Mechanical stress induces biotic and abiotic stress responses via a novel cis-element

Abstract

Plants are continuously exposed to a myriad of abiotic and biotic stresses. However, the molecular mechanisms by which these stress signals are perceived and transduced are poorly understood. To begin to identify primary stress signal transduction components, we have focused on genes that respond rapidly (within 5 min) to stress signals. Because it has been hypothesized that detection of physical stress is a mechanism common to mounting a response against a broad range of environmental stresses, we have utilized mechanical wounding as the stress stimulus and performed whole genome microarray analysis of Arabidopsis thaliana leaf tissue. This led to the identification of a number of rapid wound responsive (RWR) genes. Comparison of RWR genes with published abiotic and biotic stress microarray datasets demonstrates a large overlap across a wide range of environmental stresses. Interestingly, RWR genes also exhibit a striking level and pattern of circadian regulation, with induced and repressed genes displaying antiphasic rhythms. Using bioinformatic analysis, we identified a novel motif overrepresented in the promoters of RWR genes, herein designated as the Rapid Stress Response Element (RSRE). We demonstrate in transgenic plants that multimerized RSREs are sufficient to confer a rapid response to both biotic and abiotic stresses in vivo, thereby establishing the functional involvement of this motif in primary transcriptional stress responses. Collectively, our data provide evidence for a novel cis-element that is distributed across the promoters of an array of diverse stress-responsive genes, poised to respond immediately and coordinately to stress signals. This structure suggests that plants may have a transcriptional network resembling the general stress signaling pathway in yeast and that the RSRE element may provide the key to this coordinate regulation.

Figure 1. Verification and Functional Classification of the RWR Genes

(A) RT-qPCR expression analysis of selected genes normalized to the 60S ribosomal protein L14 (At4g27090) measured in the same samples. The resulting relative expression was then used to calculate fold change upon wounding. Data are means of n = 3. (B) Functional classification of RWR genes using GO annotations.

Figure 2. In Vivo Monitoring of RWR Gene Induction Following Wounding

(A) Image of an individual P_ERF18:LUC #3 transgenic plant over time. (B) Luciferase activity of three independent transgenic lines expressing transcriptional P_ERF18:LUC fusions. Luciferase activity was calculated in wounded leaves (single leaf per plant) and NW leaves (on NW plants). Data are means of n = 12 ± SEM. (C) Image of an individual P_CAF1L:LUC transgenic plant over time. (D) Luciferase activity of three independent transgenic lines expressing transcriptional P_CAF1L:LUC fusions. Luciferase activity was calculated in wounded leaves (single leaf per plant) and NW leaves (on NW plants). Data are means of n = 18 ± SEM.

Figure 3. Selected RWR Genes Displaying a Rapid and Stable Expression Pattern Following Mechanical Wounding

Total RNA was extracted from 3-wk-old mechanically wounded tissue and subject to RT-qPCR analysis. AtMPKK9, WRKY40, AtPP2-A13, BAP1, AT1G32920, and CML38 transcripts were normalized to the 60S ribosomal protein L14 measured in the same samples. Data are means of n = 3 ± SEM.

Figure 4. Selected RWR Genes Displaying a Rapid and Transient Expression Following Mechanical Wounding

Total RNA was extracted from 3-wk-old mechanically wounded tissue and subject to RT-qPCR analysis. ERF#018, CAF1-like, AP2C1, MPK3, and TIR-NBS transcripts were normalized to the 60S ribosomal protein L14 while SPLAYED transcripts were normalized to TIP41-like measured in the same samples. Data are means of n = 3 ± SEM.

Figure 5. Comparison of RWR Genes with the Transcript Profile of Other Environmental Stimuli

An asterisk denotes a statistically significant overrepresentation of RWR genes in the transcript profile of the indicated stress (p < 0.0001). Arabidopsis plants were challenged with M. persicae for 48 h and 72 h; P. rapae early for 5 h; P. rapae late and P. syringae for 12 h and 24 h; B cinerea early and late for 18 h and 48 h, respectively; and OGA early and late for 1 h and 3 h, respectively [–61].

Figure 6. In Vivo Monitoring of RWR Gene Induction upon Addition of Biotic Elicitors

One leaf per plant was either NW or wounded (W) and then treated with oligouronides (OGA), IR, or double-distilled H₂O. (A) Image of an individual P_ERF18:LUC #3 transgenic plant over time. (B) Local expression in the treated leaves of P_ERF18:LUC #3 transgenic plants. Data are means of n = 9 ± SEM. (C) Systemic expression of P_ERF18:LUC #3 plants monitored in the shoot apex. Data are means of n = 9 ± SEM. (D) Image of an individual P_CAF1L:LUC #2 transgenic plant over time. (E) Local expression in the treated leaves of P_CAF1L:LUC #2 transgenic plants. Data are means of n = 12 ± SEM. (F) Systemic expression of P_CAF1L:LUC #2 plants monitored in the shoot apex. Data are means of n = 12 ± SEM.

Figure 7. Circadian Regulation of RWR Genes

These data are comprised of circadian-regulated RWR genes. Upregulated RWR genes (blue) peak at dusk while downregulated RWR genes (pink) peak at dawn. Plants were entrained in light/dark cycles for 7 d and then released into constant light. Samples were collected every 4 h after plants were moved to constant light.

Figure 8. The RSRE Confers Wound-Induced Reporter Gene Expression

Independent T1 4xRSRE:LUC and 4xmtRSRE:LUC lines were mechanically wounded. Luciferase activity was then monitored in the wounded leaf. Data are means of n = 24 ± SEM.

Figure 9. The RSRE Responds to Biotic Elicitors

(A) Image of an individual 4xRSRE:LUC transgenic plant over time that was wounded and treated with OGA, IR, or H₂O. (B) Local expression in the treated leaves of 4xRSRE:LUC transgenic plants. (C) Systemic expression of 4xRSRE:LUC in the shoot apex. Data are means of 12 ± SEM.

Figure 10. The RSRE Confers Cold-Induced Reporter Gene Expression

(A) Image of an individual 4xRSRE:LUC transgenic plant incubated at 5 °C or 22 °C over time. (B) Luciferase activity of 4xRSRE:LUC plants. Data are means of 16 ± SEM.