Long-term cadmium exposure influences the abundance of proteins that impact the cell wall structure in Medicago sativa stems.

Cadmium (Cd) is a non-essential, toxic heavy metal that poses serious threats to both ecosystems and human health. Plants employ various cellular and molecular mechanisms to minimise the impact of Cd toxicity and cell walls function as a defensive barrier during Cd exposure. In this study, we adopted a quantitative gel-based proteomic approach (two-dimensional difference gel electrophoresis) to investigate changes in the abundance of cell wall and soluble proteins in stems of Medicago sativa L. upon long-term exposure to Cd (10 mg·Cd·kg-1 soil as CdSO4 ). Obtained protein data were complemented with targeted gene expression analyses. Plants were affected by Cd exposure at an early growth stage but seemed to recover at a more mature stage as no difference in biomass was observed. The accumulation of Cd was highest in roots followed by stems and leaves. Quantitative proteomics revealed a changed abundance for 179 cell wall proteins and 30 proteins in the soluble fraction upon long-term Cd exposure. These proteins are involved in cell wall remodelling, defence response, carbohydrate metabolism and promotion of the lignification process. The data indicate that Cd exposure alters the cell wall proteome and underline the role of cell wall proteins in defence against Cd stress. The identified proteins are linked to alterations in cell wall structure and lignification process in stems of M. sativa, underpinning the function of the cell wall as an effective barrier against Cd stress.

Towards a synthetic view of potato cold and salt stress response by transcriptomic and proteomic analyses.

Potato can suffer from several abiotic stresses such as cold temperature, high soil salinity, lack of water or heavy metal exposure, to name a few. They are known to affect plant growth as well as productivity, with differential regulations at several levels. Potato response to cold and salt exposure was investigated at both transcriptomic and proteomic levels in a growth chamber experiment. Cold exposure in potato resulted in a higher number of significantly differentially regulated genes compared to salt exposure, whereas there were nearly three times more differentially regulated proteins after salt exposure when compared to cold exposure. The allocation of up and down-regulated genes at the functional category level also differed between salt and cold exposure although common trends, previously described in various abiotic stresses, were observed. In both stresses, the majority of photosynthesis-related genes were down-regulated whereas cell rescue and transcription factor-related genes were mostly up-regulated. In the other functional categories no common trend was observed; salt exposure results displayed a strong down-regulation of genes implicated in primary metabolism, detoxication apparatus and signal transduction, whereas upon cold exposure, up and down-regulated genes were similar in number. At the proteomic level, the abundance of the majority of identified proteins was increased except for the photosynthesis-related proteins, which were mostly less abundant after both salt and cold exposure. Common responses between salt and cold stress and specific responses inherent to these abiotic stresses are described.

A parentage study of closely related Ukrainian wine grape varieties using microsatellite markers.

Four bred grapevine varieties released for commercial cultivation in Ukraine, namely 'Antey Magarachskii', 'Rubinovyi Magaracha', 'Granatovyi Magaracha' and 'Rubin Golodrigi', and their putative parental forms were genotyped using six microsatellite loci. Genotypes were compared with breeding records to verify genetic relationships among varieties. Results of the analysis confirmed four of six parent-offspring relationships. Results of the analysis allow to assume that genotype 'Seyve Villard 20347' is the direct parent of 'Antey Magarachskii' instead of its grandparent. The first-studied accession believed to be that of Granatovyi Magaracha was identified as impurity. In order to verify the parentage of Granatovyi Magaracha, rest accessions of that variety and its putative parent Antey Magarachskii were additionally genotyped at 13 nuclear loci and at three chloroplast loci. The parent-offspring relationship was confirmed, as all Granatovyi Magaracha accessions had a common allele with the parent variety Antey Magarachskii at each locus and the same chlorotype A. Different Granatovyi Magaracha accessions could have been obtained via vegetative propagation of two seedlings which arose from one crossing.

Chloroplast DNA phylogeography of European ashes, Fraxinus sp. (Oleaceae): roles of hybridization and life history traits.

