Multiple Transcription-Factor Genes Are Early Targets of Phytochrome A Signaling
James M. Tepperman, Tong Zhu, Hur-Song Chang, Xun Wang, and Peter H. Quail
Proc. Natl. Acad. Sci. USA 98 (16), 9437-9442 (2001)
Supplemental Material
Supplemental Material
phyA - regulated Genes in Arabidopsis
Tables
- Table 2. Early-induced genes:
Histograms.
- Table 3. Early-induced
genes: Time-course profiles.
-
Table 4. Early-repressed genes: Histograms.
- Table 5. Early-repressed genes: Time-course profiles.
-
Table 6. Late-induced genes:
Time-course profiles.
- Table 7.
Late-repressed genes: Time-course profiles.
Figures
- Figure 5. Scatterplot of dark control RNAs
- Figure 6. Transcription-factor/target-gene pair: CCA/CAB
- Figure 7. Transcription-factor/target-gene pair: HY5/CHS
- Figure 8. Transcription-factor/target-gene pair: DOF/GDCH
Notes and References
- Total RNA was prepared using either Qiagen RNeasy columns (Valencia,
CA) or the method of Chang et al. (3) and precipitated overnight at 4 degrees C
after the addition of 0.25 volumes of 10M LiCl2. Pellets were washed with 70%
EtOH, air dried and resuspended in RNase-free water. First strand cDNA synthesis
was accomplished using 5 micrograms of total RNA, 100 pmol of an oligo dT(24) primer
containing a 5' T7 RNA polymerase promoter sequence, 200 units of SuperScript II
reverse transcriptase (Gibco/BRL) in a reaction with 50 mM Tris-HCl (pH 8.3), 75
mM KCl, 3 mM MgCl2, 10 mM dithiotreitol (DTT), and 0.5 mM dNTPs. The second cDNA
strand was synthesized using 40 units of E. coli DNA polymerase, 10 units of E.
coli ligase, and 2 units of RNase H in a reaction containing 25 mM Tris-HCl (pH
7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)SO4, 0.15 mM beta-NAD+, 1 mM dNTPs, and 1.2
mM DTT. The reaction proceeded at 16 degrees C for 2 hours and was terminated using
EDTA. Double-stranded cDNA products were purified by phenol/chloroform extraction
and ethanol precipitation. Approximately 0.1 microgram of these cDNAs was used as a
template to produce biotinylated cRNA probes by in vitro transcription using T7
RNA Polymerase (ENZO BioArray High Yield RNA Transcript Labeling Kit). Labeled
cRNAs were purified using affinity resin (Qiagen RNeasy Spin Columns) and
randomly fragmented to produce molecules of approximately 35 to 200 bases.
Fragmentation was achieved by incubating at 94 degrees C for 35 minutes in a buffer
containing 40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM
magnesium acetate. High-density oligonucleotide arrays containing probes of more than 8200 different
Arabidopsis genes (Affymetrix, Santa Clara, CA) were used for gene expression
detection. Fragmented cRNAs were mixed with sonicated herring sperm DNA at 0.1
mg/ml in a hybridization buffer containing 100 mM 2-N-Morpholino-ethane-sulfonic
acid (MES), 1 M NaCl, 20 mM EDTA, 0.01% Tween 20. The hybridization mixture was
heated at 99 degrees C for 5 min and equilibrated at 45 degrees C for 5 min before the
hybridization mixture was transferred to the probe array cartridge. Hybridization
was carried out at 45 degrees C for 16 hours with mixing on a rotisserie at 60 rpm.
After hybridization, the hybridization mixture was removed and the array
cartridge was washed and stained in a fluidics station (Affymetrix). The
cartridge was rinsed with wash buffer A (6X SSPE (0.9M NaCl, 0.06 M NaH2PO4,
0.006 M EDTA), 0.01% Tween 20, 0.005% Antifoam) at 25 degrees C for 10 min and incubated
with wash buffer B (100 mM MES, 0.1 M NaCl, 0.01% Tween 20) at 50 degrees C for 20 min.
