Jan Pallon

Speciation and distribution of arsenic and localization of nutrients in rice grains

Arsenic (As) contamination of rice grains and the generally low concentration of micronutrients in rice have been recognized as a major concern for human health. Here, we investigated the speciation and localization of As and the distribution of (micro)nutrients in rice grains because these are key factors controlling bioavailability of nutrients and contaminants. Bulk total and speciation analyses using high-pressure liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICP-MS) and X-ray absorption near-edge spectroscopy (XANES) was complemented by spatially resolved microspectroscopic techniques (micro-XANES, micro-X-ray fluorescence (micro-XRF) and particle induced X-ray emission (PIXE)) to investigate both speciation and distribution of As and localization of nutrients in situ. The distribution of As and micronutrients varied between the various parts of the grains (husk, bran and endosperm) and was characterized by element-specific distribution patterns. The speciation of As in bran and endosperm was dominated by As(III)-thiol complexes. The results indicate that the translocation from the maternal to filial tissues may be a bottleneck for As accumulation in the grain. Strong similarities between the distribution of iron (Fe), manganese (Mn) and phosphorus (P) and between zinc (Zn) and sulphur (S) may be indicative of complexation mechanisms in rice grains.

IDENTIFICATION AND CHARACTERIZATION OF A STREPTOCOCCUS PYOGENES ABC TRANSPORTER WITH MULTIPLE SPECIFICITY FOR METAL CATIONS

Metal ions are crucial trace elements for bacteria infecting the human host. The LraI (lipoprotein receptor‐associated antigen I) transporter in Streptococcus spp. belongs to the superfamily of ABC transporters. The transporter consists of a lipoprotein, an ATP‐binding protein and a hydrophobic integral membrane protein. Here, we describe a new member of the LraI family in the important human pathogen Streptococcus pyogenes. The system was identified in silico by analysis of the S. pyogenes Genome Sequencing Project. The S. pyogenes operon exhibits an atypical organization compared with equivalents in other Streptococcus spp. The presence and atypical organization of the operon was verified in a number of S. pyogenes strains of different serotypes. Transcriptional analysis of the LraI operon demonstrates a polycistronic transcription attenuated by a stable stem–loop structure, which allows the lipoprotein to be expressed in larger quantities than the other two components. The localization of the native lipoprotein at the bacterial surface was shown by proteolytic digestion of S. pyogenes bacteria and NH2‐terminal sequencing of a released lipoprotein fragment. Recombinant lipoprotein was expressed as a GST fusion protein, and studies of molecular interactions with metal radioisotopes demonstrated that the protein has affinity for Zn(II), Fe(III) and Cu(II). Zn(II) and Cu(II) were found to compete for the same binding site, whereas Fe(III) uses a second site. Also, proton‐induced X‐ray analysis of lipoprotein samples identified iron, copper and zinc. Finally, a mutant strain lacking a functional mtsABC operon was generated and showed reduced uptake of 55Fe and 65Zn compared with the wild‐type strain. The operon encoding this novel ABC transporter with multiple specificity for metal cations is designated mtsABC, for metal transporter of Streptococcus.

Tit for tat? A mycorrhizal fungus accumulates phosphorus under low plant carbon availability

The exchange of carbohydrates and mineral nutrients in the arbuscular mycorrhizal (AM) symbiosis must be controlled by both partners in order to sustain an evolutionarily stable mutualism. Plants downregulate their carbon (C) flow to the fungus when nutrient levels are sufficient, while the mechanism controlling fungal nutrient transfer is unknown. Here, we show that the fungus accumulates nutrients when connected to a host that is of less benefit to the fungus, indicating a potential of the fungus to control the transfer of nutrients. We used a monoxenic in vitro model of root organ cultures associated with Glomus intraradices, in which we manipulated the C availability to the plant. We found that G. intraradices accumulated up to seven times more nutrients in its spores, and up to nine times more in its hyphae, when the C pool available to the associated roots was halved. The strongest effect was found for phosphorus (P), considered to be the most important nutrient in the AM symbiosis. Other elements such as potassium and chorine were also accumulated, but to a lesser extent, while no accumulation of iron or manganese was found. Our results suggest a functional linkage between C and P exchange.

