An untargeted metabolomic approach for the putative characterization of metabolites from Scenedesmus obliquus in response to cadmium stress.

Cadmium (Cd) is a widespread contaminant in aquatic systems and has a variety of toxicological implications on freshwater microorganisms. In this study, the green algae Scenedesmus obliquus was exposed to increasing Cd concentrations that inhibited growth by 20% (12.6 µmol L-1), 30% (39.8 µmol L-1) and 40% (83.2 µmol L-1) and the metabolite profiles of released and cellular biomolecules were explored using an untargeted direct infusion high resolution Fourier transform ion cyclotron resonance mass spectrometry approach. In Cd untreated cultures, intrinsic differences in composition existed between released biomolecules and freeze-dried cells. Based on putatively characterized compound groups, a greater proportion of Cys-GSH isomers and carboxyamides were present in exudates whereas sugar isomers and phosphonic acids comprised most cellular metabolites. In cultures exposed to 83.2 µmol L-1 Cd, an overall shift in metabolomic response across both released biomolecules and cellular components resulted in an increase of lipid-based esters, and Cys-GSH isomers. These two important metabolites are used in antioxidant defense mechanisms and reactive oxygen species prevention during cellular stress. The diversity of metabolites also decreased as Cd concentrations increased when compared to untreated cultures, suggesting that overall metabolites specialize upon metal stress. We show systemic shifts from sugar and carboxylic isomers to specialized proteins and lipid isomers to help S. obliquus cope with stress. These findings highlight the potential use of this green algae as a potential biosorbent and sheds light into the metabolomics of Cd toxicology and insights into microbial metal adaptation.

Linking molecular and optical properties of dissolved organic matter across a soil-water interface on Akimiski Island (Nunavut, Canada).

Dissolved organic matter (DOM) plays a crucial role in terrestrial and aquatic carbon and biogeochemical cycles; however, molecular transformations between aquatic and terrestrial systems remain poorly understood due to the complexity and heterogeneity of DOM. In this study, we investigated the molecular diversity of aquatic DOM and adjacent soil derived water extractable organic matter (WEOM) from seven locations on Akimiski Island, Nunavut using a combination of absorbance spectroscopy and Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS). Assigned elemental formula and Van Krevelen compositional analysis reveal compositional similarities in river, inland ponds and coastal pool sites for aquatic DOM and WEOM. More aromatic, oxygenated polyphenolic carbon rich molecules were found in aquatic DOM whereas WEOM was abundant in highly unsaturated aliphatic material. A total of 276 phenolic, unsaturated aliphatic, and vascular plant-derived polyphenolic molecules were identified as being conserved between WEOM and aquatic DOM at one river and two inland pond locations suggesting similar CHO sources from adjacent soils. Moreover, contributions of polyphenolic compounds in aquatic DOM and WEOM were greater at inland ponds than coastal pools, congruent with a greater aromaticity at inland sites. Our results highlight the similarities and differences in WEOM to aquatic DOM composition and how they range across surrounding watersheds that provide insight into the biogeochemical dynamics across a Canadian subarctic terrestrial-aquatic-continuum.

The regulatory dendritic cell marker C1q is a potent inhibitor of allergic inflammation.

The complement subunit C1q was recently identified as a marker for monocyte-derived regulatory dendritic cells supporting the differentiation of interleukin (IL)-10-secreting CD4+ T cells with a suppressive activity. Furthermore, C1q expression is upregulated in peripheral blood mononuclear cells of allergic patients in the course of successful allergen immunotherapy. Herein, we investigated a potential direct role of C1q in downregulating allergic inflammation. In mice with ovalbumin (OVA) or birch pollen (BP)-induced allergic asthma, C1q is as efficacious as dexamethasone to reduce both airway hyperresponsiveness (AHR), eosinophil, and ILC2 infiltrates in bronchoalveolar lavages, as well as allergen-specific T helper 2 cells in the lungs. Administration of C1q does not expand IL-10+/Foxp3+ regulatory T cells in the lungs, spleen, or in the blood. Depletion of plasmacytoid dendritic cells (pDCs) abrogates the capacity of C1q to reduce AHR and eosinophilic infiltrates in OVA-sensitized mice. Also C1q treatment inhibits the activation of human and mouse pDCs by CpGs, thereby demonstrating a critical role for pDCs in the anti-inflammatory activity of C1q. We conclude that regulatory dendritic cells can mediate a potent direct anti-inflammatory activity via the expression and/or secretion of molecules such as C1q, independently of their capacity to expand the pool of regulatory T cells.

