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A reverse-genetic screen for N-regulators

A reverse-genetic screen for N-regulators. UNDERLYING ASSUMPTIONS Transcription factors (TFs) are involved in N-regulation Some of these TFs are regulated at the transcriptional level by N availability SCREENING STRATEGY Identify N-regulated TF genes by real-time RT-PCR

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A reverse-genetic screen for N-regulators

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  1. A reverse-genetic screen for N-regulators • UNDERLYING ASSUMPTIONS • Transcription factors (TFs) are involved in N-regulation • Some of these TFs are regulated at the transcriptional level by N availability • SCREENING STRATEGY • Identify N-regulated TF genes by real-time RT-PCR • Over-express suspect TF genes in Arabidopsis (using constitutive and inducible promoters) • Obtain TF knockout mutants for genes of interest • Determine the phenotype of mutant and transgenic lines • Test expression of N-assimilation genes in mutants and transgenic lines • Confirm physical interaction between TF and promoters (e.g. ChIP-PCR)

  2. Real-time RT-PCR profiling of >1400 Arabidopsis transcription factor genes 90% coverage with high specificity, sensitivity, dynamic range, and robustness Czechowski et al. (2004) Plant J.38, 366-379.

  3. Real-time RT-PCR is more precise than Affy chips Real-time RT-PCR Intra-assay (A) and Interassay (B) variation Affy Chips Interassay variation

  4. N-regulated TF genes: Suspects for global control of N acquisition 10 Axenic culture: +N to –N to NO3- 6 2 0 Log2 TF ratio X/+N -2 -6 -10 +N -N2d NO3-30min Inducible over-expression to identify target genes/phenotypes 40 N-regulated TFs, including regulators of anthacyanin biosynthesis and flowering (FD). Scheible et al. (2004) Plant Physiol. (in press).

  5. The role of transcription factors in abiotic stress tolerance and plant development

  6. Overview of TF projects -N NO3- -P Redox Real-time RT-PCR/ Reverse genetics -S Heterosis Seed Develop. Salt+H2O stress

  7. Seed/silique-specific TFs: potential tools for seed biotechnology TF gene expression level relative to ubi10 Shoots (log2) 50-fold induced: candidates Siliques (log2)

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