We investigated range-wide phylogeographic variation in three European ash species (Fraxinus sp., Oleaceae). Chloroplast DNA (cpDNA) microsatellites were typed in the thermophilous Fraxinus angustifolia and Fraxinus ornus and the observed haplotypes and the geographic distribution of diversity were compared to cpDNA data previously obtained in the more cold-tolerant Fraxinus excelsior. We found wide-ranging haplotype sharing between the phylogenetically close F. angustifolia and F. excelsior, suggesting hybridization (i) in common glacial refuges in the Iberian Peninsula, northern Italy, the eastern and/or Dinaric Alps and the Balkan Peninsula, and/or (ii) during postglacial recolonization. The data allowed us to propose additional glacial refuges for F. angustifolia in southern Italy and in Turkey, and populations from the latter region were particularly polymorphic. There was evidence for refuge areas in Italy, the Balkan Peninsula and Turkey for F. ornus, which did not share any single chloroplast haplotype with the other species. In both F. angustifolia and F. ornus, cpDNA diversity (h(S) = 0.027 and h(S) = 0.009, respectively) was lower and fixation levels (G(ST) = 0.964 and G(ST) = 0.983, respectively) higher than in sympatric F. excelsior (h(S) = 0.096, G(ST) = 0.870). These diversity patterns could be due to temperature tolerance or the demographic history.

Metabolic responses to water deficit in two Eucalyptus globulus clones with contrasting drought sensitivity.

We compared the metabolic responses of leaves and roots of two Eucalyptus globulus Labill. clones differing in drought sensitivity to a slowly imposed water deficit. Responses measured included changes in concentrations of soluble and insoluble sugars, proline, total protein and several antioxidant enzymes. In addition to the general decrease in growth caused by water deficit, we observed a decrease in osmotic potential when drought stress became severe. In both clones, the decrease was greater in roots than in leaves, consistent with the observed increases in concentrations of soluble sugars and proline in these organs. In roots of both clones, glutathione reductase activity increased significantly in response to water deficit, suggesting that this enzyme plays a protective role in roots during drought stress by catalyzing the catabolism of reactive oxygen species. Clone CN5 has stress avoidance mechanisms that account for its lower sensitivity to drought compared with Clone ST51.

Polyamine, carbohydrate, and proline content changes during salt stress exposure of aspen (Populus tremula L.): involvement of oxidation and osmoregulation metabolism.

Excess salt in the soil solution affects the plant either through osmotic or ionic effects. Poplar trees, as fast growing pioneer tree species, are thought to be potential suitable candidates for afforestation on saline soils. Osmotic and oxidative stress induced by salinity could be reduced by the production and accumulation of compatible solutes and osmoprotectants in the plant. In this respect, metabolites of this type could be interesting markers for the improvement of salt stress tolerance in breeding programmes. Results have shown that Populus tremula was able to cope with up to 150 mM NaCl without any effect on plant survival. During stress application, the endogenous level of malondialdehyde did not vary significantly between the treatments, indicating that the level of lipid peroxidation was similar in the control and in the stressed plants. However, proline, spermine, sucrose, mannitol, and raffinose levels increased temporarily or throughout the salt treatment. All these molecules are more or less closely related to antioxidant or osmoprotectant mechanisms during stress, suggesting a key role for these compatible solutes, osmoprotectants, and their metabolism for salt stress resistance. The accumulation of free proline, sucrose and mannitol, and the transitory increase in spermine level observable during low and high NaCl application must be considered as general salt stress reaction markers.

Responses of poplar to chilling temperatures: proteomic and physiological aspects.

The effects of cold acclimation on primary metabolism in actively growing poplar ( Populus tremula L. x P. tremuloides Michaux) were studied. Three-month-old poplar plants were exposed to chilling stress (4 degrees C) and compared to plant material kept at a control temperature (23 degrees C). This treatment did not affect the survival of the plants but growth was almost stopped. The freezing tolerance of the adult leaves increased from -5.7 degrees C for the control plants to -9.8 degrees C after 14 days of exposure to 4 degrees C. During acclimation, the evolution of soluble carbohydrate contents was followed in the leaves. Sucrose, glucose, fructose and trehalose accumulated rapidly under chilling conditions, while raffinose content increased after one week at 4 degrees C. Proteomic analyses, by bidimensional electrophoresis, performed during this stage revealed that a large number of proteins had higher expression, while much less proteins disappeared or had a lower abundance. MALDI-TOF-MS analyses enabled ca. 30 spots to be proposed for candidate proteins. Among the accumulating or appearing proteins proposed, about a third presented similarities with chaperone-like proteins (heat shock proteins, chaperonins). In addition, dehydrins and other late embryogenesis abundant proteins, i.e., stress-responsive proteins, detoxifying enzymes, proteins involved in stress signalling and transduction pathways were also activated or newly synthesised. Finally, cold exposure induced a decrease in the candidate proteins involved in cell wall or energy production.

Estimating seed vs. pollen dispersal from spatial genetic structure in the common ash.

