Functional genomics of the Dof transcription factor family genes in suspension-cultured cells of Arabidopsis thaliana

Abstract

Harnessing plant metabolites and biomass not only for food, feed, and medicines, but also for feedstock for biofuels and industrial materials, appears promising as a strategy to provide sustainable and renewable resources. Biochemical compositions, yield, and yield stability of these plant materials are subject to transcriptional regulation of a diverse array of genes involved in regulatory networks of many metabolic pathways, growth and differentiation, and response to environmental stimuli. Since transcription factors control gene expression by binding directly or indirectly to the promoters of target genes in a sequence-specific manner to either activate or repress the transcription of downstream target genes, they naturally act as master regulators of many biological processes that are associated with quality and quantity of plant metabolites and biomass. Several pioneering studies demonstrated the potential of transcription factors for the manipulation of complex metabolic pathways in plants (Broun 2004). Therefore, understanding of function of transcription factors is an important step towards to develop the transcription factor-based technology for improvement of value and productivity of the plant materials (Broun 2004;Century et al. 2008;Grotewold 2008).With the completion of the Arabidopsis sequences, it became possible to identify putative genes for Abstract The Dof (DNA-binding with one finger) genes are members of a family of plant-specific transcription factors that have a highly conserved DNA-binding domain, namely, Dof domain. The Dof domain is a particular class of zinc finger domain that has been demonstrated to bind specifically to DNA sequences with a T/AAAAG core. In the Arabidopsis genomic database, 36 Dof genes have been identified, whereas the functions of most of the members still remain to be studied. Therefore, we attempted to comprehensively and systematically investigate functions of Arabidopsis Dof genes. As the first step, we isolated cDNAs of all the Arabidopsis Dof genes based on the coding sequences identified on the genomic database. Then, we selected genes, which are subjected to further functional analysis, through a phylogenetic analysis, and transformed Arabidopsis cultured cells (line T87) using cDNAs corresponding to the selected genes. After that, we examined transcriptional profiles in the Dof gene-overexpressed calli using an Arabidopsis DNA microarray. From the results, possible involvements of the Dof genes in regulation of metabolic pathways are discussed. Functional genomics of the Dof transcription factor family genes in suspension-cultured cells of Arabidopsis thalianaKey words: Arabidopsis thaliana, cultured cells, Dof family, metabolic regulation, microarray, transcription factors, transgenic callus.Plant Biotechnology 26, 15-28 (2009) Original PaperAbbreviations: CaMV35S, cauliflower mosaic virus 35S; CDS, coding sequence; Dof, DNA-binding with one finger; exGUS, callus ectopically expressing GUS; GUS, b-galacturonidase; LC/PDA/MS, liquid chromatography/ph...

Increase in pectin deposition by overexpression of an ERF gene in cultured cells of Arabidopsis thaliana

“…ecotype Columbia suspension-cultured cells, line T87 (Axelos et al 1992), were cultured as described previously (Tsujimoto-Inui et al 2009). Briefly, the culture was grown under continuous illumination (50-100 lmol m -2 s -1 ) at 22°C with shaking at 120 rpm in a 300 ml Elrenmeyer flask containing 80 ml JPL medium (pH 5.8) containing 1 lM 1-naphthaleneacetic Acid.…”

“…To identify transcription factors that are involved in the regulation of metabolic pathway and/or useful for metabolic engineering, we have undertaken the functional genomic study of transcription factors in Arabidopsis using the cultured cells overexpressing transcription factor genes (Tsujimoto-Inui et al 2009). Since the overexpression of transgenes is expected to confer dominant gain-of-function phenotypes and/or unexpected beneficial traits, the overexpression strategy appears particularly effective for functional analysis of transcription factors (Zhang 2003).…”

“…A cell culture that consists of homogeneous cells provides a favorable model for functional genomics as simplified experimental systems in Arabidopsis. A unique cell line, namely, T87, which has photosynthetic ability under light irradiation (Axelos et al 1992), was selected for the study (Tsujimoto-Inui et al 2009). …”

A batch processing protocol for construction of expression vector plasmids from a cDNA collection and Agrobacterium-mediated transformation of suspension-cultured cells of Arabidopsis

“…The 33 cDNAs for predicted CDSs of 36 DOF genes were amplified by PCR using the gene-specific primers and cloned into the Gateway entry vector, pENTR (Invitrogen, Carlsbad, CA, USA) as previously described (Tsujimoto-Inui et al 2009). In this study, we selected 10 of the cDNA collection for transformation of T87 cells (Supplemental Table 1).…”

“…ecotype Columbia suspension-cultured cells, line T87 (Axelos et al 1992), were maintained according to the previously described methods (Tsujimoto-Inui et al 2009). The Arabidopsis T87 cells were transformed by co-cultivation with the LBA4404 cells of A. tumefaciens harboring the pK2GW7 plasmids containing cDNAs for the 10 DOF genes according to the previously described methods (Tsujimoto-Inui et al 2009). Cell clusters, which are resistant to kanamycin, were picked up and assigned to a single transgenic cell line.…”