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Estimation of Stomatal Aperture in Arabidopsis thaliana Using Silicone Rubber Imprints
使用硅橡胶印记估测拟南芥气孔开度   

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Journal of Experimental Botany
Jun 2016

Abstract

Estimation of stomatal aperture using low viscosity silicone-base impression material has the advantage of working with the whole leaf. The developmental stage and the environment strongly affect the stomatal aperture. Therefore, it is mandatory to have accurate estimations of the stomatal aperture of intact leaves under different situations. With this technique, it is possible to get the real picture at any moment. The outputs of the data include studies on cell area and morphology, epidermis cell and stomata lineages, among others. This protocol is useful for the accurate estimation of stomatal aperture in many samples of intact leaves in Arabidopsis thaliana.

Keywords: Drought (干旱), Epidermis (表皮), Leaf development (叶发育), Oxidative stress (氧化胁迫), Photorespiration (光呼吸), Stomatal density (气孔密度)

Background

The epidermis of all leaves has specialized cells, the guard cells, surrounding microscopic pores. The guard cells and pores are called stomata, and they permit gas exchange and diffusion of water vapor between the atmosphere and the interior of the leaf. Stomata are products of an intracellular program, which generates the specific stomatal patterns during their development (Kagan et al., 1992). Stomata are found both in the abaxial and adaxial sides of the leaf, although the stomatal density (and starch accumulation) is higher on the abaxial side of the leaf sheath in Arabidopsis thaliana (Schlüler et al., 2003; Tsai et al., 2009). The stomatal density is controlled by endogenous and exogenous factors in Arabidopsis thaliana (Berger and Altmann, 2000; von Groll et al., 2002). Stomatal aperture actively responds to changes in the environment and regulates leaf transpiration rates (Santelia and Lawson, 2016). An accurate estimation of the stomatal aperture provides insight into the impact of environmental stress on plants.

Materials and Reagents

  1. 10-cm pots
  2. Plastic spatula
  3. Microscope glass slide (Glass Klass, Yancheng Huisheng Medical Instrument Factory, catalog number: 7102 )
  4. Silicone low viscosity impression material (Polysiloxane) and hardener/catalyst (Zhermack, catalog number: U113368/G )
  5. Leaves of Arabidopsis (Arabidopsis thaliana)
  6. Clear nail varnish (Colorama, Maybelline, Argentina)

Equipment

  1. Plant growth chamber: closed cabinet with controlled environmental conditions of photoperiod, light intensity, temperature, and humidity
  2. Light microscope with a 60x objective lens (Olympus, model: BH-2 ) equipped for photomicrography (Nikon Instruments, model: DS-Fi1 )

Software

  1. ImageJ (http://rsb.info.nih.gov/ij/)
  2. Microsoft Excel
  3. Sigma Plot (version 11.0)

Procedure

  1. Sampling
    Arabidopsis seeds were sown on soil in 10-cm pots and grown in a closed cabinet under a 16 h light/8 h dark photoperiod using fluorescent light at 120 µmol m-2 sec-1 at 23 ± 2 °C, and a relative humidity of 65-70%. For measurements, the younger, fully expanded and flat leaves of a 21-d-old Arabidopsis plant (i.e., third and fourth leaves) were harvested for immediate use.
    Notes:
    1. Leaf sampling should be done at the same time of the day to avoid any potential rhythmic effects.
    2. Leaves used for impressions are from plants grown in parallel under identical conditions.
  2. Leaf impressions
    1. Mix silicone low viscosity impression material (Polysiloxane) and catalyst/hardener in the approximate ratio 1:0.75. In case the whole leaf is covered with the material, mix 2 cm of silicon low viscosity impression material and 1.5 cm of catalyst/hardener. Mix very well using a spatula for about 30 sec (Figure 1A).
    2. Apply the mixture immediately to the abaxial surface of the leaves using a spatula and minimal pressure (Figures 1B and 1C). At 23 °C, hardening will usually take 4-5 min.
    3. After hardening, clip the tip of the leaf and peel along with the direction of the main leaf vein (Figures 1D and 1E).
    4. Make a positive impression by covering the silicone imprints with a thin layer of a clear nail varnish and leaving it to dry for at least one hour at room temperature (Figure 1F).
    5. Place the silicon rubber impression on a glass slide with the positive impression (surfaced covered with varnish) facing down. Transfer the thin layer of nail varnish to the glass slide by gently pressing the silicone rubber onto it (Figures 1G and 1H).
    Notes:
    1. High temperature or greater amount of catalyst/hardener will speed up hardening.
    2. Close hardening material properly after use.
    3. Silicone imprints can be stored for a long time at room temperature and be reused.