Evaluation of free/labile concentrations of trace metals in Athabasca oil sands region streams (Alberta, Canada) using diffusive gradient in thin films and a thermodynamic equilibrium model.

The Athabasca's oil sands exploitation is controversial due to its potential risks to water quality but little is known about the temporal changes in the most bioavailable fraction of metal, the free/labile species. In this study, diffusive gradient in thin films (DGT) and the Windermere Humic Aqueous Model (WHAM VII) equilibrium model were used to examine the temporal changes in free/labile metal (Cu, Ni, Zn, Pb) species in three tributaries of the north-flowing Athabasca River in the Athabasca oil sands region (AOSR). The influence of dissolved organic matter (DOM) composition (i.e. fulvic: humic ratio) on modeled Cu and Ni speciation showed a negligible effect on the labile concentration. The best agreements (92 ± 8%) between DGT-labile and WHAM calculated labile concentrations were found assuming the formation of iron oxyhydroxides (FeO(OH)). The agreement was only 70 ± 7% in the presence of inorganic colloidal aluminum oxyhydroxides (AlO(OH)) and in the absence of any inorganic colloids. Together these results suggest that a change in DOM composition had limited impacts on modeled free metal ion concentrations. Although the concentration of the main metal ligand (i.e. DOM), varied from 9 to 40 ppm, no significant temporal differences in the abundance of WHAM-modeled labile species were found, suggesting mobility and bioavailability of Cu, Ni, Pb and Zn were comparable over the 2003-2012 period.

Assessing cadmium and vanadium accumulation using diffusive gradient in thin-films (DGT) and phytoplankton in the Churchill River estuary, Manitoba.

Diffusive gradient in thin films (DGT) and phytoplankton communities were evaluated for the measurement of Cd and V at environmentally relevant concentrations in laboratory settings and in the Churchill River estuary (Manitoba, Canada) during an annual spring melt. Despite rapid changes in hydrology and water quality, DGT samplers and intracellular Cd and V concentrations were positively correlated (0.79 < r(2) < 0.99), suggesting comparable accumulation trends between both DGT-labile and intracellular monitoring techniques. The largest accumulated concentrations of both Cd and V by DGT and phytoplankton accumulation methods were found later into the river discharge period. In controlled settings, accumulated Cd and V concentrations by the diatom Attheya septentrionalis displayed a strong correlation with metals accumulated by DGTs (r(2) > 0.99). Principal component analysis (PCA) reinforced similarities between both metal monitoring techniques and assessed how changing environmental variables during the river discharge period influenced each monitoring technique. Cd accumulation was influenced by DOC concentrations and protein-like DOM whereas ionic strength (i.e. conductivity) and humic-like DOM influenced V accumulation. The present findings suggest that (1) DGT is a versatile tool for monitoring bioaccumulation of Cd and V in highly dynamic environmental systems and (2) DOC concentration, DOM composition, conductivity, pH, and river discharge influence the bioavailability of Cd and V in estuarine and riverine waters.

Combining parallel factor analysis and machine learning for the classification of dissolved organic matter according to source using fluorescence signatures.

