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      Metabolomics Suggests That Soil Inoculation with Arbuscular Mycorrhizal Fungi Decreased Free Amino Acid Content in Roots of Durum Wheat Grown under N-Limited, P-Rich Field Conditions

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          Abstract

          Arbuscular mycorrhizal fungi (AMF) have a major impact on plant nutrition, defence against pathogens, a plant’s reaction to stressful environments, soil fertility, and a plant’s relationship with other microorganisms. Such effects imply a broad reprogramming of the plant’s metabolic activity. However, little information is available regarding the role of AMF and their relation to other soil plant growth—promoting microorganisms in the plant metabolome, especially under realistic field conditions. In the present experiment, we evaluated the effects of inoculation with AMF, either alone or in combination with plant growth–promoting rhizobacteria (PGPR), on the metabolome and changes in metabolic pathways in the roots of durum wheat ( Triticum durum Desf.) grown under N-limited agronomic conditions in a P-rich environment. These two treatments were compared to infection by the natural AMF population (NAT). Soil inoculation with AMF almost doubled wheat root colonization by AMF and decreased the root concentrations of most compounds in all metabolic pathways, especially amino acids (AA) and saturated fatty acids, whereas inoculation with AMF+PGPR increased the concentrations of such compounds compared to inoculation with AMF alone. Enrichment metabolomics analyses showed that AA metabolic pathways were mostly changed by the treatments, with reduced amination activity in roots most likely due to a shift from the biosynthesis of common AA to γ-amino butyric acid. The root metabolome differed between AMF and NAT but not AMF+PGPR and AMF or NAT. Because the PGPR used were potent mineralisers, and AMF can retain most nitrogen (N) taken as organic compounds for their own growth, it is likely that this result was due to an increased concentration of mineral N in soil inoculated with AMF+PGPR compared to AMF alone.

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          Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen.

          Transcriptome analysis, using Affymetrix ATH1 arrays and a real-time reverse transcription-PCR platform for >1,400 transcription factors, was performed to identify processes affected by long-term nitrogen-deprivation or short-term nitrate nutrition in Arabidopsis. Two days of nitrogen deprivation led to coordinate repression of the majority of the genes assigned to photosynthesis, chlorophyll synthesis, plastid protein synthesis, induction of many genes for secondary metabolism, and reprogramming of mitochondrial electron transport. Nitrate readdition led to rapid, widespread, and coordinated changes. Multiple genes for the uptake and reduction of nitrate, the generation of reducing equivalents, and organic acid skeletons were induced within 30 min, before primary metabolites changed significantly. By 3 h, most genes assigned to amino acid and nucleotide biosynthesis and scavenging were induced, while most genes assigned to amino acid and nucleotide breakdown were repressed. There was coordinate induction of many genes assigned to RNA synthesis and processing and most of the genes assigned to amino acid activation and protein synthesis. Although amino acids involved in central metabolism increased, minor amino acids decreased, providing independent evidence for the activation of protein synthesis. Specific genes encoding expansin and tonoplast intrinsic proteins were induced, indicating activation of cell expansion and growth in response to nitrate nutrition. There were rapid responses in the expression of many genes potentially involved in regulation, including genes for trehalose metabolism and hormone metabolism, protein kinases and phosphatases, receptor kinases, and transcription factors.
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            Nitrogen transfer in the arbuscular mycorrhizal symbiosis.

            Most land plants are symbiotic with arbuscular mycorrhizal fungi (AMF), which take up mineral nutrients from the soil and exchange them with plants for photosynthetically fixed carbon. This exchange is a significant factor in global nutrient cycles as well as in the ecology, evolution and physiology of plants. Despite its importance as a nutrient, very little is known about how AMF take up nitrogen and transfer it to their host plants. Here we report the results of stable isotope labelling experiments showing that inorganic nitrogen taken up by the fungus outside the roots is incorporated into amino acids, translocated from the extraradical to the intraradical mycelium as arginine, but transferred to the plant without carbon. Consistent with this mechanism, the genes of primary nitrogen assimilation are preferentially expressed in the extraradical tissues, whereas genes associated with arginine breakdown are more highly expressed in the intraradical mycelium. Strong changes in the expression of these genes in response to nitrogen availability and form also support the operation of this novel metabolic pathway in the arbuscular mycorrhizal symbiosis.
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              Genome-wide reprogramming of metabolism and regulatory networks of Arabidopsis in response to phosphorus.

              Affymetrix ATH1 arrays, large-scale real-time reverse transcription PCR of approximately 2200 transcription factor genes and other gene families, and analyses of metabolites and enzyme activities were used to investigate the response of Arabidopsis to phosphate (Pi) deprivation and re-supply. Transcript data were analysed with MapMan software to identify coordinated, system-wide changes in metabolism and other cellular processes. Phosphorus (P) deprivation led to induction or repression of > 1000 genes involved in many processes. A subset, including the induction of genes involved in P uptake, the mobilization of organic Pi, the conversion of phosphorylated glycolytic intermediates to carbohydrates and organic acids, the replacement of P-containing phospholipids with galactolipids and the repression of genes involved in nucleotide/nucleic acid synthesis, was reversed within 3 h after Pi re-supply. Analyses of 22 enzyme activities revealed that changes in transcript levels often, but not always, led to changes in the activities of the encoded enzymes in P-deprived plants. Analyses of metabolites confirmed that P deprivation leads to a shift towards the accumulation of carbohydrates, organic acids and amino acids, and that Pi re-supply leads to use of the latter. P-deprived plants also showed large changes in the expression of many genes involved in, for example, secondary metabolism and photosynthesis. These changes were not reversed rapidly upon Pi re-supply and were probably secondary in origin. Differentially expressed and highly P-specific putative regulator genes were identified that presumably play central roles in coordinating the complex responses of plants to changes in P nutrition. The specific responses to Pi differ markedly from those found for nitrate, whereas the long-term responses during P and N deprivation share common and non-specific features.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                11 June 2015
                2015
                : 10
                : 6
                : e0129591
                Affiliations
                [1 ]Dipartimento di Scienze Agrarie e Forestali, Università degli Studi di Palermo, Palermo, Italy
                [2 ]Fondazione A. e S. Lima Mancuso, Università degli Studi di Palermo, Palermo, Italy
                Estación Experimental del Zaidín (CSIC), SPAIN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: SS PR GA FM. Performed the experiments: SS PR GDM FM. Analyzed the data: SS VF GDM. Contributed reagents/materials/analysis tools: GDM GA. Wrote the paper: SS PR GA FM.

                Article
                PONE-D-14-53343
                10.1371/journal.pone.0129591
                4466249
                26067663
                244eaa63-70e4-469d-b1f0-28c872fc81cb
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 27 November 2014
                : 11 May 2015
                Page count
                Figures: 4, Tables: 1, Pages: 15
                Funding
                The present research was funded by the project "Sviluppo tecnologico e innovazione per la sostenibilità e competitività della cerealicoltura meridionale (PON01_01145-ISCOCEM)" (person in charge: Dr. Giuseppe Di Miceli - Fondazione A. e S. Lima Mancuso) of the Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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                Research Article
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                All relevant data are within the paper and its Supporting Information files.

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