Optical properties of biosilicas in rice plants

Abstract

Although biogenic amorphous silicas (biosilicas) accumulated in rice plants play many important roles, such as enhancing mechanical strength, preventing disease, feeding damage, and encouraging photosynthesis, details of their properties have not been studied with an engineered approach. In the present work, we investigated the optical properties of two kinds of biosilicas in matured rice plants: silicified long cells, called “silica plates”, that cover the surfaces of leaf blades, and fan-shaped silicas aligned inside the blades. The path of light in the biosilicas was analyzed using two-dimensional optical simulation and actual optical experiments. We found that silica plates that have micrometric projections on their surfaces diffuse visible light effectively in the leaf blade, and fan-shaped silicas have a role in guiding light to chloroplasts. Thus, biosilicas would act as a solar diffuser panel and a window for promoting photosynthesis.

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1. Introduction

In evolutionary history, organisms have developed inorganic materials that have a wide variety of functions that require minimal resources and energy.¹ Biogenic inorganic materials, called biominerals, are mainly composed of calcium carbonate (CaCO₃),² hydroxyapatite (HAp),³ and silica (SiO₂).⁴ Their hierarchical architectures, organized from nanoscale to macroscale, contribute to the strengthening and toughening of the bodies.^5–9 Thus, many studies have been conducted on the relationship between the structures and mechanical properties of various biominerals.

Recently, excellent optical features of several kinds of biominerals, as well as their protective functions, have been revealed. Microlenses consisting of a microarray of single crystalline calcite in a brittle star exhibit specific optical properties, such as guiding and focusing light within the tissue.¹⁰ Plants more closely resemble light harvesters than any other living organism. However, a limited number of studies have been conducted on the optical properties of botanical minerals. Cystoliths, which are amorphous calcium carbonate and calcium oxalate druse crystals in the leaves of a banyan tree, were reported to possess a light-scattering effect.¹¹ Unfortunately, the optical roles of biominerals in most higher plants have not yet been revealed. Although biosilicas in rice plants were suggested to act as a window for light harvesting,¹² their detailed properties as an optical material have not yet been studied with an engineered approach. In the present work, we conducted optical analyses of biosilicas in mature rice plants with the aim of showing their roles in light harvesting.

In terms of optical materials, amorphous silica or silica glass is especially important due to its high transparency and high durability and is widely used in industrial products, such as optical fibers, windows, and lenses. Biogenic amorphous silicas (biosilicas) are produced under ambient conditions by diatoms,^13–18 marine sponges,^19–27 and some higher plants^28–36 using organic molecules, such as silaffin,^17,18,37 silicatein,^27,38,39 and long-chain polyamines.^40,41 The formation mechanisms of biosilicas were studied by mimicking the biogenic processes for environmentally friendly silica production.⁴² The optical properties were reported for several kinds of biosilicas. The silica frustule of a diatom focuses light on a focal plane in the cytoplasm under varying light conditions.¹³ Siliceous spicules from marine sponges show light-guiding properties similar to those of artificial optical fibers.²³ Several kinds of higher plants, such as bamboos,³⁰ equisetums,^29,35 and rice plants,^32,33 produce biosilicas called plant opal. Plant opals are formed by the concentration and polymerization of silicic acid which is taken up by roots from soil.⁴⁵ Although most part of the mechanisms for the formation of biosilicas has yet to be revealed, some sort of organic molecules might be concerned in biosilica formation in rice plants as well as diatoms and marine sponges. Rice plants accumulate a large amount of amorphous silica (20–30 wt%) on the surfaces of and inside leaf blades, stems, and husks.³³ Various shapes, such as plates, fans, prickle hairs, and dumbbells, were observed on biosilicas in rice plants.³⁴ In the plant kingdom, where the process of effectively collecting light for photosynthesis is an exceedingly important topic, biosilicas should play important roles in the light harvesting of plants. As a matter of fact, the content of silica in soil for cultivation affects the actual growth of rice plants.^31,32 Moreover, the photosynthetic rate is significantly increased with increasing silicon contents of rice leaves.⁴⁶ However, the optical properties of biosilicas in rice plants with their precise structures have hardly been studied.

