Project description:An altered response to methylamine (MA) has been used to identify genes involved in nitrogen metabolism in many microbes and as an uncoupler of proton motive force in both prokaryotes and eukaryotes. The aim of this study was to attempt the use of MA to identify critical genes for response to exogenous nitrogen sources in Arabidopsis thaliana (thale cress). EMS mutagenesis and selection on MA were used to identify a series of mutants that showed increased sensitivity to MA but failed to identify any MA resistant mutants. The eight sensitive mutants were allelic as judged by segregation and map location so one mutant, gsr1-1, was selected for intensive analysis. Co-segregation of gsr1 with DNA markers showed the gene was on chromosome 5 between markers CA72 and nga151, an interval of about 0.42 Mbp. The co-segregating mutant phenotypes associated with gsr1 included; yellow color caused by a reduction in chloroplast number; reduced chlorophyll content; decreased nitrate reductase (EC 1.7.1.3) activity in the root; and increased glutamate dehydrogenase (EC 1.4.1.2) activity in root and shoot. Decreased transcripts included those encoding several nitrogen metabolism enzymes. In contrast, the mutation increased only 4 and 7 transcript abundances by more than two fold. RT-PCR showed that in the dark, regulation of transcript abundance by exogenously applied glutamine and to a lesser extent sucrose were altered. Gene action of the gsr1 mutant was recessive for chloroplast number but co-dominant for transcript abundances and co-dominant for enzyme activities. Supplementary data from the analysis of preliminary microarray data from AFGC used 10,560 targets and two reverse labeled slides to show that more than 351 ESTs were increased in transcript abundance by more than 2 fold in the green plants. In yellow plants only seventy-three ESTs were up-regulated in transcript abundance by more than 2 fold. Therefore, the gsr1 locus may regulate responses to endogenous and exogenous glutamine in an organ-specific fashion that could coordinately regulate the activity of genes enzymes and metabolic pathways that determine cellular nitrogen, carbon and bioenergetic (redox) status. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments.

Project description:An altered response to methylamine (MA) has been used to identify genes involved in nitrogen metabolism in many microbes and as an uncoupler of proton motive force in both prokaryotes and eukaryotes. The aim of this study was to attempt the use of MA to identify critical genes for response to exogenous nitrogen sources in Arabidopsis thaliana (thale cress). EMS mutagenesis and selection on MA were used to identify a series of mutants that showed increased sensitivity to MA but failed to identify any MA resistant mutants. The eight sensitive mutants were allelic as judged by segregation and map location so one mutant, gsr1-1, was selected for intensive analysis. Co-segregation of gsr1 with DNA markers showed the gene was on chromosome 5 between markers CA72 and nga151, an interval of about 0.42 Mbp. The co-segregating mutant phenotypes associated with gsr1 included; yellow color caused by a reduction in chloroplast number; reduced chlorophyll content; decreased nitrate reductase (EC 1.7.1.3) activity in the root; and increased glutamate dehydrogenase (EC 1.4.1.2) activity in root and shoot. Decreased transcripts included those encoding several nitrogen metabolism enzymes. In contrast, the mutation increased only 4 and 7 transcript abundances by more than two fold. RT-PCR showed that in the dark, regulation of transcript abundance by exogenously applied glutamine and to a lesser extent sucrose were altered. Gene action of the gsr1 mutant was recessive for chloroplast number but co-dominant for transcript abundances and co-dominant for enzyme activities. Supplementary data from the analysis of preliminary microarray data from AFGC used 10,560 targets and two reverse labeled slides to show that more than 351 ESTs were increased in transcript abundance by more than 2 fold in the green plants. In yellow plants only seventy-three ESTs were up-regulated in transcript abundance by more than 2 fold. Therefore, the gsr1 locus may regulate responses to endogenous and exogenous glutamine in an organ-specific fashion that could coordinately regulate the activity of genes enzymes and metabolic pathways that determine cellular nitrogen, carbon and bioenergetic (redox) status. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. replicate_design

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Evidence that class I glutamine amidotransferase, GAT1_2.1, acts as a glutaminase in roots of Arabidopsis thaliana (part II)

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