Tatsuhiro Ezawa

Community structure of arbuscular mycorrhizal fungi associated with pioneer grass species Miscanthus sinensis in acid sulfate soils: Habitat segregation along pH gradients

Abstract Acid sulfate soil has an extremely low pH and revegetation of the soil is difficult because of the high concentration of toxic elements, such as aluminum, and poor nutrient availability. Community compositions of arbuscular mycorrhizal (AM) fungi that associate with Miscanthus sinensis, a pioneer grass species that occurs in acid sulfate soil, were investigated to clarify the environmental factors that regulate the community structure. The rhizosphere soils of M. sinensis growing in acid sulfate soils were collected from three sites distributed in subarctic, temperate and subtropical zones. Rhizosphere soils of plants growing in a sandy soil site in a subarctic zone were also collected. Miscanthus sinensis seedlings were grown on these soils in a greenhouse for 2 months and a large subunit ribosomal RNA gene of the fungi was amplified from DNA extracted from the roots. Based on the nucleotide sequences of the gene, 20 phylotypes across six genera were detected from the four sites. The similarity indices of AM fungal communities among the sites did not correlate with geographical distance. Ordination analysis (principal component analysis) on the communities suggested that the first principal component reflected edaphic factors, particularly soil pH. Plotting of soil pH data at which respective phylotypes occurred and subsequent statistical analysis revealed that the ranges of preferential pH were significantly different among the phylotypes. The distribution of AM fungal phylotypes along pH gradients was further recognized by plotting the first principal component scores of the phylotypes against their preferential pH. The phylotypes that showed higher scores along the second principal component were detected from three or more sites and occurred over a wide range of pH values. These observations suggest that the preference and range of substrate pH to which the fungi can adapt are different among the phylotypes and soil pH might be a likely driving force for structuring AM fungal communities in acid sulfate soils.

How does arbuscular mycorrhizal colonization vary with host plant genotype? An example based on maize (Zea mays) germplasms

Colonization of plant roots by arbuscular mycorrhizal (AM) fungi is a primary factor determining mycorrhizal associations. This study aimed to investigate the variation in AM colonization among maize genotypes and in response to plant breeding programs. Three types of maize (Zea mays) germplasms composed of 141 inbred lines, 38 hybrids, and 76 landraces were grown in replicated field experiments in Sapporo, Japan, for two cropping years to evaluate the percentage of root length colonized by indigenous AM fungi. The percent colonization varied greatly and continuously among maize genotypes. Inbred lines that originated (released) in particular locations (e.g., Tokachi, Japan) and years (e.g., 1960s) showed significantly larger values than other lines. Inter-location differences were also observed for landraces. The direction of the year-of-release effect on colonization depended on the origin. No significant differences were observed between leaf-blight-disease-resistant near-isogenic inbred lines and their parents. Modern hybrids showed significantly greater values than inbred lines and older landraces. Evaluating numerous, diverse genotypes demonstrated that AM colonization of maize plants varies with germplasm type, origin (country and location), and year of release, and that modern plant breeding programs do not necessarily lead to the suppression of colonization.

Comparison of phosphatase localization in the intraradical hyphae of arbuscular mycorrhizal fungi, Glomus spp. and Gigaspora spp.

The localization of acid and alkaline phosphatases in the intraradical hyphae of the arbuscular mycorrhizal fungi, Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (Gm), Gl. etunicatum Becker and Gerd. (Ge) and Gigaspora rosea Nicol. and Schenck (Gir) were compared. Marigold (Tagetes patula L.) and leek (Allium porrum L.) were inoculated with each of the three fungi. The mycorrhizal roots were harvested at 3, 4, 5 and 6 weeks after sowing (WAS), treated with a digestion solution containing cellulase and pectinase, and then stained for phosphatase activities at pH 5.0 and pH 8.5. The development of fungal structures in the host root was also examined. Gm formed fine-branched (mature) arbuscules only at the early phase of infection (3 to 4 WAS). Mature arbuscules of Ge and Gir were observed from the early phase (4 WAS) up to the end of experiment. At pH 5.0, the localization of the phosphatase activities of the three fungi were similar irrespective to host plant species. The activity appeared in mature arbuscules and intercellular hyphae, whereas the collapsed arbuscules were inactive. Ten millimolar NaF, an acid phosphatase inhibitor, inhibited the phosphatase activities of Gm and Ge but did not affect that of Gir. At pH 8.5, a difference among the fungal species was found in the localization of phosphatase activity while that between host species was not. The mature arbuscules were also the active sites in all three species. Only Gir showed the activity in the intercellular hyphae while the two Glomus spp. did not. Five millimolar EDTA inhibited the activity of Gir at pH 8.5 while the activities of Ge and Gm were not affected by either 5 mM EDTA or 10 mM KCN (both are alkaline phosphatase inhibitors).

