Research

The wound response in tomato – a paradigm for plant stress responses.

Life is dangerous ... especially for a plant that cannot simply run away to escape harmful environments. Plants are exposed to a plethora of pests such as insects, nematodes, fungi, bacteria and viruses. Furthermore, they are exposed to abiotic forms of stress like drought, heat, cold, high salinity, and ultraviolet light. Unfavorable environmental conditions shift equilibria in ecosystems and result in enormous reductions in crop productivity. To cope with these stresses plants have evolved remarkable defensive and protective strategies that allow them to develop into mature and fertile organisms. Plant defense responses are continuously evolving to cope with pests that modify their invasive strategies to consume plants. During this evolutionary battle, plants change their genetic make-up to combat newly arising pests. This gave rise to an enormous arsenal of plant weapons. Many of the thousands of known plant secondary metabolites are thought to function in plant defense. On top of this multitude of defense compounds, plants evolved a complex signaling network that regulates the synthesis of these defense compounds. This network is highly adaptable to respond to challenges by newly arising pests.

The Tomato Wound Response

We are employing tomato as a model plant. Stress responses in tomato are very thoroughly studied, including the wound response to herbivorous insects. When tomato plants are attacked and wounded by chewing insects (e.g. caterpillars), signals derived from insects and the plant are generated that can be perceived by plant cells. One of these wound signals is a small plant peptide called systemin which is thought to be processed from a precursor protein, called prosystemin ¹. By binding to a membrane-bound receptor, systemin initiates an intracellular signal transduction pathway that relays and amplifies the wound signal leading to the activation of defense genes. Many plant defense genes encode antinutritive proteins such as proteinase inhibitors and enzymes that break down essential amino acids. When ingested, these proteins cause adverse effects on growth and development of the attacking insects by preventing uptake of essential amino acids in the insect gut ². Amazingly, this wound response does not only occur at wound sites, but all over the plant ³. What is the long-distance signal that is generated at wound sites and reaches distant target tissues in other parts of the plant? Systemin regulates the synthesis of jasmonic acid, and this plant hormone acts not only as a second messenger within the cells, but also as a long-distance signal that moves within the plant to distant leaves where it induces the wound response ⁴.

Signal perception and signal transduction through MAPK pathways

The first step for a successful stress response is the perception of stress signals by plant cells. The perceived signal is then relayed in a chain of events that leads to an amplification of the signal, and eventually to gene activations and a cellular response.

Mitogen-activated protein kinases (MAPKs) are important elements in eukaryotic stress signal transduction chains. A MAPK is part of the 'MAPK cascade' which consists of three functionally linked kinases. MAP3Ks (or MAPKKKs) are often activated by extracellular signals in a receptor-dependent manner. MAP3Ks activate MAPKKs (or MEKs) by phosphorylation, which in turn activate MAPKs (or MPKs) by phosphorylation. MAPK cascades regulate the activity of transcription factors or cytosolic enzymes. The Arabidopsis genome contains ~ 60 putative MAP3Ks, only 10 MAPKKs, and 20 MAPKs.

Research projects in the Stratmann group

Our aim is to characterize the molecular mechanisms of stress signal transduction, and the highly complex plant signaling network that enables a plant cell to integrate multiple stress signals and launch an appropriate cellular defense response.

We have shown previously that MAPK activity in tomato leaves is induced by multiple stress signals including herbivorous insects, systemin, oligosaccharide elicitors ⁵ and ultraviolet-B (UV-B) radiation ⁶. UV-B radiation does not fully induce the wound response. However, when cells were irradiated with UV-B and subsequently wounded, the accumulation of defense proteins was increased in a synergistic manner when compared to non-irradiated plants ⁶. This provides an excellent system to study how a plant cell integrates more than one stress signal to activate a cellular defense response. Moreover, it indicates that changes in the global environment such as increased ground level UV-B radiation due to stratospheric ozone depletion (ozone hole) might have unexpected consequences for plant-consumer interactions ⁷.

We identified three specific MAPKs that respond to herbivorous insects, mechanical wound signals, systemin, oligosaccharide elicitors and UV-B ^8,9,10. Employing virus-induced gene silencing (VIGS) to silence the three MAPK genes, we demonstrated that these MAPKs regulate jasmonic acid and ethylene synthesis in response to systemin and wounding. Since synthesis of these plant hormones is required for defense gene activation, the silenced plants had not only a severely impaired wound response but also a strongly reduced resistance against herbivorous insects ¹⁰. Chewing insects generate other MAPK-activating signals in addition to systemin, such as mechanical signals 5,10 and fatty acid-amino acid conjugates (FACs) ¹¹. The corresponding signaling pathways all converge on the same three MAPKs, which can explain why silencing of these MAPKs prevented a successful defense response against the attacking insect larvae.

Current projects address the regulation of several tomato MAPKKs. We have isolated 4 tomato MAPKKs and shown in protoplast transient gene expression assays that two of them activate the three stress-responsive MAPKs. Since MAPKKs function as convergence points for multiple stress signals, it is critical to understand how they contribute to signaling specificity in MAPK pathways. Although there are 10 MAPKKs and 20 MAPKs in the Arabidopsis genome, only a few of them respond to multiple stress signals. How can signaling components that are shared by multiple input signals lead to signal-specific responses? Regulation of the MAPK cascade at the level of all three kinases is likely to contribute to signaling specificity. We study how MAPKKs are activated and inactivated in response to multiple stress signals.

Another project is the characterization of the systemin receptor SR160. A surprising recent finding in the plant sciences was that the systemin receptor is identical to the brassinosteroid receptor ^12,13. However, recently, it was shown that brassinosteroid receptor null mutants in tomato have a wild type-like response to systemin indicating that SR160 is not the systemin receptor ¹⁴. Our results are consistent with these findings but show that SR160, when overexpressed in tobacco cells, can function as a systemin binding protein. However, binding of systemin to BRI1 does not relay the signal into the cell and does not trigger a defense response (in review). It will now be important to identify the elusive systemin receptor.

These projects utilize state-of-the-art techniques in molecular biology and biochemistry.

In the long run, our studies will provide clues for biotechnology on how to alter signaling networks in crop plants to make them more resistant to the stresses they encounter in their agricultural environment.

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