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Estimation of Silica Cell Silicification Level in Grass Leaves Using in situ Charring Method
使用原位炭化法估算草叶中二氧化硅细胞的硅化水平   

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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(硅化), Silica cells(二氧化硅细胞), Grasses(草), Sorghum bicolor(高粱), Spodogram(灰象), Light microscope(光学显微镜)

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

  1. Glass slides
  2. Paper towel
  3. Aluminium foil
  4. Plastic disposable droppers
  5. Scalpel
  6. Ceramic tile
  7. Young Sorghum bicolor plants
  8. Double distilled water
  9. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
  10. 1 N Hydrochloric acid (HCl) (see Recipes)

Equipment

  1. Scalpel
  2. Forceps
  3. Muffle furnace (Alfi Laboratory supplies)
  4. Binocular microscope (Motic, model: SMZ168 Series )
  5. Light microscope (Nikon Instruments, model: Eclipse 80i )

Procedure

  1. Sampling
    1. Young plants (up to one month old) where silica cell silicification level needs to be estimated are brought to the lab. The length of youngest leaf visible from outside is recorded.
      Note: We name this youngest, visibly emerging leaf as leaf-2 (Kumar et al., 2017a) (Figure 1).
    2. Under a binocular microscope and using a scalpel and forceps, the older leaves are carefully removed from leaf-2. Leaf-2 is then cut into 10 equal segments, along the length of the leaf.


      Figure 1. Sketch showing a young sorghum plant. Leaf-2 is indicated with the arrow. Figure was adapted from Kumar et al. (2017a) with permission from the John Wiley and Sons publications. Original authors of the article and the publisher retain the copyright of this figure.

  2. Spodogram preparation
    1. Segments of leaf-2 are laid flat on a glass slide. Segments from the base of leaf-2 are always rolled; hence 1-2 drops of double distilled water are put on the glass slide and the segment is pressed against the drop. This way, the segment lies flat and sticks to the slide. A small piece of aluminium foil is folded 2-3 times and is kept near the two shorter sides of the slide (Figure 2). Another fresh slide is kept on top of the slide holding the sample in place. This step is repeated until all the segments are put on slides. All the slides are kept on a clean ceramic tile, put in a muffle furnace and burnt at 550 °C between 3 h to overnight. The furnace is turned off and allowed to cool down to room temperature which usually takes more than 12 h.
      Note: The aluminium foil in between the two slides makes sure that the two slides do not touch each other when the tissue sample is burnt. We do not go above 580 °C, as the glass slides may crack, distort, melt or stick together. We use the ceramic tile that is used in flooring in houses. The ceramic tile functions as a solid base where all the slides can be put together on one base. We do not write anything on the slides with a marker pen as burning erases all the marks. Rather, before putting the samples for burning, we note down the sample names and their orientation with respect to one of the sides of the tile. Burning for three hours is sufficient for quantitative estimation of silica cell silicification in leaves.


      Figure 2. Image showing a leaf-2 segment in between two glass slides for spodogram preparation

    2. Slides with burnt leaf material are put on paper towel without disturbing them. Using a plastic dropper, the ash is very carefully and gently washed with about 1-1.5 ml of 1 N HCl from one of the sides of the slides (Figure 3). The slides are then washed with about 1-1.5 ml of double distilled water to remove the HCl solution and solutes. The two slides are then separated and dried at room temperature.
      Note: Sample names can be written on dried slides.


      Figure 3. A burnt leaf-2 segment being washed with 1 N HCl

  3. Data acquisition
    1. After the slides dried, the bottom slides (without cover slip) are taken to the transmitted light microscope and visually scanned under low magnification (4x) to look for areas having silicified cells. The area on the slide with the longest, unbroken stretch of silica cells is identified and chosen for data acquisition. Ten sequential images are acquired following the rows of silica cells in one direction.
      Note: All the images for data analysis must be acquired using a common high magnification objective; otherwise the data will not be uniform. Images must be taken in such a way that the rows of silica cells are parallel to a side of the screen.
    2. We use 20x objective for all our images and the dimensions of all our images are 690 x 506 µm.

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.

Recipes

  1. 1 N HCl
    The strength of commercially available HCl is 12.1 N. We added 8.26 ml of commercially available HCl to 91.74 ml of double distilled water to make the final concentration of HCl to 1 N

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

  1. Kumar, S. and Elbaum, R. (2017). Interplay between silica deposition and viability during the life span of sorghum silica cells. New Phytol.
  2. Kumar, S., Milstein, Y., Brami, Y., Elbaum, M. and Elbaum, R. (2017a). Mechanism of silica deposition in sorghum silica cells. New Phytol 213(2): 791-798.
  3. Kumar, S., Soukup, M. and Elbaum, R. (2017b). Silicification in grasses: Variation between different cell types. Front Plant Sci 8: 438.