Enhancement of the Effectiveness of Indigenous Arbuscular Mycorrhizal Fungi by Inorganic Soil Amendments

Abstract The influence of inorganic soil amendments on the effectiveness of indigenous arbuscular mycorrhizal (AM) fungi was investigated in pot experiments. Intact or ground perlite, Kanumatsuchi (volcanic ash soil), vermiculite, or rice-hull charcoal was mixed with uncultivated soil in which Glomus sp. was dominant, and marigold (Tagetes patula L.) was sown to the soil mixtures. AM colonization of the host roots increased by the incorporation of ground materials but not by that of intact materials. The growth promotive effect of the indigenous fungi on the host was enhanced by both the intact and ground materials. The inorganic materials improved the soil physical properties: the intact materials increased the gaseous phase of the media and the ground materials increased the aqueous phase. It was suggested that the inorganic soil amendments might not only provide a less-competitive habitat for the fungi but also improve the physical environment.

A New Hypothesis on the Strategy for Acquisition of Phosphorus in Arbuscular Mycorrhiza: Up-Regulation of Secreted Acid Phosphatase Gene in the Host Plant

The mycorrhiza-responsive phosphatase of Tagetes patula in symbiosis with Glomus etunicatum was detected by electrophoresis, was purified by column chromatography, and was characterized as acid phosphatase that was secreted into rhizosphere. The N-terminal amino acid sequence was determined by a gas-phase sequencer, and a cDNA fragment of the phosphatase gene (TpPAP1) was amplified by degenerate primers designed based on the N-terminal amino acid sequence. The full-length cDNA was obtained by the rapid amplification of cDNA ends technique. The TpPAP1 was of host origin, and the cDNA was 1,843 bp long with a predicted open reading frame of polypeptide of 466 amino acids. Phylogenetic analysis revealed that the gene fell into the cluster of plant high-molecular-weight purple acid phosphatase. Expression analysis of the TpPAP1 in T. patula in symbiosis with Archaeospora leptoticha showed that the levels of transcripts increased eightfold by mycorrhizal colonization. Western blot analysis revealed that the 57-kDa protein corresponding to the mycorrhiza-responsive phosphatase increased by mycorrhizal colonization. The present study proposes a new strategy for acquisition of P in arbuscular mycorrhizal associations in which the fungal partner activates a part of the low-P adaptation system of the plant partner, phosphatase secretion, and improves the overall efficiency of P uptake.

Polyphosphate has a central role in the rapid and massive accumulation of phosphorus in extraradical mycelium of an arbuscular mycorrhizal fungus

Title Polyphosphate has a central role in the rapid and massive accumulation of phosphorus in extraradical mycelium of an arbuscular mycorrhizal fungus Author(s) Hijikata, Nowaki; Murase, Masatake; Tani, Chiharu; Ohtomo, Ryo; Osaki, Mitsuru; Ezawa, Tatsuhiro Citation New Phytologist, 186(2), 285-289 https://doi.org/10.1111/j.1469-8137.2009.03168.x Issue Date 2010 Doc URL http://hdl.handle.net/2115/48540 Type article (author version) File Information Hijikata_et_al_HUS.pdf

Difference in Striga-susceptibility is reflected in strigolactone secretion profile, but not in compatibility and host preference in arbuscular mycorrhizal symbiosis in two maize cultivars.

Strigolactones released from plant roots trigger both seed germination of parasitic weeds such as Striga spp. and hyphal branching of the symbionts arbuscular mycorrhizal (AM) fungi. Generally, strigolactone composition in exudates is quantitatively and qualitatively different among plants, which may be involved in susceptibility and host specificity in the parasite-plant interactions. We hypothesized that difference in strigolactone composition would have a significant impact on compatibility and host specificity/preference in AM symbiosis. Strigolactones in root exudates of Striga-susceptible (Pioneer 3253) and -resistant (KST 94) maize (Zea mays) cultivars were characterized by LC-MS/MS combined with germination assay using Striga hermonthica seeds. Levels of colonization and community compositions of AM fungi in the two cultivars were investigated in field and glasshouse experiments. 5-Deoxystrigol was exuded exclusively by the susceptible cultivar, while the resistant cultivar mainly exuded sorgomol. Despite the distinctive difference in strigolactone composition, the levels of AM colonization and the community compositions were not different between the cultivars. The present study demonstrated that the difference in strigolactone composition has no appreciable impact on AM symbiosis, at least in the two maize cultivars, and further suggests that the traits involved in Striga-resistance are not necessarily accompanied by reduction in compatibility to AM fungi.

