Based-mass Spectrometry Quantitative Proteomics Of Light Regulation Mechanisms In Phyllostachys Edulis

Bamboo is one of the most important non-timber forestry products in the world.Moso bamboo(Phyllostachys edulis)has a wide distribution and in Asia,especially in the East Asian region of China.It has been endowed with ecological,ecomomical and cultural value from ancient times.Light is not only the most critical source of energy for plant photosynthesis,but also involved in regulating the biological processes of plants.Red and blue light in light quality is involved in regulating numerous light responses in plants,while the main research objects on the light regulation mechanism of red and blue light are Arabidopsis thaliana,rice and tomato,etc.However,little has been reported in Phyllostachys edulis.Besides,there is no mature transformation system in Phyllostachys edulis,so it is difficult to analyse the function of proteins of moso bamboo from the perspective of molecular genetics.Specifically,it is difficult to analyse the protein function of moso bamboo from a molecular genetic point of view because there is no mature transformation system for moso bamboo.However,due to the difficulty and time-consuming nature of the transformation,it is less efficient to study the protein function of moso bamboo by molecular genetic means compared to the model plant.Secondly,there are numerous proteins involved in the regulation of plant growth and development,and there is a lack of reliable means to study protein quantification on a large scale.With the development of research technology,mass spectrometrybased quantitative proteomics has become an important tool for large-scale studies on the regulation of plant growth and development.Therefore,in this experiment,moso bamboo was treated with a single light source,blue light and red light,and then treated with Tandem Mass Tag(TMT)to quantify moso bamboo proteins on a large scale,and to explore the differences in moso bamboo protein levels after blue and red light treatment by bioinformatics.The aim was to identify the key pathways and proteins in the light response of moso bamboo,and to verify the results of TMT protein quantification with the changes in physiological indicators after light treatment and the quantification of target proteins by Parallel Reaction Monitoring(PRM)at a later stage.The main findings of this experiment are as follows:(1)After moso bamboo was treated with blue and red light,moso bamboo seedlings differed significantly from the dark treatment in plant height,plant fresh weight,plant dry weight and nodal length,and the fresh and dry weights of moso bamboo seedlings under the red light treatment were significantly higher than those under the blue light treatment.The stomatal conductance and transpiration rate of moso bamboo plants were significantly higher in the red light treatment than in the blue light treatment.Secondly,there were no significant differences in the chlorophyll fluorescence parameters of the moso bamboo plants after both red and blue light treatments.However,a significant increase in the relative chlorophyll content of the moso bamboo plants occurred under the blue light treatment,while there was almost no change under the red light treatment.At the same time,the enzyme activities of NAD-malate dehydrogenase and NADP-malate dehydrogenase were significantly higher in blue-treated moso bamboo than in red-treated cases.Finally,the nitrogen content of moso bamboo leaves consistently decreased after light treatment,and the decrease was more pronounced in blue light,while the carbon content level of moso bamboo increased significantly after red light treatment.(2)After the red and blue light treatment of moso bamboo,large-scale protein quantification using TMT labelling techniques and mass spectrometry,supplemented by bioinformatics,resulted in the quantification of 8693 proteins out of 11481 proteins.In this study,a total of 770 differential proteins were obtained,of which 698 differential proteins were under blue light conditions,139 proteins among them were up-regulated and 559 down-regulated.A total of 234 were obtained under red light conditions,of which 98 were up-regulated and 136 downregulated,and 162 differential proteins were present in both together.Subsequently,Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of the different blue and red light differential proteins revealed that the differential proteins under blue light treatment were mainly involved in photosynthesis,photosynthesis-antenna The study found that the differential proteins were mainly involved in photosynthesis,photosynthesis-antennae and oxidative phosphorylation pathways under blue light treatment.In contrast,the sucrose and starch metabolism pathways were significantly enriched under red light treatment.(3)Weighted Gene Co-expression Network Analysis(WGCNA)revealed that the correlation of photosynthesis and NAD synthesis modules with blue light was higher than that with red light and darkness,while the correlation of cell wall anabolism-related modules with red light was higher than that with blue light and darkness.The correlation between the modules of photosynthesis and NAD synthesis was higher in blue light than in red light and darkness.The protein-protein interactions(PPIs)analysis revealed that the blue light protein interactions were more active and there were more protein interactions,including photosynthesis-related,nitrogen anabolism,oxidative phosphorylation and other obvious interactions.In contrast,red light protein interactions were only present in a few proteins and core regions,including cell wall synthesis,carbohydrate synthesis and other core regions of interactions.Subcellular localization revealed that blue light differential proteins were mainly concentrated in the nucleus,chloroplasts and mitochondria,while red light differential proteins were mainly localized in the nucleus,chloroplasts,cytoplasm and vesicles.The quantitative validation of the targeted PRM proteins revealed that the quantitative changes were almost identical to the TMT quantification,indicating that the TMT quantification is more accurate.