A novel method to characterize silica bodies in grasses
© Dabney III et al. 2016
Received: 30 September 2015
Accepted: 23 December 2015
Published: 21 January 2016
The deposition of silicon into epidermal cells of grass species is thought to be an important mechanism that plants use as a defense against pests and environmental stresses. There are a number of techniques available to study the size, density and distribution pattern of silica bodies in grass leaves. However, none of those techniques can provide a high-throughput analysis, especially for a great number of samples.
We developed a method utilizing the autofluorescence of silica bodies to investigate their size and distribution, along with the number of carbon inclusions within the silica bodies of perennial grass species Koeleria macrantha. Fluorescence images were analyzed by image software Adobe Photoshop CS5 or ImageJ that remarkably facilitated the quantification of silica bodies in the dry ash. We observed three types of silica bodies or silica body related mineral structures. Silica bodies were detected on both abaxial and adaxial epidermis of K. macrantha leaves, although their sizes, density, and distribution patterns were different. No auto-fluorescence was detected from carbon inclusions.
The combination of fluorescence microscopy and image processing software displayed efficient utilization in the identification and quantification of silica bodies in K. macrantha leaf tissues, which should applicable to biological, ecological and geological studies of grasses including forage, turf grasses and cereal crops.
The epidermal cells of grasses (Poaceae) are arranged in parallel rows with combinations of diverse cell types . Some of these cells are specific for silicon (Si) deposition and are called silica cells. The Silicon accumulated in the silica cells develops into the mineral structures of amorphous hydrated silica (SiO2·nH2O) having various shapes and properties called silica bodies, silica phytoliths, or plant opal [2–4].
Silica bodies are one of the most durable structures in grass tissues that remain as particles in the soil even after all other organic parts of plant have naturally decayed or degraded. These particles in the soil and ash can be very important research tools for systematic botanists [2, 5], environmental biologists , archeologists [4, 7, 8], paleontologists/paleobotanists [9–14], and geologists [15–17].
The amounts of silica in plant tissues suggest that silicon has a very important role in growth and development. For example, in rice (Oryza sativa L.), several fold more Si can be detected in shoots compared with the amounts of nitrogen, phosphorus, or potassium , reaching up to ten percent of its dry mass [19, 20].
Functional analyses of plant silica have shown that silicon is critical for mitigating stressors such as fungal infection [21, 22], herbivory [23, 24], wear [25, 26], and drought [27–30]. Mature silica bodies have been found to deter herbivory and increase the abrasiveness of grass leaf blades [31–33]. In addition, ample silica bodies have been associated with photosynthetic activities [29, 34, 35], although the mechanism for this response remains unclear .
Because we are interested in improving stress tolerance response in turf grasses, we wanted to develop a method to efficiently identify and quantify silica bodies in perennial grasses. Such a method could also be extended to other grass species, such as important forage grasses and cereals. In searching for an easy, economical, and fast method to study the morphology and distributional patterns of silica bodies in turf grasses and other plants, we found a number of available techniques. These include dry ash method, wet oxidation method, scanning electron microscopy (SEM) method, and X-ray image analysis. Among which, dry ash-imaging is one of the most commonly used methods for studying silica bodies in modern plants. To study grass leaves, ash imaging has been a method-of-choice to many researchers; however, this method is extremely labor intensive when analyzing the size, density, and distribution patterns using brightfield light microscopy and researchers have to manually measure a great number of silica bodies in order to perform a statistically meaningful analysis [2, 36]. This method can be accomplished by placing samples in porcelain crucibles and into a muffle furnace, or an oven, for 1–2 h at 500 °C, but some morphological changes might occur to certain, lightly silicified phytoliths when the temperature exceeds 600 °C [2, 4, 36, 37]. The wet oxidation method was developed to examine the isolated silica bodies and is suitable for measuring the abundance of silica bodies in plant tissues, but does not work well for analyzing the distribution patterns of silica bodies [2, 4, 38]. In comparison to the dry ash method, the wet oxidation method results in less damaged silica bodies, especially when the samples are exposed in an environment of 600 °C or higher . Due to the limitation of applying light microscopy to examine surface morphology at extra high magnification, scanning electron microscopy (SEM) can also be used to study silica bodies [40, 41]. The SEM method can be combined with X-ray analysis to provide information on surface structure and composition of silica bodies [42–44]. Here we report a method to study silica bodies using fluorescence microscopy to visualize green autofluorescence in combination with the dry ash-imaging technique. This method was developed using a perennial grass species, Koeleria macrantha, commonly known as junegrass, and has potential to be used in all fields of paleobotany and modern plant sciences on silica body research.
Results and discussion
Combination of fluorescence microscopy and image software provides an opportunity to study plant silica bodies with high efficiency
Silica bodies presented in both abaxial and adaxial epidermis of K. macrantha
We propose a method of combining fluorescence microscopy and image processing software for the quantification of silica bodies in Koeleria macrantha leaf tissue, which can be applied to biological, ecological and geological studies of grass species. We observed differences between junegrass accessions for both size and density of silica bodies in leaf epidermis. In addition, we identified differences between accessions for carbon inclusions. This study outlines a means to investigate silica bodies in grass models utilizing a novel high throughput method.