In this article, we show the micrometric structures and optical properties of biosilicas in rice plants (Oryza sativa L.). Here, we especially focus on the main two species contained in mature leaf blades: one is the silicified long cell, called silica plate, that covers most of the surfaces of leaf blades, and another is the silicified bulliform cell, called fan-shaped silica, that is aligned inside leaf blades like a backbone.⁴² Analyses by two-dimensional optical simulation and actual optical experiments suggest that silica plates that have micrometric projections on their surface effectively diffuse visible light in leaf blades. Our results suggest that leaf blades should be installed with solar diffuser panels and transparent windows of biogenic amorphous silica to promote photosynthesis.

2. Experimental

2.1. Structural characterization of biosilicas in rice plants

Leaf blades of rice plants (Oryza sativa L.) were collected from paddy fields in Akita, Japan. Leaf blades were calcined at 300–500 °C for 3 h in air to remove organic materials. The morphologies of biosilicas were investigated using scanning electron microscopes (SEM, Hitachi S-4700, FEI-SIRON, JEOL JSM-7600F) at an accelerating voltage of 5 kV and an optical microscope (Keyence VH-Z500R). The distribution of chloroplasts was visualized using a fluorescence microscope (Olympus BX51) with an excitation filter (Olympus U-MWU2). Elemental analysis was performed using energy dispersive spectroscopy (EDS, FEI-SIRON) operated at 20 kV, and fluorescent X-ray analysis (Horiba XGT-2700) was conducted at 15 kV.

2.2. Optical analyses of biosilicas

2.2.1. Numerical simulations. Two-dimensional (2D) numerical simulations based on the beam propagation method (BPM) were performed to analyze the path of light in leaf blades using computer software (BeamPROP, RSoft). The simulation principle was explained in Ferrara et al.¹³ Refractive index maps of the objects used for simulation were built using computer-aided design (CAD) software (RSoft CAD) based on the morphological observation of a leaf blade. Amplitude maps of light through biosilicas were obtained after the simulation.

2.2.2. Transmission measurements. The beam divergence of light that passed through the real silica plates, which were extracted from a leaf blade, was retrieved by means of the experimental set-up described in detail in Fig. 1. The silica plates were tentatively fixed on a silica glass slide with a drop of pure water. Partially coherent radiation of 853.2 nm light was given by a light emission diode (LED) (Precise Gauges LDS1007) through a 105 μm core-sized optical fiber (Thorlabs FG105LCA, NA = 0.22 ± 0.02). The transmitted light was analyzed as the angular dependence of the intensity with the far-field pattern (FFP) measuring system (Hamamatsu A3267-12).

Fig. 1 A schematic illustration of the experimental optical set-up. Silica plates extracted from a leaf blade were fixed on a silica glass slide.

3. Results and discussion

3.1. Morphology of biosilicas in rice plants

The shape of an Oryza sativa L. rice leaf blade is shown in Fig. 2. Most of the surfaces of the leaf blade were covered with thin silica layers ∼1 μm thick, called silica plates, which had many micrometric projections ∼2–3 μm in diameter (Fig. 2b and c). The thin plates were interdigitated with each other like a tortoiseshell (Fig. 2c). From enlarged SEM images, we found 50–100 nm particles to be biosilica building blocks (Fig. 2d and g). The top layer and round projections were composed of tightly packed particles (Fig. 2e and f). On the other hand, loosely aggregated particles were observed on the side and back surfaces (Fig. 2d and g). Since fibrous matter was observed around the particles before calcination (Fig. S1, ESI†), organic components, such as cellulose, are inferred to fill the pores of the silica plates.

Fig. 2 Biosilicas on the surface of an Oryza sativa L. leaf blade. A photo of rice leaf blades (a), an optical micrograph of the surface (b), and SEM images of obverse surfaces (c, e and f), a side surface (d), and lower surface (g) of a leaf blade after the removal of organic components by calcination at 300 °C for 3 h. A schematic illustration of silica plates (h).