Elemental composition of arbuscular mycorrhizal fungi at high salinity.

We investigated the elemental composition of spores and hyphae of arbuscular mycorrhizal fungi (AMF) collected from two saline sites at the desert border in Tunisia, and of Glomus intraradices grown in vitro with or without addition of NaCl to the medium, by proton-induced X-ray emission. We compared the elemental composition of the field AMF to those of the soil and the associated plants. The spores and hyphae from the saline soils showed strongly elevated levels of Ca, Cl, Mg, Fe, Si, and K compared to their growth environment. In contrast, the spores of both the field-derived AMF and the in vitro grown G. intraradices contained lower or not elevated Na levels compared to their growth environment. This resulted in higher K:Na and Ca:Na ratios in spores than in soil, but lower than in the associated plants for the field AMF. The K:Na and Ca:Na ratios of G. intraradices grown in monoxenic cultures were also in the same range as those of the field AMF and did not change even when those ratios in the growth medium were lowered several orders of magnitude by adding NaCl. These results indicate that AMF can selectively take up elements such as K and Ca, which act as osmotic equivalents while they avoid uptake of toxic Na. This could make them important in the alleviation of salinity stress in their plant hosts.

Dinophysis blooms in the deep euphotic zone of the Baltic Sea: do they grow in the dark?

In situ growth rates of the toxin-producing dinoflagellate Dinophysis norvegica collected in the central Baltic Sea were estimated during the summers of 1998 and 1999. Flow cytometric measurements of the DNA cell cycle of D. norvegica yielded specific growth rates (μ) ranging between 0.1 and 0.4 per day, with the highest growth rates in stratified populations situated at 15–20 m depth. Carbon uptake rates, measured using 14C incubations followed by single-cell isolation, at irradiances corresponding to depths of maximum cell abundance were sufficient to sustain growth rates of 0.1–0.2 per day. The reason for D. norvegica accumulation in the thermocline, commonly at 15–20 m depth, is thus enigmatic. Comparison of depth distributions of cells with nutrient profiles suggests that one reason could be to sequester nutrients. Measurements of single-cell nutrient status of D. norvegica, using nuclear microanalysis, revealed severe deficiency of both nitrogen and phosphorus as compared to the Redfield ratio. It is also possible that suitable prey or substrate for mixotrophic feeding is accumulating in the thermocline. The fraction of cells containing digestive vacuoles ranged from 2 to 22% in the studied populations. Infection by the parasitic dinoflagellate Amoebophrya sp. was observed in D. norvegica in all samples analysed. The frequency of infected cells ranged from 1 to 3% of the population as diel averages, ranging from 0.2 to 6% between individual samples. No temporal trends in infection frequency were detected. Estimated loss rates based on observed infection frequencies were 0.5–2% of the D. norvegica population daily, suggesting that these parasites were not a major loss factor for D. norvegica during the periods of study. (Less)

Elemental composition of ectomycorrhizal mycelia identified by PCR-RFLP analysis and grown in contact with apatite or wood ash in forest soil