Parallel factor (PARAFAC) analysis of dissolved organic matter (DOM) fluorescence has facilitated a surge of investigation into its biogeochemical cycling. However, rigorous, PARAFAC-based methods for holistically distinguishing DOM sources are lacking. This study classified 1029 PARAFAC-analyzed excitation-emission matrices (EEMs) measured using DOM isolated from 24 different leaf leachates, rivers, and organic matter standards using four machine learning methods (MLM). EEMs were also divided into subsets to assess the impact of experimental treatments (i.e. whole EEMs, size fractionation, mixtures, quenching) and dataset properties (i.e. different numbers of EEMs from each leachate/river) on classification. A split-half validated, 10-component PARAFAC model was extended to 12 components to remove consistent peaks evident in model residuals. The 12-component model performed better than the 10-component model, correctly classifying up to 80 additional EEMs, when the dataset included size-fractionated DOM or several different sources (i.e. many leaf species and rivers); however, the 10-component model performed better for whole-sample EEMs when comparing leaf leachates to rivers. The MLM correctly classified whole EEMs of riverine DOM by source with up to 87.0% accuracy, leachates with up to 92.5% accuracy, and distinguished leachates from rivers with 97.2% accuracy. A difference of up to 17.3% in classification accuracy was observed depending on the MLM method used with the following order: multilayer perceptron = support vector machine > k-nearest neighbours â‰« decision tree; however, performances differed widely depending on the data subset. Classification accuracy for whole and size-fractionated rivers compared to whole and size-fractionated leachates using N-way partial least-squares discriminant analysis (NPLS-DA; 97.7%) was similar to that achieved using MLM. Combining MLM with PARAFAC is an effective method for classifying DOM based on its fluorescence signature because PARAFAC can isolate meaningful fluorescent species and unlike PLSDA, MLM constructs a single model which simultaneously classifies EEMs as belonging to one of several categories. A complete accounting of carbon flows through ecosystems should include the processes and sources that contribute to the disparate fluorescence signatures of riverine and leached DOM.

Snowpack deposition of trace elements in the Athabasca oil sands region, Canada.

The total recoverable and dissolved concentrations of 29 metals and metalloids were analyzed in snowpack collected at 91 sites in the Athabasca oil sands region, Canada in winter 2011. Based on deposition pattern from geographical centre, three groups were found: Type-1 metals (i.e. dissolved and total recoverable V; Mo) showed a significant exponential decrease with distance, suggesting oil sands development sources; Type-2 elements (e.g. Al, Sb, As, Ba, Fe, Ni, Tl, and Ti and Zn) showed exponentially decline patterns but with some local point sources; Type-3 elements (e.g. Cd, Cl, Cr, Mn, Sr and Th) deposition pattern represented local sources. A self-organizing map showed that sites with the highest elemental concentrations (Cluster I) were mainly located in the vicinity of upgrading facilities and along the north-south transects. The lowest elemental concentration sites (Cluster III) were the most distal sites or located in the western region of the study area.

Effects of molecular weight on the diffusion coefficient of aquatic dissolved organic matter and humic substances.

In situ measurements of labile metal species using diffusive gradients in thin films (DGT) passive samplers are based on the diffusion rates of individual species. Although most studies have dealt with chemically isolated humic substances, the diffusion of dissolved organic matter (DOM) across the hydrogel is not well understood. In this study, the diffusion coefficient (D) and molecular weight (MW) of 11 aquatic DOM and 4 humic substances (HS) were determined. Natural, unaltered aquatic DOM was capable of diffusing across the diffusive gel membrane with D values ranging from 2.48×10(-6) to 5.31×10(-6) cm(2) s(-1). Humic substances had diffusion coefficient values ranging from 3.48×10(-6) to 6.05×10(-6) cm(2) s(-1), congruent with previous studies. Molecular weight of aquatic DOM and HS samples (∼500-1750 Da) measured using asymmetrical flow field-flow fractionation (AF4) strongly influenced D, with larger molecular weight DOM having lower D values. No noticeable changes in DOM size properties were observed during the diffusion process, suggesting that DOM remains intact following diffusion across the diffusive gel. The influence of molecular weight on DOM mobility will assist in further understanding and development of the DGT technique and the uptake and mobility of contaminants associated with DOM in aquatic environments.