The cross section of a leaf blade is shown in Fig. 3. The presence of silica plates covering the surfaces was confirmed using EDS. We also observed a silica body with a shape like a fan, called a fan-shaped silica, inside the leaf blade (Fig. 3c and d). Fan-shaped silicas were located in a line like the backbone of the leaf blade (Fig. 3a and b), and their bottom surfaces seemed dimpled like a golf ball. From enlarged SEM images, fan-shaped silicas were found to be composed of 50–100 nm particles tightly packed in the entire body (Fig. 3e and f). Using fluorescent X-ray analysis, the silica content was estimated to be more than 99 mol%. Chloroplasts (red fluorescent parts in Fig. 3h) were distributed between nervures and around fan-shaped silicas in a horseshoe shape. The leaf blades were wholly corrugated because of the existence of nervures in regular intervals (Fig. 3g). Fig. 3i shows a schematic illustration of part of the cross section of a leaf blade.

Fig. 3 Micrographs and a schematic illustration of biosilicas inside a leaf blade. An optical micrograph of the surface of a leaf blade after calcination (a) and a schematic illustration of fan-shaped silicas embedded inside the leaf blade like a backbone (b). SEM images of the cross section of an untreated leaf blade (c), and a whole shape (d), the front surface (e), and side surface (f) of a fan-shaped silica. The distribution silicon (red parts) obtained by EDS is superimposed in (c). An optical micrograph (g), a fluorescence micrograph (h), and a schematic illustration (i) of the cross section of part of a leaf blade.

3.2. Optical analyses of biosilicas

3.2.1. Numerical simulations. The conditions of the numerical simulations for analyzing the path of light in a leaf blade are shown in Fig. 4. On the basis of the average size of biosilicas, the shapes of the fans and plates that had micrometric projections were simplified as a model system. The wavelength of 660 nm was chosen for the incident light because the maximum absorption band of chloroplasts is around 660 nm.⁴³ The incident light angle (θ in Fig. 4) was changed from 50 to 90 degrees by rotating the objects in the simulation. The widths of the incident light source were set to 10 μm in order to investigate the effects of multiple projections of the light's path in the leaf blade.

Fig. 4 A schematic illustration showing the conditions of the numerical simulation. Incident light angles (θ) were fixed at 50, 70, and 90°, and the light wavelength was set at 660 nm. The refractive index (n) of biosilicas was approximated by amorphous silicas (n = 1.46) and cellulose (n = 1.46–1.47).⁴⁴ The refractive indices inside the leaf blade were approximated by the average value of those of water and plant cells⁴³ (n = 1.40), and the other area was approximated by that of air (n = 1.0).

From SEM images, we see that biosilicas have porous structures consisting of nanoparticles (Fig. 2d and g). However, organic components, such as cellulose, exist in their pores. Therefore, the refractive index (n) of biosilicas can be estimated to be 1.46, which was approximated by amorphous silica (n = 1.46) and cellulose (n = 1.46–1.47).⁴⁴ The other refractive indices were 1.0 for air and 1.40 for the matrix of a leaf blade, which was approximated by the averages of water and plant cells.⁴³ We confirmed that the results were not essentially changed when the refractive indices of biosilicas and the matrix were varied from 1.42–1.50 and 1.33–1.46, respectively (Fig. S2†). Thus, the estimation of the refractive indices in our simulation conditions was not a serious parameter. The refractive index map for the cross section of a leaf blade for the simulation is shown in Fig. 5a.

Fig. 5 Refractive index (n) map used to simulate the path of light through the silica plates (a). Amplitude maps of light entering the silica plates at an angle of 90° (b–d). A schematic illustration of light diffusion in the leaf (e).

Panels b–d in Fig. 5 show the amplitude maps of the light that entered the silica plates at an angle of 90° and passed through them. Light that passed through silica plates was found to be extensively diffused for light harvesting by chloroplasts, as shown in Fig. 5e. We conducted the simulation by changing the incident angle, as shown in Fig. 6a. The diffusion of the light was not influenced by changing the angle of the incident light. Therefore, micrometric projections on the silica plates play a role of diffusing light from all angles.

Fig. 6 Refractive index (n) map used to simulate the path of light through the silica plates (a). Amplitude maps of light entering the silica plates at an angle of 50° (b–d). A schematic illustration of light diffusion in the leaf (e).

Fan-shaped silicas (Fig. 3) are formed as the leaves become mature. We performed the simulation in the presence of fan-shaped silicas in a mature leaf blade. As shown in Fig. 7, light that passed through both silica plates and fan-shaped silicas were diffused at a level comparable to a case with only silica plates (Fig. 5b). Since there is little difference between their refractive indices, the light-diffusing effects of the fan-shaped silicas were hardly observed (Fig. 7b).

