Phosphorus nutrition-mediated effects of arbuscular mycorrhiza on leaf morphology and carbon allocation in perennial ryegrass

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.     

Disentangling CO2 fluxes: direct measurements of mesocosm‐scale natural abundance 13CO2/12CO2 gas exchange, 13C discrimination,and labelling of CO2 exchange flux components in controlled environments

A ¹³C/¹²C mass spectrometer was interfaced with a open gas exchange system including four growth chambers to investigate CO₂ exchange components of perennial ryegrass (Lolium perenne L.) stands. Chambers were fed with air containing CO₂ with known δ¹³C (δ_CΟ2?2.6 or ?46.8‰). The system did not fractionate C isotopes and no extraneous CO₂ leaked into chambers. The on‐line ¹³C discrimination (Δ) of ryegrass stands in light was independent of δ_CΟ2 when δ_CΟ2 was constant. The δ of CO₂ exchanged by the stands in light (δ_Nd) and darkness (δ_Rn) differed by 0.7‰, suggesting some Δ in dark respiration at the stand‐level. However, Δ decreased by ～ 10‰ when δ_CΟ2 was switched from ?46.8 to ?2.5‰, and increased by ～ 10‰ following a shift from ?2.6 to ?46.7‰ due to isotopic disequilibria between photosynthetic and respiratory fluxes. Isotopic imbalances were used to assess (non‐photorespiratory) respiration in light and the replacement of the respiratory substrate pool(s) by new photosynthate. Respiration was partially inhibited by light, but increased during the light period and decreased in darkness, in association with temperature changes. The labelling kinetics of respiratory CO₂ indicated the existence of two major respiratory substrate pools: a fast pool which was exchanged within hours, and a slow pool accounting for ～ 60% of total respiration and having a mean residence time of 3.6 d.     

Arbuscular mycorrhizal fungi benefit from 7 years of free air CO2 enrichment in well-fertilized grass and legume monocultures

Rising atmospheric carbon dioxide partial pressure (pCO₂) and nitrogen (N) deposition are important components of global environmental change. In the Swiss free air carbon dioxide enrichment (FACE) experiment, the effect of altered atmospheric pCO₂ (35 vs. 60 Pa) and the influence of two different N‐fertilization regimes (14 vs. 56 g N m^?2 a^?1) on root colonization by arbuscular mycorrhizal fungi (AMF) and other fungi (non‐AMF) of Lolium perenne and Trifolium repens were studied. Plants were grown in permanent monoculture plots, and fumigated during the growth period for 7 years. At elevated pCO₂ AMF and non‐AMF root colonization was generally increased in both plant species, with significant effects on colonization intensity and on hyphal and non‐AMF colonization. The CO₂ effect on arbuscules was marginally significant (P=0.076). Moreover, the number of small AMF spores (≤100 μm) in the soils of monocultures (at low‐N fertilization) of both plant species was significantly increased, whereas that of large spores (>100 μm) was increased only in L. perenne plots. N fertilization resulted in a significant decrease of root colonization in L. perenne, including the AMF parameters, hyphae, arbuscules, vesicles and intensity, but not in T. repens. This phenomenon was probably caused by different C‐sink limitations of grass and legume. Lacking effects of CO₂ fumigation on intraradical AMF structures (under high‐N fertilization) and no response to N fertilization of arbuscules, vesicles and colonization intensity suggest that the function of AMF in T. repens was non‐nutritional. In L. perenne, however, AM symbiosis may have amended N nutrition, because all root colonization parameters were significantly increased under low‐N fertilization, whereas under high‐N fertilization only vesicle colonization was increased. Commonly observed P‐nutritional benefits from AMF appeared to be absent under the phosphorus‐rich soil conditions of our field experiment. We hypothesize that in well‐fertilized agricultural ecosystems, grasses benefit from improved N nutrition and legumes benefit from increased protection against pathogens and/or herbivores. This is different from what is expected in nutritionally limited plant communities.     