Element interconnections in Lotus japonicus: a systematic study of the effects of element additions on different natural variants.

Abstract Lotus japonicus was used to study the distribution and interconnections of 15 elements in plant tissues, including essential and non-essential elements: boron (B), sodium (Na), magnesium (Mg), potassium (K), calcium (Ca), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), strontium (Sr), molybdenum (Mo), cadmium (Cd) and cesium (Cs). Large amounts of B and Ca accumulated in plant leaves, while Fe, Na, Ni, As and Cd tended to mainly occur in the roots, and Mo was the only element to accumulate in the stems. The elemental compositions within plants were severely disturbed by treatment with toxic elements. Competition between element pairs in the same group (e.g. K and Cs; Ca and Sr) was not found. Iron, Cu and Zn accumulation were induced by Cd and Ni addition. When natural variants grew in a nutrition solution with subtoxic levels of As, Cd, Cs, Ni, Mo and Sr, intriguing relationships between the elements (such as Fe, As and K; Mg and Ni; Mn and Ca) were revealed using principal-component analysis. This study on the plant ionome offers detailed information of element interactions and indicates that chemically different elements might be closely linked in uptake or translocation systems.

ATP-Dependent but Proton Gradient-Independent Polyphosphate-Synthesizing Activity in Extraradical Hyphae of an Arbuscular Mycorrhizal Fungus

ABSTRACT Arbuscular mycorrhizal (AM) fungi benefit their host plants by supplying phosphate obtained from the soil. Polyphosphate is thought to act as the key intermediate in this process, but little is currently understood about how polyphosphate is synthesized or translocated within arbuscular mycorrhizas. Glomus sp. strain HR1 was grown with marigold in a mesh bag compartment system, and extraradical hyphae were harvested and fractionated by density gradient centrifugation. Using this approach, three distinct layers were obtained: layers 1 and 2 were composed of amorphous and membranous materials, together with mitochondria, lipid bodies, and electron-opaque bodies, and layer 3 was composed mainly of partially broken hyphae and fragmented cell walls. The polyphosphate kinase/luciferase system, a highly sensitive polyphosphate detection method, enabled the detection of polyphosphate-synthesizing activity in layer 2 in the presence of ATP. This activity was inhibited by vanadate but not by bafilomycin A1 or a protonophore, suggesting that ATP may not energize the reaction through H+-ATPase but may act as a direct substrate in the reaction. This report represents the first demonstration that AM fungi possess polyphosphate-synthesizing activity that is localized in the organelle fraction and not in the cytosol or at the plasma membrane.

Polyphosphate accumulation is driven by transcriptome alterations that lead to near-synchronous and near-equivalent uptake of inorganic cations in an arbuscular mycorrhizal fungus

Arbuscular mycorrhizal (AM) fungi accumulate a massive amount of phosphate as polyphosphate to deliver to the host, but the underlying physiological and molecular mechanisms have yet to be elucidated. In the present study, the dynamics of cationic components during polyphosphate accumulation were investigated in conjunction with transcriptome analysis. Rhizophagus sp. HR1 was grown with Lotus japonicus under phosphorus-deficient conditions, and extraradical mycelia were harvested after phosphate application at prescribed intervals. Levels of polyphosphate, inorganic cations and amino acids were measured, and RNA-Seq was performed on the Illumina platform. Phosphate application triggered not only polyphosphate accumulation but also near-synchronous and near-equivalent uptake of Na(+) , K(+) , Ca(2+) and Mg(2+) , whereas no distinct changes in the levels of amino acids were observed. During polyphosphate accumulation, the genes responsible for mineral uptake, phosphate and nitrogen metabolism and the maintenance of cellular homeostasis were up-regulated. The results suggest that inorganic cations play a major role in neutralizing the negative charge of polyphosphate, and these processes are achieved by the orchestrated regulation of gene expression. Our findings provide, for the first time, a global picture of the cellular response to increased phosphate availability, which is the initial process of nutrient delivery in the associations.