Profiles Of Genes Expressed During Regeneration Of The Secondary Vascular System In Poplar (Populus Tomentosa Carr.)

Wood formation is a complex process composing many biological events, occurs as a result of growth and differentiation of the vascular cambium. This secondary growth is a highly ordered developmental process, which involves patterned division of vascular cambium cells and a subsequent regulated differiation of cambium derivatives into secondary xylem and phloem tissues. To access key developmental stages of wood formation, we have established a regeneration system that can mimic the initiation and differentiation of cambium cells for Chinese white poplar (Populus tomentosa.). Transcript profiling was accessed by microarray analysis and differentially expressed genes in key regeneration stages were identified. The main results are as the followings: A regeneration system of secondary vascular system for poplar was set up. Anatomical studies showed that new cambium initiated at day 10th after being debarked, and began to differentiate into xylem tissue at day 18th, while the xylem re-appeared subsequently at day 22nd. The whole regeneration of secondary tissues completed within a few weeks after being debarked. This provides the opportunity to follow key stages of wood formation by sampling clonal trees at different regeneration stages. We have constructed a cambium zone specific substracted cDNA library through suppressive subtractive hybridization between mRNA samples from cambium zone and leaf tissues. This cambium zone specific cDNA library was used to generate microarray chips representing 1,197 cDNA clones. Two hundred and twenty seven differentially expressed genes were obtained by comparing the expression profiles at different regeneration stages by microarray analysis. The differentially expressed genes were partially sequenced and their functions were deduced by Blast search in poplar genomic and PopulusDB databases. During the early stages of the regeneration when vascular cambium initiated and developed, the differentially expressed genes included calmodulin, auxin induced proteins, nodulin like proteins, other unknown function transcription factors and proteins in signal pathway which may involve in vascular system development. Some primary cell wall related proteins, such as expansin, extensin, and hydroxyproline-rich glycoprotein, were also expressed. During the late stages of regeneration when immature and mature xylem developed, Myb-related and other transcription factors, protein kinase and proteins involved in signal pathway were among the differentially expressed genes. Arabidopsis thaliana undergoes secondary growth and produces extensive secondary xylem when properly treated. Thus we used Affymetrix whole-transcriptome ATH1 chips to analyze gene expression profiles during regeneration of secondary vascular system in poplar. By the comparison between each two adjacent stages, 149 differentially expressed genes were identified. While by comparison of gene expression at all subsequent regeneration stages with the first stage at day 6th, 368 genes with dramatically changed expressing pattern during regeneration were obtained by self-organizing map analysis based on their expression data. We also obtained differentially expressed genes by Arabidopsis ATH1 chip analysis that might involve in cambium initiation, differentiation and xylem development. These included signal pathway proteins as auxin induced proteins, protein kinase, G protein, and transcription factors as Myb, AP2, Zinc finger protein, bHLH. In addition, genes involved in cell wall biosynthesis and cell wall related proteins, such as cellulose synthase, pectin methylesterase, pectin acetylesterase, expansin, extensin, hydroxyproline-rich glycoprotein were also on the list. The results are very similar to those obtained using our cDNA microarray, which indicates similar genes expressed during development of secondary vascular system in both species, and Arabidopsis could be also used to study wood formation. Most of the genes obtained by the microarray-based approaches were assigned as "function unknown". Some of these genes specifically expressed during the early stage of generation, may involve in cambial initiation and development; and some at the late stages of regeneration, may involve in xylem development. Due to the fact that fewer genes involved in the development of secondary vascular systems in woody plants exist in the database, these "unknown function"genes might represent new genes specific to woody plants that play important roles in wood formation. Transcriptome analysis during regeneration of the secondary vascular system in poplar notonly provides new insight into the transcriptional regulatory networks of wood forming tissues, but also highlights candidate genes may be responsible for variable traits of wood property. To isolate and characterize these key genes is needed to understand their biological function.