Role of salicylic acid in plants

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CURRENT STATE OF KNOWLEDGE

Role of salicylic acid in plants

In 1979 SA was identified as a compound capable to decrease the severity of infection when applied on plants before inoculation (the process was later called priming; Durrant and Dong, 2004). The majority of our knowledge about its mode of action comes from the studies of plant innate immunity. However, SA is involved in other physiological processes including adaptation to stress and development and thus can be seen as a plant hormone.

Systemic acquired resistance and SA

In the 1960s, it was shown that tobacco plants challenged with tobacco mosaic virus (TMV) subsequently developed increased resistance to secondary infection in distal tissues (Durrant and Dong, 2004). This spread of resistance throughout the plant tissues was termed SAR (Ross, 1961). Flor (1971) proposed a molecular model for plant infection called ‘gene-for-gene’. In this model, pathogen bears a gene of avirulence (Avr) and plant bears the corresponding gene of resistance (R). When the R protein recognizes the corresponding Avr protein, an incompatible reaction is triggered leading to localized programmed cell death (PCD) and eventually to SAR development. The localized PCD is SA-dependent and it is called hypersensitive response (HR). During HR reactive oxygen species (ROS) are produced and this slows down or even stops the infection. Interestingly, ROS generated by Arabidopsis respiratory burst oxidase (AtRBOH) proteins can antagonize SA-dependent pro-death signals. This is a good example of how both SA and ROS control localized PCD (Torres et al., 2005). When R protein does not recognize its Avr partner, compatible interaction is triggered. Compatible reaction often leads to development of infection and eventually to death of the challenged plant. We now know that SAR can be activated in many plant species by pathogens that cause necrosis. The process of SAR development is SA-dependent and it can also be activated by SA alone. The role of SA in SAR has been discussed extensively in a number of reviews (Bostock, 2005; Dempsey et al., 1999; Dong, 2001; Durrant and Dong, 2004; Nürnberger and Scheel, 2001; Ryals et al., 1996; Shah and Klessig, 1999). The obtained resistance is long-lasting, sometimes for the lifetime of the plant, and effective against a broad spectrum of pathogens including viruses, bacteria, fungi, and oomycetes representing various parasitic strategies (i.e. biotrophs, hemibiotrophs, and necrotrophs).
Molecularly, SAR is characterized by the increased expression of a large number of pathogenesis-related genes (PR genes), in both local and systemic tissues. PR proteins were first described in the 1970s when accumulation of various novel proteins after infection of tobacco with TMV was observed (Durrant and Dong, 2004). Although many PR proteins have antimicrobial properties in vitro, the function of some of them in the defence response has not been clearly defined (Table I). It is generally thought that SAR results from concerted interplay of many PR proteins rather than from a specific PR protein. PR genes serve as useful molecular markers of the onset of SAR.
Another type of systemic resistance is the jasmonic acid (JA) -mediated induced systemic resistance (ISR). ISR protects plants against a range of pathogens, but it is independent of SA and PR gene induction. It is usually associated with the soil bacteria, often strains of Pseudomonas fluorescens. It can be triggered by various volatile oxylipins including JA and methyljasmonate (MeJA).
SAR has been studied using many approaches including analyses of concentration changes of SA and its conjugates in plant tissues prior to, during or after the resistance is expressed. Various mutants constitutively expressing resistance genes or affected in development of systemic resistance were the most useful tools. Alternative ways of inducing plant resistance were sought to facilitate its study including treatment of plants with SA or various SA-mimicking, but less phytotoxic, compounds, such as benzothiadiazole S-methyl ester (BION; BTH; Actiguard®) and 2,6-dichloroisonicotinic acid (INA). Although these compounds are thought to be equivalent to SA treatment (they induced similar sets of PR genes), a more detailed study on the whole transcriptome scale revealed that many genes are regulated in a different manner, especially between SA and BTH treated plants (von Rad et al., 2005).
