Isolation of poplar genes involved in xylem vessel differentiation
Ontology highlight
ABSTRACT: Downstream genes of PtVNS genes were explored with inducible expression system using glucocorticoid receptor (GR). Transgenic poplar plants expressing 35S:AtVND7-VP16-GR were treated with dexamethazone (DEX). A number of genes related to the formation of xylem vessels were induced by DEX.
ORGANISM(S): Populus tremula x Populus tremuloides Populus sp.
Project description:Downstream genes of PtVNS genes were explored with inducible expression system using glucocorticoid receptor (GR). Transgenic poplar plants expressing 35S:AtVND7-VP16-GR were treated with dexamethazone (DEX). A number of genes related to the formation of xylem vessels were induced by DEX. Total RNAs of the transgenic plants carrying 35S:AtVND7-VP16-GR treated with and without DEX were compared.
Project description:Direct target genes of VND7 were explored with inducible expression system using glucocorticoid receptor (GR). Transgenic plants expressing 35S:VND7-VP16-GR were treated with dexamethazone (DEX) and/or protein synthesis inhibitor cycloheximide (CHX). A number of genes related to the formation of vascular vessel was induced by DEX even in the presence of CHX. Total RNAs of the transgenic plants expressing 35S:VND7-VP16-GR treated with DEX plus CHX and those treated with CHX only were compared. As a control experiment, transgenic plants harboring empty vector were treated similarly and the total RNAs were compared similarly to identify genes merely induced by DEX treatment itself.
Project description:Lignocellulosic feedstock (i.e., wood) is gaining popularity as a source of fermentable sugars for liquid fuel production. To improve the quantity and quality of woody biomass, the developing xylem (DX) cell-specific genetic modification is desired. Bioinformatic analyses followed by the validation of cell type-specific transcriptomes led to the identification of 37 transcripts specifically expressed in DX. After further confirmation of DX-specific expression, we selected four genes (DX5, DX8, DX11 and DX15) to demonstrate the feasibility of our strategy. The promoter regions of selected DX genes were isolated and produced stable transformants of poplar by using transcriptional promoter:β-glucuronidase (GUS) fusion constructs. The GUS expression patterns of DX5 and ANAC073 (orthologous gene of Arabidopsis) revealed that these promoters were active in xylem cells in poplar at early seedling growth, and showed strongest expression in the developing xylem cells in the wood formation/development at later growth stages of poplar. DX specific and strong expression patterns of all the other DX promoters (DX8, DX11 and DX15) tested suggests that these promoters may be useful to control transgene expression in the DX cells of woody plants with the aim of the feedstock improvement.
Project description:We take the two year old plant for sampling. Use the Affymetrix poplar gene chip to elucidate the gene functions and mechanisms in Populus tomentosa newly formed developing xylem and lignified xylem. We used microarrays to detail the global programme of gene expression in newly formed developing xylem and lignified xylem.
Project description:Lignocellulosic feedstock (i.e., wood) is gaining popularity as a source of fermentable sugars for liquid fuel production. To improve the quantity and quality of woody biomass, the developing xylem (DX) cell-specific genetic modification is desired. Bioinformatic analyses followed by the validation of cell type-specific transcriptomes led to the identification of 37 transcripts specifically expressed in DX. After further confirmation of DX-specific expression, we selected four genes (DX5, DX8, DX11 and DX15) to demonstrate the feasibility of our strategy. The promoter regions of selected DX genes were isolated and produced stable transformants of poplar by using transcriptional promoter:?-glucuronidase (GUS) fusion constructs. The GUS expression patterns of DX5 and ANAC073 (orthologous gene of Arabidopsis) revealed that these promoters were active in xylem cells in poplar at early seedling growth, and showed strongest expression in the developing xylem cells in the wood formation/development at later growth stages of poplar. DX specific and strong expression patterns of all the other DX promoters (DX8, DX11 and DX15) tested suggests that these promoters may be useful to control transgene expression in the DX cells of woody plants with the aim of the feedstock improvement. In order to identify specifically and strongly expressed genes from DX cells of woody plant, we generated the tissue/cell type-specific transcriptomes from poplar stem by using whole genome GeneChip technology (Affymetrix).
