Exploring the Mechanisms of Humidity Responsiveness in Plants and Their Potential Applications

Abstract

Plant structures exhibit complex behaviors through unique shape changes and movements closely related to moisture factors. When the plants absorb moisture, their inside has a higher tension than their outside, so the entire structure is folded to closure or opened. The principle and property could be applied to bio-inspired technology, which is the process of fusion mimicking the structure, function, metabolism, mechanism, and ecological system of those creatures adapted to their environments. In this study, we analyzed the functions and physical characteristics of environment-sensing plants to demonstrate the principles of plants with opening-and-closing and curling-and-uncurling mechanisms and to better understand these behavior principles. From a biological and ecological viewpoint, the target sensory and cognitive plants that respond to external humidity and vibration were found to undergo structural changes in the size of the xylem and the degree of adhesion of the leaf and stem, as well as the opening, closing, and curling of the external shapes of the plants. The phenomenon of external form changes based on the microstructural characteristics of plants showed a promising direction for addressing issues in existing technology, such as non-powered operation. Therefore, in this study, we presented a biomimetic humidification model that was biocompatible and reversible. Pinecone samples with the applied opening-and-closing mechanism were to apply these biological properties to biomimetics. The results provide biomimetic knowledge for understanding the functions of biological and ecological features underlying the morphological changes in humidity-sensing plants and plant bioacoustics. These bio-inspired plant resources could provide sustainable new-growth power and valuable scientific information for advancing the research and technological development of biomimetics.

1. Introduction

Plants have excellent sensory systems, behaviors, adaptations, and cognitive functions [1,2,3] and are highly sensitive to the mutual network between the environment and plant body. Hygroscopic behavior is defined as the phenomenon wherein water molecules are drawn in from the surrounding environment through absorption. The main factors that contribute to the hygroscopic movement in plants include the driving force of turgor pressure, the mechanism of swelling-driven deformation, and the trajectory of movement. As plants that exhibit hygroscopic behavior show atypical external mechanisms due to internal structural changes, studying their structural aspects from both a biological and ecological standpoint is valuable. Although the various angles of hygroscopic behavior of plants have been researched [4,5,6] in order to apply biomimicry in engineering technology [7,8], there is limited knowledge about the adaptive anatomy related to the altered shape, structure, and physiology of plants [9]. It is widely known that plants use their own unique materials and structures to adapt to and survive in their surroundings. In other words, living things in nature have the characteristic of responding to external environmental stimuli for environmental sensing, food search, and reproduction. With recent rapid industrial growth, environmental issues have come to the forefront, emphasizing the importance of environmental sensing technology.

Biomimetics refers to the advancement in sustainable technology developed by applying the basic principles and structures of biological and ecological resources. It is a field of application that aims to solve social problems by mimicking nature’s unique characteristics and is a new-growth power in the Fourth Industrial Revolution [10,11]. Biomimetic research is focused on approaches from the engineering field for solving technical problems [12,13]. However, acquiring the sources of biological and ecological knowledge needed for technology development remains challenging [14,15].

The genera Selaginella, Erodium cicutarium, and Pinus are examples of humidity-sensing plants, and the related studies mainly focus on engineering theory [16,17,18]. In particular, the mechanisms of xerophytes, hygrophytes, and semi-hygrophytes are distinct [19,20], but hygroscopic movement in response to moisture, as mentioned in this study, is considered a common function. In Selaginella involvens, the leaves curl toward the central axis (stem) in dry conditions and uncurl under sufficient relative humidity [21]. In Pyrrosia lingua, a perennial evergreen herb that grows on tree stems or rock surfaces, a similar phenomenon is observed.

To understand the functional and behavioral mechanisms underlying the hygroscopic behavior of plants, research on plant species is necessary to select target species allowing biomimetic materialization and to appreciate their structural specifications. Consequently, there is a growing trend in demand for environmental sensing sensors designed to detect prominent environmental factors such as humidity, temperature, air pollution, and harmful gases. Especially humidity has direct and indirect impacts on everyday life and industrial environments. Technologies are being developed, leveraging the principles of plant humidity detection and the integration of engineering techniques. These include temperature-responsive sensors based on eco-friendly materials, enabling information transmission without the need for external measuring equipment or power sources.