Fig. 7 Refractive index (n) map used to simulate the path of light through the silica plates with fan-shaped silicas (a). Amplitude map of light entering both silica plates and fan-shaped silicas at 90° angles (b).

3.2.2. Transmission measurements. We conducted actual optical experiments, in which the intensities of light passing through real silica plates were measured, to evaluate the appropriateness of the numerical simulation. We chose 105 μm instead of 10 μm for the beam diameter in order to increase the detection sensitivity. Maps of the intensity of light that has passed through a silica glass slide without any silica plates and with silica plates are shown in Fig. 8. The round shape in Fig. 8a corresponds to the light radiated from the optical fiber, whose core diameter was 105 μm. The larger and darker circular form in Fig. 8b indicates that light is diffused by passing through silica plates on the glass slide. Comparing the profiles of the intensity distribution in the angles of light that has passed through the silica glass slide without any silica plates (blue dashed curve A) and with silica plates (red solid curve B) in Fig. 8c, light that passed through silica plates attached to the glass slide was definitely diffused.

Fig. 8 Angular intensity distributions of light that passed through the glass slide without any silica plates (a), with silica plates (b), and intensity profiles of X and Y axes of (a) (blue dashed curve A) and (b) (red solid curve B) (c). Simulated amplitude map in a case with a number of projections, and the light source is 105 μm (d); the profile of the light amplitude at the entrance (Z = 0) and at a point 200 μm distant from the entrance (Z = 200) (e).

We performed the numerical simulation by enlarging the diameter of the incident light beam to 105 μm in order to analyze the consistency of the optical experiment and the simulation. In Fig. 8d, we observed that light was diffused by a number of projections. From the profile of the light amplitude (Fig. 8e), it was confirmed that light was more diffused at a point 200 μm distant from the entrance (Z = 200 in Fig. 8d) than at the entrance (Z = 0 in Fig. 8d). Hence, the diffusing effect of a number of projections of silica plates was certified by both the numerical simulations and actual optical experiments.

3.2.3. Roles of biosilicas. Silica plates that have many micrometric projections cover most of the surfaces of the leaf blade and play a role in effectively diffusing light.

Although fan-shaped silicas are considered to have less effect on diffusing light, they have such high light permeability that they play a role as a window for light. As shown in panels (g) and (h) in Fig. 3, it is possible to see through fan-shaped silicas regardless of the leaf's thickness (∼1 mm). Here we considered the window effect of fan-shaped silicas. If there were no fan-shaped silicas, light could not reach the bottom of the leaf blades (Fig. 9a); or, if the fan-shaped silicas were replaced by water and plant cells, the transmission of incoming light would be disturbed by scattering and absorption (Fig. 9b). Therefore, fan-shaped silicas with high permeability can be considered to play a role in delivering light diffused by silica plates to chloroplasts without damping (Fig. 9c).

Fig. 9 Schematic illustrations indicating the path of light in the leaf blade in the case without fan-shaped silicas (a and b) and the case with fan-shaped silicas (c).

In this study, we revealed the optical properties of two kinds of biosilicas in a mature rice plants: silica plates and fan-shaped silicas. Sunlight coming into the leaves is diffused by a number of micrometric projections on the thin silica plates and then is delivered to chloroplasts by fan-shaped silicas without loss through scattering and absorption (Fig. 9c). Also, the distribution of chloroplasts makes it possible to use light delivered by biosilicas more effectively. Since chloroplasts are not distributed in a plane but in a horseshoe shape, the number of chloroplasts that can absorb light increases. Therefore, this most effective light use promotes photosynthesis in rice plants. In order to confirm the actual morphological effects of biosilicas on rice plant photosynthesis, biological substantive experiments are needed in the future.

4. Conclusion

The microstructures and optical properties of biosilicas in rice plants were investigated. Both numerical simulations and transmission measurements confirmed that silica plates with a number of micrometric projections play a role in diffusing light. Fan-shaped silicas were considered to effectively deliver light to chloroplasts. These results indicate that silica plates as light diffusers and fan-shaped silicas as windows should promote photosynthesis by delivering light into the leaf blade.

Acknowledgements

This work was supported by Grant-in-Aid for Scientific Research (A) (16H02398) from Japan Society for the Promotion of Science.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra24449a

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