Quantitative changes in protein expression of cadmium-exposed poplar plants

Cadmium (Cd) pollution is a worldwide major concern having, among others, deleterious effects on plants. In the present work, the effects of a 20 microM Cd exposure in hydroponics culture during 14 days were evaluated in young poplar leaves. Proteins were analysed by 2-D DIGE, followed by MALDI-TOF-TOF identification. Additionally, growth and other physiological parameters were monitored during the experiment. Treated plants exhibited an inhibition of growth and visual symptoms appeared after 7 days. A significant accumulation of Cd in all organs was recorded by ICP-MS analysis. A number of changes in the expression of proteins with various functions were identified; in particular a decreased abundance of oxidative stress regulating proteins, whereas pathogenesis-related proteins showed a drastic increase in abundance. Furthermore, a large number of proteins involved in carbon metabolism showed a decrease in abundance, while proteins involved in remobilizing carbon from other energy sources were upregulated. In conclusion, the negative effect of Cd could be explained by a deleterious effect on protein expression from the primary carbon metabolism and from the oxidative stress response mechanism. Accumulation of Cd in stems of poplar, coupled with a low impact of Cd on physiological parameters, promotes the use of poplar trees for phytoremediation purposes.     

The Expression of Cyclin D1 during Adipogenesis in Pig Primary Stromal–Vascular Cultures

Objective: Our objective in this study was to measure the expression of cyclin D1 in pig primary stromal‐vascular (S‐V) cells as they differentiate into adipose cells and to identify which factors may alter cyclin D1 expression. Research Methods and Procedures: Western blot analysis was performed on cultured S‐V cells using 8% sodium dodecyl sulfate‐polyacrylamide gels, mouse monoclonal cyclin D1 antibody, and anti‐mouse IgG secondary labeled with horseradish peroxidase. For immunocytochemistry, cultures were fixed with 4% paraformaldehyde and incubated with anti‐CCAAT/enhancer binding protein (C/EBPα) and anti‐cyclin D1 together. Cyclin D1 expression was evaluated in 105‐day fetal dorsal subcutaneous tissues using paraffin sections. Results: Our results with Western blot analysis showed that cyclin D1 was found in freshly isolated S‐V cells and continued to be expressed during the first 3 days of adipose cell development with a significant increase in late development at day 9. Elevated cyclin D1 levels were colocalized with C/EBPα beginning at day 3 and remained colocalized with C/EBPα through day 9. Removing insulin from cultures resulted in a reduction in differentially elevated levels of cyclin D1. Discussion: The elevated level of cyclin D1 expression colocalized with C/EBPα expression is unexpected because differentiated adipocytes would be expected to have reduced proliferative potential. The elevated levels of cyclin D1 expression we observed in mature adipocytes depend on insulin. In addition, cyclin D1 is absent from lipid‐filled fetal adipose cells in vivo, where insulin levels are very low. The activity of cyclin D1 in differentiated adipocytes may be directed toward proteins outside of the cell cycle.     

Concepts in plant stress physiology. Application to plant tissue cultures

Because the term stress is used, most often subjectively, with variousmeanings, this paper first attempts to clarify the physiological definition,andthe appropriate terms as responses in different situations. The flexibility ofnormal metabolism allows the development of responses to environmental changeswhich fluctuate regularly and predictably over daily and seasonal cycles. Thusevery deviation of a factor from its optimum does not necessarily result instress. Stress begins with a constraint or with highly unpredictablefluctuations imposed on regular metabolic patterns that cause bodily injury,disease, or aberrant physiology. Stress is the altered physiological conditioncaused by factors that tend to alter an equilibrium. Strain is any physicaland/or chemical change produced by a stress, i.e. every established condition,which forces a system away from its thermodynamic optimal state. The papersecondly summarises the Strasser's state-change concept which is preciselythat suboptimality is the driving force for acclimation (genotype level) oradaptation (population level) to stress. The paper continues with the actualknowledge on the mechanisms of stress recognition and cell signalling. Briefly:plasma membranes are the sensors of environmental changes; phytohormones andsecond messengers are the transducers of information from membranes tometabolism; carbon balance is the master integrator of plant response; betwixtand between, some genes are expressed more strongly, whereas others arerepressed. Reactive oxygen species play key roles in up- and down-regulation ofmetabolism and structure. The paper shows finally that the above concepts canbeapplied to plant tissue cultures where the accumulating physiological andgenetical deviations (from a normal plant behaviour) are related to thestressing conditions of the in vitro culture media and ofthe confined environment. The hyperhydrated state of shoots and the cancerousstate of cells, both induced under conditions of stress in invitro cultures, are identified and detailed, because they perfectlyillustrate the stress-induced state-change concept. It is concluded that stressresponses include either pathologies or adaptive advantages. Stress may thuscontain both destructive and constructive elements : it is a selection factoraswell as a driving force for improved resistance and adaptive evolution.     

Visualization of Differential Gene Expression – Using Fluorescence‐Based cDNA‐AFLP

cDNA‐AFLP is one of the techniques developed to study differentially expressed genes. This recent technique is advantageous because it does not need prior sequence knowledge and is reliable due to highly stringent PCR conditions. The traditional cDNA‐AFLP method uses radioactively labelled products and is characterised by high sensitivity and resolution. Here, the use of Cy5‐labelled primers to detect products on polyacrylamide gels is reported. This non‐radioactive method, based on fluorescence, is shown to be faster and the recovery of interesting bands is easier. The study of the differential gene expression of the interaction between potato and Phytophthora infestans was used for the valuation of this method. Different gene expression profiles – such as up‐regulation, down‐regulation or point expression – were obtained. Moreover, this technique was shown to be highly reproducible.     