Relationships between molecular weight and fluorescence properties for size-fractionated dissolved organic matter from fresh and aged sources.

Relationships between the molecular weight (MW) and fluorescence properties of dissolved organic matter (DOM) are important considerations for studies seeking to connect these properties to water treatment processes. Relationships between the size and fluorescence properties of nine allochthonous DOM sources (i.e. leaf leachates, grass, and headwaters) were measured using asymmetrical flow field-flow fractionation (AF4) with on-line absorbance and fluorescence detectors. Correlations between optical properties and MW were readily apparent using parallel factor analysis (PARAFAC) coupled to self-organizing maps (SOM): protein/polyphenol-like fluorescence (peaks B and T) was highest at lower molecular weights (<0.5 kDa), fulvic/humic-like fluorescence (peaks A, C, and M) was highest at mid-weights (0.5-1 kDa), and humic-like fluorescence (Peaks A + C) was highest at larger molecular weights (>1 kDa). Proportions of peaks B, T, and A + C were significantly correlated with MW (p < 0.001). The first principal component (PC1, 42% of variation in fluorescence properties) was a significant predictor of sample MW (R² = 0.63, p < 0.05), while scores on PC2 (27% of total variance) traced a source-based gradient from deciduous leachates/headwaters through to coniferous leachates/headwaters. PC3 (13% of var.) was also correlated with MW (p < 0.005). A secondary peak in peak T fluorescence was associated with larger size fractions in aged sources, and scores on PC1 also traced a path from the leachates of fresher leaves, through more humified leaves, to headwaters. Findings are consistent with the hypothesis that the structure of aged DOM arises through supramolecular assembly.

Assessing the multisite binding properties of multiple sources of dissolved organic matter at nanomolar copper concentrations using piecewise linear regression and parallel factor analysis of fluorescence quenching.

This study reports on the development and application of a piecewise linear model for the determination of copper-binding parameters at concentrations in the nanomolar range using fluorescence quenching. L-Tyrosine, Suwannee River natural organic matter, and two leaf leachates with similar fluorescence signatures were used as test compounds, and results were compared with those of the standard Ryan-Weber model. The piecewise model was also applied to and compared with data from an earlier study. Parallel factor analysis (PARAFAC) was used to identify three to five independent fluorophores in each test compound, and copper-binding parameters were estimated for one to three binding sites for each fluorophore. The binding properties of similar and different fluorophores were also compared. The conditional binding strengths (log K') estimated using the piecewise approach were similar to those obtained using the Ryan-Weber approach (p > 0.05); however, the piecewise linear model provided superior results compared to models based on the Ryan-Weber equation in several ways, including (1) capable of distinguishing more binding sites for a single fluorophore, (2) capable of extracting binding parameters at environmentally relevant, nanomolar concentrations of copper, where fluorescence changes are often observed as enhancement, (3) greater precision over repeated titrations, and (4) no severe underestimation of complexing capacities. Finally, the copper-binding properties of PARAFAC components with similar optical signatures were found to be similar, both in sources with dramatically different and similar total fluorescence signatures.

Distinguishing dissolved organic matter at its origin: size and optical properties of leaf-litter leachates.

Dissolved organic matter (DOM) was leached from eight distinct samples of leaves taken from six distinct trees (red maple, bur oak at three times of the year, two sugar maple and two white spruce trees from disparate soil types). Multiple samples were taken over 72-96h of leaching. The size and optical properties of leachates were assessed using asymmetrical flow field-flow fractionation (AF4) coupled to diode-array ultraviolet/visible absorbance and excitation-emission matrix fluorescence detectors (EEM). The fluorescence of unfractionated samples was also analyzed. EEMs were analyzed using parallel factor analysis (PARAFAC) and principal component analysis (PCA) of proportional component loadings. Both the unfractionated and AF4-fractionated leachates had distinct size and optical properties. The 95% confidence ranges for molecular weight distributions were determined as: 210-440Da for spruce, 540-920Da for sugar maple, 630-800Da for spring oak leaves, 930-950Da for senescent oak, 1490-1670 for senescent red maple, and 3430-4270Da for oak leaves that were collected from the ground after spring thaw. In most cases the fluorescence properties of leachates were different for individuals from different soil types and across seasons; however, PCA of PARAFAC loadings revealed that the observed distinctiveness was chiefly species-based. Strong correlations were found between the molecular weight distribution of both unfractionated and fractionated leachates and their principal component loadings (R(2)=0.85 and 0.95, respectively). It is concluded that results support a species-based origin for differences in optical properties.