SAR needs a systemic signal to be emitted and perceived in distant tissues. The nature of the systemic signal is yet unknown, but according to the literature it is not species-specific and it is transmitted via phloem (Durrant and Dong, 2004). The detection of increased SA levels in systemic leaves and in the phloem led many researchers to believe that SA might be a systemic signal for SAR. Malamy et al. (1992) showed that the endogenous SA concentration raises both in local and systemic tissues after infection of tobacco with TMV and this increase correlates with PR gene induction. Métraux et al. (1990) found that cucumber plants infected with either Colletotrichum lagenarium or tobacco necrosis virus (TNV) have considerably elevated levels of SA in the phloem sap. Radiolabelling studies in TMV-infected tobacco showed that most of the SA (69%) accumulating systemically was made and exported from the inoculated leaf (Shulaev et al., 1995). Similarly, in cucumber infected with TNV, SA found in systemic leaves was both imported from the infected leaf and synthesized de novo (Durrant and Dong, 2004). A number of experiments argue against SA being the systemic signal. One of them shows that detachment of Pseudomonas syringae-infected cucumber leaves before SA levels had increased in the petiole did not block the development of SAR (Rasmussen et al., 1991). Alternative hypotheses have been raised pointing to nitric oxide (NO) or methylsalicylate (MeSA; Forouhar et al., 2005). Feed-back loop probably exists in the SA-NO relationship as NO triggered SA production (Wendehenne et al., 2004) and it was produced after SA treatment of Arabidopsis seedlings (Zottini et al., 2007). MeSA as a volatile compound could induce resistance not only in the uninfected parts of the same plant but also in neighbouring plants. In a very interesting field study MeSA emitted from soybean infested with soybean aphid (Aphis glycines) was identified as the major olfactory attractant for predacious seven-spotted lady beetles (Coccinella septempunctata; Zhu and Park, 2005). NO indeed functions in local pathogen perception where it stimulates ROS production during the oxidative burst and it induces the general phenylpropanoid pathway (Zeier et al., 2004a). Unfortunately, NO can easily be quenched in contact with the cellular environment (Baudouin et al., 2006) so it would be difficult to imagine it as a long distance signal. Others have suggested members of the lipid transfer protein (LTP) family (Durrant and Dong, 2004). These are small proteins which specifically bind fatty acids and which can be easily transported through phloem. They have been studied in various species but their exact role is not clear. Recently LTP covalently modified by an oxylipin has been found in barley (LTP1b) thus opening interesting perspectives regarding the roles of LTPs in plant defence and development (Bakan et al., 2006).

SA signalling pathway

Locally produced SA can act both in the cell where it was produced and in neighbouring cells. A mechanism was proposed in which pathogen elicitor caused apoplast alkalization which in turn promoted pH-independent passive export of SA. Released SA moves through the plant tissue in a wave preceding the infection and it enters cells which still have acid apoplasts to trigger defence responses there (Clarke et al., 2005). SA uptake was higher at lower extracellular pH and took place probably via an H+ symport mechanism in Arabidopsis cell suspensions. As its pKa is 2.97 a small portion remains in non-ionized state and can be retained inside the cell by ion-trap mechanism. Accordingly, SA treatment caused transient cytoplasmic acidification. However, the SA uptake occurred even at pH close to neutrality. The mobility of SA in the phloem of Ricinus communis seems to be facilitated by a pH-dependent carrier system translocating aromatic monocarboxylic acids (Rocher et al., 2006).
Till now, no canonical receptor molecule for SA has been identified in plants despite the numerous efforts. Nevertheless, several high affinity binding proteins have been identified in tobacco (e.g. catalase, ascorbate peroxidase, and carbonic anhydrase; Kumar and Klessig, 2003). One of them, called SA binding protein 2 (SABP2), was first shown to have a lipase activity and later its MeSA hydrolyzing esterase activity was described (Forouhar et al., 2005). Furthermore, silencing the SABP2 gene by RNAi diminished both local resistance and SAR (Kumar et al., 2006). These observations led authors to propose that SABP2 is the receptor for SA; however, the exact position of SABP2 in the SA signalling pathway is not clear yet. Interestingly, its tomato orthologue is rather a MeJA esterase (MJE; Shah, 2005).
