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Simple Methods for Screening and Statistical Analysis of Leaf Epidermal Cells in Dicotyledonous Plants
双子叶植物中叶表皮细胞筛选和统计分析的简单方法   

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Abstract

Leaf epidermal cell size and number are positively correlated to leaf area. Stomata are specialized epidermal cells vital for gas exchange and water transpiration. So, observation and statistical analysis of the leaf epidermal cells are valuable for the study of leaf development and response to environmental stimulus. The classical method is using the scanning electron microscope (SEM), which is an expensive and time-consuming method, thus makes the large-scale screening of epidermis impractical. Here we provide simple but effective methods (agarose-based epidermal imprinting and tape-based epidermis tearing) for solving this problem without using the SEM.

Keywords: Epidermal cell(表皮细胞), Stomata(stomata), Epidermal imprinting(表皮印记), Epidermis tearing(表皮撕裂), ImageJ(ImageJ)

Materials and Reagents

  1. Glass slide (Leica, model: 3809299 )

  2. Scotch tape (1/2") (3M, Scotch®, model: 600 )

  3. Tobacco (one month old) and soybean seedlings (two weeks old)
  4. Distilled water

  5. Low melting point agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16520-050 )
  6. 3% agarose solution (see Recipes)

Equipment

  1. Thermostatic water bath (40 °C)
  2. Pointed tweezer (Tweezerman, model: 4091833 )
  3. Refrigerator (4 °C) (Haier, model: HT18TS77SP )
  4. Optical microscope (Olympus, model: BX51 )
  5. Objective used on microscope (Olympus, model: UPlanFI )

Software

  1. ImageJ (version 1.38x or above)

Procedure

  1. Leaves for screening of epidermis are photographed with a digital camera (Figure 1). The total Leaf Area (LA) is measured using ImageJ. For measuring area using ImageJ, use the straight lines tool to create a line selection corresponding to the scale ruler, and enter that distance in the Analyze > Set Scale dialog box to set the scale. Then use the Polygon selections tool to encircle the target region, and press the M key to measure the area.


    Figure 1. A photograph of leaves used for screening of epidermis. Leaves with a scale ruler were photographed with a digital camera. Leaf area was measured using ImageJ.

  2. For statistical analysis of leaf epidermis, leaf is divided into several parts along leaf veins (Figure 2), and the area of each leaf part (ALP) is measured using ImageJ.


    Figure 2. Leaf partition for screening of epidermis. For measuring the epidermal cell size and number, leaf (tobacco) was divided into several parts (partitioned by yellow lines), and the area of each leaf part (ALP) was measured using ImageJ. Blue cycles indicate the leaf pieces selected for observing epidermis. Arrow indicates the central part selected for screening of stomata. Bar = 2 cm.

  3. One to three leaf pieces (must be uniformly distributed) in each leaf part are selected for observing epidermis using the following methods (Figure 2). 

  4. For observing fragile and less hairy leaves (such as tobacco), the agarose-based epidermal imprinting method is carried out as follows:
    1. 3% agarose solution is melted thoroughly in a boiling water bath (about 10 min for 50 ml solution) or in a microwave (30 sec for 50 ml agarose solution under 1,000 W) and kept in a 40 °C water bath. 

    2. Agarose is laid on the surface of a clean glass slide with a pipette to form a thin layer (about 1 mm thick) (Video 1).

      Video 1. Sample preparation for epidermal imprinting onto agarose

    3. Leaf blade to be observed is quickly and gently laid on the agarose layer (Video 1).
    4. Put the slide immediately at 4 °C for 3 min in a refrigerator.
    5. The leaf blade is carefully peeled off using a pointed tweezer (Video 2).
    6. The specimen is observed and photographed under the optical microscope (Figure 3).

    Video 2. Demonstration of how to peel off leaf blade after imprinting

  5. For observing hairy leaves (such as soybean), the tape-based epidermis tearing method is carried out as follows:
    1. Scotch tape is cut to pieces (about 0.3 x 0.3 cm). 