Influence of water chemistry and dissolved organic matter (DOM) molecular size on copper and mercury binding determined by multiresponse fluorescence quenching.

The effects of water chemistry (i.e. pH and Ca(2+) concentration) dissolved organic carbon (DOC) concentration) and DOM quality (i.e. composition and molecular weight) on metal complexation were successfully investigated by a combination of tangential flow filtration, excitation-emission matrix fluorescence, parallel factor analysis (PARAFAC), and fluorescence quenching on four freshwater samples and one extracted Suwannee River fulvic acid (SRFA). Two terrestrial and one microbial humic-like components were found in this study. Despite strong correlation between the Ryan-Weber model and the multiresponse model, the latter is more appropriate for the calculation of binding parameters in multiple-ligand DOM system. Decreasing pH from 6 to 4 significantly reduced logK-Cu(2+) from 5.22±0.24 to 4.60±0.30 at pH 6 and 4, respectively (p<0.001), while the impacts of Ca(2+) and DOC were not discernible at concentrations<100ppm and<2.06ppm, respectively. For natural freshwater DOM binding, the three humic-like components had similar logK values for both metals. High molecular weight (>1kDa) DOM generally had higher logK and binding fluorophore abundance than bulk (unfractionated) and low molecular weight (<1kDa) DOM for both metals. This trend however was not always true for Hg(2+) where the binding parameters were quite variable. Overall the combined results provide evidence that binding parameters are not only affected by water chemistry, but also depend on DOM molecular weight.

Binding interactions of algal-derived dissolved organic matter with metal ions.

The nature and composition of dissolved organic matter (DOM) strongly influences its binding properties to heavy metals and thus their fate, mobility and toxicity in aquatic environments. Fluorescence spectroscopy with parallel factor analysis (PARAFAC) was used to characterize DOM exuded by the cosmopolitan freshwater green algae Scenedesmus acutus during early exponential growth phase. One protein-like (peak T; C2) and two humic-like components (peaks A+C and A+M, C1 and C3, respectively) were split half validated on 122 emission-excitation matrices (EEMs). Our data show that both humic-like could be associated with biological activities. Unlike Cd, Pb and Zn, Cu strongly binds to algogenic DOM with conditional stability constants (logK) averaging 5.26±0.29 (from 4.85 to 5.36). Significant differences in logK values were found between humic-like PARAFAC components, indicating clear differences in the binding properties of humic-like components with copper.

BAFF and BAFF-R levels are associated with risk of long-term kidney graft dysfunction and development of donor-specific antibodies.

There are lines of evidence that B cells may play a role in transplantation. B cell activating factor, BAFF, is a homotrimer that has been shown to play a role in B cell survival, maturation and activation. To date, little is known of the role of BAFF and its receptors in transplantation. We analyzed the level of BAFF mRNA and its soluble protein, as well as transcripts coding for its receptors, BAFF-R, TACI and BCMA, in the blood of 143 patients with stable kidney transplant function 5 years or more posttransplantation. Three endpoints were analyzed: the time to renal dysfunction, the time to appearance of anti-HLA antibodies and the time to development of donor-specific antibodies. We established threshold values for BAFF and BAFF-R and showed that (1) stable patients with high BAFF-R levels had a higher risk of developing graft dysfunction, (2) patients with lower levels of BAFF transcripts or a higher level of soluble BAFF had a significantly higher risk of developing donor-specific antibodies. These data suggest that BAFF constitutes a risk factor for renal graft dysfunction and development of donor-specific antibodies. They also suggest that agents targeting BAFF-R interactions may offer new therapeutic opportunities in transplantation.