SA treatment triggers protein phosphorylation cascades. These involve MAP kinases (reviewed by Innes, 2001), specifically the wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK) both being described in tobacco. WIPK is activated by wounding and SIPK is activated by both NO and SA. Neither of them responds to JA or ethylene. Moreover SIPK is strongly activated by ROS and it appears to modulate the cellular response to ROS and the level of WIPK. Arabidopsis MPK4 negatively regulated SA-mediated defences and its direct binding partners from the family of MAPKK and MAPKKK have been identified by yeast two-hybrid screen (Innes, 2001).
Ca2+-dependent protein kinases (CDPKs) are also involved in plant immune response. They serve as important sensors of Ca2+ fluxes. Specific isoforms of this multigene family are implicated in signalling pathways leading to abiotic and biotic stress resistance, including disease resistance. CDPKs that are up-regulated by wounding, elicitors, and/or infection, including Avr/R gene interactions have been characterized from tobacco, tomato, and maize (Bostock, 2005). However, further research is needed to determine if and how these kinases operate in connection with induced resistance pathway regulation and signal cross-talk.
Perception of the unknown systemic signal and/or perception of SA lead to induction of early transcription factors (e.g. WRKY or basic leucine zipper, bZip, family) and to a shift of cytoplasmic redox potential to more reductive state. Changes in the redox state affect the activity of SAR-related transcription factors and of enzymes regulating levels of reactive oxygen species (ROS) in the cell. SA is capable of modulating pools of major cellular antioxidants such as glutathione, thioredoxin, and ascorbate. The redox modulating and antioxidant capacity of SA may be its major link with other stress responses where it is involved because ROS occur during many stress situations. On the other hand, failure to control ROS is one of the causes leading to cell death. Potential SA targets in cellular redox management involve hydrogen peroxide scavengers, SA binding proteins, catalase and ascorbate peroxidase, and chloroplastic carbonic anhydrase. The ability of SA to interact with these proteins may be partially caused by its affinity to transition metals (e.g. heme and nonheme iron; Bostock, 2005).
Reductive state of the cytoplasm leads to monomerization of homotrimeric transcription factor ‘non-expressor of PR-1’ (NPR1; Bostock, 2005). NPR1 is considered as the main switch in the signalling network of plant induced resistance (Pieterse and van Loon, 2004). It is also well conserved between monocots and dicots (Yuan et al., 2007). NPR1 is expressed throughout the plant at low levels and its mRNA level raises two- to three-fold after pathogen infection or treatment with SA. NPR1 expression is likely mediated by WRKY transcription factors as mutation of the WRKY binding sites (W-boxes) in the NPR1 promoter inhibited it. Monomers of NPR1 can easily enter the nucleus and in combination with other transcription factors trigger the next wave of gene expression (Bostock, 2005; Yuan et al., 2007). Such self-amplification of the initial signal is typical for many signalling pathways.
The absence of any obvious DNA-binding domain and the presence of protein-protein interaction domains in NPR1 prompted several laboratories to carry out yeast two-hybrid screens for NPR1-interacting proteins. In one of the screens, three small structurally similar proteins named NIMIN1, NIMIN2, and NIMIN3 (non-inducible immunity interactor) were identified. NIMIN1 and NIMIN2 interact with the C-terminus of NPR1, while NIMIN3 interacts with the N-terminus. NIMINs contain stretches of acidic amino acids and are hypothesized to be transcription factors; however, more experiments are required to demonstrate their biological activity (Durrant and Dong, 2004). Other NPR1 interactors found in the yeast two-hybrid screens were members of the TGA family of bZip transcription factors. NPR1 interacts indeed with the Arabidopsis TGA factors, TGA2, TGA3, TGA5, TGA6, and TGA7 but only weakly or not at all with TGA1 and TGA4. Interaction with TGA1 and TGA4 was dependent on their redox status (Durrant and Dong, 2004). TGA2, TGA5 and TGA6 were functionally redundant and only their triple mutant was compromised in SAR induction. All these TGA factors were also important for SA tolerance and for the negative regulation of the basal expression of PR1 (Zhang Y et al., 2003). 51 TGA2-binding elements in the promoters of Arabidopsis genes were identified and, indeed, SA-induced genes were significantly over-represented among the genes neighbouring putative TGA2-binding sites (Thibaud-Nissen et al., 2006). Using truncated or mutant forms of NPR1, the ankyrin-repeat domain in the middle of the protein was shown to be essential for binding TGA factors, while the N-terminal region appears to enhance the binding (Durrant and Dong, 2004). The exact mode of interaction between NPR1 and TGA2 was studied in detail. It was postulated that TGA2 and NPR1 are recruited independently to the promoter of PR1 and upon SA stimulation they combine to form an enhanceosome which triggers PR1 expression. TGA2 itself (without NPR1 or SA stimulus) functions as a repressor of PR1 expression in the proposed model (Rochon et al., 2006). NPR1 also interacts with TGA factors from tobacco and rice (Durrant and Dong, 2004).
