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

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Notes and References

  1. 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.


  2. 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.

  3. 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).

  4. 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.