In this study, the morphological structure and function of the genus Selaginella and Pyrrosia lingua were analyzed to understand the intricate deformation of internal structure caused by humidity changes, with the goal of developing technological applications through biomimetics. Finite element analysis was performed to appreciate the deforming tendency caused by hygroscopic swelling. The characteristics of moss and telegraph trees were also analyzed, as their shapes and external structures change in response to the external environment. This study aims to find an ideal model scheme for an eco-friendly humidifying system through experimentation based on the absorption and release of moisture using materials that are harmless to both the environment and humans. By understanding the movement principles of plants, this study hopes to contribute to the development of biomimetic technology.

2. Materials and Methods

2.1. Plant Materials and Pretreatments

Mature plant samples of Selaginella involvens (Sw.) Spring, Selaginella rossii (Baker) Warb, Pyrrosia lingua (Thunb.) Farwell, Racomitrium canescens, and Codariocalyx motorius were purchased from a local farm (Secheon-gun, Republic of Korea) from February to June 2022. The plants were planted on a seedbed (50 cm× 30 cm) placed at room temperature. Water was supplied as required for experiments on moisture and dry conditions because there was minimal variation in plant growth during the experimental analysis period.

2.2. Light Microscopy Analysis

To determine internal structures in plants, light microscopy analysis was conducted. The samples were soaked in fixation solution (2% glutaraldehyde and 2% paraformaldehyde in 0.05 M cacodylate buffer pH 7.2) at room temperature for 4 h. After removing the fixation solution with a pipette, the samples were cleaned thrice at 30 min intervals with 0.05 M cacodylate buffer, followed by dehydration by successive immersion in 30%, 50%, 70%, 90%, and 95% ethyl alcohol, and twice in 100% ethyl alcohol, changing every 30 min. The samples were then immersed in mixtures of 100% ethyl alcohol and LR white resin (polar monomer polyhydroxylated acromatic acrylic resin) in the ratio of 2:1, 1:1, and 1:2, substituted every 6 h, followed by resin permeation. After two rounds of substitution with 100% LR white resin, the samples along with LR White resin were placed into gelatin capsules, which were sealed and hardened at 60 °C for 24 h. An ultramicrotome equipped with a glass cutter was used to obtain 1–2 µm thick sections, which were placed on a glass slide and dried at 75 °C for fixing the samples. The fixed samples were stained using 0.5% toluidine blue O and observed under a light microscope (Zeiss Smartzoom 5, Jena, Germany).

2.3. Three-Dimensional X-ray Imaging System Analysis

The morphological changes in leaf shapes were determined by using a 3D X-ray system. For sample analysis, the following conditions are set: voltage, 30 kV; power, 2 W; optical magnification, 4×; exposure time, 18 s; and pixel size, 1.7 µm. The 3D image system (Versa 520, Carl Zeiss, Oberkochen, Baden-Württemberg, Germany) equipped with a luminous source was used for determining structural changes without any sample destruction.

2.4. Field Emission Scanning Electron Microscopy (FE-SEM) Analysis

The samples were placed into a microcentrifuge tube (Eppendorf, Hamburg, Germany) containing 1 mL of fixing solution, and a vacuum was applied for 30 min. The samples were kept at room temperature for 4 h for fixation. Further, the fixing solution was removed, and the samples were washed every 20 min thrice with 0.05 M cacodylate buffer. Next, the samples were fixed with 1% OsO₄ for 1 h at room temperature and then cleaned every 20 min thrice with 0.05 M cacodylate buffer pH 7.2. The fixing solution was removed again, and the sample was dehydrated by sequential immersion every 20 min in 30%, 50%, 70%, 90%, 95%, and 100% ethanol. After critical point drying and platinum coating, the surface structures of the sample leaves were observed under a scanning electron microscope (Helios G4 PFIB CXe DualBeam, FEI company, Brno, Czech Republic) with a 1–3 kV acceleration voltage.