The probe array was stained with Streptavidin Phycoerythrin (SAPE) (100 mM MES,
1M NaCl, 0.05% Tween 20, 0.005% Antifoam, 10 micrograms/ml SAPE, 2 mg/ml BSA) at 25 degrees C for
10 min, washed with wash buffer A at 25 degrees C for 20 min and stained with
biotinylated anti-streptavidin antibody at 25 degrees C for 10 min. The probe array was
stained with SAPE at 25 degrees C for 10 min and washed with wash buffer A at 30 degrees C for
30 min. The probe array was scanned twice and the intensities were averaged with
a Hewlett-Packard GeneArray Scanner. Genechip Suite 3.2 (Affymetrix) was used for
image analysis. The average intensity of all probe sets was used for
normalization and scaled to 100 in the absolute analysis for each probe array.
- Expression data for all gene sequences on the microarrays were
analyzed using software written by Guangzhou Zou (Novartis Agricultural Discovery
Institute, Inc.) and Microsoft Excel. For each gene, the level of expression in
the wild-type, dark-control seedlings at time zero of the time-course was defined
as the reference level to which the expression levels at all other timepoints on
the curve for that gene were compared. Initially, all genes displaying a
two-fold or greater deviation in expression from this reference value at one or
more timepoints on the curve for FRc-irradiated, wild-type seedlings were
identified. The data for these genes were then examined separately at each
timepoint for both wild type and mutant. For the 1-hour FRc timepoint,
triplicate hybridization-intensity values for each of four seedling samples (wild
type and phyA mutant, each at time zero (dark controls) and after 1 hour of FRc
irradiation) were analyzed. These values were obtained using three independent
RNA samples, separately prepared from three different tissue samples for each of
the four treatments, each hybridized to a separate microarray chip (12 in total).
The triplicate values for each gene were averaged and the standard errors (±
S.E.) calculated. Genes for which the mean expression level in the
FRc-irradiated wild type at 1 hour deviated two-fold or more from the mean
time-zero, wild-type, dark-control level, and for which the S.E. bars for the
FRc-irradiated wild type did not overlap with those of the other three
treatments, were defined as "early-response" genes. For the 3- to 24-hour
timepoints, only genes that deviated in expression by two-fold or more from that
of the FRc-irradiated phyA mutant at that timepoint were retained, and combined
into a single category termed "late-response" genes. Finally, for all genes
identified by the above criteria, the full time-course curves were visually
inspected individually for coherence and continuity. Genes displaying profiles
that appeared to lack rational continuity or internal consistency, frequently due
to apparent random fluctuations in signal intensity, especially with genes
expressed at low levels, were eliminated.
- Chang, S., Puryear, J., & Cairney, J. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11, 113-116 (1993).
- All time-courses for
individual genes included here comply rigorously with the objective, quantitative
selection criteria described in the text (Supplemental Material, Note 2). Visual inspection of these individual
curves indicates, however, that a number of genes, particularly in the
down-regulated category, exhibit overall patterns of expression that might
intuitively suggest only marginal, if any, true regulation by phyA. It is likely,
therefore, that the present data overestimate the number of phyA-regulated genes
to some extent. We have retained these data here, nevertheless, in the interests
of consistency with the objective criteria used, and to ensure their availability
as the basis for future more refined analysis by interested researchers. In
addition, some genes appear to exhibit a certain degree of responsiveness
to FRc in the phyA mutant, as well as in the wild type, albeit generally to
a lower extent than in the wild type. The reason for this observation is
undetermined, but possibilities might include the participation of another
phy with previously undescribed activity under FRc conditions and
specificity for this limited subset of genes, photodynamic stress induced
by the prolonged irradiation of the incompletely developed photosynthetic
apparatus with FRc, or other undefined variables involved in the
experimental procedure that selectively affected these genes. The basis
for this phenomenon requires further investigation.