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

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

Gravitational and Space Research
Volume 3 (2015): Issue 2 (December 2015)
By: C. Adam Cook,  Avery Tucker,  Kaiyu Shen and  Sarah E. Wyatt  
Open Access
|Dec 2015

Figures & Tables

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 min4 min 10 min30 min
GeneLocus IDLFC*P-valueLFCP-valueLFCP-valueLFCP-value
WRKY26AT5G07100  1.35  0.11-1.76  0.00-0.53  0.31-0.94  0.09
AtAIBAT2G46510  0.50  0.16-1.03  0.04-0.16  0.67-0.24  0.32
WRKY33AT2G38470-0.34  0.18-1.00  0.02-0.01  0.99  0.13  0.76
BT2AT3G48360-0.41  0.74-1.41  0.10-1.59  0.01-1.26  0.08
WRKY18AT4G31800-0.11  0.86-1.08  0.05-1.83  0.02-1.24  0.03
DOI: https://doi.org/10.2478/gsr-2015-0008 | Journal eISSN: 2332-7774
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Language: English
Page range: 20 - 29
Published on: Dec 1, 2015
Published by: American Society for Gravitational and Space Research
In partnership with: Paradigm Publishing Services
Publication frequency: 2 issues per year
Keywords:
Gravitropism,
Gravity Signaling,
Arabidopsis,
GPS Treatment,
Transcription Factors,
Microarray,
RT-qPCR
Related subjects:
Life sciences,
Life sciences, other,
Materials sciences,
Materials sciences, other,
Physics,
Physics, other

© 2015 C. Adam Cook, Avery Tucker, Kaiyu Shen, Sarah E. Wyatt, published by American Society for Gravitational and Space Research
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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