2.5. Micro-Computational Tomography (CT) Analysis

To show the changes in leaf shapes, micro-CT was used. With the plant sample fitting volume-of-interest (VOI) secured, the dried samples were placed on a sample plate, and the moisturized sample was placed inside a coating film. Both were treated by heating, sealing, and fixing. The Skyscan1272 (CT Analyzer, Bruker, Kontich, Belgium) program was used to set the subsequent analysis conditions, such as resolution with X-ray beams and VOI screen monitoring, as follows: Amount of beamed X-ray, 40 kV (dry) and 50 kV (moisturized) per 200 µA; camera binning, 1 × 1 (4032 × 2688); pixel size, 6.7 µm (dry) and 6.24 µm (moisturized); rotation step, 0.3 degrees. After scanning (dry sample 1 h:16 min:46 s, moisturized sample 1 h:16 min:32 s), the NRecon program was used to reconstruct the scanned image into the tomographic image. Using CTvox, the tomographic images were layered to generate a 3D view, followed by a video recording.

2.6. Biomimetic Application

To develop an environmentally friendly humidifier with a high potential for non-toxic materials, minimal energy usage, and a design that mimics the opening and closing behavior of plants, we conducted finite element analysis to assess the deformation tendencies of wood samples. Cypress wood was chosen for the experiment as it has phytoncide and is water-resistant, which helps prevent decay [22]. Additionally, its hygroscopic swelling rate is higher than that of any hard wood, making it suitable for studying the bending behavior of wood when exposed to moisture [23]. The cypress wood was prepared in the same size with a 300 mm width, 12 mm length, and 7 mm thickness. To measure the differences in bending behavior, two dissimilar materials were attached to each face of cypress wood. To practically implement a model with humidifying functions (expansion and contraction) using wood, superglue was used, as it adheres well to both wood and selected materials. For added strength in the presence of moisture, epoxy-superglue was used on one side, and only superglue was used on the other (Figure 1A). The dissimilar materials were aluminum and Acrylonitrile Butadiene Styrene (ABS) sheets, which were joined to the cypress wood using epoxy-superglue (Figure 1B). The epoxy-superglue is non-toxic when fully cured and ensures that the materials remain securely attached even after prolonged exposure to water.

3. Results and Discussion

3.1. Internal Structure of Humidity-Sensing Plants

Cross-sections of plant leaves were observed under a light microscope to determine their internal structures and the unique features of tissues related to water absorption (Figure 2A). Structurally, the epidermis was covered by a cuticle layer on the leaf surface, which facilitates survival when the external environment becomes dry; the epidermal layer was developed under the cuticle layer [24,25]. The internal structure of S. involvens was composed of xylem, phloem, and trabeculae inside a vein [26]. The interiors of S. rossii and P. lingua showed xylem and phloem surrounded by a bundle sheath. Further, the empty space at the center of the leaves was found to be air space. The structure of the internal tissue of the leaf was altered very distinctly depending on the humidity conditions. In particular, xylem enlarged markedly when moisture was absorbed. Thus, xylem and trabeculae were the main reasons underlying the change in the external shape of plants, depending on the amount of moisture in the surrounding environment. Regarding the internal structure of moss, the leaf midrib comprises irregular long squares (or hexagons) in the center (Figure 2B). The inside of the moss leaf is close to the stem, and sclerenchyma tissue development was determined to play a supporting role. Moss leaves are closely attached to the main stem and are overlapped, and short protrusions densely form on the surface. However, owing to the ambiguity of the tissue position, the names of the tissues are not accurately indicated in the data.

The internal and surface structures of leaves, such as cell density and stoma, were observed using FE-SEM. When the environmental conditions were dry and moist, the stoma on S. involvens leaves closed and opened, respectively (Figure 3A).

In S. rossii, leaves stuck closely to the stems in dry conditions, whereas under moist conditions, leaves were away from the stems (Figure 3B). Further, the xylem size of the stem was dependent on the water conditions. The xylem under wet conditions showed a dense structure, whereas the xylem under dry conditions was loose. Although this study mainly focused on xylem tissue, the plant cell walls are known to expand and loosen under wet and dry conditions, respectively [27]. A clear change in the leaf surface of P. lingua was observed depending on humidity and dryness (Figure 3C). To determine the structural specificity of moss leaves (Figure 3D), the density and surface structures of moss leaves were analyzed; contaminants such as moisture and dust were determined to be adsorbed and easily removed, respectively, because of the surface tension of numerous microscopic protrusions.

