Abstract
Silica cells are specialized leaf epidermal cells in grasses with almost the whole cell volume filled with solid silica. In sorghum, silica deposition in silica cells takes place in young, elongating leaves around the mid-length of the leaf. We developed a protocol for estimating the level of silica cell silicification in Sorghum bicolor leaves using in situ charring method (Kumar et al., 2017a). Here, we provide greater details on our protocol and method of image analysis. Although we based our protocol on sorghum, this protocol can be extended for estimating silica cell silicification level in any grass species.
Keywords: Silicification, Silica cells, Grasses, Sorghum bicolor, Spodogram, Light microscope
Background
Silica deposition is central to grasses. Grasses deposit up to 10% of their dry weight as silica. The major sites of silica deposition in plants are root endodermal cells, abaxial epidermal cells of inflorescence bracts and silica cells in leaves (Kumar et al., 2017b). Almost the entire volume of silica cells is filled with solid, amorphous silica. Silica deposition in silica cells is physiologically controlled and takes place around the middle length of young leaves (Kumar et al., 2017a), which we named leaf-2 (Figure 1). Silica cell silicification is also a fast process completed within hours (Kumar and Elbaum, 2017), hence in the same leaf there are areas of high and low silicification intensity depending upon which area of leaf we are examining. To study the deposition process, we need a way to quantify the silica cell silicification level in different parts of the same leaf. In situ charring or spodogram preparation of plant material is an easy and cheap way to study silica deposition in plants. The plant material is burnt at temperatures typically above 500 °C for 3 h to overnight that oxidises all of the organic material. The ash remaining after the charring process contains silica and other minerals. The non-silicate minerals from the ash are dissolved by 1 N HCl and the remaining insoluble substance is silica. The in situ charring method can also be used to quantify the silica cell silicification level (Kumar et al., 2017a). The leaf piece is kept in between two glass slides to keep the leaf specimen flat (Figure 2), and then the specimen is burnt, ash washed with HCl (Figure 3) and subsequently with double distilled water to remove the mineral salts. The slide is then taken to a light microscope to count the number of silica cells silicified per unit length. Figure 4 shows a spodogram prepared from the middle of a young leaf. This part was analyzed to quantify silica deposition levels. Our method can be extended to quantify the silica cell silicification level in any leaf piece, but best results are obtained with young and still silicifying leaves.
Materials and Reagents
Equipment
Procedure
Data analysis
Numbers of silicified silica cells are counted in each row of an image and only the row with the highest number of silicified silica cells is considered for data analysis. In the beginning of silica cell silicification process, only the boundaries of silica cells look silicified and in the spodogram only the cell boundary is lightly visible. Those cells are also counted as silicified cells. Sometimes, in the more mature segment of leaf-2, many silica cells join together and form of polylobate bodies. In this case, three lobes are regarded as one silica cell. We show our method with an example (Figure 4) below. In this spodogram image, there are three rows of silica cells, the top row (row-1) with 8 silica cells, the row in the middle (row-2) with 7 silica cells, whereas the row in the bottom (row-3) has 13 silica cells. Hence, the silicification score of this image is 13. The silicification score from each of the ten sequential images of a sample was averaged. The average number becomes the silica cells silicification status of that particular segment. Silicification scores of different segments are calculated and compared. Figure 4. A spodogram image under 20x magnification. Silica cells are encircled with dotted ovals. Scale bar = 100 µm.
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Acknowledgments
SK is thankful to the Planning and Budgeting Committee, Council of Higher Education, Israel for a post-doctoral fellowship. The research was supported by Israel Science Foundation grant 534/14. The authors declare that any conflict of interest or competing interests do not exist.
References
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