    2. Adhere a tape piece tightly to the leaf blade with proper pressure but avoid crushing the epidermis (Video 3). 

      Video 3. Sample preparation for tape-based epidermis tearing method

    3. Tear off the strip carefully from one side using a pointed tweezer (the epidermis is sticking to the tape) (Video 3). 

    4. If massive mesophyll cells adhered to the epidermis, they can be gently scraped off from one side using a tweezer. 

    5. The epidermis strip is immersed in distilled water on a glass slide, avoiding bubbles (Video 3).

    6. The specimen is observed and photographed under the optical microscope (Figure 4).
  6. The average cell size of each leaf is measured and calculated as follows:
    1. Three regions (50-100 cells) in micrographs of each leaf piece are selected using the Polygon selections tool in ImageJ (Figure 3). The region area is measured, and cell number is counted.
    2. Calculate the Cell Size of each leaf Part (CSP) (divide the total area of all regions selected in each leaf part by total cell number of all regions). 

    3. The average cell size (ACS) is calculated using the following formula: 

      ACS=Σ CSPn x ALPn/LA
  7. The Cell Number per leaf (CN) is then calculated as follows:
    CN = LA/ACS
  8. For measuring the stomatal density and the stomatal aperture, the central part of each leaf is selected for observing epidermis using the methods described in step 4 or 5. 

  9. Three regions (20-50 stomata) in micrographs are selected using the polygon selection tool in ImageJ. The region area is measured, the stomatal number is counted, and the stomatal density is calculated (dividing the stomatal number by area). The stomatal width and length are also measured using the Straight lines tool in ImageJ, and the stomatal aperture is calculated (divide the stomatal width by the stomatal length). 


Representative data



Figure 3. Screening of leaf epidermal cells by agarose imprinting. Leaf (tobacco) epidermis was imprinted onto agarose and screened under the optical microscope. The polygon with yellow edges indicates a selected area for measuring the cell size of a leaf part. The black dots are marks (created using the Paintbrush Tool in ImageJ) for counting cell numbers. Bar = 200 μm.


Figure 4. Screening of leaf epidermal cells by tearing the epidermis. Leaf (soybean) epidermis was torn off using the scotch tape and screened under the optical microscope. Bar = 50 μm.

Notes

  1. Using the agarose imprinting method, the leaf blade must be flat and laid on the melting agarose very gently. Otherwise, some detail structure of the epidermis will be lost or even failed to be imprinted on the agarose. 

  2. Using the epidermal tearing method, the mesophyll cells should be scraped gently from one side for one time only to avoid destroying the structure of epidermis. 

  3. Young and flat leaves are better than old and rounded leaves for epidermis screening.

Recipes

  1. 3% agarose solution (50 ml)
    Add 1.5 g low melting point agarose to 50 ml distilled water
    Boil until the agarose is melted completely
    Use when the temperature is about 40 °C
    Seal and store at RT (only need melting when necessary to use)

Acknowledgments

This protocol was improved from the previously published studies (Mathur and Koncz, 1997; De Veylder et al., 2001; Tao et al., 2015). This work was supported by the National Natural Science Foundation of China (grant No.91317306) and the State Key Lab of Plant Genomics.

References

  1. De Veylder, L., Beeckman, T., Beemster, G. T., Krols, L., Terras, F., Landrieu, I., van der Schueren, E., Maes, S., Naudts, M. and Inze, D. (2001). Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis. Plant Cell 13(7): 1653-1668.
  2. Mathur, J. and Koncz, C. (1997). Method for preparation of epidermal imprints using agarose. Biotechniques 22(2): 280-282.
  3. Tao, J. J., Cao, Y. R., Chen, H. W., Wei, W., Li, Q. T., Ma, B., Zhang, W. K., Chen, S. Y. and Zhang, J. S. (2015). Tobacco translationally controlled tumor protein interacts with ethylene receptor tobacco histidine kinase1 and enhances plant growth through promotion of cell proliferation. Plant Physiol 169(1): 96-114.

