Evelien Vandevenne

A pectin-methylesterase-inhibitor-based molecular probe for in situ detection of plant pectin methylesterase activity.

In the quest of obtaining a molecular probe for in situ detection of pectin methylesterase (PME), the PME inhibitor (PMEI) was biotinylated and the biotinylated PMEI (bPMEI) was extensively characterized. Reaction conditions for single labeling of the purified PMEI with retention of its inhibitory capacity were identified. High-performance size-exclusion chromatography (HPSEC) analysis revealed that the bPMEI retained its ability to form a complex with plant PME and that it gained the capacity to strongly bind an avidin species. By means of dot-blot binding assays, the ability of the probe to recognize native and high-temperature or high-pressure denatured plant PMEs, coated on an absorptive surface, was investigated and compared to the binding characteristics of recently reported anti-PME monoclonal antibodies. Contrary to the antibodies, bPMEI only detected active PME molecules. Subsequently, both types of probes were used for PME localization in tissue-printing experiments. bPMEI proved its versatility by staining prints of carrot root, broccoli stem, and tomato fruit. Applying the tissue-printing technique on carrot roots after thermal treatment demonstrated the complementarity of bPMEI and anti-PME antibodies, with the former selectively detecting the remaining active PME and the latter staining both native and inactivated PME molecules.

Development and evaluation of monoclonal antibodies as probes to assess the differences between two tomato pectin methylesterase isoenzymes.

The enzyme pectin methylesterase (PME) was purified from red ripe tomatoes (Lycopersicon esculentum) and through affinity chromatography two isoenzymes were fractionated (t1PME and t2PME). Further analysis of these two isoenzymes, both having a molar mass of 34.5kDa, revealed a difference in the N-terminal sequence and in amino acid composition. t1PME was identified as the major isoenzyme of PME in tomato fruit. In this study the aim was to develop a toolbox, consisting of monoclonal antibodies, that allows to gain insight into the in situ localization of PME in plant based food systems like tomatoes. A panel of six interesting monoclonal antibodies was raised against both isoenzymes, designated MA-TOM1-12E11, MA-TOM1-41B2, MA-TOM2-9H8, MA-TOM2-20G7, MA-TOM2-31H1 and MA-TOM2-38A11. The differences in epitopes between these monoclonal antibodies were determined using affinity tests towards denatured PME, cross-reactivity tests and inhibition tests. Characterization of these antibodies indicated an immunological difference between t1PME and t2PME, also revealing a conserved epitope on t2PME, carrot PME and strawberry PME. Different epitopes are recognized by the generated antibodies making them excellent probes for immunolocalization of PME by tissue printing. In tomato, t1PME and t2PME showed a pronounced co-localization, especially in the pericarp and the radial arms of the pericarp. Three of the generated antibodies could be used for immunolocalization of PME in carrots (Daucus carota L.), which was only present in the cortex and not in the vascular cylinder of carrots.

Advances in understanding pectin methylesterase inhibitor in kiwi fruit: an immunological approach

In order to gain insight into the in situ properties and localisation of kiwi pectin methylesterase inhibitor (PMEI), a toolbox of monoclonal antibodies (MA) towards PMEI was developed. Out of a panel of MA generated towards kiwi PMEI, three MA, i.e. MA-KI9A8, MA-KI15C12 and MA-KI15G7, were selected. Thorough characterisation proved that these MA bind specifically to kiwi PMEI and kiwi PMEI in complex with plant PME and recognise a linear epitope on PMEI. Extract screening of green kiwi (Actinidia deliciosa) and gold kiwi (Actinidia chinensis) confirmed the potential use of these MA as probes to screen for PMEI in other sources. Tissue printing revealed the overall presence of PMEI in pericarp and columella of ripe kiwi fruit. Further analysis on the cellular level showed PMEI label concentrated in the middle lamella and in the cell-wall region near the plasmalemma. Intercellular spaces, however, were either completely filled or lined with label. In conclusion, the developed toolbox of antibodies towards PMEI can be used as probes to localise PMEI on different levels, which can be of relevance for plant physiologists as well as food technologists.