TGA factors bind to activator sequence-1 (as-1; TGACG) or as-1-like promoter elements, which have been found in several plant promoters activated during defence, including Arabidopsis PR1, an important SAR marker. Linker scanning mutagenesis of the PR1 promoter identified two as-1-like elements, LS7 and LS5. LS7 is a positive regulatory element required for induction by INA, whereas LS5 is a weak negative regulatory element. It was shown that both TGA2 and TGA4 could bind to LS7, whereas only TGA2 could bind to LS5. Furthermore, binding of TGA2 but not TGA4 was enhanced by the addition of NPR1, consistent with the yeast two-hybrid interaction data (Durrant and Dong, 2004).
Although NPR1 is clearly a positive regulator of PR genes, it may exert its function by either enhancing a transcriptional activator or inhibiting a transcriptional repressor. The presence of multiple as-1-like elements in the PR1 promoter and the differential binding affinities of each TGA factor to these elements as well as to NPR1 highlight the complexity of the regulatory mechanism. The NPR1/TGA system seems to be operative even in monocots as demonstrated in the experiment where the ectopic expression of the Arabidopsis NPR1 in rice enhanced resistance to the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Bostock, 2005). NPR1 controlled the expression of the protein secretory pathway genes. Up-regulation of these genes was essential for SAR and was ensured through a previously undescribed cis-element activated by another transcription factor that translocated into the nucleus upon SAR induction (Wang et al., 2005). However the NPR1 is considered as the major transcriptional switch in the network of induced resistance, there is growing evidence for a SA-dependent NPR1-independent pathway(s) that contributes to defence gene induction (Durrant and Dong, 2004).
Transcription factors bind to their respective boxes or cis-elements in promoters of target genes. Several cis-elements are known to be associated with SAR. Interestingly, only 17 of 26 genes from the PR1 regulon (a cluster of genes similarly regulated as PR1) have an as-1 element in their promoters, whereas W-boxes were overrepresented, occurring an average of 4.3 times per promoter. This suggests that WRKY factors rather than TGA factors are important for coregulation of PR1 regulon genes. The LS4 W-box in the PR1 promoter acts as a strong negative cis-element, leading some authors to propose that WRKY factors repress the expression of PR1 regulon genes (Durrant and Dong, 2004). This issue was clarified in an elegant study where 8 WRKY transcription factors were directly regulated by NPR1 and also by TGA  transcription factors. Among these WRKY58 was the negative regulator and four others were positive regulators of PR gene expression (Wang D et al., 2006). WRKY25 was another identified negative regulator of SA-mediated induced resistance to bacterial pathogen (Zheng et al., 2007). It is not surprising because it directly interacts with MKS1, a substrate for MPK4, which is a repressor of SA-dependent defence responses (Andreasson et al., 2005). Some WRKY transcription factors (e.g. WRKY33) are specific for some types of pathogens (Zheng et al., 2006). WRKY transcription factors may seem specific for plant-pathogen interaction, however, they are not. For instance, one of the positive SAR regulators, WRKY70, is also a negative regulator of developmental senescence (Ulker et al., 2007). Another promoter analysis on 1058 genes that were induced by pathogen infection, SA, MeJA, or ozone revealed that as-1 elements, W-boxes, abscisic acid (ABA) response elements (ABRE), and G-boxes were overrepresented across all treatments, whereas the Myc motif (CANNTG) was overrepresented only in the SA-induced genes (Mahalingam et al., 2003). Upon activation of SAR, NPR1-dependent derepression would occur, possibly through the inactivation of transcription factor ‘suppressor of NPR1, inducible’ (SNI1) which is required to maintain the low basal expression of PR genes (Durrant and Dong, 2004). The signalling cascade which triggers PR1 expression is summarized in Figure 1.