    Figure 1. Leaf impression procedure

  3. Measurement of stomatal apertures with a light microscope
    1. Observe imprints under a light microscope using a 60x objective lens and take micrographs of the magnified image.
    2. Measure the width and length of stomatal aperture (Figure 2) using the image processing software ImageJ (http://rsb.info.nih.gov/ij/).
    Notes:
    1. Since the imprints are usually not totally flat, not all regions of a leaf section are in focus. The leaf section is considered to be in focus when the edges of the cells are clear and sharp. To solve this problem, several photographs of the leaf section can be taken at different focal depths.
    2. Do not use images for measurements that are out of focus.


      Figure 2. Snapshot of ImageJ software showing a representative measurement of length and width of a stomata. Scale bar = 3 µm.

Data analysis

The number of stomata analyzed on a typical silicon imprint was 30-40. These stomata were randomly selected in the separate microscopic fields in the same slide. Ten leaves per Arabidopsis line were analyzed.

  1. Compile the data and calculate the ratio between the width and length of all selected stomata using Excel spreadsheet software.
  2. The data obtained were statistically evaluated using ANOVA (Sigma Plot software, version 11.0). Consider a P-value less than 0.05 as statistically significant. Perform the experiment at least three times.
  3. An example with ten measurements carried out using three leaves of Arabidopsis accession Columbia (Col-0) is shown in Table 1.
    Note: Columbia (Col-0) is the most widely-used accession of Arabidopsis thaliana.

    Table 1. Measurement and calculation of stomatal aperture from three leaves of Arabidopsis Col-0

Note: Other results of stomatal aperture were published in Scarpeci et al., 2017.

Acknowledgments

This work was supported by ANPCyT and CONICET, Argentina. This protocol was modified from previous work (Weyers and Johansen, 1985).

References

  1. Berger, D. and Altmann, T. (2000). A subtilisin-like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana. Genes Dev 14(9): 1119-1131.
  2. Kagan, M. L., Novoplansky, N., and Sachs, T. (1992). Variable cell lineages form the functional pea epidermis. Ann Bot 69(4): 303-312.
  3. Santelia, D. and Lawson, T. (2016). Rethinking guard cell metabolism. Plant Physiol 172(3): 1371-1392.
  4. Scarpeci, T. E., Frea, V. S., Zanor, M. I. and Valle, E. M. (2017). Overexpression of AtERF019 delays plant growth and senescence, and improves drought tolerance in Arabidopsis. J Exp Bot 68(3): 673-685.
  5. Schlüler, U., Muschak, M., Berger, D. and Altmann, T. (2003). Photosynthetic performance of an Arabidopsis mutant with elevated stomatal density (sdd1-1) under different light regimes. J Exp Bot 54(383): 867-874.
  6. Tsai, H. L., Lue, W. L., Lu, K. J., Hsieh, M. H., Wang, S. M. and Chen, J. (2009). Starch synthesis in Arabidopsis is achieved by spatial cotranscription of core starch metabolism genes. Plant Physiol 151(3): 1582-1595.
  7. Von Groll, U., Berger, D. and Altmann, T. (2002). The subtilisin-like serine protease SDD1 mediates cell-to-cell signaling during Arabidopsis stomatal development. Plant Cell 14(7): 1527-1539.
  8. Weyers, J. D. B. and Johansen, L. G. (1985). Accurate estimation of stomatal aperture from silicone rubber impressions. New Phytol 101: 109-115.