3.2. Leaf Morphological Changes

To analyze the leaf movements of humidity-sensing plants, all of which change shape upon absorbing moisture, the leaves were examined using micro-CT (Figure 4A). Under dry conditions, the leaf bases of plants begin to change first. The upper tip of the leaf in S. involvens curled inward; the leaf of S. rossii showed a certain dry aspect when dehydration occurred widely from the leaf; and the whole leaf of P. lingua was found to curl both lengthwise and breadthwise. However, upon absorbing moisture, the leaves of all three plants uncurled. In this study, we focused on leaf-external changes rather than on how long the leaf curls and uncurls in dry and moist conditions because part of a leaf was used as the experimental material. When the leaves of S. involvens and S. rossii curled toward the central axis (stem) under dry conditions, the lower part spread further than the upper part of the leaves. P. lingua does not show foliate opening and closing depending on humidity, but its leaves curl up under dry conditions; therefore, the change in curling and uncurling length depending on humidity could not be measured. In other words, the degree of leaf curling or rolling under dry conditions was subject to the extent of leaf attachment to the stem and the changes in the leaf surface structure depending on humidity.

To comprehend the morphological changes in leaf shape depending on humidity, a 3D x-ray imaging system was used. The length of the cross-section was measured when the leaf curled and uncurled according to the external dry and moist environments, respectively. This experiment was performed only with S. involvens, which clearly showed changes in external shapes according to relative humidity. The average length of the leaf cross-section in S. involvens was measured to be approximately 1256–1786 µm under dry conditions and approximately 1825–2311 µm under moist conditions, clearly demonstrating changes of at least over 68–77% in leaf size because of the opening-and-closing mechanism (curling-and-uncurling).

As the leaflets are moved by large vibrations (wavelength) rather than external sound, the relevant structural changes were observed (Figure 4B). The leaflets at the base of the telegraph tree responded to vibration and moved spirally, straightened out, and then rotated. Other than leaflets, no other tissue responded to vibration.

3.3. Biomimetic Application

A biomimetic approach could also be used to create designs that are connected to natural models by mimicking the integrated aspects of biological forms, functions, structures, and materials [28,29]. Wood materials were modeled for analyzing deformation tendencies in different tangents and radial directions [30,31].

The hygroscopic swelling rate, a key parameter of the material, was measured after determining how the shape of the sample glued with dissimilar materials was bent upon moisture absorption. The dissimilar materials were glued such that the glued parts would be flexible upward and downward without any longitudinal deformation. The hygroscopic swelling of wood cut in the radial and tangent directions [32] was analyzed (Figure 5A). The hygroscopic swelling analysis of the wood with thicknesses of 3 mm, 6 mm, and 15 mm revealed that the degree of deformation increased with decreasing wood thickness, resulting in a bow-like shape upon deformation. The wood thicknesses were selected based on the visually observable degree of deformation. The hygroscopic swelling rate of 3 mm thick wood was five times higher than that of 15 mm thick wood. Bending changes occur more quickly for thin layers, which leads to an additional increase in curvature amplitude [33].

Similar to radial-direction wood, a thinner tangent-direction wood was also found to undergo a larger degree of deformation with bow-shaped bending. In general, compression is required for proper bending of wood; in particular, pressure at the end of the wood is essential. This could explain why thin wood has a greater bending angle than that of thick wood [34]. The degree of bending was determined after the wood sample had absorbed sufficient moisture (Figure 5B). The tables and graphs showed that the hygroscopic swelling rates of the samples were the largest when aluminum was glued. However, because these swelling rates were not small in other samples, strength and durability were considered when choosing the desirable samples rather than the degree of deformation. When only the epoxy-superglue was applied to wood, the deformed shape caused by hygroscopic swelling was restored to its original shape even after being wet for a long time, possibly because the absorption of water into the epoxy-superglue causes expansion. ABS superglue tends to permeate deeply into wood and harden. As the hygroscopic swelling rate inside wood changes upon superglue permeation into it, the ABS superglue was considered an appropriate material.