Spatial genetic structure was analysed with five highly polymorphic microsatellite loci in a Romanian population of common ash (Fraxinus excelsior L.), a wind-pollinated and wind-dispersed tree species occurring in mixed deciduous forests over almost all of Europe. Contributions of seed and pollen dispersal to total gene flow were investigated by analysing the pattern of decrease in kinship coefficients among pairs of individuals with geographical distance and comparing it with simulation results. Plots of kinship against the logarithm of distance were decomposed into a slope and a shape component. Simulations showed that the slope is informative about the global level of gene flow, in agreement with theoretical expectations, whereas the shape component was correlated with the relative importance of seed vs. pollen dispersal. Hence, our results indicate that insights into the relative contributions of seed and pollen dispersal to overall gene flow can be gained from details of the pattern of spatial genetic structure at biparentally inherited loci. In common ash, the slope provided an estimate of total gene dispersal in terms of Wright's neighbourhood size of Nb = 519 individuals. No precise estimate of seed vs. pollen flow could be obtained from the shape because of the stochasticity inherent to the data, but the parameter combinations that best fitted the data indicated restricted seed flow, sigmas pound 14 m, and moderate pollen flow, 70 m pound sigmap pound 140 m.

Assessment of genetic structure within and among Bulgarian populations of the common ash (Fraxinus excelsior L.).

We analysed genetic variation within and between populations of the common ash from Bulgaria in order to extract biological information useful in the context of conservation management of eastern European genetic resources of noble hardwood species. A total of 321 trees from three regions of Bulgaria were typed at six highly polymorphic microsatellite loci. Analysis of within-population inbreeding suggests an upper boundary value of 2.7% for the selfing rate. Significant spatial genetic structure consistent with models of isolation by distance was detected within four out of 10 populations as well as among populations. Estimates of neighbourhood size in the range 38-126 individual trees were obtained based on spatial genetic structure analyses at either the intrapopulation or interpopulation level. Differentiation among populations explained only about 8.7% of total genetic diversity. These results are discussed in comparison with data from social broad-leaved species such as oak and beech.

Are hyperhydric shoots of Prunus avium L. energy deficient?

The content of oxidized and reduced pyridine nucleotides and some enzymatic activities of the oxidative pentose phosphate and glycolytic pathways were compared in normal (NS, growing on agar) and hyperhydric (HS, growing on gelrite) shoots of Prunus avium L. after 4 weeks of in vitro culture. The chlorophyll fluorescence from leaves and the redox capacity of the plasma membrane (reduction of exogenously added ferricyanide) of both types of shoots were recorded. The pool of oxidized and reduced pyridine nucleotides was lower in HS than in NS. These results suggested a reduced metabolism of HS in comparison to normal ones. This hypothesis was also supported by other observations. First, chlorophyll fluorescence measurements showed a lower chlorophyll content and a slight reduction of the photosynthetic capacity in HS. Second, the low activity of some enzymes of oxidative pentose phosphate pathway (OPP) and glycolysis indicated a decline of these biochemical pathways in HS with the consequence of a reduced production of chemical energy in the form of NAD(P)H and ATP. Finally, the lower reduction of ferricyanide by HS suggested a lower rate of redox reactions at the level of the plasma membrane of these shoots in comparison to NS.

Compared responses of poplar cuttings and in vitro raised shoots to short-term chilling treatments.

In woody plants, chilling stress occurs during the early spring growth and can have important economic consequences. The aim of this study was to compare the reliability of two different experimental systems, 3-month-old softwood cuttings and in vitro-grown shoots, to study chilling effects in a poplar clone (Populus tremula×P. tremuloides cv. Muhs1). Different parameters were recorded: lignin content, sucrose concentration and protein and fresh weight variation, during a 2-week treatment at 10  °C. Two families of polypeptides of high molecular weight (110 and 116 kDa) were shown to accumulate in response to chilling in both cuttings and microshoots. For some of the parameters studied, i.e. appearance of some groups of polypeptides and reduction of fresh weight gain, both in vitro and ex vitro systems were suitable and produced similar results. In contrast, for some other observations, i.e. on sucrose concentration and lignin content, the systems led to different conclusions. While sucrose and lignin contents were shown to increase in cuttings submitted to chilling, no variation in lignin and only a small temporary peak of sucrose could be observed in microshoots kept under chilling. These parameters seem not to be suitable for studying the response of poplar in the in vitro system in such a short-term study.

Characteristics of demand for pharmaceutical products: an examination of four cephalosporins.

We model demand for four cephalosporins and compute own- and cross-price elasticities between branded and generic versions of the four drugs. We model demand as a multistage budgeting problem, and we argue that such a model is appropriate to the multistage nature of the purchase of pharmaceutical products, in particular the prescribing and dispensing stages. We find quite high elasticities between generic substitutes and also significant elasticities between some therapeutic substitutes.

Changes in the concentrations of auxins and polyamines during rooting of in-vitro-propagated walnut shoots.