简介

使用低粘度硅胶印模材料估算气孔开孔具有与整叶合作的优点。 发育阶段和环境对气孔的影响很大。 因此,在不同情况下,必须准确估计完整叶片的气孔孔径。 使用这种技术,可以随时获取真实的图片。 数据的输出包括细胞面积和形态学研究,表皮细胞和气孔谱系等。 该协议对准确估计拟南芥中许多完整叶样品的气孔孔径是有用的。
【背景】所有叶子的表皮都有特殊的细胞,保卫细胞,周围的微细孔。保卫细胞和毛孔称为气孔,它们允许气体在气氛和叶子内部之间进行气体交换和扩散。 Stomata是细胞内程序的产物,其在其发育过程中产生特定的气孔模式(Kagan等,1992)。虽然气孔密度(和淀粉积累)在拟南芥叶片的背轴上较高,但在叶片的背面和近轴侧都发现了气孔(Schlüler等,2003; Tsai et al。,2009 )。气孔密度由拟南芥中的内源和外源因子控制(Berger和Altmann,2000; von Groll et al。,2002)。气孔孔积极响应环境变化并调节叶蒸腾速率(Santelia和Lawson,2016)。气孔孔径的准确估计可以了解环境胁迫对植物的影响。

关键字:干旱, 表皮, 叶发育, 氧化胁迫, 光呼吸, 气孔密度

材料和试剂

  1. 10厘米盆
  2. 塑料铲
  3. 显微镜玻璃片(玻璃科,盐城惠生医疗器械厂,目录号:7102)
  4. 硅氧烷低粘度压印材料(聚硅氧烷)和硬化剂/催化剂(Zhermack,目录号:U113368/G)
  5. <拟南芥(Arabidopsis thaliana)的叶子
  6. 清除指甲油(Colorama,美宝莲,阿根廷)

设备

  1. 植物生长室:具有光周期,光强度,温度和湿度可控环境条件的封闭柜体
  2. 装有显微照相(Nikon Instruments,型号:DS-Fi1)的60x物镜(Olympus,型号:BH-2)的光学显微镜

软件

  1. ImageJ( http://rsb.info.nih.gov/ij/
  2. Microsoft Excel
  3. Sigma Plot(版本11.0)

程序

  1. 抽样
    将拟南芥种子播种在10厘米盆中的土壤上,并在封闭的橱柜中在16小时光/8小时暗光周期下使用120μmol/平方米的荧光灯生长。 sec -1 ,23±2℃,相对湿度65-70%。对于测量,21日龄的拟南芥植物(即,第三和第四叶)的年轻,完全膨胀和平坦的叶子被收获以立即使用。
    注意:
    1. 叶采样应在白天同时进行,以避免任何潜在的节奏效应。
    2. 用于印象的叶子来自在相同条件下并行生长的植物。
  2. 叶印
    1. 混合硅氧烷低粘度压印材料(聚硅氧烷)和催化剂/硬化剂,其比例约为1:0.75。在整个叶子被材料覆盖的情况下,混合2厘米的硅低粘度压印材料和1.5厘米的催化剂/硬化剂。使用刮刀混合很好约30秒(图1A)。
    2. 使用刮铲和最小压力将混合物立即施加到叶子的背面(图1B和1C)。在23°C时,硬化通常需要4-5分钟。
    3. 硬化后,夹住叶片的尖端,并与主叶静脉的方向一起剥离(图1D和1E)。
    4. 通过用透明的指甲油薄层覆盖硅胶印记并使其在室温下干燥至少1小时,形成积极的印象(图1F)。
    5. 将硅橡胶印象放在玻璃片上,正面印象(表面覆盖有清漆)面朝下。通过轻轻地将硅橡胶压在其上,将薄薄的指甲油转移到玻璃载玻片上(图1G和1H)。
    注意:
    1. 高温或更高量的催化剂/硬化剂将加速硬化。