Elevated SA levels trigger increased NPR1 transcription via WRKY factors binding to two W-boxes in the 5’-UTR of NPR1. In resting Arabidopsis cells, NPR1 oligomers are sequestered in the cytoplasm. Elevated SA levels trigger a biphasic change in the cellular redox environment leading to the reduction of the intermolecular disulfide bounds of NPR1 oligomers. The resulting NPR1 monomers localize to the nucleus where they activate binding of TGA factors to TGA boxes (LS5 and LS7) in the PR1 promoter. The TGA box LS5 and LS4, a W-box that probably interacts with WRKY factors, are negative elements that repress PR1 expression. The TGA box LS7 and LS10, which appears to interact with unknown transcription factors, are positive elements. The GCC box, LS8, is likely to interact with ERF transcription factors. The effects of this element on PR1 expression are not known. However, SAR induction stimulates binding of protein factors to this element. Additional WRKY factors might contribute to PR1 regulation indirectly or by interacting with putative W-boxes upstream from LS4. Causal interactions are indicated by grey arrows. ‘+’ means activating mechanisms, ‘-’ means repressing mechanisms. Coding regions of genes are represented by squares, cis-elements by upright rectangles and transcription factors as well as NPR1 by ovals. Transcription start sites of genes are marked by black arrows. Adopted from Eulgem (2005).
Once produced, PR proteins are probably down-regulated by the proteasome complex as documented for pepper where CaRFP1, encoding the C3-H-C4 type RING-finger protein, was isolated from pepper leaves infected by Xanthomonas campestris pv. vesicatoria. CaRFP1 physically interacted with the basic PR1 protein (CaBPR1) and it acted as an E3 ligase for polyubiquitination of target PR proteins. It was induced by several stresses including SA treatment and its over-expression negatively regulated disease resistance (Hong et al., 2006). These results could be partly explained in terms of negative regulation of SA production as documented for Arabidopsis. The SIZ1 gene, which encodes an Arabidopsis small ubiquitin-like modifier (SUMO) E3 ligase, regulated innate immunity. Mutant siz1 plants exhibited constitutive SAR characterized by elevated accumulation of SA, increased expression of PR genes and increased resistance to a bacterial pathogen. Transfer of the nahG gene to siz1 plants resulted in reversal of these phenotypes back to wild type. SIZ1 interacted epistatically with PAD4, phytoalexin deficient, to regulate PR gene expression and disease resistance in NPR1-independent manner and it was required for SA and PAD4-mediated R gene signalling pathway (Lee et al., 2007). Characterization of mutants of two ubiquitin-activating enzyme genes revealed that ubiquitination pathway was crucial in the activation and downstream signalling of several R-proteins in Arabidopsis (Goritschnig et al., 2007).

