Seeds of Amaranthus hypochondriacus L. are known to accumulate a trypsin-inhibitor (ATI) member of the potato-I inhibitor family and an a-amylase inhibitor (AAI), possessing a knottin-like fold. They are believed to have a defensive role due to their inhibition of trypsin-like enzymes and aamylases of insect pests. In this work, both inhibitory activities were found in leaves of young A. hypochondriacus plants. High constitutive levels of foliar inhibitory activity against bovine trypsin and insect a-amylases were detected in in vitro assays. Trypsin inhibitory activity was further increased by exposure to diverse treatments, particularly water stress. Salt stress, insect herbivory and treatment with exogenous methyl jasmonate (MeJA) or abscisic acid (ABA) also induced trypsin inhibitor activity accumulation, although to a lesser degree. In gel and immunoblot analyses showed that foliar trypsin inhibitor activity was constituted by at least three different inhibitors of approximately 29, 8 (including ATI) and 3 kDa, respectively. These inhibitors showed differing patterns of accumulation in response to diverse treatments. On the other hand, significant increases in a-amylase inhibitor activity and AAI levels were detected in leaves of insectdamaged, MeJA-and ABA-treated A. hypochodriacus plantlets, but not in those subjected to water-or salt-stress. A differential induction of trypsin inhibitor activity and a-amylase inhibitor accumulation in response to insect herbivory by two related species of lepidopterous larvae was observed, whereas mechanical wounding failed to induce either inhibitor. The overall results suggest that trypsin and a-amylase inhibitors could protect A. hypochondriacus against multiple types of stress.
BackgroundThe identification of proteins by mass spectrometry is a standard method in biopharmaceutical quality control and biochemical research. Prior to identification by mass spectrometry, proteins are usually pre-separated by electrophoresis. However, current protein staining and de-staining protocols are tedious and time consuming, and therefore prolong the sample preparation time for mass spectrometry.Methodology and Principal FindingsWe developed a 1-minute covalent pre-gel staining protocol for proteins, which does not require de-staining before the mass spectrometry analysis. We investigated the electrophoretic properties of derivatized proteins and peptides and studied their behavior in mass spectrometry. Further, we elucidated the preferred reaction of proteins with Uniblue A and demonstrate the integration of the peptide derivatization into typical informatics tools.Conclusions and SignificanceThe Uniblue A staining method drastically speeds up the sample preparation for the mass spectrometry based identification of proteins. The application of this chemo-proteomic strategy will be advantageous for routine quality control of proteins and for time-critical tasks in protein analysis.
Plant peroxidases (PODs) are involved in diverse physiological processes, including defense against pathogens and insects. Contrary to their biological importance, only very few plant PODs have been proven on protein level, because their low abundance makes them difficult to detect in standard proteomics work-flows. A statistically significant positive correlation between POD activity and post-harvest insect resistance has been found for maize (Zea mays, p84C3) kernels. In combining activity-directed protein purification, genomic and proteomic tools we found that protein B6T173 (ZmPrx35) is responsible for the majority of the POD activity of the kernel. We successfully produced recombinant ZmPrx35 protein in Escherichia coli and demonstrate both, in vitro activity and the presence of a haem (heme) cofactor of the enzyme. Our findings support the screening for insect resistant maize variants and the construction of genetically optimized maize plants.
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