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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
Double-stranded Template Copy Number
100 101 102 103 104 105 106
CT V
alu
e
15
20
25
30
35
40
45
R2=0.999
A
R2=0.996
Fraction of Root cDNA
0.0 0.2 0.4 0.6 0.8 1.0
Ex
pre
ss
ion
Le
ve
l (2
(40
-CT) )
0
25000
50000
75000
100000
125000
R2=0.998
R2=0.993
R2=0.994
B
Fraction of Shoot cDNA
1.0 0.8 0.6 0.4 0.2 0.0
R2=0.997
90% coverage with high specificity, sensitivity, dynamic range, and robustness
Czechowski et al. (2004) Plant J. 38, 366-379.
Real-time RT-PCR is more precise than Affy chips
Real-time RT-PCRIntra-assay (A) and
Interassay (B) variation
Affy ChipsInterassay variation
N-regulated TF genes: Suspects for global control of N acquisition
Axenic culture: +N to –N to NO3-
40 N-regulated TFs, including regulators of
anthacyanin biosynthesis and flowering (FD).
Inducible over-expressionto identify target
genes/phenotypes
Log
2 T
F ra
tio X
/+N
02
-2
6
10
-6
-10+N -N2d NO3
-30min
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
-N NO3-
-P
-S
Redox
Heterosis
Seed Develop.
Salt+H2Ostress
Seed/silique-specific TFs: potential tools for seed biotechnology
TF gene expression level relative to ubi10
Siliques (log2)
Shoots
(lo
g2)
50-fold induced: candidates
