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)

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,

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

N-regulated TF genes: Suspects for global control of N acquisition Axenic culture: +N to –N to NO N-regulated TFs, including regulators of anthacyanin biosynthesis and flowering (FD). Inducible over-expression to identify target genes/phenotypes Log 2 TF ratio X/+N N-N 2d NO min Scheible et al. (2004) Plant Physiol. (in press).

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

Overview of TF projects Real-time RT-PCR/ Reverse genetics -NNO 3 - -P -S Redox Heterosis Seed Develop. Salt+H 2 O stress

Seed/silique-specific TFs: potential tools for seed biotechnology TF gene expression level relative to ubi10 Siliques (log 2 ) Shoots (log 2 ) 50-fold induced: candidates