简介

叶表皮细胞大小和数量与叶面积正相关。 气孔是对气体交换和水蒸腾至关重要的专门的表皮细胞。 因此,叶表皮细胞的观察和统计分析对于叶发育和对环境刺激的反应的研究是有价值的。 经典的方法是使用扫描电子显微镜(SEM),这是一种昂贵和耗时的方法,因此使得大规模筛选表皮不切实际。 在这里,我们提供简单但有效的方法(基于琼脂糖的表皮印记和基于胶带的表皮撕裂),以解决这个问题,而不使用SEM。

关键字:表皮细胞, stomata, 表皮印记, 表皮撕裂, ImageJ

材料和试剂

  1. 玻璃载玻片(Leica,型号:3809299)
  2. 苏格兰胶带(1/2")(3M,Scotch,型号:600)
  3. 烟草(一个月大)和大豆幼苗(两周大)
  4. 蒸馏水
  5. 低熔点琼脂糖(Thermo Fisher Scientific,Invitrogen TM ,目录号:16520-050)
  6. 3%琼脂糖溶液(见配方)

设备

  1. 恒温水浴(40°C)
  2. 指尖镊子(Tweezerman,型号:4091833)
  3. 冰箱(4℃)(海尔,型号:HT18TS77SP)
  4. 光学显微镜(Olympus,型号:BX51)
  5. 显微镜(Olympus,型号:UPlanFI)上使用的物镜

软件

  1. ImageJ(版本1.38x或更高版本)

程序

  1. 用于筛选表皮的叶子通过数码相机拍照(图1)。 使用ImageJ测量总叶面积(LA)。 对于使用ImageJ的测量区域,使用直线工具创建线选择 对应于刻度尺,并在分析>中输入该距离。设置缩放对话框以设置缩放。然后使用多边形选择工具包围目标区域,然后按M键测量区域

    图1.用于筛选表皮的叶的照片。用数码相机拍摄带有标尺的叶子。使用ImageJ测量叶面积。

  2. 对于叶表皮的统计分析,叶沿着叶脉分成几个部分(图2),并且使用ImageJ测量每​​个叶部分(ALP)的面积。


    为了测量表皮细胞大小和数量,将叶(烟草)分成几个部分(由黄线分隔),并且将每个叶部分的面积ALP)。蓝色周期表示选择用于观察表皮的叶片。箭头表示选择用于筛选气孔的中心部分。酒吧= 2厘米。

  3. 使用以下方法选择在每个叶部分中的一至三片叶片(必须均匀分布)用于观察表皮(图2)。
  4. 为了观察脆弱和少毛的叶(例如烟草),基于琼脂糖的表皮印记法如下进行:
    1. 将3%琼脂糖溶液在沸水浴中(对于50ml溶液约10分钟)或在微波(在1,000W下对于50ml琼脂糖溶液30秒)完全熔化并保持在40℃水浴中。
    2. 用移液管将琼脂糖置于干净的载玻片的表面上以形成薄层(约1mm厚)(视频1)。

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    3. 将观察到的叶片快速且温和地铺在琼脂糖层上(视频1)。
    4. 将载玻片在冰箱中立即在4℃放置3分钟
    5. 使用尖锐的镊子小心地剥离叶片(视频2)。
    6. 在光学显微镜下观察样品并拍照(图3)

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  5. 为了观察毛叶(例如大豆),基于带的表皮撕裂方法如下进行:
    1. 将苏打带切成片(约0.3×0.3cm)。
    2. 在适当的压力下将胶带片紧紧地贴在叶片上,但避免压碎表皮(视频3)。

