| Copper (Cu) is an essential micronutrient for plant growth and development, however, excess Cu is extremely toxic to plants. Plants possess several potential metal tolerance mechanisms including chelation, compartmentation and exclusion. Rice(Oryza sativa L.) is considered as the main staple food in many countries of the world, it is also monocotyledon model plant. Rice Cu-tolerant variety (No.1139) and Cu-sensitive variety (No.1195), which was previously screened and identified by our research group, was used as the plant material throughout the experiments. Total protein of rice roots were separated by 2-DE, then the differentially expressed proteins in response to copper stress was identified by MALDI-TOF MS and PMF. Copper-immobilized metal affinity chromatography (Cu-IMAC) combine with 2-DE and MALDI-TOF/TOF MS was used to analyse copper-binding protein in response to copper stress in rice roots. Our gole of this study is to explain further the mechanisms of tolerance to copper stress through total protein and copper-binding protein in response to copper stress in rice roots.Sample preparation, which effect on the resolution and reproducibility of map, is a crucial step in 2-DE analysis. The proteins of rice roots contain copper ions and secondary metabolites under copper stress, hence it is necessary to develop protein extraction methods. The improved TCA/acetone precipitation extraction method (method B) and improved phen-based extraction method (method D) had higher resolution and number of protein spots (862 and 659 spots, respectively) than method A and method C (283 and 239 spots? respectively), respectively increased 2 and 1.8 times in connection with bumber of protein spots. Especially, the improved TCA/acetone extraction (method B), which can obtain higher number of protein spots, more even distribution and higher resolution of 2-DE profile, is more suitable for rice root proteomic analysis under copper stress.The 7 days rice seedlings were treated with 8 μmol L-1 Cu2+ for 3 days. Differences in protein expression in the roots of two rice varieties with differential Cu tolerance were identified using 2-DE and peptide mass fingerprints (PMF) obtained by MALDI-TOF MS. Thirty-four protein spots exhibited significant absolute variation, greater than 1.5-fold, between control and copper treated samples in at least one variety. These Cu-response proteins included those involved in antioxidative defense, redox regulation, stress response, sulfur and GSH metabolism, carbohydrate metabolism, signal transduction, and some other proteins with various functions. A total of 24 common proteins tended to be up-regulated in both varieties in response to copper stress. Among these, twelve proteins were linked to antioxidative defense, redox regulation, sulfur assimilation, and GSH biosynthesis. Nine proteins, including putative cysteine synthase, probable serine acetyltransferase 3, L-ascorbate peroxidase 1, putative glutathione S-transferase 2, and thioredoxin-like 3-3, exhibited a greater increase in the Cu-tolerant variety B1139 compared to the Cu-sensitive variety B1195. Three proteins, including L-ascorbate peroxidase 1 and putative glutathione S-transferase, were markedly induced in B1139, while they were slightly down-regulated or not detectable in B1195. These results will enhance our understanding of plant molecular responses to Cu stress.Many proteins possess cysteine, methionine, and histidine which have high affinity to divalent metal ions, thus copper-binding proteins play a key role in maintaining intracellular copper homeostasis. Proteomic analysis of copper-binding protein in response to copper stress in rice root, which based on Cu-IMAC,2-DE and MS/MS. However, some copper-binding protein can be incompletely separated under copper stress, because some of the copper-binding protein may not be available sites to bind copper which is immobilized on sepharose-IDA. The purification efficiency of copper-binding protein can increase owing to removal metal ions from copper-binding sites of proteins before Cu-IMAC.Sepharose-EDDS and TED, which were synthesized, were compared to remove metal ability with commercialized Sepharose-IDA and NTA. The results show four kinds of media can remove more than 95% of copper, especially sepharose-EDDS is best; Sepharose with IDA, NTA and EDDS can remove more than 90% Zn2+ from proteins; Sepharose with NTA and EDDS can remove more than 90% Mn2+ from proteins; Sepharose with IDA and EDDS can remove more than 90 and 93% Cd2+ from proteins respectively. The result shows that sepharose-EDDS can remove more than 93% Cu2+, Zn2+, Mn2+ and Cd2+, thus it apply to remove heavy metal from protein of several heavy metal-stressed plant.Then sepharose-EDDS and IDA were applied to the separation and purification of copper-binding proteins. The results show that the removal of metal ions from proteins can significantly increase the peak area of the copper-binding proteins. Metal ions were removed by sepharose-EDDS or IDA before Cu-IMAC, which remarkably increased the peak area of specific copper-binding protein, moreover the latter was uncommonly higher than the former. The metal ions were removed from proteins by sepharose-IDA before Cu-IMAC, which lead to abundance of 38 protein spots to significantly increase in 2-DE map. Thirteen protein spots were randomly selected and identified by MS/MS. Among of them, twelve protein spots possess putative copper-binding motif which was speculated by Smith et al. Although putative caffeoyl-CoA-O-methyltransferase 1 does not contain putative motif, crystal structure analysis displays that it has the conserved metal binding sites.Difference in copper-binding proteins expression in the root with differantial Cu tolerance were identified using Cu-IMAC,2-DE and MALDI-TOF/TOF MS. The 7 days rice seedlings were treated with 8 μmol L-1 Cu2+ for 3 days. The abundance of specific copper-binding protein uncomonly increased in both copper-stressed rice varieties root compared to control, especially B1139 is remarkably higher than B1195. Thirty-five protein spots exhibited significant absolute variation, greater than 1.5-fold, between control and copper treated samples in at least one variety. A total of 18 common proteins tended to be up-regulated in both varieties in response to copper stress. Among them, seven proteins, including putative peroxidase, CHP-rich zinc finger protein-like and glutathione S-transferase II, exhibited a greater increase in the Cu-tolerant variety B1139 compared to the Cu-sensitive variety B1195. However four protein spots, including Copper/zinc superoxide dismutase, Germin-like protein 6 and Glutamine synthetase shoot isozyme, inversely exhibited. Six proteins, including putative PR, PR-10a, putative ubiquitin-conjugating enzyme spm2 and putative cold shock protein-1, were markedly induced in B1139, while they were not detectable in B1195. Four protein spots, including methionine sulfoxide reductase A2-1, putative caffeoyl-CoA O-methyltransferase 1 and calcium-binding protein, were significantly up-regulated in B1139, while they did not change. Glutamine synthetase shoot isozyme (spot 11) and glutamine synthetase root isozyme (spot 12) were down-regulated in B1139, while they were not detected in B1195. Treatment with 8μM Cu resulted in an commonly decreased intensity of three spots in two varieties compared to control, these proteins belong to translation initiation factor 5A.The foregoing analysis shows that ten spots was only induced in the rice B1139 in response to copper stress and the up-regulation of 7 protein spots was more pronounced in B1139 than in B1195. These Cu-binding proteins included those involved in antioxidant defence and detoxification, regulation of gene transcription, stress response, lignin biosynthesis, and signal transduction. |
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