Characterizing the stress/defense transcriptome of Arabidopsis

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.

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.

Figure 2

Differential screening of clones from the stress libraries generated using SSH. Subtracted cDNA fragments obtained by the SSH procedure were cloned (see Materials and methods for details) and maintained as bacterial cultures in 96-well plates for each library. Quadruplicate colony dot blots were prepared and the membranes hybridized with labeled unsubtracted cDNA probes derived from (a) the driver, (b) the unsubtracted cDNA probes from the tester, (c) the forward subtracted cDNAs or (d) the reverse-subtracted cDNA.

Figure 3

Recovery of differentially expressed genes as a function of the number of clones screened. To reduce the redundant sequencing of clones, we pooled DNA from previously sequenced clones and used it as probes on new filters prepared from the stress libraries. As more clones were screened within a library, the fraction of genes that had not yet been recovered decreased.

Figure 4

Representative northern blot analyses of stress-modulated genes using cloned cDNA fragments from the SSH libraries. (a) Acute ozone treatment; (b) comparison of treatment with salicylic acid, virulent bacteria or avirulent bacteria; (c) chronic ozone treatment; (d) treatment with virulent oomycete. Control leaves (C) were infiltrated with 10 mM MgCl₂. Tissue for RNA isolations was harvested at the indicated time points post-treatment.

Figure 5

A pie chart showing the fraction of stress-modulated genes in each of the functional categories described in Bevan et al. [38].