Plant material and sample collection
To examine the structure and properties of silica bodies, mature Koeleria macrantha leaf blades were collected from plants grown in the greenhouse in a 2:1 mixture of Sunshine MVP (Sungro Horticulture) and MVP:Turface (PROFILE Products LLC) substrates with no additional fertilizer. Plant material was derived from: (a) populations from the University of Minnesota turfgrass breeding program derived from material collected in either Colorado (KM-CO), Nebraska (KM-NE) or Minnesota (KM-MN) [55–57]; (b) the cultivar ‘Barkoel’; (c) several accessions from the United States Department of Agriculture National Plant Germplasm System including PI 430287 (Ireland), PI 387927 (Canada), W6 33040 (Russia Federation), PI 207489 (Afghanistan), W6 13043 (China), and PI 302912 (Spain).
Dry ash-imaging sample preparation
A microslide with the leaf samples on was then covered with another glass microslide in an effort to not disturb the placement of the leaves and to add appropriate weight to keep the ash sample intact (Fig. 6b). The slides were then heated on either a Corning Hot Plate Stirrer PC-351 (Fig. 6a) or a Tek-Pro Heat-Stir 36 H2397-1 (not shown) placed in a fume hood. The hot plate temperature was gradually increased every 5 min up to 320 °C. The temperature was approximated using an infrared thermometer Ryobi IR001 (CW0938) read at >608.2 °F/320 °C. The ash process usually took 2–3 h depending on the accession. Grass leaf samples first turned dark brown or black (Fig. 6c), and then gray (Fig. 6d) when the ashing process completed. To end the heating process, we turned off the hot plate and kept the slides on the plate for 1 h or longer to slowly cool them down. (Note: The hot plate stirrer and glass slides are extremely hot while preparing the ash samples; do not move the slides directly to a cooler place while they are still hot, which often result in broken slides.) Slow heating and cooling down prevents the microslides from cracking. For the dry ash samples preparation, we recommend to use a hot plate instead of a coiled electric stove, where the slides often break due to its uneven heating surface.
After the slides were cooled down for at least 1 h, the top microslide was then carefully removed and discarded. A 1 ml plastic transfer pipette (Fisher Scientific, Pittsburgh, PA, USA) was cut at the 0.5 ml measurement to make an ease cedar wood oil application, and a single drop of cedar wood oil (Electron Microscopy Sciences, Hatfield, PA, USA) was applied. A cover slip was then placed on the microscopy slide without disrupting the sample. Cedar wood oil was allowed to diffuse fully under the cover slip with slightly warming the slide on an alcohol burner. The slides were then imaged using an Ernst Leitz Wetzlar 307143.004 microscope (Wetzlar, Germany) and photographed with a SPOT Insight 4 Camera (Diagnostic Instruments, USA). The auto-fluorescence was detected using a Green Fluorescence Protein filter cube (SN: 31001, excitation at 480 nm, beamsplitter at 505 nm, and emission at 535 nm) that was manufactured by Chroma (Chroma Technology Corp, Bellows Falls, VT, USA). The camera interference program used to take the images was SPOT Basic v4.6. Up to 5 sets of brightfield and fluorescent images per object on the slide were taken at 200× and 800× magnification rate. Duplicate fluorescent images were analyzed using Photoshop CS5 (Adobe Systems Incorporated, San Jose, CA, USA) and/or Image J (imagej.net) for silica body occupancy rate of leaf surface (percentage of surface area), the size of silica bodies, and the pattern of silica body distribution (number of silica bodies per unit) on epidermis. Granule-like structures in silica bodies are carbon inclusions, which were counted and recorded using ImageJ, Analyzing Particles tool. The data collected from 20 silica bodies per accession, each with the number of carbon inclusions and their spatial distribution patterns, such as tightly clustered, loosely clustered, randomly distributed, or single granule.
Image and data analysis
First, a random selected fluorescence image was imported into Adobe Photoshop CS5 (Fig. 1a). Second, we used the Photoshop Magic Wand Tool to select the dark image background without silica bodies. Third, the Select-Inverse tool was utilized to select all the silica bodies (Fig. 1b). Fourth, we used the Window-Histogram function to read the pixel counts of the entire image (Fig. 1c) and the counts of selected silica bodies (Fig. 1d). To count the number of silica bodies in an image, we used the Window->Tools->Count Tool in the Photoshop CS5, the number of silica bodies was automatically shown. For those who do not have a licensed Adobe Photoshop CS5 or advanced version, the freeware ImageJ is available (http://imagej.nih.gov/ij/download.html) with tutorials videos on YouTube (youtube.com). Using the Analyzing Particles tool in imageJ we could count the number of silica bodies automatically as well. All statistically significant differences were tested at P < 0.05 level. The results were analyzed by ANOVA using R 3.1.2. . The Tukey multiple comparison test was used to test the significant differences of silica bodies among all accessions studied .
CD, EW, and CC designed the experiments and wrote the manuscript; CD and JO performed the experiments. All authors read and approved the final manuscript.
We would like to thank Dr. Stefanie Dukowic-Schulze for technical assistance, and Andrew Hollman for plant care. We deeply appreciated two reviewers’ comments, which helped us to improve the protocol significantly. This project was sponsored by funds from the United States Golf Association (2007-16-357) and the Minnesota Agricultural Experimental Station (MIN-21-041, MIN-21-031). The authors are grateful to the University of Minnesota Libraries for funds to support this open access publication.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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