AMF colonization would affect such relationships through the improvement of the P status of the plants.
All the effects of mycorrhizal symbiosis on plant growth observed in this study were strictly correlated with effects of AMF on P capture. Furthermore, the relationships between RPUR and all leaf morphological characters analysed, and between RPUR and plant C allocation, were very similar in mycorrhizal and nonmycorrhizal plants. No evidence for P status-independent effects of AMF was found on undisturbed perennial ryegrass plants.
Adjustment in leaf morphology (LMA) rather than plant C allocation (LMR) was the main response observed. Higher LMA in P-deficient plants was caused by increases in both tissue density and thickness. However, variation in tissue density was almost entirely caused by passive variations in soluble C, while that in thickness involved structural changes.
We especially thank Anja Schmidt, Monika Breitsamter and Brigitte Schilling for chemical analyses. Angela Ernst-Schwärzli, Wolfgang Feneis, Melitta Sternkopf and all members of the Lehrstuhl für Grünlandlehre (TUM) provided invaluable assistance at different phases of this work. Valuable comments from Rudi Schäufele, Astrid Lux-Endrich, Thomas Gebbing, Yuncai Hu, Reinhold Gutser and Alastair Fitter helped in early stages of this work. We also thank Andreas Heinemeyer (University of York) for kindly providing AMF inoculum. This study was supported by Deutsche Forschungsgemeinschaft (SFB 607).