Gepubliceerd op 11 juli 2005
Prof. dr. M.A. Haring, Dr. R.C. Schuurink, Dr. T. Munnik
We are studying two signal transduction cascades that help plants cope with biological and environmental stress conditions: phospholipid-based cellular signalling and volatile-based external communication.
The main character in the first pathway is the lipid second messenger, phosphatidic acid (PA), which is produced via activation of phospholipase D (PLD), hydrolyzing structural phospholipids such as phosphatidylcholine, or via the combined action of phospholipase C (PLC) and diacylglycerol kinase (DGK). By manipulating the activity of individual PLC, DGK and PLD genes in tomato and Arabidopsis plants we aim to elucidate their role in stress signalling and development. How PA exerts its effects is still unknown, mainly due to the lack of characterised PA targets. We are using PA-coated Sepharose beads together with mass spectrometry to isolate and identify such targets.
As it is mostly unknown where lipid signalling events take place sub-cellularly, we are using fusions of specific lipid-binding domains with YFP as fluorescent "biosensors" that visualize changes in phospholipid pools during stress treatments.
In our second theme, we are using a tritrophic system comprising tomato (Lycopersicon esculentum), spider mites (Tetranychus urticae) and predatory mites (Phytoseiulus persimilis) to study the molecular basis of the direct and indirect defence mechanisms of plants against herbivores. Both a micro-array based approach and a metabolomics approach are applied to analyse this system. We have adopted Arabidopsis as a model system for the genetic analysis of volatile perception and aim to characterize those genes that are important for the response to volatile signals. In addition, we are studying the release of floral scent (benzenoids) in Petunia with the aim of understanding how the biosynthesis of pollinator-attracting volatiles is regulated at the molecular level.
In collaboration with Prof. dr. P. de Wit and Dr. M. Joosten (Phytopathology, WUR), evidence has been provided that the lipid second messenger PA is involved in the plant's defence response against pathogens. Using transgenic tobacco cells expressing the tomato Cf-4+ resistance gene as a model system to study Cf-4/Avr4 signalling, PA was shown to be triggered within minutes of elicitation with AVR4 from C. fulvum, but not with AVR9 (De Jong et al., 2004). Most PA was produced via the phosphorylation of DAG and not via PLD. Moreover, inhibitors of PLC activity effectively blocked the AVR4-induced PA accumulation and blocked the AVR4-induced oxidative burst. In addition, treating cells with a water-soluble synthetic PA analogue could induce an oxidative burst response. To identify which PLC(s) and DGK(s) is/are involved in the early PA response and to characterise their function in plant defence, multiple T-DNA insertion lines will be analyzed with regard to pathogen resistance.
The fungal elicitor xylanase is one of the exceptions, in that it also activates a PLD pathway. PLD b1-silenced cells were hypersensitive to xylanase and secreted a compound that coloured the culture medium. Overexpression of this PLD fused to GFP, revealed a cytosolic localisation, while in response to xylanase huge amounts of GFP-labelled vesicular structures accumulated. Using a PA-affinity approach, several putative PA-binding proteins have been isolated. Targets characterized so far include proteins involved in general processes (PEP carboxylase, HSP90) as well as signal transduction (protein kinases, phosphatases; Testerink et al., 2004; Anthony et al., 2004).
Our studies of the tritrophic system 'tomato - spider mite - predatory mite' have shown that upon spider mite-infestation volatile terpenoids (MeSA) are emitted, to which predatory mites are attracted. This indirect defence is jasmonate (JA)-dependent and its induction occurs a few days later than the direct defence (proteinase inhibitor production), which is rapidly induced by spider mite-herbivory. JA is also essential for the induction of salicylic acid methyltransferase, the enzyme that converts salicylic acid (SA) to MeSA. JA might possibly antagonise SA through stimulation of MeSA emission (Kant et al., 2004 and Ament et al., 2004). Using these tools, we have selected for spider mite cultivars that do not induce the direct and indirect defenses in tomato.