Partitioning of sugars in Lolium perenne (perennial ryegrass) during drought and on rewatering

Simulated swards of perennial ryegrass ( Lolium perenne ) growing in 1-m³ soil blocks in the glasshouse were either well watered or deprived of water for 57 d and then rewatered. The first aim was to measure effects of drought on sugar (water-soluble carbohydrate) composition of laminae and sheaths of mature laminae, and bases and laminae of young (growing) leaves. The second aim was to use pulse labelling with ¹⁴CO₂ to follow the partitioning of recently-fixed assimilates, and the assembly and consumption of reserve sugars (fructans). Over the last 7 d of drought growth almost stopped, old leaves died faster than they were replaced, and total sugar (which had doubled in concentration during drought) was rapidly consumed. Leaf laminae had lower content of total sugars and of large fructan (DP>5) than did growing bases and sheaths. Drought greatly reduced the rate at which sugar was exported from the laminae to the sheaths and growing leaf bases, and the rate at which it was converted to fructan. Nevertheless, fructan accumulated over the first 50 d of drought. Rewatering did not result in depolymerization and remobilization of sugars that had been formed during the last 7 d of drought, but stimulated their further assembly into high-DP fructans. Our hypothesis, that accumulation of neo-kestose (a DP-3 fructan) in droughted laminae was a symptom of sugar remobilization just before death, was disproved. It is concluded that sugar reserves contribute to drought resistance only under extreme conditions. The specific role of fructan in dry environments might be to improve regrowth when drought is relieved, rather than to enhance growth during drought.     

Mechanistic understanding of interspecific interaction between a C4 grass and a C3 legume via arbuscular mycorrhizal fungi,as influenced by soil phosphorus availability using a 13C and 15N dual-labelled organic patch

Arbuscular mycorrhizal fungi (AMF) can improve plant nutrient acquisition, either by directly supplying nutrients to plants or by promoting soil organic matter mineralization, thereby affecting interspecific plant relationships in natural communities. We examined the mechanism by which the addition of P affects interspecific interactions between a C4 grass (Bothriochloa ischaemum, a dominant species in natural grasslands) and a C3 legume (Lespedeza davurica, a subordinate species in natural grasslands) via AMF and plant growth, by continuous ¹³C and ¹⁵N labelling, combined with soil enzyme analyses. The results of ¹⁵N labelling revealed that P addition affected the shoot uptake of N via AMF by B. ischaemum and L. davurica differently. Specifically, the addition of P significantly increased the shoot uptake of N via AMF by B. ischaemum but significantly decreased that by L. davurica. Interspecific plant interactions via AMF significantly facilitated the plant N uptake via AMF by B. ischaemum but significantly inhibited that by L. davurica under P-limited soil conditions, whereas the opposite effect was observed in the case of excess P. This was consistent with the impact of interspecific plant interaction via AMF on arbuscular mycorrhizal (AM) benefit for plant growth. Our data indicate that the capability of plant N uptake via AMF is an important mechanism that influences interspecific relationships between C4 grasses and C3 legumes. Moreover, the effect of AMF on the activities of the soil enzymes responsible for N and P mineralization substantially contributed to the consequence of interspecific plant interaction via AMF for plant growth.     

Long-term steady-state labelling of wheat plants by use of natural 13CO2/12CO2 mixtures in an open,rapidly turned-over system

A photosynthate labelling method is presented which takes advantage of the natural difference in carbon-isotope composition () which exists between atmospheric CO₂ (-8) and commercially available compressed CO₂. Carbon dioxide with -4.0 and –27.9%., respectively, has been used for labelling. A plant growth cabinet served as the labelling compartment. CO₂-free air was continuously injected at a rate of up to 54m³·h^–1. Dilution of cabinet CO₂ by CO₂-free air was counterbalanced by addition of CO₂ with known constant . Since the labelling-cabinet atmosphere was continuously exchanged at a high rate, photosynthetic carbon-isotope discrimination was fully expressed. In order to study the distribution of carbon acquired by the plant during a defined growth period, the of CO₂ was modified by replacing, for example, atmospheric CO₂ by CO₂ with –27.9%. and the weight and 5 of plant carbon pools was monitored over time. In such an experiment the change of CO₂ was followed by a rapid change of the of sucrose in mature flag-leaf blades of wheat (Triticum aestivum L.). The 5 of sucrose stabilized near –51%., indicating complete exchange by current photosynthate. In contrast 83% of the total carbon in mature flag-leaf blades was not exchanged after 14 d continuous labelling. Differential labelling of pre- and post-anthesis photosynthate indicated that 13% of grain carbon originated from pre-anthesis photosynthesis. Carbon-isotope discrimination and its consideration in experimentation and labelling data evaluation are discussed in detail. Since the air supplied to the labelling cabinet is dry and free of CO₂, carbon-isotope discrimination and carbon turnover and partitioning can be studied over a wide range of CO₂ concentrations (0–2600 cm³ · m^–3) and vapor-pressure deficits.Abbreviation and Symbol PPFD photosynthetic photon flux density - carbon-isotope composition Dr. G. Schleser (Forschungszentrum Jülich, FRG) and Professor S. Hoernes (Mineralogisch-Petrologisches Institut, Universität Bonn) for valuable help and advice during the initial stages of the project and Professor W. Kühbauch (Institut für Pflanzenbau, Universität Bonn) for continuing support. Technical assistance of Ute Labusch, Petra Biermann, Ludwig Schmitz and Thomas Gebbing is gratefully acknowleged.