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      视频3.基于胶带的表皮撕裂方法的样品准备
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    3. 使用尖锐的镊子从一侧小心地撕下条(表皮粘在磁带上)(视频3)。
    4. 如果大量叶肉细胞粘附到表皮,可以使用镊子从一侧轻轻地刮掉。
    5. 将表皮条浸在载玻片上的蒸馏水中,避免气泡(视频3)。
    6. 在光学显微镜下观察样品并拍照(图4)。
  6. 每片叶的平均细胞大小如下测量和计算:
    1. 使用ImageJ中的多边形选择工具(图3)选择每个叶片的显微照片中的三个区域(50-100个细胞)。测量区域面积,并计数细胞数量。
    2. 计算每个叶部分(CSP)的单元格大小(除以所有区域的总面积 在每个叶部分中由所有区域的总细胞数选择)。
    3. 平均泡孔尺寸(ACS)使用下式计算:
      ACS =ΣCSPn×ALPn/LA
  7. 然后计算每片的细胞数(CN)如下:
    CN = LA/ACS
  8. 为了测量气孔密度和气孔孔径,使用步骤4或5中描述的方法选择每个叶的中心部分用于观察表皮。
  9. 使用ImageJ中的多边形选择工具选择显微照片中的三个区域(20-50个气孔)。测量区域面积,计数气孔数,并计算气孔密度(将气孔数除以面积)。还使用ImageJ中的直线工具测量气孔宽度和长度,并计算气孔孔径(将气孔宽度除以气孔长度)。

代表数据



图3.通过琼脂糖印迹筛选叶表皮细胞将叶(烟草)表皮印在琼脂糖上,并在光学显微镜下筛选。具有黄色边缘的多边形表示用于测量叶部分的单元尺寸的选择区域。黑点是用于计算单元格编号的标记(使用ImageJ中的画笔工具创建)。 Bar =200μm。


图4.通过撕裂表皮来筛选叶表皮细胞。使用透明胶带撕下叶(大豆)表皮,并在光学显微镜下筛选。 Bar =50μm。

笔记

  1. 使用琼脂糖印迹方法,叶片必须是平的,并放在熔化的琼脂糖非常温柔。否则,表皮的一些细节结构将丢失或甚至不能印在琼脂糖上。
  2. 使用表皮撕裂方法,叶肉细胞应从一侧轻轻刮一次,以避免破坏表皮的结构。
  3. 年轻和扁平的叶子比用于表皮筛选的老叶和圆叶更好

食谱

  1. 3%琼脂糖溶液(50ml) 将1.5g低熔点琼脂糖加入到50ml蒸馏水中 煮沸直到琼脂糖完全融化
    当温度约为40°C时使用
    密封和储存在室温(只需要熔化,当需要使用)

致谢

该方案从先前公开的研究(Mathur和Koncz,1997; De Veylder等人,2001; Tao等人,2015)得到改进。这项工作是由中国国家自然科学基金(拨款号:91317306)和植物基因组学国家重点实验室支持。

参考文献

  1. De Veylder,L.,Beeckman,T.,Beemster,GT,Krols,L.,Terras,F.,Landrieu,I.,van der Schueren,E.,Maes,S.,Naudts, 。(2001)。  细胞周期蛋白依赖性激酶的功能分析拟南芥的抑制剂。 植物细胞13(7):1653-1668。
  2. Mathur,J.和Koncz,C。(1997)。  使用琼脂糖制备表皮印记的方法 Biotechniques 22(2):280-282。
  3. ------------------------------------------------------------- 插入文件"href ="http://www.ncbi.nlm.nih.gov/pubmed/25941315"target ="_ blank">烟草翻译控制的肿瘤蛋白与乙烯受体烟草组氨酸激酶1相互作用,并通过促进细胞增殖来增强植物生长 。 Plant Physiol 169(1):96-114。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tao, J., Chen, S. and Zhang, J. (2016). Simple Methods for Screening and Statistical Analysis of Leaf Epidermal Cells in Dicotyledonous Plants. Bio-protocol 6(17): e1916. DOI: 10.21769/BioProtoc.1916.
  2. Tao, J. J., Cao, Y. R., Chen, H. W., Wei, W., Li, Q. T., Ma, B., Zhang, W. K., Chen, S. Y. and Zhang, J. S. (2015). Tobacco translationally controlled tumor protein interacts with ethylene receptor tobacco histidine kinase1 and enhances plant growth through promotion of cell proliferation. Plant Physiol 169(1): 96-114.
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