In contrast, UV superglue lost its adhesiveness upon repeated hygroscopic swelling and contraction (restoration). We found that upon gluing two kinds of wood with their annual rings crossed with each other and only applying the wooden material to a humidifier, bending like a bow was observed as if dissimilar materials were glued to wood. However, controlling the changeability of shape caused by hygroscopic swelling was found to be difficult due to complex folding in different homogeneous [35] materials. As dissimilar materials glued to the wood have some uniformity, they have the advantage of allowing controlled hygroscopic swelling.

Finally, the wood glued with dissimilar materials was examined. Although an aluminum sheet could be glued to cypress wood, metal was considered undesirable as it showed extreme swelling and contraction upon temperature changes. Considering the uniformity of the wood sample deformation, deformation controllability, and commercial value, ABS plastic was considered the most appropriate material for gluing on wood. This environment-friendly material is also used in children’s toys, is very durable, and is easy to process. Polyoxymethylene (POM) can also be considered even though it was not tested, as it is stiff, has less flexibility than ABS, and can result in less bending of the sample at the time of hygroscopic swelling than that with ABS. Further, because POM is 40% heavier than ABS, it can increase the sample weight.

Based on the experimental results obtained in this study, we have conceived an initial model for a user-friendly humidifier that can be easily used in everyday life, approaching more practical applications. The wings of the pinecone humidifier performed the main humidifying function. Unlike other humidifiers, this operates based on the humidity of the surrounding environment, eliminating the need for additional energy consumption and thus offering the potential for energy savings. Of course, the engineering application and commercialization of a humidifier incorporating the characteristics of pinecone scales may pose challenges. However, in this study, we proposed a pinecone humidifier model based on fundamental research data on plant moisture absorption, expansion, and related factors. As an initial humidifier model with an opening-closing mechanism, we present the model of an environment-friendly humidifier applying the principle of a pinecone opening and closing based on humidity (Figure 6). The wing of the pinecone humidifier was separated into a straight-shaped upper part (A) and an oval-shaped lower part (B). The wings mimicked the scales of a pinecone. The upper part was divided into two parts so that different fabrics could be examined. For the upper part of the wing, which absorbs and emits water to regulate the surrounding humidity, fabric materials were employed. For the lower part, a sponge material that undergoes volume changes owing to the surrounding humidity was used so that the opening-closing mechanisms could be achieved. The central part of column (C), as the body of the humidifier, functioned to replenish water. A hard-frame plastic material was employed as the encircling part. The two passages inside the column, which allow the water to flow, result in wing wetting for the evaporation of water. In future biomimetic applications, extracting the characteristics of moss structure with super hydrophobicity and the movement of the telegraph tree in response to external sound could also be valuable. The results of this study demonstrated the potential for an efficient mechanical model that responds to humidity by mimicking plant characteristics, thereby not using electrical energy and contributing to energy savings. The data from the results could serve as foundational information for future applied research aimed at developing environmentally friendly, reversible, and high-performance biomimetic environmental-sensing sensors, especially in humid environments.

4. Conclusions

The hygroscopic behavior of plants is based on a unique mechanism that manifests as a change in internal structure in response to changes in relative humidity. Recognizing the potential for engineering applications based on this unique property of plants, the structural principles and behavior of humidity-sensing plants in response to changes in humidity conditions were studied. By utilizing the properties of pinecones, a finite element method was used to study the deformation characteristics of wood during hygroscopic swelling. Based on this principle, we developed a prototype model for a natural humidifier made from cypress wood, which is safe for human use. This study highlights the potential for developing environment-friendly and energy-efficient technologies based on the principle of biological characteristics and applied engineering research. This study also shows the importance of continued research into the biological and ecological characteristics of different plant species, which can help to further establish the physical and structural principles of plants with opening-and-closing mechanisms in response to external humidity.