Abstract The aim of this study was to identify ectomycorrhizal species with a potential to release elements from apatite and wood ash and accumulate them in the mycelia. Fungal rhizomorphs and mycelia were sampled from sand-filled mesh bags with or without amendment of apatite or wood ash. The mesh bags were buried in forest soil in the field for 13 or 24 months. Elemental composition of the samples was analyzed with particle-induced X-ray emission and the fungus was identified by polymerase chain reaction-restriction fragment length polymorphism analysis of the ITS-region of ribosomal DNA. The majority of rhizomorphs and mycelia collected from the mesh bags were of mycorrhizal origin with Paxillus involutus being the most common species (31%). Other identified species were Thelephora terrestris, Suillus granulatus and Tylospora fibillosa. S. granulatus contained 3-15 times more K (3 mg g(-1)) than the other species and had large calcium-rich crystals deposited on the surface of rhizomorphs when grown in contact with apatite. P. involutus contained the largest amount of Ca (2-7 mg g(-1)). Wood ash addition increased the amount of Ti, Mn and Pb in the rhizomorphs while apatite addition increased the amount of Ca in the rhizomorphs. The high concentration of K in S. granulatus rhizomorphs suggests that this fungus is a good accumulator of K while P. involutus appeared to accumulate heavy metals originating from wood ash.

Elemental Analysis Mirrors Epidermal Differentiation

Using a scanning nuclear microprobe, the distribution of elements and trace elements of skin cross sections of normal skin, non-lesional psoriatic skin and in dry atopic skin have been mapped. In non-lesional psoriatic skin and in dry atopic skin the epidermal Ca-gradient is higher than that of normal skin. In addition, abnormally high Fe and Zn levels were recorded in the stratum granulosum and corneum regions in the pathological skin. It is suggested that these findings correlate to an increased cell turnover in the basal cell layer of the psoriatic and atopic skins. The ratio of Ca/Zn in stratum corneum of paralesional psoriatic skin is approximately 8:1 compared to 12: 1 in normal skin and 15: 1 in atopic skin. This suggests that the differentiation process in paralesional psoriatic skin may actually be an example of disturbed programmed cell death.

Dedicated accelerator and microprobe line

Abstract The development of a dedicated facility for nuclear microprobe analysis and the experiences from using it are discussed. The general properties of the present Lund nuclear microprobe will be described and the advantages of using a dedicated accelerator discussed.

PIXE analysis to estimate the elemental composition of ectomycorrhizal rhizomorphs grown in contact with different minerals in forest soil

The aim of this study was to investigate possible interactions between minerals and ectomycorrhizal (EM) mycelia. Fungal rhizomorphs growing in association with apatite and/or biotite were sampled both from a laboratory experimental system (Rhizopogon sp. and Pinus muricata) and from mesh bags buried in forest soil in the field. The elemental composition of the samples was analyzed with particle-induced X-ray emission (PIXE). Many EM rhizomorphs associated with apatite in laboratory systems and in mesh bags contained larger amounts of Ca (mean ranges between 12 and 31 mg Ca g(-1)) than similar rhizomorphs connected to acid-washed sand (range 0.3-3.5 mg Ca g(-1)). Ca originating from apatite was deposited as calcium oxalate crystals on the surface of the rhizomorphs. EM mycelium produced in mesh bags had a capacity to mobilize 0.6 mg P kg(-1) year(-1) from apatite-amended sand (which is 0.04% of the added apatite). A high concentration of K in some rhizomorphs (up to 11 mg K g(-1)) suggests that these fungi are good accumulators of K and may have a significant role in transporting K to trees

Calibration and long-term stability of a PIXE set-up

A general mass calibration procedure of a PIXE set-up is described. As an example, the results of the calibration of the PIXE set-up in Lund are given. For the calibration commercially available standards were used. The parameters of a physical model were adjusted to fit a calibration curve. To decrease errors due to inhomogeneities of the standard foils, the average from four different pieces of each foil was taken. The accuracy of the calibrated system in Lund is estimated to be better than 5% for individual elements and still less for ratios of adjacent elements. For long-term quality control of the results, a special sample has been designed. This consists of a thick silver plate with a small copper spot in the centre. It is very sensitive to small changes in a PIXE set-up. By analysing this sample periodically, mistakes in the arrangement are promptly detected-a feature which is much appreciated in a mixed research, development and routine analysis laboratory. Also a measure of the long-term stability is obtained. For the Lund system, the long-term stability is 2.5% for a homogeneous sample and 4% for a small (inhomogeneous) sample.