The walnut shell network: 3D visualisation of symplastic and apoplastic transport routes in sclerenchyma tissue.

MAIN CONCLUSION: High symplastic connectivity via pits was linked to the lignification of the developing walnut shell. With maturation, this network lessened, whereas apoplastic intercellular space remained and became relevant for shell drying. The shell of the walnut (Juglans regia) sclerifies within several weeks. This fast secondary cell wall thickening and lignification of the shell tissue might need metabolites from the supporting husk tissue. To reveal the transport capacity of the walnut shell tissue and its connection to the husk, we visualised the symplastic and apoplastic transport routes during shell development by serial block face-SEM and 3D reconstruction. We found an extensive network of pit channels connecting the cells within the shell tissue, but even more towards the husk tissue. Each pit channel ended in a pit field, which was occupied by multiple plasmodesmata passing through the middle lamella. During shell development, secondary cell wall formation progressed towards the interior of the cell, leaving active pit channels open. In contrast, pit channels, which had no plasmodesmata connection to a neighbouring cell, got filled by cellulose layers from the inner cell wall lamellae. A comparison with other nut species showed that an extended network during sclerification seemed to be linked to high cell wall lignification and that the connectivity between cells got reduced with maturation. In contrast, intercellular spaces between cells remained unchanged during the entire sclerification process, allowing air and water to flow through the walnut shell tissue when mature. The connectivity between inner tissue and environment was essential during shell drying in the last month of nut development to avoid mould formation. The findings highlight how connectivity and transport work in developing walnut shell tissue and how finally in the mature state these structures influence shell mechanics, permeability, conservation and germination.

Simple and rapid methods for SEM observation and TEM immunolabeling of rubber particles.

We developed a method involving air-drying of a rubber suspension after fixation in glutaraldehyde-tannic acid and postfixation in osmium tetroxide for SEM observation. For TEM immunolabeling the suspension was air-dried after osmium-only fixation. Whereas conventional methods failed to satisfactorily stabilize rubber particles, the methods described here proved successful in preserving their integrity.

A novel approach for FE-SEM imaging of wood-matrix polymer interface in a biocomposite.

Understanding the interface between polymer and biomass in composite products is important for developing high performance products, as the quality of adhesion at the interface determines composite properties. For example, with greater stiffness compared to polymer matrix, such as that of high density polyethylene, the wood component enhances stiffness of wood-polymer composites, provided there is good adhesion between composite components. However, in composites made from wood flour (wood particles) and synthetic resins it is often difficult to clearly resolve particle-matrix interfaces in the conventionally employed microscopy method that involves SEM examination of fractured faces of composites. We developed a novel approach, where composites made from high density polyethylene and wood flour were examined and imaged with a FE-SEM (field emission scanning electron microscope) in transverse sections cut through the composites. Improved definition of the interface was achieved using this approach, which enabled a more thorough comparison to be made of the features of the interface between wood particles and the matrix in composites with and without a coupling agent, as it was possible to clearly resolve the interfaces for particles of all sizes, from large particles consisting of many cells down to tiny cell wall fragments, particularly in composites that did not incorporate the coupling agent used to enhance particle adhesion with the matrix polymer. The method developed would be suitable particularly for high definition SEM imaging of a wide range of composites made combining wood and agricultural residues with synthetic polymers.

The micromorphology and protein characterization of rubber particles in Ficus carica, Ficus benghalensis and Hevea brasiliensis.

Rubber biosynthesis takes place on the surface of rubber particles. These particles are surrounded by a monolayer membrane in which the rubber transferase is anchored. In order to gain better insight into whether rubber particles from different plant species share common structural characteristics, the micromorphology of rubber particles from Ficus carica, Ficus benghalensis, and Hevea brasiliensis was examined by electron microscopy. Rubber particles of all three species were spherical in shape, and the size of rubber particles of H. brasiliensis was much smaller than those of F. carica and F. benghalensis. In addition, investigations were undertaken to compare the cross-reactivity of the antibody raised against either the H. brasiliensis small rubber particle protein (SRPP) which is suggested to be involved in rubber biosynthesis, or the cis-prenyltransferase (CPT) which has an activity similar to rubber transferase. Both western analysis and TEM-immunogold labelling studies showed that rubber particles of F. carica and F. benghalensis do not contain the SRPP. None of the rubber particles in F. carica, F. benghalensis and H. brasiliensis contained the CPT, suggesting that the CPT itself could not catalyse the formation of high molecular weight rubber. These results indicate that rubber particles in the three different plant species investigated share some degree of similarity in architecture, and that the SRPP and CPT themselves are not the core proteins necessary for rubber biosynthesis.