The first monoterpene synthase genes cloned from tmato were characterized: LeMTS1 can generate linalool in vitro, and LeMTS2 generates mycrene and limonene. While wounding, jasmonate and spider mite-herbivory induce LeMTS1, LeMTS2 does not respond to these challenges. However, LeMTS1 responds in the stems and roots, in which it is specifically expressed, to Fusarium infection. LeMTS1 transcripts are mainly present in leaves. We have also identified a geranylgeranyl pyrophosphatase synthase (GGPPS), which is strongly induced by spider mite-herbivory and jasmonic acid. We are testing whether this GGPPS provides the precursor for the homoterpene TMTT, the major volatile emitted by tomato upon spider mite-herbivory. In order to establish a role for volatiles in plant-plant communication we have screened EMS-populations of Arabidopsis and have identifeid several mutants that are insensitive to C6-volatiles. We are taking a map-based cloning approach to identify the mutated genes, one of which is located on chromosome 3.
Through targeted transcriptome analyses of Petunia flowers we have identified several biosynthetic genes such as benzoic acid methyltransferase (BMT) and benzylbenzoate transferase (BEBT), which are transcriptionally upregulated at the onset of scent production. Moreover, we have identified an R2R3-MYB type transcription factor of which the transcript levels follow the circadian rhythm of benzenoid emission. Down-regulation of this MYB in transgenic Petunia Mitchell plants reduced volatile benzenoid emission severely.
It appeared that this MYB specifically regulates the floral shikimate pathway that leads to the precursors for benzenoid synthesis. For instance, benzoic acid levels are 10-times lower in the RNAi plants than in Mitchell. This MYB therefore seems to be a key-regulator of floral benzenoid synthesis.
T-DNA insertion mutants of PLC, PLD and DGK genes in Arabidopsis as well as silencing of PLD isoforms in tomato plants and cell cultures resulted in intriguing phenotypes, which will be further investigated. We will also start unravelling the role of PA and its biosynthetic enzymes in cold acclimation and freezing tolerance. With regard to the PA targets, we aim to delimit the actual PA binding region and investigate the role of individual PA targets in PA-dependent signalling processes.
The role of volatile terpenoids produced by the terpene synthases LeMTS1 and 2 will be investigated in over-expressing plant lines, focussing on plant-herbivore and plant-pathogen interactions. We will try to determine whether the GGPPS1 gene codes for the enzyme that produces precursors for the most prominent tomato volatile, TMTT. With a metabolomics and transcriptomics approach we will investigate which molecular processes are activated or suppressed in tomato by the stealthy mite lines that we have discovered. The gene that is responsible for the C6-volatile insensitive phenotype of Arabidopsis will be identifeid by BAC-based complementation. With regard to the MYB transcription factor that regulates floral fragrances of Petunia, we will try to obtain over-expressing plant lines.
2005:
Verdonk JC, Haring MA, Van Tunen AJ, Schuurink RC (2005) ODORANT1 Regulates Fragrance Biosynthesis in Petunia Flowers. Plant Cell. 17(5):1612-24
2004:
Anthony RG, Henriques R, Helfer A, Mészáros T, Rios G, Testerink C, Munnik T, Deák M, Koncz C, Bögre L. (2004) A protein kinase target of a PDK1 signalling pathway is involved in root hair growth in Arabidopsis. EMBO J. 23, 572-581
Ament K, Kant MR, Sabelis MW, Haring MA and Schuurink RC (2004) Jasmonic acid is a key regulator of spider mite-induced volatile terpenoid and methyl salicylate emission in tomato. Plant Physiology 135, 2025-2037
De Jong CF, Laxalt AM, Bargmann BOR, de Wit PJGM, Joosten MHAJ, Munnik T. (2004). Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction. Plant J, 39, 1-12
Kant MR, Ament K, Sabelis MW, Haring MA and Schuurink RC(2004) Differential timing of spider mite-induced direct and indirect defenses in tomato plants. Plant Physiology 135, 483-495
Van Leeuwen W, Okresz L, Bögre L, Munnik T. (2004) Learning the lipid language of plant signalling. Trends Plant Sci. 9, 378-384
Testerink C, Dekker HL, Lim Z-Y, Johns MK, Holmes AB, de Koster CG, Ktistakis NT, Munnik T. (2004) Isolation and identification of phosphatidic acid targets from plants. Plant J, 39, 527–536
Zonia LE, Munnik T. (2004) Osmotically-induced cell swelling versus cell shrinking elicits specific changes in phospholipid signals in tobacco pollen tubes. Plant Physiol. 134, 813-823
Bron: SILS
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