Proteomics Research On Populus Simonii×P. Nigra Leaves With Different Dark Treatments

To better understand the regulative mechanism of P. simonii×P. nigra in darkness, we investigated dynamic proteomic differences among P. simonii×P. nigra leaves grown under darkness for 0 day,1 day,3 days and 7 days using a label-free quantitative proteomics approach based on nanoscale ultraperformance liquid chromatography-ESI-MSE.97 proteins were significantly altered during dark treatment and these proteins were involved in 9 pathways including photosynthesis,redox regulation and amino acid metabolism. Of these 97 proteins,61 were identified as membrane proteins that seldom had been identified with two-dimensional electrophoresis methods, indicating the power of our label-free method for membrane protein identification. To confirm the label-free quantitative results, Western blot was performed to compare expression of proteins in P. simonii×P. nigra leaves with different dark treatment. Antibodies against Lhcb1,Lhcb2 and Lhcb6(CP24) were used in the Western blot analysis. Western blot results for these proteins agreed well with the quantification of the label-free method.GO analysis and PPDB database annotation of these differential proteins showed that 40 proteins were involved in photosynthesis and most of them drastically increased after dark treatment. These proteins increased after dark treatment located in thylakoid membrane, indicating that the levels of some proteins located in thylakoid membranes were highly sensitive to light. Amino acid metabolism pathways play an important role in plant development, Cysteine synthase and Glutamine synthetase involved in sulfur and nitrogen metabolism were identified in this study. Furthermore, Superoxidedismutase[Cu-Zn] and peroxidase drastically increased after dark treatment indicating that reactive oxygen species(ROS) free radicals in P. simonii×P. nigra leaves might be induced in abundance under darkness.Moreover, although some differential proteins likely regulated by light were identified in this study, functions of these proteins in P. simonii×P. nigra remain unknown, there is still a long way to understand the light regulatory mechanism in forest trees. Taken together, these results represent a comprehensive dynamic protein profile and light-regulated network of P. simonii×P nigra leaves in dark condition and provide a basis for further study of the mechanism of gene function and regulation in light-induced development of forest trees.