Investigating the in situ localisation of pectin methylesterase and its inhibitor: Development of an immunological toolbox

Investigating the in situ localisation of pectin methylesterase and its inhibitor: Development of an immunological toolboxDuring processing of plant-based food products the aim is to guarantee food safety and quality among which texture forms one of the most prominent quality attributes. The plant cell wall, one of the characteristic components of plant-based food products, greatly contributes to texture. Amongst the numerous compounds building the structurally heterogeneous cell wall, pectin is of interest because of its functional properties in the cell wall with regard to tissue integrity and rigidity. Moreover, pectin is an important target in texture engineering during food processing, in which also the enzyme pectin methylesterase (PME) and its inhibitor (PMEI) play an important role. In order to fully exploit the possibilities of the pectin conversions imposed by PME in texture engineering, it is important to understand these mechanisms prior to and during processing. Ex situ analysis alone is subjected to some restrictions and should ideally be complemented with in situ analysis. Thus, it is in this context that probes have been developed in the current work to enhance the in situ insight into PME and PMEI. During processing, endogenous PME in plant-based foods plays an important role in attaining the desired food structure. Because of the interest in endogenous PME, plant PME was here purified from red ripe tomato fruit (Solanum lycopersicon) and used for the production of monoclonal antibodies (MAs) as probes. Two isoenzymes were fractionated from tomato fruit (t1PME and t2PME), both having a molar mass of 34.5 kDa and displaying differences in amino acid sequence. t1PME was identified as the major isoenzyme of PME in tomato fruit. Both isoenzymes were used for the generation of MAs, resulting in a panel of six interesting MAs designated MA-TOM1-12E11, MA-TOM1-41B2, MA-TOM2-9H8, MA-TOM2-20G7, MA-TOM2-31H1 and MA-TOM2-38A11. Characterisation of these antibodies, including the evaluation of the cross-reactivity towards PME from tomato, carrot, strawberry and Aspergillus aculeatus, indicated an immunological difference between t1PME and t2PME and also revealed a conserved region on t2PME, carrot PME and strawberry PME. The PME specificity of the developed antibodies makes them excellent probes for immunolocalisation of PME. Tomato fruit tissue printing revealed a pronounced co-localisation of t1PME and t2PME on tissue level, especially in the pericarp and the radial arms of the pericarp. This co-localisation persisted on cellular level where both isoenzymes were detected only in the primary cell wall and not in the middle lamella or intercellular junctions, using immunofluorescence microscopy. PME can also be added exogenously during food processing for texture improvement of plant-based foods. In this case, the exogenously added PME is often of fungal origin. Therefore PME from Aspergillus aculeatus (fPME) was used as antigen for the production of MAs. Three of the generated MAs, named MA-ASP-23G10, MA-ASP-35F6 and MA-ASP-38H6, bind exclusively to fPME and were used for the immunolocalisation of exogenous fPME upon infusion into plant tissue. Three different infusion techniques were compared for three different types of plant tissue, revealing a homogenous distribution of exogenous PME upon pressure-and vacuum-assisted infusion in contrast to passive osmotic infiltration as was detected by tissue printing. The remaining two MAs, MA-ASP-25F7 and MA-ASP-32A1, recognise fPME as well as PME from different plant sources like tomato, carrot, strawberry, broccoli and apple. The use of these antibodies allowed detection of endogenous PME in tomato which revealed an overall presence of PME in the pericarp of tomato fruit. Moreover, the PME detected by immunofluorescence microscopy using these MAs coincided with the localisation of the isoenzymes of tomato PME (t1PME and t2PME) in the plant cell wall of tomato fruit, as detected by the isoenzyme-specific MAs, MA-TOM1-41B2 and MA-TOM2-9H8. The obtained results highlight the versatility and applicability of the developed antibodies as probes to detect PME in the context of food processing. In order to gain insight into the in situ properties and localisation of kiwi PMEI, a toolbox of MAs towards PMEI was developed. Three MAs were selected from a panel of MAs generated towards kiwi PMEI, i.e. MA-KI9A8, MA-KI15C12 and MA-KI15G7. Thorough characterisation proved that these MAs bind specifically to kiwi PMEI and kiwi PMEI in complex with plant PME and recognise a linear epitope on PMEI. Extract screening of fruits and vegetables among which green kiwi (Actinidia deliciosa) and gold kiwi (Actinidia chinensis) confirmed the potential use of these MAs 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 cellular level by immunofluorescence microscopy showed that PMEI-label was 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. The three sets of antibodies developed towards endogenous PME, exogenous PME and PMEI have proven their use as toolset for the immunolocalisation of PME and PMEI. Importantly, the applications performed in this work represent only the start of a multitude of applications in which these probes can be applied for pectin research in plant-based food processing or even in a wider context.status: publishe