简介

二氧化硅细胞是草中特化的叶表皮细胞,几乎全部细胞体积充满固体二氧化硅。 在高粱中,二氧化硅细胞中的二氧化硅沉积发生在叶子中部周围的年轻的伸长叶片中。 我们开发了使用原位炭化法(Kumar等人,2017a)在高粱叶中估计二氧化硅细胞硅化水平的方案,。 在这里,我们提供了更多关于我们的协议和图像分析方法的细节。 虽然我们根据我们的高粱协议,这个协议可以扩展到估计任何草种的硅藻细胞硅化水平。
【背景】二氧化硅沉积是草的中心。禾草以二氧化硅的形式沉积至其干重的10%。植物中二氧化硅沉积的主要部位是根内胚层细胞,花序苞片的背面表皮细胞和叶中的二氧化硅细胞(Kumar等人,2017b)。几乎整个二氧化硅电池体积都填充有固体无定形二氧化硅。二氧化硅细胞中的二氧化硅沉积是生理控制的,发生在幼叶的中间长度周围(Kumar等人,2017a),我们将其命名为leaf-2(图1)。二氧化硅细胞硅化也是几个小时内完成的一个快速过程(Kumar and Elbaum,2017),因此在同一叶片中,硅化强度高低的区域取决于我们正在检查的叶片的哪个区域。为了研究沉积过程,我们需要一种方法来量化同一片叶子不同部位的二氧化硅细胞硅化水平。炭化或植物原料的原位制备是研究植物中二氧化硅沉积的简单而廉价的方法。植物材料通常在500℃以上的温度下燃烧3小时至过夜,氧化所有的有机材料。炭化过程后剩余的灰分含有二氧化硅和其他矿物质。来自灰的非硅酸盐矿物被1N HCl溶解,而剩余的不溶物质是二氧化硅。原位炭化法也可以用于量化硅石硅化物的水平(Kumar等人,2017a)。将叶片保持在两片载玻片之间以保持叶片样品平整(图2),然后将样品燃烧,用HCl(图3)和随后的双蒸水除去矿物盐。然后将该载玻片放入光学显微镜以计算每单位长度硅化的硅石细胞的数量。图4显示了从幼叶中间制备的节点图。分析该部分以量化二氧化硅沉积水平。我们的方法可以扩展到量化任何叶片中的二氧化硅细胞硅化水平,但是获得最好的结果是年轻和仍然硅化叶片。

关键字:硅化, 二氧化硅细胞, 草, 高粱, 灰象, 光学显微镜

材料和试剂

  1. 玻璃幻灯片
  2. 纸巾
  3. 铝箔
  4. 塑料一次性滴管
  5. 手术刀
  6. 瓷砖
  7. 年轻的高粱植物
  8. 双蒸水
  9. 盐酸(HCl)(Sigma-Aldrich,目录号:258148)
  10. 1 N盐酸(HCl)(见食谱)

设备

  1. 手术刀
  2. 镊子
  3. 马弗炉(Alfi实验室用品)
  4. 双目显微镜(Motic,型号:SMZ168系列)
  5. 光学显微镜(尼康仪器,型号:Eclipse 80i)

程序

  1. 采样
    1. 将需要估计硅石硅化水平的年轻植物(达1个月大)带到实验室。记录从外面可见的最年轻叶子的长度。
      注意:我们把这个最年轻,明显出现的叶命名为leaf-2(Kumar等,2017a)(图1)。
    2. 在双目显微镜下,使用手术刀和钳子,将老叶子从叶子2上小心地取出。叶2然后沿着叶的长度切成10等份。


      图1.年轻高粱植物示意图叶子2用箭头表示。图由Kumar等人改编。 (2017a),经John Wiley and Sons出版社许可。文章的原创作者和出版商保留这个图的版权。

