| Wood formation is a complex process involving many genes expressed coordinately at the different developmental stages of secondary vascular system (SVS). Among the complicated pathways of gene expression, proteins, rather than mRNAs, play the direct role in the morphogenesis and its regulation of organisms. Therefore, the deep analysis of the dynamic process of the protein profiles is helpful to find the key genes and elucidate their functions during the wood foramtion.The regeneration system of SVS previously established in Populus tomentosa by our laboratory was used to investigate protein profiling during this process. A developmental stage-dependent sampling scheme and a gel-based nanoLC-ESI-MS/MS were combined to analyze the proteins expressed in the samples collected from developing SVS and its different regeneration stages. Totally 1467 non-redundant proteins were identified, the functions of which were categoried into 23 groups, such as cell cycle and cell division, cell wall and envelope biogenesis, transcription related proteins, signal transduction etc. Among the identified proteins, the most components were groups related to amino acid transport and metabolism, carbohydrate transport and metabolism, energy production and transition, posttranslational modification and chaperones, as well as translation and ribosome biogenesis. Morevoer,13% of the identified proteins were funtionally unknown. More basal proteins (28%) were identified than which of our previous works performed by 2DE-MALDI-TOF MS and HPCF/HPRP-MALDI-TOF MS. Among the 1467 nono-redundant proteins identified,207 were expressed specified in normal developing SVS, while 592 in the regenerative SVS and 668 in both normal developing and regenerative SVS, which fully indicated the similarity and difference of protein expression between the nomal developing and regenerative SVS.Our previous work showed calli formed on the surface of trunks at 2 days after girdling, followed by discontinuous and continuous meristem cell layers sequencially. Afterwords, cambium zone in a consecutive and regular arrangement manner shaped and differentiated, eventually an entire structure of wood formation produced at 22 days after girdling. During the whole regeneration process, cellular processes including division and differentiation took place at different developmental stages, followed with replication, transcription, and translation of genes and the regulation of these processes. Totally,30 proteins related to transcription and its regulation,16 proteins related to cell cycle and division,25 proteins related to cytoskeleton,60 proteins related to signal transduction and 40 proteins related to cell wall components were identified in this study.Moreover, part of proteins identified here were compared to the transcriptome analysis and real-time PCR validation. Among the 3 proteins selected for comparison, only one protein showed the same expression pattern of the corresponding gene, whereas the other two presented different expression patterns with their transcripts analysis and real-time PCR validation respectively. This difference could be due to the selectivity or alternative splicing in transcriptional level, and the posttranslational modifications as well as different detectabilities between the transcriptional and proteomic analysis.In this study, an effective system by the manner of protein fraction and identification for proteomics analysis by 1D gel-based nanoLC-ESI-MS/MS of poplar cambium was established. And a large number of proteins related to wood formation were identified, which could be used for candidate genes of separating and identifying key genes of wood formation. The result from this study provided a theoretical basis and genetic resources for tree breeding by means of biotechnology.
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