Project description:Secondary vascular system (SVS) development resulted from cambial growth is a currently not well understood process. Therefore, more studies are needed to shed more lights on the molecular mechanisms underpinning the cambial activity. The regeneration of SVS from debarked trunk that can mimic the vascular cambium-driven wood formation has developed and could be used to revealed a larger number of differentially expressed genes during the stages of cambium formation and xylem differentiation in Populus tomentosa. We used microarrays to detail the global programme of gene expression in 6 time points during the regeneration of SVS.
Project description:Regeneration is a common strategy for plants to repair their damaged body plans after attack from other organisms or physical assaults. Trees with bark girdling on a large scale will grow new bark within one month and this bark regeneration after girdling system has been proven to be an efficient method to study secondary vascular development as well as plant tissue regeneration in vivo. We herein show the molecular features of differentiating xylem cell fate switch process during secondary vascular tissue (SVT) regeneration in Populus. Based on our data, we propose a working model to illustrate the molecular dynamics underlying xylem cell fate switch process during SVT regeneration, which is significant to understand the pattern formation during the SVTs regeneration and also would shed light on the mechanisms of tissue regeneration in plants.
Project description:Regeneration is a common strategy for plants to repair their damaged body plans after attack from other organisms or physical assaults. Trees with bark girdling on a large scale will grow new bark within one month and this bark regeneration after girdling system has been proven to be an efficient method to study secondary vascular development as well as plant tissue regeneration in vivo. We herein show the molecular features of differentiating xylem cell fate switch process during secondary vascular tissue (SVT) regeneration in Populus. Based on our data, we propose a working model to illustrate the molecular dynamics underlying xylem cell fate switch process during SVT regeneration, which is significant to understand the pattern formation during the SVTs regeneration and also would shed light on the mechanisms of tissue regeneration in plants. Specific regenerated tissues of Populus at different stages were isolated by tangential cryo-sectioning. Total RNA from cryo-sections representing different regenerating tissues was extracted for Affymetrix Poplar Whole Genome Array hybridization. Five samples (two replicates for each sample) were used for gene expression analysis: differentiating xylem (diX, Stage 0), dedifferentiating xylem cells (deX, Stage I), regenerated phloem (rPh, Stage II), differentiating regenerated cambium (diC, Stage II) and regenerated cambium (rC, Stage III). In addition, one pooled genomic DNA sample from cryo-sections of differentiating xylem from two trees was isolated for DNA hybridization to produce a new CDF file that was used to mask out some potentially cross-hybridizing probesets from the standard Affymetrix Poplar Genome Array. Supplementary file: poplar.cdf
Project description:The formation of vascular tissue occurs when cellulose, hemicellulose, lignin and other wall components are deposited within the primary cell wall. These secondary thickened cells then undergo programmed cell death producing a network of empty cells with which water and ions can be transported throughout the plant. The hormones auxin and cytokinin are the principle signals for vascular tissue initiation. As a consequence cells cultured in-vitro can be converted into vascular tissue with the addition of exogenous auxin and cytokinin. We have created an in-vitro cell system, using callus produced from leaves that can be induced to form vascular tissue. Leaves are callused on induction media for two weeks. The callus is then transferred to liquid media and incubated under optimum conditions resulting in an increase in vascular tissue formation. Approximately 20% of cells will differentiate during the incubation period. The alteration of cytokinin concentration affects the ability of the cultured cells to undergo differentiation. Consequently callus incubated in liquid media, containing lower cytokinin concentrations, will undertake relatively little differentiation. Samples have been isolated from cell cultures at different time points and different hormone concentrations during incubation. Quantitative PCR using the marker AtCesA7, which encodes a cellulose synthase subunit specific to secondary wall deposition, was used as a guide to determine periods of high and low vascular differentiation. This system provides an opportunity to compare gene expression between differentiating and non differentiating cells and allow the identification of genes up regulated during vascular tissue formation.
Project description:Currently little is known about the genetic mechanisms regulating the vascular cambium or the secondary growth of stems. We show here that the Populus Class I KNOX homeobox gene ARBORKNOX2 (ARK2) regulates both cell division in the cambium region and the differentiation of daughter cells in secondary xylem and phloem. ARK2 is expressed in the shoot apical meristem, and the vascular cambium region, reflecting some overlap in the regulation of these meristems. ARK2 is expressed broadly in the cambium region and in differentiating lignified cells types before becoming progressively restricted to the cambium. Populus overexpressing ARK2 present stem phenotypes with precocious cambium formation, delayed differentiation of cambium daughter cells, a wider cambium region, and ultimately less phloem fibers and secondary xylem. In contrast, Populus expressing RNAi or amicroRNA that target ARK2 transcripts present precocious differentiation of secondary phloem fibers and xylem, and ultimately more secondary xylem tissue and thicker secondary cell walls in phloem fibers and secondary xylem cells. These phenotypes in turn correlate with changes in the expression of genes affecting cell division, auxin, and cell wall synthesis and lignification that indicate that ARK2 primarily affects woody tissue development by regulation of cell differentiation. Notably, wood properties associated with secondary cell wall synthesis are negatively associated with ARK2 expression, including lignin and cellulose content. Together, our results suggest that ARK2 functions primarily by negatively regulating cell differentiation during secondary growth. We propose that ARK2 may identify a co-evolved regulatory module that influences complex wood properties relevant to ecological, industrial, and biofuels applications.