Determination of relative molecular weights of fluorescent components in dissolved organic matter using asymmetrical flow field-flow fractionation and parallel factor analysis.

Dissolved organic matter in aquatic systems is of variable structure and composition. Asymmetrical flow field-flow fractionation coupled to UV/vis diode array and fluorescence detectors (AF4-DAD-EEM) was used to assess the size and optical properties of dissolved organic matter. The results were analyzed using parallel factor analysis (PARAFAC) and statistical fractogram deconvolution to correlate fluorescing components with molecular weight fractions. This coupling, which is shown for the first time in this work, is a powerful method capable of revealing novel information about the size properties of PARAFAC components. Tyrosine/polyphenol-like fluorescence (peak B) was significantly correlated (p<0.05) with the smallest size group (relative molecular weight=310±10 Da), microbial humic-like and terrestrial visible humic-like fluorescence (peaks M, C, A) with the intermediate size group (1600±150 Da), and terrestrial fulvic-like and tryptophan/polyphenol-like fluorescence (peaks A and T) with the largest size group (4300±660 Da).

Chemical speciation and partitioning of trace metals (Cd, Co, Cu, Ni, Pb) in the lower Athabasca river and its tributaries (Alberta, Canada).

Concentrations of Cd, Co, Cu, Ni and Pb were measured in particulate and dissolved phases at 11 sites located upstream and near Athabasca oil sands development. The in situ discrimination between non-labile and labile dissolved metals was done using diffusive gradients in thin-films (DGT) devices. The DGT-labile fraction of Co and Ni was 30% lower near development sites whereas Cu, Cd and Pb showed minor changes spatially. It was found that an 8-fold increase in dissolved organic matter (DOM) near development induced a rapid decrease in DGT-labile metals. Dissolved metal concentrations were used along with DOM, major ions, nutrients, pH and conductivity to calculate the distribution of dissolved metal species using the speciation model WHAM. Labile-DGT metal concentrations agreed well with WHAM-predicted concentrations. It was also found that a significant amount of metals were associated with the non-DGT labile fraction (i.e. colloidal DOM) and colloid abundance was more important than suspended particulate matter abundance in influencing metal mobility near Athabasca oil soils development. Since changes in colloidal DOM levels are likely to be the result of surface mining activities, this confirms the serious effects of oil sands activities on metal biogeochemical cycles in the lower Athabasca River.

Spatio-temporal variation in the characteristics of dissolved organic matter in the streams of boreal forests: impacts on modelled copper speciation.

Dissolved organic matter (DOM) is an important constituent of natural waters that controls numerous biogeochemical processes such as the toxicity and mobility of metals. In order to predict how metals behave in the presence of DOM, it is necessary to understand its acidic properties. In this study, we report the variations in the acid character of aquatic organic acids using 30 years of sampling data collected from three boreal streams. Based on a charge balance model, significant spatial and temporal variation in carboxylic acid site density was observed. The seasonal average carboxylic group density ranged from 8.01 +/- 1.47 to 12.0 +/- 1.90 microeq mg C(-1) and the overall (multi-site) average site density (winter excluded) was 9.66 +/- 0.125 microeq mg C(-1) (n = 3193). Both different sources of DOM and seasonal differences in source availability were found to contribute to variations in site density. In the deciduous catchment where wetland contributions to DOM were negligible, the seasonal variability in site density was highly marked with increases of up to 50% observed between spring and fall. Less seasonal variation was noted at the coniferous sites, which had relatively high wetland source contributions. The geochemical equilibrium speciation model MINTEQA2 showed that the increase in site density observed from winter to fall coincided with a decrease in free copper concentration. We conclude that some source-based differences in DOM that may result from variation in both catchment characteristics and seasonal DOC loadings necessitate the determination of location-specific and/or seasonal site density values due to the resulting variability in metal speciation that has been predicted through modelling.

