Atomic force microscope (AFM) study on enzymatic degradation of polysaccharides structure in cell wall of fruit

Title: Atomic force microscope (AFM) study on enzymatic degradation of polysaccharides

structure in cell wall of fruit

2011/01/B/NZ9/00787

Leader: Artur Zdunek

Period: 2011-2015

Financed by Narodowe Centrum Nauki (NCN)

Goal of the project is to describe structural changes of individual cell wall polysaccharides and entire cell walls, as polysaccharides assemblies, combined with their mechanical properties as a result of enzymatic degradation. Atomic force microscope (AFM) will be used in this study due to possibility of imaging and investigation of mechanical properties of biological materials at nano-scale. An outcome of the project will be knowledge on structural changes of individual macromolecules (cellulose, pectins and hemicelluloses) and cell walls (assemblies of the polymers) as a result of activity of following enzymes, which are active during fruits maturation: pectate lyaze (PL), polygaracturonase (PG), pectin-methyloesterase (PME), endo (1-4) β-D-glucanase and xyloglucan endotransglycosylase (XET). Another outcome will be knowledge on changes of mechanical properties of cell walls as result of the above enzymes.

Model of primary cell wall (type I) consists of polysaccharides (cellulose, hemicelluloses and pectins) and structural proteins. Properties of the polysaccharides and mutual interactions determine physico-chemical properties of the cell wall, in particular their mechanical properties. In fruits cell walls mechanical properties change extensively during maturation and the changes are governed by an enzymes activity. In general, enzymes cause structural changes of the polysaccharides and in result softening of fruit. It occurs particularly during maturation, which is observed as firmness loss or texture decay. From the point of consumer or famer, this process is very undesirable, when occurs too fast after harvest.

Knowledge on enzymatic changes of polysaccharides structure and mechanical properties of cell walls, studied in this project, has high significance for explanation of mechanisms causing changes of fruit properties during maturation, ripening and senescence. This knowledge and developed methodology will allow for next studies considering: genetic factors, cultivation, storage, etc., and in consequence for optimization of growing, storage and post-harvest processing. It is expected that experiments planned in this project will allow also for verification of sometimes contrary reports on a role of enzymes in fruit texture and will complete existing knowledge on fruits maturation mechanisms.

Cell wall polymers are investigated after selective extraction of particular fractions. Isolated cell wall polysaccharides can be analyzed using chemical methods or by using cytochemical staining to visualize the polysaccharide types and gather information about their quantity and location. In addition, imaging techniques like SEM or FTIR can be used. In this project we propose to use atomic force microscope (AFM) which since 90’s is used for investigation of biological samples structure and interactions at molecular level. However, at present one can find very little number of papers on AFM use for fruits investigation. From this point of view, the project is significant for further development of the AFM technique for investigation of biological samples.

AFM measures changes in the magnitude of interaction repulsive and attractive forces between the tip and sample surface as the surface is scanned beneath the probe.  The tip of few nanometer in diameter is placed on cantilever of a certain springiness constant. The cantilever is deflected when interaction between surface and the tip occurs. Available tips allows now to obtain nanometer resolution. It was shown that the resolution is sufficient for imaging of single polysaccharides. One of AFM advance is imaging in fluid or in liquid which makes it very useful for biological study. Most often, no sample preparation is needed.  Additionally, AFM allows for evaluation of mechanical properties in each point of the image (at nm resolution) such as: adhesion, viscosity, Young modulus. Another AFM mode is force spectroscopy where a macromolecule is tensioned between tip and substrate. It allows for evaluation of macromolecule conformation. In food research AFM was used for study of polymers structure and its changes as result of technological processes, supplying data not possible to obtain with other methods. AFM was used for investigation of fruit polysaccharides (mainly pectins and cellulose) with consideration storage and calcium effect. The technique seems to be very useful for study of polysaccharides structure of cell walls from the point of view fruit softening because AFM gives direct view on both structure and mechanical properties of sample. However, the use of AFM is still at preliminary stage. Until now, there is lack of AFM study on structural enzymatic degradation of cell wall polysaccharides of their assemblies. This project considers this topic.

We propose in this project to work on apples and pears due to high importance of these fruits in Poland and Europe, and extensive softening with relatively high initial firmness.

The project will be realized in following tasks:

• Adopting (standardization) protocols for sequential extraction to obtain fractions of: water-soluble pectins, chelate-soluble pectins, sodium-carbonate soluble pectins, hemicelluloses and cellulose with biochemical analyses.
• Developing protocols for obtaining consistent AFM topography images of different polysaccharides fractions
• Developing image analysis protocols for quantitative evaluation of AFM topography images.
• Developing protocols for investigation of mechanical properties of cell walls by AFM.
• In vitro AFM study on changes of plant cell walls topography and mechanical properties as result of controlled enzymes action
• In vitro AFM study on depolimerization of individual polysaccharides structure as result of controlled enzymes action
• AFM study on changes of plant cell walls topography and mechanical properties during fruits development, maturation and senescence
• AFM study on depolimerization of individual polysaccharides structure during fruits development, maturation and senescence

The above studies will be completed with evaluation of enzymatic activity of fruits and with a set of basic maturity indexes (firmness, starch index, soluble solid content, titratable acidity and sensory analysis (only for mature fruits)). The data set will allow for relation of ripening dynamic with structural changes in cell walls.

Publications

1. Kozioł A., Cybulska J., Pieczywek P.M., Zdunek A., 2015. Evaluation of Structure and Assembly of Xyloglucan from Tamarind Seed (Tamarindus indica L.) with Atomic Force Microscopy, Food Biophysics, DOI 10.1007/s11483-015-9395-2
2. Zdunek A., Kozioł A., Pieczywek P.M., Cybulska J. 2014. Evaluation of the Nanostructure of Pectin, Hemicellulose and Cellulose in the Cell Walls of Pears of Different Texture and Firmness. Food and Bioprocess Technology, 7, 12, 3525-3535.  http://dx.doi.org/10.1007/s11947-014-1365-z
3. Zdunek A., Kurenda A., 2013. Determination of the Elastic Properties of Tomato Fruit Cells with an Atomic Force Microscope. Sensors, 13, 12175-12191. link
4. Cybulska J., Zdunek A. , Kozioł A. 2015. The self-assembled network and physiological degradation of pectins in carrot cell walls, Food Hydrocolloids, 43, 41–50. http://dx.doi.org/10.1016/j.foodhyd.2014.04.032.