Calcium Oxalate Crystals in Eucalypt Ectomycorrhizae: Morphochemical Characterization

et al. (2013) Calcium Oxalate Crystals in Eucalypt Ectomycorrhizae: Morphochemical Characterization. PLoS ONE 8(7): e67685. doi:10.1371/journal.pone.0067685 Calcium Oxalate Crystals in Eucalypt Ectomycorrhizae: Morphochemical Characterization Victor Satler Pylro 0 1 Andre Luiz Moreira de Freitas 0 1 Wagner Campos Otoni 0 1 Ivo Ribeiro da Silva 0 1 Arnaldo Chaer Borges 0 1 Maurcio Dutra Costa 0 1 Agustin Guerrero-Hernandez, Cinvestav-IPN, Mexico 0 The experiments were performed at Mycorrhizal Associations Laboratory/BIOAGRO, Microbiology Department of Federal 1 1 Departamento de Microbiologia, Universidade Federal de Vic osa, Vic osa, Minas Gerais, Brazil, 2 Departamento de Biologia Vegetal, Universidade Federal de Vic osa, Vic osa, Minas Gerais, Brazil, 3 Departamento de Solos, Universidade Federal de Vic osa , Vic osa, Minas Gerais , Brazil Ectomycorrhizal fungi are ubiquitous in forest ecosystems, benefitting plants principally by increasing the uptake of water and nutrients such as calcium from the soil. Previous work has demonstrated accumulation of crystallites in eucalypt ectomycorrhizas, but detailed morphological and chemical characterization of these crystals has not been performed. In this work, cross sections of acetic acid-treated and cleared ectomycorrhizal fragments were visualized by polarized light microscopy to evaluate the location of crystals within cortical root cells. Ectomycorrhizal sections were also observed by scanning electron microscopy (SEM) coupled with energy dispersive x-ray (EDS) microprobe analysis. The predominant forms of crystals were crystal sand (granules) and concretions. Calcium, carbon and oxygen were detected by EDS as constituent elements and similar elemental profiles were observed between both crystal morphologies. All analyzed crystalline structures were characterized as calcium oxalate crystals. This is the first report of the stoichiometry and morphology of crystals occurring in eucalypt ectomycorrhizas in tropical soils. The data corroborates the role of ectomycorrhizae in the uptake and accumulation of calcium in the form of calcium oxalate crystals in hybrid eucalypt plants. - Funding: This work was funded by FAPEMIG (www.fapemig.br) grant APQ-00403-09, CNPq (www.cnpq.br), and CAPES (www.capes.gov.br). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The occurrence of calcium oxalate crystals (CaC2O4 or CaOx) has been observed in plants of several botanical families [1], and they contribute a large portion of the total calcium of these plants. CaOx deposits have been described in most tissues and organs as either intracellular (usually associated with vacuoles of specialized cells; idioblasts) or extracellular [14] deposits. The biological function of CaOx crystals in plants is neither completely understood nor characterized. Several functions have been attributed to them, largely based upon the amount, distribution and morphology of the crystals as well as the inherent characteristics of the cells where they are produced [1]. Some studies imply that CaOx may serve different biological functions such as a calcium reservoir, deposit of secondary metabolites and sequestration of potentially toxic metal ions [5,6], formation of aerenchyma in aquatic plants [7], providing structural support [8], or protection against herbivory by association with stinging substances or proteolytic toxins [911]. Eucalyptus is the most important genus of exotic plants in Brazilian planted forests, with great economic and environmental significance [12]. Generally, the soils under eucalypt cultivation in Brazil are highly weathered, with pH values below 5.5, an Aluminum saturation of 90%, and a low content of organic matter and limiting concentrations of Phosphorus, Nitrogen and Calcium (Ca2+) [1316]. Soil exchangeable calcium is often at or below 8 kg ha21 and insufficient to fulfil the plant demand [17]. Soil microorganisms play an essential role in biochemical cycles and contribute to edaphic homeostasis. In the current understanding that biological and functional diversity is a crucial factor in maintaining ecosystems [18] are included ectomycorrhizal fungi associated with the roots, that benefit plants by increasing the volume of the soil explored by roots, and thus the amount of absorbed nutrients and water [19]. The presence of CaOx crystals in ectomycorrhizal hyphae is limited to temperate soils containing high concentrations of calcium [2022]. In Brazil, the number of studies aiming at evaluating the accumulation of calcium crystals in eucalypt ectomycorrhizae is scarce [23]. However, the existing report has suggested a paramount role of ectomycorrhizal fungi in supplying Ca to eucalyptus in Brazilian soils poor in Ca, since putative CaOx crystals have been shown to be predominantly present in ectomycorrhizae rather than in non-mycorrhizal fine roots. Understanding the morphochemical patterns of crystalline structures in Eucalyptus ectomycorrhizae can help generate new information regarding the role of ectomycorrhizal fungi in Ca2+ uptake by plants. Therefore, the aim of this study was to evaluate the location, morphology and chemical composition of crystals present in eucalypt ectomycorrhizae. Materials and Methods University of Vicosa (UFV). Analyses of Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray (EDS) were performed at the Microscopy Center of the Federal University of Minas Gerais. All samples were processed at the Center of Microscopy and Microanalysis at UFV. Characterization of Sampling Site Samples were taken from within a 2.5 year old planted stand of clonal E. grandis X E. urophylla hybrids located in the experimental field of UFV (20u 469 27.399 S and 42u 519 36.299 W; elevation: 697 m). Ten soil cores (2.0 cm diameter, 20 cm depth) were collected at random points within the study area, and the cores were combined to form a composite sample. Physicochemical analyses (Table 1) were performed according to routine methods. Fine lateral roots colonized by ectomycorrhizal fungi were collected from the 010 cm layer of soil around randomly selected trees within the plantation. The soil was excavated and the roots were collected with fine-tipped forceps and razor blades. Samples were placed in a moist chamber and transported to the laboratory, for analysis. Ectomycorrhizal Eucalypt Preparation, Acid Digestion and Image Processing Ectomycorrhizal samples were washed in phosphate buffer, pH 7.0, to remove soil particles adhered to the surface, and then dehydrated in an ethanol series [10, 30, 50, 70 and 90% ethanol (vol/vol)] for 10 min in each solution and finally kept in 95% ethanol (vol/vol) for 12 h. Subsequently, part of the samples was cleared in a 1.8% (vol/vol) sodium hypochlorite solution for 1 h. The cleared samples containing cry (...truncated)