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Characterizing the stress/defense transcriptome of Arabidopsis.

Mahalingam R, Gomez-Buitrago A, Eckardt N, Shah N, Guevara-Garcia A, Day P, Raina R, Fedoroff NV - Genome Biol. (2003)

Bottom Line: Several stress-responsive cis-elements showed a statistically significant over-representation in the promoters of the genes in the stress cDNA collection.These include W- and G-boxes, the SA-inducible element, the abscisic acid response element and the TGA motif.This set of stress-, pathogen- and hormone-modulated genes is an important resource for understanding the genetic interactions underlying stress signaling and responses and may contribute to the characterization of the stress transcriptome through the construction of standardized specialized arrays.

View Article: PubMed Central - HTML - PubMed

Affiliation: Life Sciences Consortium, Pennsylvania State University, State College, PA 16802, USA.

ABSTRACT

Background: To understand the gene networks that underlie plant stress and defense responses, it is necessary to identify and characterize the genes that respond both initially and as the physiological response to the stress or pathogen develops. We used PCR-based suppression subtractive hybridization to identify Arabidopsis genes that are differentially expressed in response to ozone, bacterial and oomycete pathogens and the signaling molecules salicylic acid (SA) and jasmonic acid.

Results: We identified a total of 1,058 differentially expressed genes from eight stress cDNA libraries. Digital northern analysis revealed that 55% of the stress-inducible genes are rarely transcribed in unstressed plants and 17% of them were not previously represented in Arabidopsis expressed sequence tag databases. More than two-thirds of the genes in the stress cDNA collection have not been identified in previous studies as stress/defense response genes. Several stress-responsive cis-elements showed a statistically significant over-representation in the promoters of the genes in the stress cDNA collection. These include W- and G-boxes, the SA-inducible element, the abscisic acid response element and the TGA motif.

Conclusions: The stress cDNA collection comprises a broad repertoire of stress-responsive genes encoding proteins that are involved in both the initial and subsequent stages of the physiological response to abiotic stress and pathogens. This set of stress-, pathogen- and hormone-modulated genes is an important resource for understanding the genetic interactions underlying stress signaling and responses and may contribute to the characterization of the stress transcriptome through the construction of standardized specialized arrays.

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Analysis of subtraction efficiency using PCR. Tester cDNA was prepared from the poly(A)+ RNA of plants sprayed with the virulent oomycete P. parasitica and the driver cDNA was from water-treated control plants. The unsubtracted and subtracted pools of cDNA were amplified using primers for the pathogen-inducible PR1 gene or the constitutively expressed G3PDH gene. Aliquots of the samples were taken after 15, 20, 25 and 30 cycles of PCR amplification and the products were analyzed on a 2% agarose gel.
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Figure 1: Analysis of subtraction efficiency using PCR. Tester cDNA was prepared from the poly(A)+ RNA of plants sprayed with the virulent oomycete P. parasitica and the driver cDNA was from water-treated control plants. The unsubtracted and subtracted pools of cDNA were amplified using primers for the pathogen-inducible PR1 gene or the constitutively expressed G3PDH gene. Aliquots of the samples were taken after 15, 20, 25 and 30 cycles of PCR amplification and the products were analyzed on a 2% agarose gel.

Mentions: The efficiency of subtraction was evaluated by PCR amplification of a housekeeping gene, that for gyceraldehyde-3-phosphate dehydrogenase (G3PDH), and one of several differentially expressed genes. If subtraction is efficient, transcripts of housekeeping genes should be reduced, while those of differentially expressed genes should be substantially enriched in the population of cDNA fragments used for library construction. Figure 1 shows that the G3PDH fragment is barely detectable even after 30 cycles of amplification in the subtracted sample, while it is clearly detectable in the unsubtracted sample after 20 cycles. To test enrichment for differentially expressed genes, we amplified the PR1 gene for the biotic stressors and SA treatment, the plant defensin gene PDF1.2 for MJ treatment, and the amino-cyclopropane synthase gene (ACS1) for the ozone treatments [9,19,35]. The genes tested showed strong amplification in the subtracted samples after 15 cycles of PCR, whereas in the unsubtracted samples the PCR product was seen only after 10 additional cycles (Figure 1). On the basis of the number of PCR cycles required for equal amplification of the corresponding PCR products from the subtracted and unsubtracted cDNA samples, we estimated that the subtracted libraries were 32-64-fold enriched for differentially expressed genes.