Adipocyte Development is Dependent Upon Stem Cell Recruitment and Proliferation of Preadipocytes

KRAS, KRYSTYNA M., DOROTHY B. HAUSMAN, GARY J. HAUSMAN, AND ROY J. MARTIN. Adipocyte development is dependent upon stem cell recruitment and proliferation of preadipocytes. Obes Res. Objectives: The ability to acquire fat cells persists over the life spans of animals. It is unknown whether adipocyte acquisition is the result of preadipocyte proliferation or stem cell recruitment to become adipocytes. The purposes of these studies were 1) to characterize early differentiation of stromal vascular (S-V) cells to preadipocytes as it is influenced by insulin, dexamethasone (DEX), and insulin-like growth factor-I (IGF-I); and 2) to determine whether new fat cells arise from stem cell recruitment or preadipocyte proliferation. Research Methods and Procedures: Freshly isolated S-V cells from rat inguinal adipose tissues were plated for 24 hours then exposed to serum-free medium. Results: Approximately 15% of freshly plated S-V cells were preadipocytes as determined by a preadipocyte specific marker, AD3. Total cell number and proportion of preadipocytes were significantly greater with 100 nM insulin treatment than with 0, 0. 1, or 1. 0 nM, but IGF-I treatment at 10 nM resulted in preadipocyte development similar to that with 100 nM insulin treatment. The addition of 5 nM DEX to the 100 nM insulin treatment resulted in a 20% increase in preadipocyte number by day 2 when compared to either treatment alone. 5-Bromo-2′-deoxyuridine treatment suppressed the increased proportion of preadipocytes from days 0–2 in non-insulin treated cells and prevented the increase typically observed with insulin. A mitosis inhibitor also significantly reduced the proportion of preadipocytes. Discussion: These results show for the first time that S-V cells are recruited as preadipocytes and that proliferation of these preadipocytes and early differentiation occur simultaneously.     

Physiological and proteome study of sunflowers exposed to a polymetallic constraint

The new energy requirements of the growing world population together with the actual ecological trend of phytoremediation have made challenging the cultivation of energetic crops on nonagricultural lands, such as those contaminated with trace elements. In this study, phenotypical characterization and biochemical analyses were combined to emphasize the global response of young sunflowers (Helianthus annuus L.) grown in hydroponic media contaminated with different Cd, Ni, and Zn concentrations. Leaves and roots of sunflowers reaching the stage “2‐extended leaves” and exposed to different trace metal concentrations were harvested and analyzed by 2D‐DIGE in order to study in depth the molecular responses of the young plants upon the polymetallic exposure. Proteomics confirmed the observed global reduction in growth and development. If photosynthetic light reactions and carbon metabolism were the most affected in leaves, in roots significant disruptions were observed in proteins involved in respiration, oxidative balance, protein and gene expression, and in the induction of programmed cell death. Elemental analyses of the plantlets indicated a profound impact of the treatment resulting in misbalance in essential micronutrients. Altogether, this study highlights the sensitivity of the sunflower to a polymetallic pollution and indicates that its use as a remediative tool of trace element polluted soils is limited.     

Cadmium and Zinc are differentially distributed in Populus tremula x P. alba exposed to metal excess

Abstract

Poplar plants were exposed during 61 days to a soil added with heavy metals so as to contain 300 mg Zn²⁺.kg^?1 soil dry weight (SDW) (Zinc) or 50 mg Cd²⁺.kg^?1 SDW (Cadmium). The Cd treatment induced a delayed growth of poplar, whereas Zn induced no change in physiological parameters. Both treatments resulted in a significant metal accumulation in plants. Zn²⁺ and Cd²⁺ exhibited contrasting distribution within tissues, indicating dissimilar handling by the plant. The main difference was the efficient compartmentalisation of Zn²⁺ in specific organ parts: old leaves and bark, while Cd²⁺ did not exhibit such a compartmentalisation. Results were also compared with a previous work where plants were exposed to 360 mg Cd²⁺.kg^?1 SDW.     

Success of maximum likelihood phylogeny inference in the four-taxon case

We used simulated data to investigate a number of properties of maximum- likelihood (ML) phylogenetic tree estimation for the case of four taxa. Simulated data were generated under a broad range of conditions, including wide variation in branch lengths, differences in the ratio of transition and transversion substitutions, and the absence of presence of gamma-distributed site-to-site rate variation. Data were analyzed in the ML framework with two different substitution models, and we compared the ability of the two models to reconstruct the correct topology. Although both models were inconsistent for some branch-length combinations in the presence of site-to-site variation, the models were efficient predictors of topology under most simulation conditions. We also examined the performance of the likelihood ratio (LR) test for significant positive interior branch length. This test was found to be misleading under many simulation conditions, rejecting too often under some simulation conditions. Under the null hypothesis of zero length internal branch, LR statistics are assumed to be asymptotically distributed chi 2(1); with limited data, the distribution of LR statistics under the null hypothesis varies from chi 2(1).