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Effect of enhanced atmospheric CO2 on mycorrhizal colonization by Glomus mosseae in Plantago lanceolata and Trifolium repens

Plantago lanceolata L. and Trifolium repens L. were grown for 16 wk in ambient (360 μmol mol⁻¹) and elevated (610 μmol mol⁻¹) atmospheric CO₂. Plants were inoculated with the arbuscular mycorrhizal (AM) fungus Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe and given a phosphorus supply in the form of bonemeal, which would not be immediately available to the plants. Seven sequential harvests were taken to determine whether the effect of elevated CO₂ on mycorrhizal colonization was independent of the effect of CO₂ on plant growth. Plant growth analysis showed that both species grew faster in elevated CO₂ and that P. lanceolata had increased carbon allocation towards the roots. Elevated CO₂ did not affect the percentage of root length colonized (RLC); although total colonized root length was greater, when plant size was taken into account this effect disappeared. This finding was also true for root length colonized by arbuscules. No CO₂ effect was found on hyphal density (colonization intensity) in roots. The P content of plants was increased at elevated CO₂, although both shoot and root tissue P concentration were unchanged. This was again as a result of bigger plants at elevated CO₂. Phosphorus inflow was unaffected by CO₂ concentrations. It is concluded that there is no direct permanent effect of elevated CO₂ on mycorrhizal functioning, as internal mycorrhizal development and the mycorrhizal P uptake mechanism are unaffected. The importance of sequential harvests in experiments is discussed. The direction for future research is highlighted, especially in relation to C storage in the soil.     

13CO2/12CO2 exchange fluxes in a clamp‐on leaf cuvette: disentangling artefacts and flux components

Leaks and isotopic disequilibria represent potential errors and artefacts during combined measurements of gas exchange and carbon isotope discrimination (Δ). This paper presents new protocols to quantify, minimize, and correct such phenomena. We performed experiments with gradients of CO₂ concentration (up to ±250 μmol mol^?1) and δ¹³C_CO2 (34‰), between a clamp‐on leaf cuvette (LI‐6400) and surrounding air, to assess (1) leak coefficients for CO₂, ¹²CO₂, and ¹³CO₂ with the empty cuvette and with intact leaves of Holcus lanatus (C₃) or Sorghum bicolor (C₄) in the cuvette; and (2) isotopic disequilibria between net photosynthesis and dark respiration in light. Leak coefficients were virtually identical for ¹²CO₂ and ¹³CO₂, but ～8 times higher with leaves in the cuvette. Leaks generated errors on Δ up to 6‰ for H. lanatus and 2‰ for S. bicolor in full light; isotopic disequilibria produced similar variation of Δ. Leak errors in Δ in darkness were much larger due to small biological : leak flux ratios. Leak artefacts were fully corrected with leak coefficients determined on the same leaves as Δ measurements. Analysis of isotopic disequilibria enabled partitioning of net photosynthesis and dark respiration, and indicated inhibitions of dark respiration in full light (H. lanatus: 14%, S. bicolor: 58%).     

Partitioning of reserve and newly assimilated carbon in roots and leaf tissues of Lolium perenne during regrowth after defoliation: assessment by 13C steady-state labelling and carbohydrate analysis

The relative significance of the use of stored or currently absorbed C for the growth of leaves or roots of Lolium perenne L. after defoliation was assessed by steady-state labelling of atmospheric CO₂. Leaf growth for the first two days after defoliation was to a large extent dependent on the use of C reserves. The basal part of the elongating leaves was mainly new tissue and 91% of the C in this part of the leaf was derived from reserves assimilated prior to defoliation. However, half of the sucrose in the growth zone was produced from photosynthesis by the emerged leaves. Fructans that were initially present in elongating leaf bases were hydrolysed (loss of 93 to 100%) and the resulting fructose was found in the new leaf bases, suggesting that this pool may be used to support cell division and elongation. Despite a negative C balance at the whole-plant level, fructans were synthesized from sucrose that was translocated to the new leaf bases. After a regrowth period of 28 d, 45% of the C fixed before defoliation was still present in the root and leaf tissue and only 1% was incorporated in entirely new tissue.     