Characterizing the stress/defense transcriptome of Arabidopsis.

Mahalingam R, Gomez-Buitrago A, Eckardt N, Shah N, Guevara-Garcia A, Day P, Raina R, Fedoroff NV - Genome Biol. (2003)

Analysis of subtraction efficiency using PCR. Tester cDNA was prepared from the poly(A)+ RNA of plants sprayed with the virulent oomycete P. parasitica and the driver cDNA was from water-treated control plants. The unsubtracted and subtracted pools of cDNA were amplified using primers for the pathogen-inducible PR1 gene or the constitutively expressed G3PDH gene. Aliquots of the samples were taken after 15, 20, 25 and 30 cycles of PCR amplification and the products were analyzed on a 2% agarose gel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC153460&req=5

Figure 1: Analysis of subtraction efficiency using PCR. Tester cDNA was prepared from the poly(A)+ RNA of plants sprayed with the virulent oomycete P. parasitica and the driver cDNA was from water-treated control plants. The unsubtracted and subtracted pools of cDNA were amplified using primers for the pathogen-inducible PR1 gene or the constitutively expressed G3PDH gene. Aliquots of the samples were taken after 15, 20, 25 and 30 cycles of PCR amplification and the products were analyzed on a 2% agarose gel.
Mentions: The efficiency of subtraction was evaluated by PCR amplification of a housekeeping gene, that for gyceraldehyde-3-phosphate dehydrogenase (G3PDH), and one of several differentially expressed genes. If subtraction is efficient, transcripts of housekeeping genes should be reduced, while those of differentially expressed genes should be substantially enriched in the population of cDNA fragments used for library construction. Figure 1 shows that the G3PDH fragment is barely detectable even after 30 cycles of amplification in the subtracted sample, while it is clearly detectable in the unsubtracted sample after 20 cycles. To test enrichment for differentially expressed genes, we amplified the PR1 gene for the biotic stressors and SA treatment, the plant defensin gene PDF1.2 for MJ treatment, and the amino-cyclopropane synthase gene (ACS1) for the ozone treatments [9,19,35]. The genes tested showed strong amplification in the subtracted samples after 15 cycles of PCR, whereas in the unsubtracted samples the PCR product was seen only after 10 additional cycles (Figure 1). On the basis of the number of PCR cycles required for equal amplification of the corresponding PCR products from the subtracted and unsubtracted cDNA samples, we estimated that the subtracted libraries were 32-64-fold enriched for differentially expressed genes.

Bottom Line: Several stress-responsive cis-elements showed a statistically significant over-representation in the promoters of the genes in the stress cDNA collection.These include W- and G-boxes, the SA-inducible element, the abscisic acid response element and the TGA motif.This set of stress-, pathogen- and hormone-modulated genes is an important resource for understanding the genetic interactions underlying stress signaling and responses and may contribute to the characterization of the stress transcriptome through the construction of standardized specialized arrays.

View Article: PubMed Central - HTML - PubMed

Affiliation: Life Sciences Consortium, Pennsylvania State University, State College, PA 16802, USA.

ABSTRACT

Background: To understand the gene networks that underlie plant stress and defense responses, it is necessary to identify and characterize the genes that respond both initially and as the physiological response to the stress or pathogen develops. We used PCR-based suppression subtractive hybridization to identify Arabidopsis genes that are differentially expressed in response to ozone, bacterial and oomycete pathogens and the signaling molecules salicylic acid (SA) and jasmonic acid.

Results: We identified a total of 1,058 differentially expressed genes from eight stress cDNA libraries. Digital northern analysis revealed that 55% of the stress-inducible genes are rarely transcribed in unstressed plants and 17% of them were not previously represented in Arabidopsis expressed sequence tag databases. More than two-thirds of the genes in the stress cDNA collection have not been identified in previous studies as stress/defense response genes. Several stress-responsive cis-elements showed a statistically significant over-representation in the promoters of the genes in the stress cDNA collection. These include W- and G-boxes, the SA-inducible element, the abscisic acid response element and the TGA motif.

Conclusions: The stress cDNA collection comprises a broad repertoire of stress-responsive genes encoding proteins that are involved in both the initial and subsequent stages of the physiological response to abiotic stress and pathogens. This set of stress-, pathogen- and hormone-modulated genes is an important resource for understanding the genetic interactions underlying stress signaling and responses and may contribute to the characterization of the stress transcriptome through the construction of standardized specialized arrays.

Show MeSH
Related in: MedlinePlus