    2. 硅胶印记可以在室温下长期储存并重新使用


    图1. Leaf impression程序

  3. 用光学显微镜测量气孔
    1. 在光学显微镜下使用60x物镜观察印记,并拍摄放大图像的照片。
    2. 使用图像处理软件ImageJ( http://rsb.info.nih.gov/ij/)。
    注意:
    1. 由于印记通常不是完全平坦的,所以叶片部分的所有区域都不在焦点。当细胞的边缘清晰锐利时,叶片部分被认为是聚焦的。为了解决这个问题,可以在不同的焦点深度拍摄叶片部分的几张照片。



    2. 图2. ImageJ软件的快照,显示气孔长度和宽度的代表性测量。比例尺= 3μm。

数据分析

在典型的硅印记上分析的气孔数量为30-40。这些气孔在相同载玻片的分开的微观领域中随机选择。分析了每条拟南芥10条叶。

  1. 使用Excel电子表格软件编译数据并计算所有选定气孔的宽度和长度之间的比例。
  2. 使用ANOVA(Sigma Plot软件,版本11.0)对获得的数据进行统计学评估。考虑小于0.05的P 值具有统计学意义。至少进行三次实验。
  3. 使用三种叶片的拟南芥加拿大哥伦比亚(Col-0)进行十次测量的例子如表1所示。
    注意:哥伦比亚(Col-0)是拟南芥最广泛使用的加拿大国家。

    表1.拟南芥Col-0的三叶气孔孔径的测量和计算

注意:其他气孔孔径的结果发表在Scarpeci等,2017年

致谢

这项工作得到阿根廷ANPCyT和CONICET的支持。这个协议是从以前的工作中修改过的(Weyers和Johansen,1985)。

参考

  1. Berger D.和Altmann,T。(2000)。参与拟南芥气孔密度和分布调节的枯草杆菌蛋白酶样丝氨酸蛋白酶。 Genes Dev 14(9):1119-1131。 br />
  2. Kagan,ML,Novoplansky,N。和Sachs,T。(1992)。可变细胞谱系形成功能性豌豆表皮。 69(4):303-312。
  3. Santelia,D.和Lawson,T.(2016)。重新思考保卫细胞代谢。植物生理学172(3):1371-1392。
  4. Scarpeci,TE,Frea,VS,Zanor,MI和Valle,EM(2017)。  AtERF019 的过表达延迟植物生长和衰老,并改善拟南芥中的耐旱性。 > 68(3):673-685。
  5. Schlüler,U.,Muschak,M.,Berger,D.and Altmann,T。(2003)。在不同光照条件下,气孔密度升高( sdd1-1 )的拟南芥突变体的光合作用。



    54(383):867-874。
  6. 蔡,HL,Lue,WL,Lu,KJ,Hsieh,MH,Wang,SM和Chen,J。(2009)。拟南芥中的淀粉合成是通过核心淀粉代谢基因的空间转录来实现的。植物生理学> 151(3):1582-1595。
  7. Von Groll,U.,Berger,D.and Altmann,T。(2002)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/12119372 "target ="_ blank">枯草杆菌蛋白酶样丝氨酸蛋白酶SDD1在拟南芥气孔发育期间介导细胞与细胞的信号传递。植物细胞 14(7) :1527-1539。
  8. Weyers,JDB和Johansen,LG(1985)。  准确估计来自硅橡胶印象的气孔孔径。 新的Phytol 101:109-115。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Scarpeci, T. E., Zanor, M. I. and Valle, E. M. (2017). Estimation of Stomatal Aperture in Arabidopsis thaliana Using Silicone Rubber Imprints. Bio-protocol 7(12): e2347. DOI: 10.21769/BioProtoc.2347.
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