Rooting was induced in in-vitro-propagated walnut (Juglans regia L.) shoots by subculturing the shoots on rooting medium containing agar and 3 mg l(-1) indole-3-butyric acid (IBA) for 7 days in darkness. Changes in the concentrations of endogenous free indole-3-acetic acid (IAA), indole-3-acetylaspartic acid (IAAsp) and free polyamines were determined during culture on root-inducing medium. In extracts of whole shoots, the concentration of free IAA showed a transient peak at 60 h (around 48 h in extracts from basal shoot portions) and then remained at a relatively low concentration for the remainder of the 7-day culture period. The concentration of IAAsp in extracts of whole shoots peaked at about the same time as the concentration of free IAA, whereas the IAAsp concentration in extracts from basal shoot portions peaked earlier, at around 12 h. The concentrations of free polyamines in extracts of whole shoots increased soon after the shoots were transferred to root-inducing medium. The concentrations of IAA and IAAsp remained stable when the rooted shoots were transferred to a vermiculite/gelrite mixture (without auxin) and grown in light.

The role of calcitonin gene-related peptide in the mouse thymus revisited.

Calcitonin gene-related peptide has been identified by immunocytochemistry within the thymus of fetal through aged adult mice. Calcitonin gene-related peptide positive nerves are observed from embryonic day 17 throughout the lifespan of the mouse. A sparse cell population positive for CGRP is first observed during the late embryonic period at the corticomedullary boundary and the medulla, and it becomes more densely distributed in this region in the adult. In the thymus of the aged mouse the number of CGRP-positive cells diminishes. Pharmacologic studies demonstrated that fresh thymocytes display a receptor Kd for CGRP of 1.17 +/- 0.06 x 10(-10)M and a Bmax of 12.7 +/- 4.7 fmol/mg protein. Functional studies indicate that CGRP is a potent inhibitor of mitogen and antigen-stimulated proliferation of T cells and that it inhibits IL-2 production in cloned splenic T cells. Recent studies suggest that endogenous CGRP may serve as a natural inhibitor of inappropriate induction of mature, antigen-sensitive cells in the thymus as well as play a role in thymocyte education. These findings are discussed in terms of the distribution of CGRP cells and nerve terminals within the thymus and their relationship to positive and negative selection of the T-cell repertoire.

Calcitonin gene-related peptide in the developing and aging thymus. An immunocytochemical study.

Calcitonin gene-related peptide (CGRP) is known to block Con A and PHA induced T cell proliferation. As a first step in determining the role of this peptide in T cell education and function we have studied the distribution of CGRP within the developing mouse thymus using immunocytochemistry. CGRP-like immunoreactivity (CGRP-IR) was found in the thymic nerves in close proximity to blood vessels in the 17-day-old embryonic mouse thymus. A discrete population of small cells at the cortico-medullary junction also stained intensely for CGRP. As the mouse thymus reached maturity (three to eight weeks) CGRP innervation became more dense, with fibers running along the vasculature at the cortico-medullary boundary, then branching into the cortical and medullary regions. Some fibers were invested in the blood vessels while a large portion formed varicosities among the cells of the thymus. In the mature thymus, the small CGRP-IR cortico-medullary cells were more numerous, and CGRP-IR was also found in subcapsular and trabecular mast cells. The pattern of innervation remained the same in the aging mouse thymus (six months), but there appeared to be somewhat fewer cortico-medullary cells and an increase in mast cell number. In the aged (eighteen months) thymus, the small CGRP-IR cortico-medullary cells were rarely seen, but mast cells were more numerous, most of which stained positively for CGRP, in the connective tissue. Nerves containing CGRP-IR generally had the same distribution as in the younger mice but appeared somewhat truncated. The distribution of CGRP-IR nerves in the mouse thymus at different stages of development was similar to that reported for cholinergic (AChE-positive) nerves. Since the brain-stem vagal nuclei have been shown by retrograde transport studies to project to the thymus as well as to contain CGRP-IR neurons, our findings suggest that CGRP-IR thymic nerves may be derived from the vagus complex.

[The effect of chronic exposure to lead on lysosomal levels in amniotic fluid in an experimental model]. / Vliv chronické expozice olovu na hladinu lysozymu v plodové vode v modelovém pokuse.

Groups of female rats were exposed for prolonged periods to different lead concentrations in drinking water; in one group was also added to the drinking water, in the amniotic fluid of exposed females greatly reduced lysozyme values were found, as compared with controls. Concurrent Zn administration considerably reduced the effect of lead. Long-term exposure to low concentrations of lead caused a greater drop of lysozyme levels in amniotic fluid than that found in previous experiments after short-term exposure to higher lead concentration.