Comparative Proteome Analysis Of The Response Of Ramie Under N, P And K Deficiency

Ramie (Boehmeria nivea) is a natural fiber crops characterized by high biomass yield (three harvests per year) and a strong root system. Large amount of NPK is required for ramie growth. The current research main focus on high-efficiency NPK utilization thourgh cultural management, however, there has been little research on the molecular mechanisms of ramie related to the absorption, utilization and metabolism of nitrogen (N), phosphorus (P) and potassium (K).The study of ramie proteomics will lead to enhanced competitiveness. Establishes a two-dimensional electrophoresis system and protein extraction method that is suitable for the proteomic analysis of ramie’s various parts (roots, stem and leave), also it is suitable for the requirement of protein spot identification by Mass Spectrum, which is key to the success of proteomics studies. Through the established two-dimensional electrophoresis system studies the molecular mechanisms of ramie related to the absorption, utilization and metabolism of NPK by comparative proteome analysis of the response of ramie under N, P and K deficiency, and it will provide important information for further study on the high-efficiency NPK utilization mechanism of ramie. In this study, the experimental matrial of Hua Zhu#5was used to improve and establish a two-dimensional electrophoresis system and study the comparative proteome analysis of the response of ramie under N, P and K deficiency. The main results were following:1. This article improves and establishes a two-dimensional electrophoresis system that is suitable for the proteomic analysis of ramie’s various parts by optimizing the traditional TCA/acetone protein extraction method: Protein lysis buffer (7M urea,2M thiourea,4%CHAPS,1%DTT), protein purification (4times volume of acetone), and IPG strips (pH4-7,17cm). This system is also applied to the roots, stems and leaves of ramie. Compared with traditional method, this system can effectively remove the non-protein impurities from ramie, the protein spots obtained from the improved (823±15) significantly higher than the traditional(320±14) method, and it is suitable for the different parts of ramie and the requirement of protein spot identification by Mass Spectrum, where clear protein two-dimensional electrophoretogram can be obtained; The number of protein in the stem, roots and leaves were280±6,1093±20and765±16respectively. Higher-abundance proteins were found in the leaves, which affected the resolution of lower-abundance proteins to some extent, the stem with less protein content is the major transport tissue and less involved in other physiological functions. Ramie protein was mainly distributed in the pH range4.5-6.5with molecular weight40-80KD;30%-38%of protein was distributed in the20-40KDa region, mostly low molecular weight protein. The Coomassie Brilliant Blue procedure was used to stain2-DE gels can suitable for the requirement of protein spot identification by Mass Spectrum, which can obtain780±17protein spots.2. The SPAD values in N-, P-and K-deficient treatments consistently declined, and the growth of ramie finally stopped. The differentially expressed proteins in the leaves of ramie were analyzed by proteome analysis after6d of N-and K-deficient treatments and3d of P-deficient treatment by using MALDI-TOF/TOF mass spectrometry and32,27and51differential proteins were obtained, respectively. The differential proteins were functionally classified into ten categories: photosynthesis, protein destination and storage, energy metabolism, primary metabolism, disease/defense, signal transduction, cell structure, transcription, secondary metabolism and protein synthesis.3. Under NPK deficiency, both carbon source and NADPH supply declined in ramie. The proteins related to photosynthesis such as ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit were downregulated to adapt to nutrient stress. At the same time, the energy metabolism was generally weakened. For the K-deficient treatment, the Nucleoside diphosphate kinase and GADPH activities increased and so enhanced the connection between glycolysis and photosynthesis, thus enhancing resistance. The P deficiency may induce hormone synthesis by enhancing hexokinase activity. Actin level was upregulated and promoted root growth and absorption of more P from soil. Both P and K deficiency cause the secretion and accumulation of citrate to dissolve the nutrients from the soil and increase the stress resistance. As a response to N deficiency, the TCA cycle was sustained to provide energy to adapt to the stress.4. Ramie generally showed higher secondary metabolism under NPK deficiency. The P deficiency may facilitate the flow and utilization of carbon and N in the cells to enhance leucyl aminopeptidase activity and to sustain plant growth. Ramie responded to K deficiency by facilitating the synthesis of cysteine using inorganic sulfides, thus increasing resistance to K deficiency.5. Large quantities of proteins of the HSP family were downregulated in ramie. Due to the nutrient deficiency, some peroxides and ROS were also produced. However, in N-and P-deficient treatments the peroxides and ROS were cleared in ramie by increased activities of copper-zinc superoxide dismutase and2-cysteine peroxiredoxin B.6. The upregulated level of profilin due to P deficiency may significantly affect fiber growth of ramie. The cell differentiation and growth of ramie were inhibited within a certain range of P. A variety of life activities of ramie were regulated by enhancing the signal transduction responsible for synthesis of ATP and DNA under N deficiency and this may help counteract the decline of protein synthase caused by P and K deficiency and increase resistance to N deficiency.