Studies of SAR transcriptome

During the past seven years, differential display, DNA microarrays, and other high throughput transcriptomic approaches have been applied to gain a more comprehensive view of changes in global gene expression associated with plant responses to diverse stressors. Information obtained on SAR transcriptome, i.e. studies where wild type or mutated plants were infected with different pathogens (Glazebrook et al., 2003), are the main source of our knowledge about the differential activation of signalling pathways during the onset of plant immune response. Other studies in Arabidopsis have focused on expression profiles induced by pathogens or pathogen-related activities using multiple conditions rather than pairwise comparisons to better assess the complexity of signal networks, e.g. transcriptional profiling of plants infected with various pathogens or treated with various plant activators including SA, SA analogues and JA. Schenk et al. (2000) performed transcription profiling of infected plants and plant activator-treated plants on a custom microarray with 2375 EST-derived clones. 323 genes were differentially expressed after 24h of SA treatment and overlaps between infection-regulated and plant activator-regulated genes were discussed. Mahalingam et al. (2003) identified 732 transcripts as differentially expressed after pathogen infection or plant activator treatment (among these 68 by SA treatment) on a custom microarray constructed from stress cDNA library of 1058 gene transcripts. Von Rad et al. (2005) used a custom microarray for transcriptional profiling of about 700 genes after treatment with commercial plant activators and found that their mode of action is rather complex and mimics neither pure JA nor pure SA action. Transient JA pathway activation was followed by a more sustained activation of the SA pathway. Pylatuik and Fobert (2005) tested several whole genome scale microarray platforms and took SA treatment as a model treatment. Several hundreds of genes differentially expressed after 2h and 8h of SA treatment were identified along with NPR1-dependent and independent gene clusters. However, the complete list of differentially expressed genes was not provided. Thibaud-Nissen et al. (2006) identified 1265 SA-induced genes after 2h of SA treatment of Arabidopsis plants and used the expression data to prove that newly-discovered TGA2-binding elements are over-represented in their promoters. However, the list of SA-induced genes was not provided either. Kliebenstein et al. (2006) also took the SA treatment as a model treatment to demonstrate gene expression diversity in Arabidopsis. They identified 439 SA-induced and only 19 SA-repressed genes in the wild type Columbia background. The SA transcriptome was not discussed at all in the study. Using various SAR-inducing and SAR-repressing conditions comparison Maleck et al. (2000) identified several regulons. Regulons share common cis-elements in their promoters and can be attributed to specific transcription factor families. Other studies focused on regulatory cis-elements in the promoters of SAR-regulated genes. Using cDNA-AFLP display Blanco et al. (2005) identified around 40 genes differentially regulated by SA treatment. They identified common cis-elements of NPR1-dependent and NPR1-independent genes. Interestingly, NPR1-dependent genes did not contain as-1 element and were mostly involved in signal transduction while NPR1-independent genes had as-1 element in their promoters and were mostly involved in cellular detoxification. Accordingly, two categories of SAR-regulated genes with distinct profiles of cis-regulatory elements in their promoters were identified in a bioinformatic study (Pan et al., 2004). Glucocorticoid receptor (GR) fused to a protein can control its nuclear localization in a dexamethasone-dependent manner. Using the whole genome transcriptomic approach on npr1 plants transformed with NPR1-glucocorticoid receptor (NPR1-GR), Wang et al. (2005) identified around 150 direct transcriptional targets of NPR1 in response to SA, pointed out the importance of the protein secretory pathway for successful development of SAR and showed that its induction is NPR1-dependent. In an expression profiling of 402 transcription factors known to be involved in various stress responses including SAR, Chen et al. (2002) identified about 40 transcription factors that were specifically up-regulated during infection with various pathogens. These transcription factors may be responsive to SA too. Dong et al. (2003) performed transcription profiling of 72 WRKY transcription factors in Arabidopsis and found that six were repressed and 43 induced by both SA treatment and incompatible reaction induced by Pseudomonas syringae pv. tomato. It confirms the central role of WRKY transcription factors during the development of plant immune response.

