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| Status |
Public on Nov 23, 2005 |
| Title |
Characterization of an Arabidopsis mutant deficient in the non-phosphorylating of GAPN gene. |
| Organism |
Arabidopsis thaliana |
| Experiment type |
Expression profiling by array
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| Summary |
Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) is a conserved protein found in higher plants. In photosynthetic cells, the enzyme is involved in a shuttle transfer mechanism to export NADPH from the chloroplast to the cytosol. In this work, we demonstrate that GAPN gene express in leaves and roots at similar levels; showing the highest level of expression in flowers. To investigate the role of this enzyme in different plant tissues, we characterized a mutant from Arabidopsis thaliana having an insertion at the GAPN gene locus. The homozygous mutant was determined to be null respect to GAPN, as it exhibited complete absence of both expression of GAPN mRNA and enzyme activity. Transcriptome analysis demonstrated that the insertion mutant plant shows altered expression of several enzymes involved in carbohydrate metabolism. Significantly, cytosolic phosphorylating (NAD-dependent) glyceraldehyde-3-phosphate dehydrogenase mRNA levels are induced in the mutant, which correlates with an increase in enzyme activity. mRNA levels and enzymatic activity of glucose-6-phosphate dehydrogenase were also elevated, correlating with an increase in NADPH concentration. Moreover, an increased levels of oxidative stress was measured in the mutant plants. Downregulation of several glycolytic and photosynthetic genes suggests that GAPN is important for the efficiency of both metabolic processes. The results presented demonstrate that GAPN has a relevant role in plant growth and development.
Keywords: 2 color experiment in triplicate including dye-swap
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| Overall design |
Arabidopsis oligonucleotide microarrays fabricated by the University of Arizona contain 26,000 oligonucleotides. The experimental and reference RNA samples were directly labeled with either Cy5-dUTP or Cy3-dUTP fluorescent dye (Amersham Pharmacia Biotech, Piscataway, NJ), using random hexamer primers (Invitrogen). Excess nucleotides and primers were removed using QIAquick PCR Purification Kit (Qiagen, Valencia, CA). Labeled samples were mixed and then hybridized to a microarray for 15 h at 60C. The slides were washed at room temperature in three wash steps: 2x SSC, 0.5% SDS; 0.5x SSC; and 0.05x SSC for 5 min each with gentle shaking. The slides were scanned with a GenePix 4000B Scanner (Axon Instruments Inc., Union City, CA). Normalization between the Cy3 and Cy5 fluorescent dye emission channels was achieved by adjusting the levels of both image intensities. The experiments were repeated three times with samples from different experiments, as biological replicates. In dye swapping experiments, the RNA samples from different experiments were labeled reciprocally, both as a biological and technical repetition for comparing the reproducibility of the experiments.
The hybridization intensities of each microarray element were measured using ScanAlyze 4.24 (available at http://genome-www4.stanford.edu/MicroArray/SMD /restech.html). The two channels were normalized in log space using the z-score normalization on a 95% trimmed data set. We removed unreliable spots according to the following criteria: spots flagged as having false intensity caused by dust or background on the array were removed; and spots for which intensity was less than three fold above background were also eliminated. Data from multiple experiments were normalized (Bolstad et al., 2003) and signals from spots from different experiments were analyzed using Significance Analysis of Microarrays using the one class response (SAM, Tusher et al., 2001, http://www-stat.stanford.edu/~tibs/SAM/.), with a false discovery rate <10%.
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| Contributor(s) |
Rius SP, Casati P, Iglesias AA, Gomez-Casati DF |
| Citation(s) |
16368875 |
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| Submission date |
Oct 31, 2005 |
| Last update date |
Mar 16, 2012 |
| Contact name |
Paula Casati |
| E-mail(s) |
casati@cefobi-conicet.gov.ar
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| Phone |
54-341-4371955
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| Organization name |
Universidad Nacional de Rosario
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| Department |
Biological Sciences
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| Lab |
Casati
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| Street address |
Suipacha 531
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| City |
Rosario |
| State/province |
Santa Fe |
| ZIP/Postal code |
2000 |
| Country |
Argentina |
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| Platforms (1) |
| GPL1941 |
Galbraith Laboratory Operon Long Oligonucleotide Microarray version 3.0 (ATv302.01.Z) |
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| Samples (3) |
| GSM81077 |
Characterization of an arabidopsis mutant deficient in the non-phosphorylating of GAPN gene. |
| GSM81094 |
Characterization of an Arabidopsis mutant deficient in the non-phosphorylating of GAPN gene. |
| GSM81148 |
Characterization of an Arabidopsis mutant deficient in the non-phosphorylating of GAPN gene-2 |
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| Relations |
| BioProject |
PRJNA93599 |
| Supplementary data files not provided |
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