Functional characterisation of nitrated and oxidised phloem proteins in the systemic wound response of pumpkin (Cucurbita maxima)
Zusammenfassung der Projektergebnisse
The unique extrafascicular phloem (EFP) of cucurbits serves in herbivore defense similar to laticifers in other plant species. Phloem latex (EFP exudates) can be easily collected from cut petioles and stems. Damaging leaves of pumpkin (Cucurbita maxima) triggered a systemic wound response (SWR) in EFP, which involved signaling through the phloem-mobile stress hormone jasmonic acid (JA) and rapid partial sieve tube occlusion by callose. Metabolomics of phloem latex (EFP exudates) by gas chromatography coupled to mass spectrometry and gel-free proteomics by stable isotope-coded protein labeling were adopted for more detailed investigations. This way, we revealed that the energy metabolism was enhanced but enzymes related to cell wall synthesis and pentosephosphate pathway were less expressed during the SWR. A 32-kD 14-3-3 protein and two Sucrose- Nonfermenting 1 (SNF1)-related protein kinases are candidate central regulators of stress metabolism in the EFP. Silverleaf Whitefly-Induced Protein 1, Mitogen Activated Protein Kinase 6 and four chaperones accumulated upon wounding and might be directly or indirectly involved in herbivore defense. In this regard cyclophilin 18 (CYP18) is a particularly prominent damage-inducible phloem protein, which can be easily detected by gel electrophoresis, and thus, might be a useful molecular marker in breeding efforts for improving crop resistance to herbivorous and phloem-sucking insects. Several lines of evidence hint at stress-induced redox- and nitric oxide (NO) signaling in the EFP of pumpkin. After leaf wounding the antioxidant enzymes dehydroascorbate reductase, glutathione reductase and ascorbate peroxidase are strongly reduced by about 50 % in enzyme activity as compared to untreated control plants. We also observed a drop in reduced glutathione levels. Taken together, these results point at oxidative stress in the EFP due to a decrease in the antioxidant capacity. Furthermore, wounding triggered an increase in tyrosine nitration of phloem latex proteins indicative for NO signaling. The major Phloem Protein 2 (PP2) was nitrated and at the same time also specifically de-carbonylated/reduced after wounding as analyzed by a western blot approaches. Hence, the lectin PP2 might be a good model for deciphering the interaction of NO and reactive oxygen species in regulating functions of defence-related proteins. Cyclic guanosine monophosphate (cGMP) can act as a second messenger of NO in stress responses. Until now the intriguing question remains unanswered if the observed wound-induced cGMP production in the EFP is NO dependent. In sum, systemic wound responses of the pumpkin EFP are regulated by JA, NO, cGMP and redox signaling. The exact sequence and mechanism of interactions between the signals will be addressed in future studies.
Projektbezogene Publikationen (Auswahl)
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(2012) Deciphering systemic wound responses of the pumpkin extrafascicular phloem by metabolomics and stable isotope-coded protein labeling. Plant Physiol 160, 2285-2299
Gaupels, Frank; Sarioglu, Hakan; Beckmann, Manfred; Hause, Bettina; Spannagl, Manuel; Draper, John; Lindermayr, Christian & Durner, Jörg
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(2012) Looking deep inside: detection of low-abundance proteins in leaf extracts of Arabidopsis and phloem exudates of pumpkin. Plant Physiol 159, 902-914
Fröhlich, Andreas; Gaupels, Frank; Sarioglu, Hakan; Holzmeister, Christian; Spannagl, Manuel; Durner, Jörg & Lindermayr, Christian
