Involvement of phospholipase D and NADPH-oxidase in salicylic acid signaling cascade

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ABA effects on in vivo phosphoinositide accumulation and PLC activity

It was previously shown that SA inhibits basal PI-PLC (Ruelland et al., 2014). To check the specificity of this process, we now wanted to check in vivo the effect of ABA on the basal PI-PLC activity and compare it to that of SA. ABA (or SA) was added to Arabidopsis cells in suspension culture and lipids were extracted 20 or 45 min later. Radioactive 33Pi-orthophosphate was added 15 min before lipid extraction. Basal PI-PLC activity can be monitored by detecting radiolabelled PA, that with a labelling time of 15 min mainly corresponds to PA produced through DGK activity (Vaultier et al., 2006), which acts on DAG produced by PI-PLC. Addition of SA resulted in an increase in radiolabelled PI4P and PI-4,5-P2, at 20 and 45 min, thus confirming previous results (Ruelland et al., 2014, Krinke et al., 2007). No such increase could be seen for ABA (Fig. 4.1A). We can however notice a decrease in this basal PA (corresponding to basal coupled PI-PLC/DGK) for both ABA and SA (Fig. 4.1B).

Remodelling of the transcriptome of Arabidopsis cell in suspension culture by ABA

Cells were treated either with 10 μM abscisic acid (ABA) or solvent (tertiary butanol 0.1%, v/v) and harvested 4 hrs later. RNAs were extracted and used for microarray experiment. It was possible to identify 380 ABA-repressed genes and 988 ABA-induced genes. These genes were then classified according to Gene Ontology categories, primarily the Biological processes (Figure 4.2.A), Cellular Components (Figure 4.2.B) or Molecular functions (Figure 4.2.C). Not surprisingly, for the ABA-induced genes, amongst the most over-represented categories within Biological Processes (except ―other biological process‖) are the ―response to stress‖ and ―response to abiotic or biotic stimulus‖. There was also a significant number of responses in the ―transport‖ and ―signal transduction‖ categories, but a relative under-representation of the ―DNA or RNA metabolism‖ categories amongst the ABA-induced genes. In ABA-repressed genes, there was over representation of ―cell organization and biogenesis‖ and ―response to abiotic or biotic stimulus” categories (Figure 4.2.A). Within the Cellular Components categories, there was a striking 5-fold over representation of the ―cell wall‖ category for the ABA-repressed genes (Figure 4.2.B). For Molecular Functions categories, there was an over-representation of the ―transporter activity‖ category for the ABA-induced genes, and of the ―nucleotide binding‖ and ―structural molecule activity‖ functions for the ABA-repressed genes (Figure 4.2.C).

Overlap of ABA-responsive genes to genes and genes responsive to inhibitors of the PI-PLC pathway

We wanted to know if the ABA response alone indeed overlaps with the response to inhibitors of the PI-LC pathway. ABA transcriptome data were compared to those of the response to 30 μM wortmannin, an inhibitor of the enzymes responsible for providing substrates for PI-PLC, i.e. type III-PI4K. The transcriptomic responses to this agent was already published (Delage et al., 2012b). A contingency table was established (Table 4.1.A). In response to either ABA or to 30 μM wortmannin, genes can be up-regulated, down-regulated or unaffected by the treatment. Comparison of responses to ABA and to the inhibitor indicated 9 overlapping categories. The genes within each of these 9 categories were counted. Assuming that ABA and SA act independently, the theoretical number of genes predicted for each response category can be calculated. From this a Representation Factor (RF, that corresponds to the ratio between observed number to theoretical number) could be calculated, and a P-value associated with this RF. There is an over representation of genes for which ABA and 30 μM wortmannin lead to the same response (Table 4.1.A). Indeed, 338 genes are induced by ABA or by 30 μM wortmannin; this being 5-fold more than expected in case of an independent distribution and this over representation is statistically significant. 177 genes are repressed by ABA or by 30 μM wortmannin; this also being 5-fold more than expected in case of a random distribution and this over representation is statistically significant. There is a clear overlap between the response to ABA and the response to 30 μM wortmannin.

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ABA and SA response overlap in Arabidopsis cells in suspension culture

We then wanted to know whether there was an overlap at the level of transcriptomic response between SA and ABA. The transcriptomic reponses to ABA or SA were crossed. A clear over-representation of genes for which both SA and ABA have the same effects was found while comparing the transcriptomic data of cellular responses to ABA to that to that of cells treated by 250 μM within 4 hrs of by SA (Krinke et al., 2007)(Table 4.2).

Table of contents :

INTRODUCTION
CHAPTER 1. Literature overview
CHAPTER 2 MATERIALS AND METHODS
2.1 Material used in the study
2.2. Pharmacological treatments
2.3. Methods concerning phospholipids analysis
2.3.1 Phospholipid radioactive labeling and lipid extraction
2.3.2. Thin-layer chromatography and scintillation counting
2.4. Methods concerning plant physiology
2.4.1 Histochemical assay
2.4.2. Stomatal assay
2.4.3 ROS visualization and measurement
2.4.4 Callose deposition evaluation
2.4.5 Pathogen assay
2.4.6 Root growth assay
2.5. Methods concerning gene expression analysis
2.5.1 RNA extraction
2.5.2 Transcript abundance evaluation by qPCR
2.5.3 Transcriptome Studies
2.5.4 Statistical Analysis of Microarray Data
2.5.5 In silico analysis of microarray data
2.5.6 Comparison of two sets of transcriptomic data
2.5.7 Data Deposition 31
2.6 Data analysis and statistics 31
CHAPTER 3 Involvement of phospholipase D and NADPH-oxidase in salicylic acid signaling cascade
CHAPTER 4 Basal phosphoinositide-dependent phospholipase C activity in Arabidopsis suspension cells as a signalling hub accounting for the overlap in transcriptome remodelling by abscisic or salicylic acid
CHAPTER 5 Phosphatidylinositol-dependent phospholipase C and diacylglycerol kinase are involved in primary responses to flagellin in plant cells
CHAPTER 6 The mutations of both type III phosphatidylinositol-4-phosphate kinases ß1 and ß2 isoforms lead to altered response to auxins and cytokinins in Arabidopsis roots
REFERENCES

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