Project description:Xylem vessels function in the long-distance conduction of water in land plants. The NAC transcription factor VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). We previously isolated seiv (suppressor of ectopic xylem vessel cell differentiation induced by VND7) mutants, which are suppressor mutants of VND7-inducible xylem vessel cell differentiation. Here, we report that the responsible genes for seiv3, seiv4, seiv6, and seiv9 are protein ubiquitination-related genes encoding PLANT U-BOX46 (PUB46), uncharacterized F-BOX protein, PUB36, and UBIQUITIN-SPECIFIC PROTEASE1, respectively. We also found the decreased expression of genes downstream of VND7 and abnormal xylem transport activity in the seiv mutants. Upon VND7 induction, ubiquitinated levels from 492 and 180 protein groups were up- and down-regulated, respectively. Proteins for cell wall biosynthesis and protein transport were ubiquitinated by the VND7 induction, whereas such active protein ubiquitination was not observed in the seiv mutants. We detected the ubiquitination of three lysine residues in VND7: K94, K105, and K260. Substituting K94 with arginine significantly decreased the transactivation activity of VND7, suggesting that the ubiquitination of K94 is crucial for regulating VND7 activity. Our findings highlight the crucial roles of target protein ubiquitination in regulating xylem vessel activity.
Project description:affy_rewatering_poplar - rewatering - Biological question (15 lines max): Xylogenesis is a complex process and is highly influenced by environmental factors. Numerous studies have shown xylem structural acclimation to moderate and long-lasting water deficits. A decrease in cell lumen size and an increase in vessel density are generally observed. Other preliminary experiments in our laboratory focusing on a short and progressive drought-rewatering cycle in poplar show a gradual decrease in fibre size with decreasing water availability and an important rise in vessel number with irrigation recovery. This suggests a reorientation during the early stages of cambial derivatives differentiation towards vessel formation. The aim of this study is to characterize global gene expression in young differentiating xylem of poplar in response to a drought-rewatering cycle. Results should provide a comprehensive genomic basis for the xylem structural modifications observed.-The experimental design consisted in a differential water supply. Experiments were done on tilted trees to induce tension wood formation. Trees were regularly irrigated at full capacitance until July 18, 2007, the beginning of the experiment. Two sets of plants were designed. For each set, irrigation was withheld until July 25, 2007. At this time, one of the two sets (treated-sample) was re-irrigated at full capacitance during 14hrs (WDR) while the other one was maintained under water deficit (WD). Samples were collected after the 14hrs for all trees. Keywords: treated vs untreated comparison
Project description:affy_rewatering_poplar - rewatering - Biological question (15 lines max): Xylogenesis is a complex process and is highly influenced by environmental factors. Numerous studies have shown xylem structural acclimation to moderate and long-lasting water deficits. A decrease in cell lumen size and an increase in vessel density are generally observed. Other preliminary experiments in our laboratory focusing on a short and progressive drought-rewatering cycle in poplar show a gradual decrease in fibre size with decreasing water availability and an important rise in vessel number with irrigation recovery. This suggests a reorientation during the early stages of cambial derivatives differentiation towards vessel formation. The aim of this study is to characterize global gene expression in young differentiating xylem of poplar in response to a drought-rewatering cycle. Results should provide a comprehensive genomic basis for the xylem structural modifications observed.-The experimental design consisted in a differential water supply. Experiments were done on tilted trees to induce tension wood formation. Trees were regularly irrigated at full capacitance until July 18, 2007, the beginning of the experiment. Two sets of plants were designed. For each set, irrigation was withheld until July 25, 2007. At this time, one of the two sets (treated-sample) was re-irrigated at full capacitance during 14hrs (WDR) while the other one was maintained under water deficit (WD). Samples were collected after the 14hrs for all trees. Keywords: treated vs untreated comparison 6 arrays - poplar
Project description:We take the two year old plant for sampling. Use the Affymetrix poplar gene chip to elucidate the gene functions and mechanisms in Populus tomentosa newly formed developing xylem and lignified xylem. We used microarrays to detail the global programme of gene expression in newly formed developing xylem and lignified xylem. Populus tomentosa newly formed developing xylem and lignified xylem were taken for RNA extraction and hybridization on Affymetrix microarrays. CB2009304-A and CB2009304-B from newly formed developing xylem, CB2009304-G and CB2009304-H from lignified xylem.