Table of contents :

1 INTRODUCTION
2 CURRENT STATE OF KNOWLEDGE
2.1 Role of salicylic acid in plants
2.1.1 Systemic acquired resistance and SA
2.1.1.1 SA signalling pathway
2.1.1.2 Studies of SAR transcriptome
2.1.2 Mutants with altered SA/SAR signalling
2.1.2.1 Mutants pointing to lipid-based signalling in SAR
2.1.2.2 Mutants affected in SA synthesis
2.1.2.3 SA signalling in redox mutants
2.1.2.4 NPR1-dependent and –independent pathways
2.1.2.5 Connections and cross-talks
2.1.2.6 Metabolic costs of induced resistance
2.1.3 SA in other physiological contexts
2.1.3.1 Temperature stress
2.1.3.2 Oxidative stress
2.1.3.3 Plant development
2.2 Phospholipid signalling in plants
2.2.1 Key phospholipid signalling molecules
2.2.1.1 Phosphoinositides
2.2.1.2 PA and DGPP
2.2.1.3 Free fatty acids and lyso-phospholipids
2.2.2 Key enzymes in phospholipid signalling
2.2.2.1 PI 3-kinase
2.2.2.2 PI 4-kinase
2.2.2.3 PIP 5-kinases
2.2.2.4 Phospholipase C
2.2.2.5 DAG- and PA-kinase
2.2.2.6 Phospholipase D
2.2.2.7 Phospholipase A
2.2.3 Possible cellular targets of phospholipid signals
2.2.3.1 FYVE domain
2.2.3.2 PH domain
2.2.3.3 C2 domain
2.2.3.4 Other phospholipid-binding domains
3 MATERIALS AND METHODS
3.1 Materials
3.1.1 Chemicals and kits
3.1.2 Plant materials
3.1.3 Characterization of T-DNA insertion mutants
3.2 Characteristics of cell suspensions
3.2.1 Growth curve
3.2.2 Viability staining
3.3 Phospholipid analysis
3.3.1 SA treatment and phospholipid analysis
3.3.2 Analysis of PI species by RP-HPLC
3.4 Gene expression analyses
3.4.1 RNA extraction for semiquantitative RT-PCR and microarray experiments
3.4.2 RNA extraction for QRT-PCR
3.4.3 Semiquantitative RT-PCR analysis
3.4.4 QRT-PCR analysis
3.4.5 Transcriptome studies
3.4.6 Statistical analysis of microarray data
3.5 Data deposition
4 RESULTS
4.1 Introductory experiments leading to characterization of the model system
4.1.1 Non-lethal and effective SA concentration
4.1.2 Marker genes of the SA pathway
4.1.3 Radiolabelling of phospholipids in vivo
4.2 SA activates PI 4-kinase in vivo
4.2.1 Radiolabelling of phospholipids during SA treatment
4.2.2 Characteristics of the SA-induced changes in labelled phospholipids
4.2.3 SA-induced PIP is PI(4)P
4.2.4 PI decrease can be impaired by inhibitors of type III PI 4-kinase
4.2.5 Phosphorylation events are involved in the activation of PI 4-kinase
4.2.6 Ca2+ influx in response to SA
4.2.7 Expression of genes involved in the phosphoinositide metabolism during SA treatment
4.3 SA transcriptome is partially regulated by a W30-sensitive pathway
4.3.1 SA induces changes in the transcriptome of Arabidopsis
4.3.2 Effect of wortmannin on the SA-regulated transcriptome
4.3.3 Common cis-elements in the promoters of SA-regulated genes
4.3.4 Expression of selected marker genes of the W30-sensitive pathway in the T-DNA mutants of PI 4-kinase β
4.4 SA activates PLD in vivo
4.4.1 Expression of several SA-regulated genes is regulated by PLD
4.4.2 PLD intervenes in the early stages of SA treatment
4.4.3 Exogenous PA application did not reverse the n-butanol inhibition of SA response
4.5 SA transcriptome is partially regulated by the PLD pathway
4.5.1 Identification of PLD-regulated genes in the SA transcriptome
4.5.2 Common cis-elements in the promoters of PLD-regulated genes in response to SA
4.5.3 Expression of PR1 and WRKY38 in the T-DNA mutants of several PLDs
4.6 Overlap of W30-sensitive and PLD-regulated SA transcriptomes
5 DISCUSSION
5.1 Arabidopsis cell suspensions are an admissible model to study SA signalling
5.2 Activation of a PI 4-kinase as an early response to SA
5.3 Phosphorylation events precede the PI 4-kinase activation in response to SA
5.4 Ca2+ signalling is not involved in the early SA response
5.5 Protein synthesis differentially affects the SA response
5.6 SA-regulated transcriptome
5.7 Effects of wortmannin on the SA-responsive transcriptome
5.8 PI4Kβ1 may be involved in the SA response
5.9 PLD influences SA-regulated gene expression
5.10 Effects of PLD on the SA-responsive transcriptome
5.11 PI(4,5)P2-dependent PLDs modulate the SA response
5.12 PI 4-kinase and PLD act synergistically in the SA signalling pathway
6 CONCLUSIONS
7 ABBREVIATIONS
8 LITERATURE CITED
9 SUPPLEMENTAL DATA

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