  2. 波形图准备
    1. 叶片2的片段平放在载玻片上。叶-2的基部段总是滚动的;因此将1-2滴双蒸水放在载玻片上并将该部分压在滴剂上。这样,该部分平坦,并坚持幻灯片。将一小块铝箔折叠2-3次,并保持在滑块的两个短边附近(图2)。将另一张新鲜的载玻片保持在载玻片的顶部,将样品固定在位。重复该步骤,直到所有的片段放在幻灯片上。所有的幻灯片保存在干净的瓷砖上,放入马弗炉中,在550℃下烧结3小时至过夜。关闭炉子并使其冷却至通常需要12小时以上的室温。
      注意:两张玻片之间的铝箔确保两张玻片在组织样本燃烧时不会相互接触。我们不会超过580°C,因为载玻片可能会开裂,变形,熔化或粘在一起。我们使用在房屋地板上使用的瓷砖。瓷砖作为一个坚实的基地,所有的幻灯片可以在一个基地放在一起。我们不会用记号笔在幻灯片上写任何东西,因为刻录会擦除所有标记。相反,在放样品燃烧之前,我们记下样品的名称及其相对于瓷砖的一侧的方向。燃烧三个小时足以定量评估叶子中的二氧化硅细胞硅化。


      图2.显示两个载玻片之间用于提供散点图的叶片2片段的图像

    2. 带有烧焦的叶子材料的幻灯片放在纸巾上,不会打扰他们。使用塑料滴管,非常小心地轻轻地从玻片的一侧(图3)用约1-1.5ml的1N HCl洗涤灰。然后用约1-1.5ml双蒸水洗涤载玻片以除去HCl溶液和溶质。然后将两张玻片在室温下分离并干燥。
      注意:样品名称可以写在干燥的幻灯片上。


      图3.用1N HCl洗涤烧过的叶片2片段

  3. 数据采集
    1. 载玻片干燥后,将底部载玻片(没有盖玻片)取到透射光显微镜,并在低倍放大(4倍)下目视扫描以寻找具有硅化细胞的区域。确定并选择载玻片上具有最长,不间断的二氧化硅细胞的区域用于数据采集。在一个方向上的二氧化硅电池行之后获得十个连续的图像。
      注意:所有用于数据分析的图像必须使用一个普通的高倍率目标来获取;否则数据将不统一。必须以这样的方式拍摄图像,即二氧化硅电池的行与屏幕的一侧平行。
    2. 我们对所有图像使用20x物镜,所有图像的尺寸均为690 x 506μm。

数据分析

在图像的每一行中计数硅化硅石细胞的数量,仅考虑硅化硅石细胞数量最多的行进行数据分析。在二氧化硅细胞硅化过程开始时,只有二氧化硅细胞的边界看起来被硅化,并且在细胞图中只有细胞边界是轻微可见的。那些细胞也被算作硅化细胞。有时,在更为成熟的leaf-2片段中,许多二氧化硅细胞连接在一起形成多聚体。在这种情况下,三个叶片被认为是一个二氧化硅单元。我们用下面的例子来展示我们的方法(图4)。在这张人形图中,有三行二氧化硅细胞,上排(第一排)有八个二氧化硅细胞,中间一排(第二排)有七个二氧化硅细胞,而下排(排 - 3)有13个硅石电池。因此,该图像的硅化分数为13.对来自样品的十个连续图像中的每一个的硅化分数进行平均。平均数量成为该特定段的硅石细胞硅化状态。计算并比较不同部分的硅化分数。


图4.放大20倍的情况下的图像。二氧化硅细胞被虚线椭圆包围。比例尺=100μm。

食谱

  1. 1 N HCl
    市售HCl的强度为12.1N。我们将8.26ml市售HCl加入到91.74ml双蒸水中,使HCl的终浓度为1N。

致谢

SK感谢以色列高等教育委员会规划和预算委员会的博士后奖学金。这项研究得到了以色列科学基金会534/14号的资助。作者声明,任何利益冲突或利益冲突都不存在。

参考

  1. Kumar,S.和Elbaum,R.(2017)。 高岭石细胞寿命期间二氧化硅沉积与生存能力之间的相互作用 新Phytol 。
  2. Kumar,S.,Milstein,Y.,Brami,Y.,Elbaum,M。和Elbaum,R.(2017a)。 二氧化硅在高粱硅质细胞中的沉积机制 New Phytol 213(2):791-798。
  3. Kumar,S.,Soukup,M.和Elbaum,R.(2017b)。 禾草中的硅化:不同细胞类型之间的变化 <前期植物科学< / em> 8:438。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kumar, S. and Elbaum, R. (2017). Estimation of Silica Cell Silicification Level in Grass Leaves Using in situ Charring Method. Bio-protocol 7(22): e2607. DOI: 10.21769/BioProtoc.2607.
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