| Copper is a widespread heavy metal in the world, as a consequence of industrial and agricultural activities in recent years, which are released to the environment. The accumulation of Cu within soils is becoming a major environmental problem. When plant grew on the copper-contaminated areas, cell wall not just provided a physical barrier between copper-contaminated soils and the internal contents of plant cells, also played a crucial role in plant defense response to toxic metals. However, the cytological mechanism in cell wall of Cu uptake, accumulation, detoxification, tolerance and signals transduction is needed for further research. Elsholtzia splendens as a copper-tolerant plant can survive extremes in a high copper content area, which may serve as a good candidate for phytoremediation of copper-contaminated soils. It can accumulate over1000mg/kg of Cu in the body. In this study, we elucidated the mechanisms of Cu uptake, accumulation, tolerance and detoxification in E. splendens cell wall at the cytological and proteome levels for the first time and proposed a possible protein interaction network in response to copper stress of cell wall. Our research would give new insights into stress response in cell wall and could provide theoretical and scientific basis for phytoremediation of copper contaminated soil. The main results were summarized as follows:1. Using of the grading centrifugation method, it was found that the subcellular distribution and the chemical form of copper ions in roots, stems and leaves under different copper concentration. The highest proportion of Cu was stored in the cell walls (68%) and vacuoles (26.6%), only a small amount of Cu was stored in cell organelles such as chloroplasts. With the increasing of Cu concentration, subcellular copper content of each component also increased. The distribution characteristic of Cu especially in roots was closely related to its tolerance. Meanwhile, Cu taken up by E. splendens existed in different chemical forms. In roots, the majority of Cu was in the forms of water-soluble and un-dissolved phosphate, while most of Cu was in the forms of un-dissolved phosphate, inorganic salts and proteins in stems and leaves. In addition, increasing Cu supply, the binding capacity of Cu to soluble salt or protein reduced but that to insoluble oxalate or un-dissolved phosphate increased. Cu ions integrated with low bio-available compounds, such as un-dissolved phosphate or oxalate which contributed to the metal tolerance and detoxification of E. splendens. Cluster analysis, based on copper abundance levels revealed that the correlations with the chemical forms and intracellular location which indicated the relationship between their migration activity and the toxicity.2. Chemical modification of cell wall, adsorption dynamic experiments and the FTIR spectroscopy analysis were combined to explore adsorption kinetic characteristics and the functional groups of the cell wall and its components under copper stress. The adsorption dynamic experiments showed that the copper content adsorbed by the root cell wall was close to its saturation level after300minutes, which was about90%of the maximum adsorption capacity. We obtained a saturated adsorption capacity of copper after500minutes, which was5.85mg Cu/g cell wall. The proportion of copper adsorption by pectin and cellulose in the root cell wall were19.85%and25.48%, respectively. The copper adsorption by the root cell wall had been significantly reduced by the ammonia and cellulose treatments on the cell wall. The FTIR spectra also revealed that hydroxyl, carboxyl and amino group are the main binding sites of Cu2+by the root cell wall in the adsorption process. Cellulose and hemicellulose provide a combination of Cu2+with carboxyl functional groups, and pectin offers carboxyl groups while the cell wall proteins provide amino functional groups for binding sites. The results showed that the root cell wall and its components had high adsorption capacity for copper, which was an important mechanism of high copper tolerance by E. splendens.3. The content, composition and distribution of cell wall polysaccharide of E. splendens and their relationship to copper tolerance were analyzied. Three typical biochemical indexes (sugar, galacturonic acid and Keto-deoxyoctulosonate) of cell wall polysaccharides were investigated. The results illustrated that the content of them was closely related to the toxicity degree. Changes of cell walls polysaccharide in cell walls fraction (such as pectin, cellulose, hemicellulose, etc.) decreased copper toxicity in plant. Expression of cell wall polysaccharides offered more carboxyl groups and provided more binding sites for copper ions. This behavior could be considered as a defense mechanism by E. splendens against heavy metal. With the increasing of Cu supply, various components in cell wall were fluctuated. The total sugar content of hemicellulose and pectin increased very significantly in roots and leaves (P<0.01), whereas that declined perceptibly in stems. The sugar content of cellulose all declined in roots, stems and leaves (P<0.05). The galacturonic acid content of pectin and cellulose increased slightly in roots, stems and leaves, while that of hemicellulose declined in roots, leaves and increased in stems. The Keto-deoxyoctulosonate (KDO) content of pectin was increased slightly by copper in roots, stems and leaves. Plants accumulated lots of copper ions in the cell wall to protect protoplast against copper toxicity, which was a physiological response by E. splendens. It also confirmed that cell wall, especially for cell wall polysaccharides played an important role in copper detoxification.4. Optimization of the extraction methods of cell wall proteins and determined the relation on correlation of copper accumulation and cell wall proteins content in E. splendens. With the increasing of Cu supple, the copper accumulation in the root cell wall was also increased gradually. The content of copper in cell wall reached its highest level (1.41mg/g cell wall) under100?mol/L Cu. The cell wall proteins content in roots first increased then decreased with the increasing of Cu supply. The content of root cell wall protein reached its highest level (26.85?g/g dry weight of cell wall) on50?mol/L copper stress. Copper accumulation in the root cell wall caused a series of physiological response including gene regulation which presented the fluctuation in the content of cell wall proteins. The same fluctuation trend between the content of cell wall proteins and the copper accumulation indicated that cell wall proteins might participate in the copper tolerance or detoxification by the cell wall. In SDS-PAGE protein spectra, more than25protein bands from the root cell wall proteins were separated, most of them falled in the standard for10-170KD molecular weight range. With50?mol/L Cu stress, the root cell wall proteins within10to25KD increased obviously compared with the contrast stripe.5. To gain a comprehensive understanding the response to Cu in plant cell wall. iTRAQ with LC-ESI-MS/MS approach were applied to identified differentially expressed cell wall proteins of E. splendens under Cu stress. A total of479proteins were identified, among them55proteins expressed significantly under48h-treatment at50?mol/L Cu. Functional classifications of the55different expressed proteins were classified based on the enrichment in two Gene Ontology (GO) categories:molecular function, and biological process. The top three categories of the identified proteins were those involved in metabolic processes (23.24%), cellular processes (16.20%), and response to stimuli (14.79%), following the top three abundant category was classified according to GO molecular annotation into binding (41.38%), catalytic activity (31.03%), and transport activity (11.49%) respectively. The observed diversity in biological function and biological processes of the identified cell wall proteins demonstrated that the response to Cu stress by the root cell wall of E. splendens was a complex process. Many physiological and biochemical characteristics were altered to counteract the adverse conditions. Among the22up-regulated proteins, most were related to the antioxidant defense pathway, cell wall polysaccharide modification and metabolisem process. However, the most highly down-regulated proteins in response to copper stress belonged to the category of signal, energy and protein synthesis. The communication between cytoskeleton and cell wall protein was one of the most characteristic features of cellular mechanism by which cells sense and respond to various extracellular signals effectively. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein interaction network associated with metal response in cell wall. |
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