Organic colloid separation in contrasting aquatic environments with tangential flow filtration.

The use of tangential flow filtration (TFF) for size fractionation of natural dissolved organic matter was investigated. The performance of regenerated cellulose membrane with a nominal molecular weight cut-off of 1 kDa was examined on 20 samples from lake, river and estuary systems, characterised by contrasting dissolved organic carbon (DOC) contents and conductivity. The evaluation was based on absorbance, fluorescence and DOC measurements. Detailed protocols of membrane cleaning and conditioning nation are proposed. The ultrafiltration membrane can efficiently be cleaned to provide low carbon blank (<0.01 mg/L). Fluorescence measurements confirmed that the higher molecular weight compounds were isolated in the retentate and the lower molecular weight remain in the permeate. Mass balance for natural samples show good recovery for DOC (109 +/- 12%, n = 20) and fluorescence measurements (106 +/- 9%, n = 13). No relation between factors of concentration (fc) and mass balance quality was observed for the fc range 1.5-11. Moreover, high ionic strength and high DOC contents did not enhance membrane fouling. These findings demonstrate that reliable fractionations by TFF of natural organic colloids in aquatic systems can be achieved.

Use of chelating resins and inductively coupled plasma mass spectrometry for simultaneous determination of trace and major elements in small volumes of saline water samples.

For some saline environments (e.g. deeply percolating groundwater, interstitial water in marine sediments, water sample collected after several steps of fractionation) the volume of water sample available is limited. A technique is presented which enables simultaneous determination of major and trace elements after preconcentration of only 60 mL sample on chelating resins. Chelex-100 and Chelamine were used for the preconcentration of trace elements (Cd, Cu, Pb, Zn, Sc) and rare earth elements (La, Ce, Nd, Yb) from saline water before their measurement by inductively coupled plasma mass spectrometry. Retention of the major elements (Na, Ca, Mg) by the Chelamine resin was lower than by Chelex; this enabled their direct measurement in the solution after passage through the resin column. For trace metal recoveries both resins yield the same mass balance. Only Chelex resin enabled the quantitative recovery of rare earth elements. The major elements, trace metals and rare earth elements cannot be measured after passage through one resin only. The protocol proposes the initial use of Chelamine for measurement of trace and major elements and then passage the same sample through the Chelex resin for determination of the rare earth elements. The detection limit ranged from 1 to 12 pg mL(-1). At concentrations of 1 ng mL(-1) of trace metals and REE spiked in coastal water the precision for 10 replicates was in the range of 0.3-3.4% (RSD). The accuracy of the method was demonstrated by analyzing two standard reference waters, SLRS-3 and CASS-3.

Structure, organization and expression of two closely related novel Lea (late-embryogenesis-abundant) genes in Arabidopsis thaliana.

We have isolated and sequenced a 9.5 kb genomic region from A. thaliana, located on chromosome 2, which contains two tandemly arranged closely related genes (AtM10 and AtM17) coding for a new family of LEA proteins. The deduced proteins have a molecular mass of 11 and 29 kDa, respectively, are extremely hydrophilic except at their N-termini and share 70% amino acid (aa) identity. A 47 aa motif containing a 6-cysteine domain is present once in AtM10 and four times in AtM17. The short intergenic region, the identical position of the intron and the overall sequence homology suggest that these two genes evolved through a duplication event. This conclusion is supported by the presence of two homologous strictosidine synthase-like (pseudo)genes downstream from AtM17 and AtM10. Expression studies, using AtM10 and AtM17 cDNAs, revealed that both transcripts accumulate exclusively in seeds from late embryogenesis until two days after imbibition. Expression of both genes in young seedlings is repressed during ABA, salt or drought treatment, whereas a cold stress induces the expression of AtM17 only. In situ hybridization revealed that AtM10 transcripts are detected throughout the embryo while those of AtM17 are more localized to cotyledon cells.