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Microarray Identifies Transcription Factors Potentially Involved in Gravitropic Signal Transduction in Arabidopsis

C. Adam Cook
C. Adam Cook
, 
Avery Tucker
Avery Tucker
, 
Kaiyu Shen
Kaiyu Shen
 e 
Sarah E. Wyatt
Sarah E. Wyatt
   | 01 dic 2015
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Article Category: Short Communication
Pubblicato online: 01 dic 2015
Pagine: 20 - 29
DOI: https://doi.org/10.2478/gsr-2015-0008
© 2015 C. Adam Cook et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1.

Selection of transcription factors for further study. A microarray analysis was performed on wild type Arabidopsis at 2, 4, 10, and 30 min after reorientation in the cold during the GPS treatment. Transcription factors were selected based on annotation from the Database of Arabidopsis Transcription Factors and evaluated based on differential gene expression at 2 and 4 min. No transcription factors were differentially expressed at 2 min. A scatter plot of gene expression of 33 transcription factors identified at the 4 min time point. Five transcription factors (circled) were selected for further analysis based on high differential expression (LFC≤-1): AtAIB, WRKY18, WRKY26, WRKY33, and BT2.
Selection of transcription factors for further study. A microarray analysis was performed on wild type Arabidopsis at 2, 4, 10, and 30 min after reorientation in the cold during the GPS treatment. Transcription factors were selected based on annotation from the Database of Arabidopsis Transcription Factors and evaluated based on differential gene expression at 2 and 4 min. No transcription factors were differentially expressed at 2 min. A scatter plot of gene expression of 33 transcription factors identified at the 4 min time point. Five transcription factors (circled) were selected for further analysis based on high differential expression (LFC≤-1): AtAIB, WRKY18, WRKY26, WRKY33, and BT2.

Figure 2.

RT-qPCR analysis. Relative gene expression of each transcription factor was quantified in inflorescence stems of wild type Arabidopsis (var. Columbia) plants either reoriented (grey bars) or held vertical (black bars) across the GPS treatment. Expression levels were assessed at 2, 4, 10, and 30 min after reorientation of the treatment group in the cold and normalized to the 2 min vertical control. Each assay was performed in triplicate. Asterisks indicate significance determined by a Student’s t-test (p-value<0.05).
RT-qPCR analysis. Relative gene expression of each transcription factor was quantified in inflorescence stems of wild type Arabidopsis (var. Columbia) plants either reoriented (grey bars) or held vertical (black bars) across the GPS treatment. Expression levels were assessed at 2, 4, 10, and 30 min after reorientation of the treatment group in the cold and normalized to the 2 min vertical control. Each assay was performed in triplicate. Asterisks indicate significance determined by a Student’s t-test (p-value<0.05).

Figure 3.

Selection of homozygous mutants defective in each transcription factor: ataib, bt2, wrky18, wrky26, or wrky33. Arabidopsis lines with T-DNA inserts in the genes of interest were obtained from publicly available seed banks. Seed was planted, and DNA extracted from both wild type and mutant lines. PCR was used to confirm homozygous insertion in the gene of interest. Gene-specific forward and reverse primers and a primer complementary to the left border of the T-DNA insert for each mutant line were used to amplify DNA extracted from Columbia wild type (left) and the mutant indicated (right). Arrows (left) indicate the amplicon from the gene specific primers; the arrow heads (right) indicated the amplicon produced by a gene-specific primer and the T-DNA left border primer. Single bands in each lane indicate homozygosity of the allele.
Selection of homozygous mutants defective in each transcription factor: ataib, bt2, wrky18, wrky26, or wrky33. Arabidopsis lines with T-DNA inserts in the genes of interest were obtained from publicly available seed banks. Seed was planted, and DNA extracted from both wild type and mutant lines. PCR was used to confirm homozygous insertion in the gene of interest. Gene-specific forward and reverse primers and a primer complementary to the left border of the T-DNA insert for each mutant line were used to amplify DNA extracted from Columbia wild type (left) and the mutant indicated (right). Arrows (left) indicate the amplicon from the gene specific primers; the arrow heads (right) indicated the amplicon produced by a gene-specific primer and the T-DNA left border primer. Single bands in each lane indicate homozygosity of the allele.

Figure 4.

GPS phenotype of ataib, bt2, wrky18, wrky26, or wrky33. Seed for mutant (open circles) and Columbia wild type (closed circles) lines were planted, and plants grown to maturity with inflorescence stems of 8-10 cm. Plants were reoriented with respect to gravity at 4°C for 1 h then returned to vertical at RT. Images were taken every 15 min from 0-90 min after return to RT, and the angle of curvature for a minimum of 15 inflorescence stems was measured. Asterisks indicate significance difference, as determined by a Student’s t-test.
GPS phenotype of ataib, bt2, wrky18, wrky26, or wrky33. Seed for mutant (open circles) and Columbia wild type (closed circles) lines were planted, and plants grown to maturity with inflorescence stems of 8-10 cm. Plants were reoriented with respect to gravity at 4°C for 1 h then returned to vertical at RT. Images were taken every 15 min from 0-90 min after return to RT, and the angle of curvature for a minimum of 15 inflorescence stems was measured. Asterisks indicate significance difference, as determined by a Student’s t-test.

Microarray expression values of the five transcription factors across the GPS treatment.

2 min 4 min 10 min 30 min
Gene Locus ID LFC* P-value LFC P-value LFC P-value LFC P-value
WRKY26 AT5G07100   1.35   0.11 -1.76   0.00 -0.53   0.31 -0.94   0.09
AtAIB AT2G46510   0.50   0.16 -1.03   0.04 -0.16   0.67 -0.24   0.32
WRKY33 AT2G38470 -0.34   0.18 -1.00   0.02 -0.01   0.99   0.13   0.76
BT2 AT3G48360 -0.41   0.74 -1.41   0.10 -1.59   0.01 -1.26   0.08
WRKY18 AT4G31800 -0.11   0.86 -1.08   0.05 -1.83   0.02 -1.24   0.03
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