Functional characterization of DUF642 genes in Arabidopsis thaliana Open Access
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University of Alberta
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Deyholos, Michael, Biological sciences
- Examining committee member and department
campbell, shelagh, Biological sciences
Department of Biological Sciences
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Doctor of Philosophy
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DOMAIN OF UNKNOWN FUNCTION 642, (DUF642), is an uncharacterized protein family in the Pfam database, a large collection of protein families. In Arabidopsis thaliana there are 10 proteins that contain DUF642 domains. DUF642 appears to be specific to plants and is present in gymnosperms, monocots and dicots. The present study was designed to investigate the biochemical function and physiological role of two Arabidopsis DUF642 genes, AT5G25460 (DGR2) and AT5G11420 (DUFB). The three dimensional structures of DGR2 and DUFB proteins were predicted by I-TASSER and validated by Ramachandran plot using RAMPAGE Server. Structural models indicated that DGR2 and DUFB showed similarity to carbohydrate binding proteins with hydrolase and carbon-oxygen lyase activity, respectively. Translation fusions with reporter genes showed a punctate pattern of subcellular localization within the cytoplasm, which did not co-localize with the Sec21 Golgi marker but was immediately adjacent to each other, suggesting DGR2 localizes to the trans-Golgi network. DGR2 and DUFB were expressed heterologously in E. coli but sufficient purified proteins could not be obtained for downstream functional assays. Although both DGR2 and DUFB have high sequence similarity (93.4% nucleotide identity), and were presumed to be paralogs, they were expressed in complementary spatial domains according to expression patterns of reporter genes and qRT-PCR analysis. GUS reporter fusions of DGR2 were expressed in the root apex and the later stages of lateral root primordial (LRP) where cells are dividing and elongating whereas DUFB was expressed in the elongating tissues of roots and not in LRP. Both genes were responsive to auxin identified by reporter gene assay where promoter region sequences of DGR2 and DUFB were fused to the GUS gene. T-DNA insertion mutants (Salk_042864 for DGR2 and Salk_094931 for DUFB), RNAi, and DGR2 and DUFB overexpressing plants showed no morphological differences from wild-type phenotypes, therefore, metabolic profiling of these mutant plants was performed by gas chromatography/mass spectrometry to reveal metabotypes. The results suggested that DGR2 and DUFB both affected TCA cycle intermediates and were involved in the carbohydrate metabolism. In addition to DGR2 and DUFB characterization in Arabidopsis, a portion of Ph.D. research work included metabolic profiling of developing flax seeds by GC/MS.
In summary, we have generated predicted 3D models of DGR2 and DUFB. Subcellular localization revealed that DGR2 possibly localizes to the trans-Golgi network. We speculated that DGR2 is required for cell elongation and division whereas DUFB is required for cell elongation in Arabidopsis. Metabolite profiling of the Arabidopsis T-DNA insertion mutants, RNAi and overexpression plants of DGR2 and DUFB reveals metabolic phenotypes previously unidentified and illustrate perturbation of TCA cycle. Both proteins may influence growth by modifying probably pectin thereby perturbed primary metabolic processes. Both genes are expressed E. coli but failed to obtain purified proteins. The metabolic profiling of developing flax seeds by GC/MS identified unique metabolites and the pathways perturbation during different stages of flax seed development.
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