Field proteomics of Populus alba grown in a heavily modified environment - An example of a tannery waste landfill.

Tannery waste is highly toxic and dangerous to living organisms because of the high heavy metal content, especially chromium [Cr(III)]. This study analysed the proteomic response of the Populus alba L. clone 'Villafranca' grown for 4years on a tannery waste landfill. In this extremely hostile environment, the plants struggled with continuous stress, which inhibited growth by 54%, with a 67% decrease in tree height and diameter at breast height compared to those of the forest reference plot, respectively. The leaves and roots of the tannery landfill-grown plants produced strong proteomic stress signals for protection against reactive oxygen species (ROS) and repair to ROS-damaged proteins and DNA as well as signals for protection of the photosynthetic apparatus. The content of HSP80 was also high. However, primary metabolic pathways were generally unaffected, and signals of increased protein protection, but not turnover, were found, indicating mechanisms of adaptation to long-term stress conditions present at the landfill. A proteomic tool, two-dimensional electrophoresis coupled with tandem mass spectrometry, was successfully applied in this environmental in situ study of distant plots (280km apart).

Response of leaf and fine roots proteomes of Salix viminalis L. to growth on Cr-rich tannery waste.

The tannery industry is a major source of anthropogenic chromium (Cr) contamination due to the large amounts of solid waste produced and its problematic management. The unique composition of tannery waste, usually high concentrations of Cr and other metals as well as organic matter and nutrients, makes it a great risk for soil and water environment but also a possible effective fertilizer for non-food plants that can tolerate metals. The goal of this study was to understand the adaptation mechanism of Salix viminalis to growth on Cr-rich tannery waste from an active landfill. We used a proteomic approach to identify leaf and fine roots proteins altered by tannery waste as compared to control soil conditions. We found no obvious symptoms of oxidative stress in leaves or fine roots. Proteomic results indicated some changes in metabolism, with increases in energy production processes and their greater efficiency for leaves rather than root development. Comparison between S. viminalis and P. × canescens response to tannery waste suggested that S. viminalis is not suitable for remediation of Cr-contaminated areas of a tannery waste landfill site.

Utilization of proteomics in experimental field conditions--A case study of poplars growing on grassland affected by long-term starch wastewater irrigation.

The presented study verified the possibility of using proteomics as a tool for investigating poplars growing on obviously separate plots. The examination covered poplars planted on grassland irrigated for 40 years with potato industry wastewater and in a plot appropriate for poplar planting, spaced at a distance of 67 km from each other (hereinafter referred to as forest). The work aimed to compare the obtained proteomic results with data on biometric and biochemical parameters and mineral composition as well as to assess, at a molecular level, the usefulness of grasslands for planting. Proteome analysis showed that most of the stress-related proteins detected were less abundant on the irrigated grassland, confirming the viability of its revegetation with poplars. Proteomic data corresponded well with the other results, highlighting the probable reason for the proteome changes; i.e. deficiency of phosphate ions detected in the forest area. Moreover, proteome analysis revealed biotic stress symptoms in plants growing on the grassland, which were also well explained by other data but would not have been detected without performing the proteomic analysis. Therefore, environmental plant proteomics is a useful and valuable tool during field studies, even when samples are taken from plots some distance apart.

Populus × canescens grown on Cr-rich tannery waste: Comparison of leaf and root biochemical and proteomic responses.

Treatment of tannery effluents generates large amounts of sediments containing concentrated doses of metals (mainly chromium). Such waste is most commonly disposed of by landfilling, which is hazardous to the ecosystem due to Cr leaching. Afforestation of disposal sites with fast growing trees could stabilize contaminants in the soil and prevent them from spreading. The aim of this study was to examine the adaptation of Populus × canescens Sm. to tannery waste using biochemical and proteomic methods. We analyzed changes in the leaves and fine roots of poplar planted in soil or tannery waste. We found no obvious symptoms of metal stress, such as: elevated hydrogen peroxide levels or lipid peroxidation, but we observed activation of many elements of antioxidative system. Comparison of 2-DE protein profiles of leaves and fine roots from poplar grown on soil or tannery waste revealed increased expression of glycolytic enzymes and proteins involved in the synthesis of cell wall components, changes in the levels of proteins associated with photosynthesis, stress-related proteins, proteasome subunits and methionine biosynthesis enzymes. This experiment demonstrated that proteomic analysis has the potential to link the effects of Cr-rich tannery waste with biological consequences.

An optimized method to extract poplar leaf proteins for two-dimensional gel electrophoresis guided by analysis of polysaccharides and phenolic compounds.