Carbon mobilization in shoot parts and roots of wheat during grain filling: assessment by 13C/12C steady-state labelling, growth analysis and balance sheets of reserves

cv, cultivar
δ, deviation of C isotope composition from a standard
Δ, C isotope discrimination
WSC, water soluble carbohydrates

Steady-state labelling of all post-anthesis photosynthate of wheat was performed to assess the mobilization of pre-anthesis C (C fixed prior to anthesis) in vegetative plant parts during grain filling. Results were compared with estimates obtained by indirect approaches to mobilization of pre-anthesis C: ‘classical’ growth analysis and balance sheets of water soluble carbohydrates (WSC) and protein. Experiments were performed with two spring wheat cultivars grown with differential nitrogen fertilizer supply in 1991 and 1992. The fraction of pre-anthesis C mobilized in above-ground vegetative biomass ranged between 24 and 34% of total C present at anthesis. Treatment effects on mobilization of pre-anthesis C in total above-ground vegetative biomass were closely related (r² = 0·89) to effects on mobilization of WSC-C plus protein-C (estimated as N mobilized × 3·15). On average, 81% of pre-anthesis C mobilization was attributable to the balance of pre-anthesis WSC (48%) and protein (33%) between anthesis and maturity. In roots, WSC and protein mobilization accounted for only 29% of the loss of pre-anthesis C. Notably, mobilization of pre-anthesis C was 1·4–2·6 times larger than the net loss of C from above-ground vegetative biomass between anthesis and maturity. This discrepancy was mainly due to post-anthesis C accumulation in glumes and stem. Post-anthesis C accumulation was related to continued synthesis of structural biomass after anthesis and accounted for a mean 15% of total C contained in above-ground vegetative plant parts at maturity. A close correspondence between net loss of C and mobilization of pre-anthesis C was only apparent in leaf blades and leaf sheaths. Although balance sheets of WSC and protein also underrated the mobilization of pre-anthesis C by ≈ 19%, they gave a much better estimate of pre-anthesis C mobilization than growth analysis.     

Kinetics and relative significance of remobilized and current C and N incorporation in leaf and root growth zones of Lolium perenne after defoliation: assessment by 13C and 15N steady-state labelling

The contribution of pre-defoliation reserves and current assimilates to leaf and root growth was examined in Lolium perenne L. during regrowth after defoliation. Differential steady-state labelling with ¹³C (CO₂ with δ¹³C = -0.0281 and -0.0088) and ¹⁵N (NO₃^? with 1.0 and 0.368 atom percentage, i.e. δ¹⁵N = 1.742 and 0.0052, respectively) was applied for 2 weeks after defoliation. Rapidly growing tissues were isolated, i.e. the basal elongation and maturation zones of the most rapidly expanding leaves and young root tips, with a biomass turnover rate > 1 d^?1. C and N weights of the elongation zone showed a transient decline. The dry matter and C concentration in fresh biomass of leaf growth zones transiently decreased by up to 25% 2 d after defoliation, while the N concentration remained constant. This ‘dilution’ of growth zone C indicates a decreased net influx of carbohydrates relative to growth-related influx of water and N in expanding cells, immediately after defoliation. Recovery of the total C and N weights of the leaf elongation zone coincided with net incorporation of currently absorbed C and N, as shown by the kinetics of δ¹³C and atom percentage ¹⁵N in the growth zones after defoliation. C isotope discrimination (Δ¹³C) in leaf growth zones was about 23‰, 1–2‰ higher than the Δ in root tips. Δ¹⁵N in the leaf and root growth zones was 10±3‰. The leaf elongation zones (at 0–0.03 m from the tiller base) and the distant root tips (about 0.2 m from the base) exhibited similar kinetics of current C and N incorporation. The amount of pre-defoliation C and N in the growth zones, expressed as a fraction of total C and N, decreased from 1.0 to 0.5 at 3 (C) and 5 (N) d after defoliation, and to 0.1 at 5 (C) and 14 (N) d after defoliation. Thus, the dependence of growth zones on current assimilate supply was significant, and stronger for C than for N. The important roles of current assimilates (as compared to pre-defoliation reserves) and ‘dilution’ of dry matter in regrowth after defoliation are discussed in relation to the method of labelling and the functional and morphological heterogeneity of shoot tissues.