¹³ C Incorporation into Signature Fatty Acids as an Assay for Carbon Allocation in Arbuscular Mycorrhiza

Abstract

The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:15 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating 13 C enrichment of 16:15 and compared it with 13 C enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [13 C]glucose. The 13 C enrichment of neutral lipid fatty acid 16:15 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for 13 C in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:15 than for the root specific neutral lipid fatty acid 18:26,9. We labeled plant assimilates by using 13 CO 2 in whole-plant experiments. The extraradical mycelium often was more enriched for 13 C than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between 13 C enrichment in neutral lipid fatty acid 16:15 and total 13 C in extraradical mycelia in different systems (r 2 ‫؍‬ 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the 13 C enrichment of 16:15. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia.Arbuscular mycorrhizal (AM) fungal mycelia acquire hexoses released by the roots of their host (3,39,42,43) and metabolize them to lipids, mainly neutral lipids, such as triacylglycerols (39). Neutral lipids are transported throughout the fungal mycelium (4), are metabolized through the glyoxalate cycle (3,24), and probably provide the major fungal energy source. The mechanisms that regulate C transfer from plant to fungus are not well understood (21). However, AM fungal colonization affects plant C metabolism (13,38,51,52) and the genes that regulate this metabolism (20,40).AM fungal neutral lipids usually are stored in intraradical vesicles or in spores and make up a large proportion of the AM fungal biomass (6,22,36). The fatty acids of these lipids have a characteristic and specific composition (7). In Glomus intraradices, 50 to 70% of the neutral lipids are the fatty acid 16:15 (19, 35), which is uncommon in other groups of fungi (28,32,47) and can be used as an AM fungal signature (31, 37).13 C nuclear magnetic resonance has been used to determine C-metabolic pathways after 13 C labeling of monoxenic root cultures (3). The incorporation and turnover of C in AM fungal mycelium can be difficult to measure because the hyphae are not easily extracted and separated from roots or soil.13 C enrichment or 13 C dilution of signature compounds, such as ...

Effect of P Availability on Temporal Dynamics of Carbon Allocation and Glomus intraradices High-Affinity P Transporter Gene Induction in Arbuscular Mycorrhiza

“…Freezedried mycelium (approximately 20 g) or ball-milled root material (approximately 100 g) was enclosed in tin capsules, and the 13 C atom% was determined with an isotope ratio mass spectrometer (20-20 stable isotope analyzer) interfaced with an ANCA-NT combustion module (PDZ Europa Scientific Instruments, Crewe, United Kingdom). Fatty acid methyl esters (prepared as described above) were analyzed with an isotope ratio mass spectrometer interfaced with a Hewlett-Packard gas chromatograph in order to determine the 13 C atom% in neutral lipid fatty acids (NLFA) and phospholipid fatty acids (25). The gas chromatograph was equipped with a 50-m HP5 capillary column (Hewlett-Packard), and He was used as the carrier gas.…”

“…Lipids were extracted from mycelia and mycorrhizal roots by vortexing (1 min) in 10 ml of a one-phase mixture containing citrate buffer, methanol, and chloroform (0.8:2:1, vol/vol/vol; pH 4.0). The lipids were fractionated into neutral lipids, glycolipids, and phospholipids on prepacked silica columns (100-mg sorbent mass; Varian Medical Systems, Palo Alto, CA) by elution with 1.5 ml chloroform, 6 ml acetone, and 1.5 ml methanol, respectively (25). The fatty acid residues in neutral lipids and phospholipids were transformed into free fatty acid methyl esters and analyzed by gas chromatography by using a 50-m HP5 capillary fused silica column (Hewlett-Packard, Wilmington, DE) with H 2 as the carrier gas (11).…”

CO2 uptake by a soil microcosm

“…Several monounsaturated FAMEs were observed that are common across taxa, but 18:1u9 is common to both fungi and Gram-positive bacteria (Bååth, 2003;Vestle and White, 1989). Further, enrichment of general fungal biomarkers such as 16:1u5 (Olsson et al, 1995(Olsson et al, , 2003, 18:1u7 (Olsson, 1999) and 18:2u9,12 (Frostegård and Bååth, 1996;Zelles, 1999;Zechmeister-Bolternstern et al, 2011) indicates that fungi (e.g., ectomycorrhizal and arbuscular mycorrhizal etc.) were possibly actively consuming biomass and/or exudates of the S 2 O 3 2À activated consortium (cross-feeding?).…”

Phosphorus Availability Influences Elemental Uptake in the Mycorrhizal Fungus Glomus intraradices , as Revealed by Particle-Induced X-Ray Emission Analysis

“…However, the nutrients stored in spores may reflect not only the need for formation of new fungal biomass but also that of the plants that are being colonized. The establishment of new symbiotic relations could thus depend on the accumulation of plant-limiting nutrients in spores and their transport into the host roots for recognition of a nonparasitic intruder.The nutrient accumulation and distribution in AM fungal spores have not been studied in as much detail as carbon accumulation has (32), although there are clear links between the nutrient availability and C metabolism (16,17,18). Quantitative analysis of elemental composition in AM fungal mycelia is crucial for our understanding of the distribution of nutritional resources.…”

“…The nutrient accumulation and distribution in AM fungal spores have not been studied in as much detail as carbon accumulation has (32), although there are clear links between the nutrient availability and C metabolism (16,17,18). Quantitative analysis of elemental composition in AM fungal mycelia is crucial for our understanding of the distribution of nutritional resources.…”