Commonly used methods for protein extraction from plant leaves, such as extraction with phenol or a combination of trichloroacetic acid and acetone, were ineffective for four tested cultivars of poplar. Moreover, multiple protocols for 2DE of the extracted proteins gave different results when protein profiles of relatively closely related plants were compared. Given that polycyclic compounds strongly hinder 2DE, we analyzed the impact of polyphenols and polysaccharides present in the plant tissues used for protein extraction, on the quality of 2DE protein profiles. Analysis of content of polyphenols and polysaccharides in leaves of poplar cultivars showed that even small differences in concentrations of analyzed metabolites accompany large differences between poplar cultivars when considering the susceptibility of samples to protein extraction for 2DE. High-quality 2DE results were correlated with decreased amounts of polyphenols. Additional analysis using MS/MS suggested that only levels of total phenolics affected the results of 2DE. Soluble total nonstructural carbohydrates also had a negative effect, but the level of starch was not important. Finally, we present an optimized method for extraction of proteins from poplar leaves, which enables reliable comparative analysis of four different poplar cultivars, that is, "Eridano," "Villafranca," "NE-42," and "Luisa Avanzo," which have not yet been used for the proteomic studies.

An optimized method to extract poplar leaf proteins for two-dimensional gel electrophoresis guided by analysis of polysaccharides and phenolic compounds.

Commonly used methods for protein extraction from plant leaves, such as extraction with phenol or a combination of TCA and acetone, were ineffective for four tested cultivars of poplar. Moreover, multiple protocols for 2DE of the extracted proteins gave different results when protein profiles of relatively closely related plants were compared. Given that polycyclic compounds strongly hinder 2DE, we analyzed the impact of polyphenols and polysaccharides present in the plant tissues used for protein extraction, on the quality of 2DE protein profiles. Analysis of content of polyphenols and polysaccharides in leaves of poplar cultivars showed that even small differences in concentrations of analyzed metabolites accompany large differences between poplar cultivars when considering the susceptibility of samples to protein extraction for 2DE. High-quality 2DE results were correlated with decreased amounts of polyphenols. Additional analysis using MS/MS suggested that only levels of total phenolics affected the results of 2DE. Soluble total nonstructural carbohydrates also had a negative effect, but the level of starch was not important. Finally, we present an optimized method (OPTI) for extraction of proteins from poplar leaves, which enables reliable comparative analysis of four different poplar cultivars i.e. 'Eridano', 'Villafranca', 'NE-42' and 'Luisa Avanzo', which have not yet been used for the proteomic studies.

Thresholds of heavy-metal toxicity in cuttings of European black poplar (Populus nigra L.) determined according to antioxidant status of fine roots and morphometrical disorders.

The effects of increasing concentrations of polluted soils collected from two different stands in the direct neighbourhood of a copper smelter were studied by analyzing the activity of lipid peroxidation and antioxidant enzymes in the fine roots of cuttings of black poplar (Populus nigra L.). Morphological parameters of affected and unaffected plants were compared and concentrations of heavy metals in their fine roots were estimated. Copper and lead were the major pollutants, and their threshold concentrations were determined on the basis of positive or negative impact on shoots and root growth, morphological disorders of the roots (thickening, decreasing of fine roots volume), stimulation or inhibition of the most important antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), guaiacol and ascorbate peroxidases (GPOX, APOX) and glutathione reductase (GR) as well as increasing MDA concentration. Finally, three thresholds of Cu and Pb concentrations resulting in increasing toxicity against plants were proposed, splitting the ranges of the non toxic presence of these metals into soil (Cu<100 ppm, Pb<30 ppm), full tolerance (Cu 100-220 ppm, Pb 30-70 ppm), limited tolerance (Cu 220-650 ppm, Pb 70-200 ppm) and breakdown of tolerance (Cu>650 ppm, Pb>200 ppm).

Changes in carbohydrate metabolism in fine roots of the native European black poplar (Populus nigra L.) in a heavy-metal-polluted environment.

Effects of copper-smelter-related deposition of heavy metals in the soil on carbohydrate metabolism of fine roots of the native European black poplar were investigated in spring and autumn. Total soluble non-structural carbohydrates in fine roots from trees growing in the polluted habitat were lower than in a control site, but this was directly associated only with a lower raffinose concentration. Neither glucose nor fructose concentrations differed significantly between polluted and unpolluted sites. In contrast, the galactose concentration was higher in the presence of heavy metals, especially in autumn. Also the stachyose concentration was higher in the polluted site, but only in autumn, suggesting it could be an alternative way of detoxification of galactose. No difference between control and polluted stands was observed in sucrose concentration. However, estimates of sucrolytic activity revealed markedly higher activities of sucrose synthase (SuSy), soluble acid (AI) and neutral (NI) invertases in the polluted stand than in the control. In contrast, the estimated glycolytic enzyme activities were not affected by the presence of heavy metals in soil.