Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.
Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism’s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.
Replication of leaf surface microstructure is a known method in the biomimetics research field1,2,3,4. The earliest replications of the leaf surface microstructure were performed using nail polish and rubber materials applied on the leaf surface for better visualization of microstructure, specifically stomata5,6,7,8,9,10. The method was then improved, and advanced polymers were used to mimic leaf surface microstructure using soft lithography, especially in the context of biomimetics of super hydrophobic surfaces2,3,4,11,12. In recent years, this method was proven as a useful tool in the study of the interaction between the leaf surface and microorganisms residing on the surface whether they are pathogenic13,14 or beneficial, as part of the natural leaf phyllosphere15. Simplification of the natural system was proven extremely useful in the study of surface-microorganism interactions even when purely synthetic systems were used as surfaces15,16,17,18.
While replication of leaf surface microstructure was shown to be a useful tool for studying the interaction occurring on the surface of the leaf with different microorganisms, no such tool exists for plant roots. Plant roots are harder to study since they reside below the ground and all interactions occur within the soil. Similar to leaves, root surface microstructure is likely to play a role in root-microorganism interactions. However, currently no method exists to isolate the specific role of root surface microstructure in the complex root-microorganism interactions. The most studied root surface microstructural feature is the root hairs19,20,21. Root hairs have an important role in increasing the surface area and by that allowing more efficient intake of nutrients and water22, however their involvement as a structural feature in root-microorganism interactions has never been tested.
The most widely used polymer for soft lithography in leaves is polydimethyl siloxane (PDMS). PDMS properties resemble those of the leaf cuticle15,23. However, in plant roots, the most abundant material is cellulose24,25 which has different properties than those of PDMS26,27,28. Using PDMS to build a synthetic platform for studying the surface microstructure effects in root-environment interactions is, hence, less than ideal.
The protocol presented here enables the formation of synthetic root surface microstructure replica from various materials. Like the method for leaf surface microstructure replication this is a two-step process. The first step uses the biological tissue (root) as a source for molding into a polyurethane mold (a negative replica). The polyurethane mold, which represents the negative image of the root surface microstructure, can then be used as a base to generate the positive replication of the root surface microstructure from a variety of materials, including PDMS and cellulose derivatives. This root surface replication can later be used as a platform to understand the surface structure role in root-microorganism interactions.
We present a novel method for the replication of root surface microstructure. This method relies on existing methods of leaf surface microstructure replication4. In order to develop this method, we had to tweak the existing method for leaves. We realized that the problematic step in copying the leaf replication method into roots involves the first step of the root molding. This is the most sensitive part of the method as it involves the biological tissue. As a result, we wanted to choose a polymer that would demand relatively gentle conditions for curing and hence causing minimal damage to the biological tissue. We chose polyurethane because it can be polymerized quickly (within 10 min) under UV light29. Additionally, it is very hard once polymerized30 and we hoped that this property would allow for the relatively easy removal of the root from the polyurethane mold.
The presented method is a two-step approach in which the negative image (negative replica) is formed in the first step and the replication is formed in the second step, based on the negative replica. This extends the range of materials we can work with. Leaf surface microstructure replication was mainly performed on PDMS or epoxy materials11,31. Some work was done with other materials, specifically materials supporting microorganism growth13,32. This is because in recent years this method has been used to study microorganism-surface interactions in the context of leaf surface structure. However, no cellulose-like materials have been used in this method in the context of leaves. We suggest the use of a polyurethane negative replica as a mold and a variety of materials for the positive replica. In other words, making the positive replica, from a variety of materials, is relatively easy once a good negative replica is made. We currently use cellulose derivatives, but are exploring the possibilities of using more relevant materials to root surface such as pectin and lignin33,34 in combination with cellulose derivatives.
The method also expands upon the existing method of leaf surface microstructure replication since the leaf is a 2D surface while the root surface is curved and hence is a 3D surface. Our method does not enable the replication of the whole surface since embedding the whole root in the polyurethane solution does not allow for its release. Therefore, one side of the root has to be chosen when replicating the root surface microstructure. The generated synthetic surface is curved and represents roughly half the surface, but not all of it. Our assumption is that the structural features of the root surface are mostly symmetrical about the axis along the root length. However, in studies where such symmetry is not assumed, one should be careful to choose the appropriate side root to replicate.
We present two options for roots to be used as molds. The first is the option of adventitious roots grown from the stem and the second is the option of germinated roots on paper. The first option is mostly meant to assist researchers in practicing the method as these roots are more robust and easier to work with. The second option represents the genetic differences that can be found between roots of different cultivars, regardless of the environmental conditions. These surfaces can be used as important research tools, however, one should be aware that the environment can have a strong influence on the root surface structure, specifically the soil in which the roots are grown35,36. Due to the mechanical stress inflicted by the soil, some morphological changes are bound to happen, in addition to wounds accruing on the surface as the root penetrates the soil37. Removal of roots from soil, as well as cleaning them, without damaging their structure is a very difficult task. Hence, we are not optimistic as to the ability to use this method to reliably mimic the root surface microstructure of roots grown in soil. However, for research that focuses on genetic differences or environmental differences where the change in microstructure is noticeably clear, this method can be used as a tool to study the influence of root surface microstructure.
Our method produces an inert surface mimicking of only the microstructural properties of the root surface. While this method is designed to separate the structural effects in root-environment interactions from all other effects, we cannot ignore the chemical compounds in those interactions. Some microorganisms may not survive or function on the surface without the addition of compounds, specifically nutrients. The next step in the development of this platform will be the controlled addition of chemical compounds to study their effects on the different interactions when combined with structure.
This method was developed as a first step in the development of a synthetic platform to study root-microorganism interactions. Here we mimic the microstructure of the root surface and this initial platform can be used to study the influence of surface microstructure on microorganism behavior. However, this platform is limited since it lacks many other elements from the natural system. This platform should be further developed with the use of the right materials to generate the surface and with the addition of other, critical, chemicals into the system. In a more advanced platform, we can also imagine spatial distribution of the chemicals. However, since currently no other method exists to isolate structural effects in root-microorganism interactions, we hope researchers could use this initial platform to ask structure-specific questions in those interactions.
The authors have nothing to disclose.
Research was supported by seed funds from The Agricultural Research Organization to MK.
2-hydroxy-2-methylpropiophenone | Sigma | 405655 | |
Diethyl phthalate | Across | 114520010 | |
Diurethane dimetharylate | Sigma | 436909 | |
Ethyl cellulose | Across | 232705000 | |
Ethyl methacrylate | Sigma | 234893 | |
Shaphir Solution | GAT fertilizer | 6-2-4 | |
Sylgard 184 kit | Polymer-G | 510018400500 |