搜索

2 users have reported that they have successfully carried out the experiment using this protocol.
Quantification of Callose Deposition in Plant Leaves
植物叶中胼胝质沉积的定量测定   

评审
匿名评审
引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
The Plant Journal
Jan 2015

Abstract

Callose is an amorphous homopolymer, composed of β-1, 3-glucan, which is widespread in higher plants. Callose is involved in multiple aspects of plant growth and development. It is synthetized in plants at the cell plate during cytokinesis, in several stages during pollen development and is deposited at plasmodesmata to regulate the cell-to-cell movement of molecules. Moreover, it is produced in response to multiple biotic and abiotic stresses (Chen and Kim, 2009). Callose is considered to act as a physical barrier by strengthening the plant cell well to slow pathogen infection and to contribute to the plant’s innate immunity. Thus the callose staining method is useful to quantify activity of plant immunity. In addition, this staining can be used to visualize structures in plant tissue, where the callose may be implied whether during the development of plants or response against pathogen infection. This method is based on the use of methyl blue which reacts with (1→3)-β-glucans to give a brilliant yellow fluorescence in UV light. Moreover, calcofluor stains chitin present in fungal cell membranes and also binds to cellulose at locations where the cuticle is damaged.

Keywords: Callose (胼胝质), Papillae (乳头), Plant resistance (植物抗性), 1,3-beta-glucans (1,3-beta-glucans), Penetration resistance (贯入阻力)

Materials and Reagents

  1. Plant materials: Arabidopsis thaliana cotyledons and leaves, Solanum lycopersicum leaves or Citrus leaves
  2. 96% ethanol
  3. Na2HPO4.2H2O
  4. NaH2PO4.2H2O
  5. Methyl blue (Sigma-Aldrich, catalog number: M6900 )
  6. Sodium phosphate buffer (see Recipes)
  7. Methyl blue solution 0.5% and 0.05% (see Recipes)

Optional

  1. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  2. Fluorescent Brightener 28 (synonym Calcofluor white) (Sigma-Aldrich, catalog number: F3543 )
  3. HCl
  4. Tris.HCl buffer (see Recipes)
  5. Fluorescent brightener solution 0.01% (see Recipes)

Equipment

  1. Leica IRB epifluorescence microscope with UV filter (BP 340 to 380 nm, LP 425 nm) equipped with a Leica DC300F camera (Leica Microsystems, model: Leica IRB )

Software

  1. Digital photographs analysis software like GIMP (http://www.gimp.org) or Adobe Photoshop (adobe.com) are recommended

Procedure

Note: Staining with fluorescent Brightener is optional. This will stain the chitin present in the sample, and can be useful to stain the fungus. However, the use of this staining can mask the fluorescence emitted by callose. For this reason, if the goal of the experiment is to quantify callose deposition, the use of fluorescent Brightener is not recommended.

  1. Staining procedure
    1. This procedure can be used for staining Arabidopsis thaliana cotyledons and leaves, Solanum lycopersicum leaves or Citrus leaves with little variation according to tissue toughness.
    2. Sample the entire leaves or the cotyledons from the plant and place them in 50 ml tubes containing 96% ethanol for chlorophyll removal. Distain the samples in 96% ethanol until they appear completely white (time can vary between 1 day for small leaves like the ones of Arabidopsis till one week for citrus leaves). The saturated distaining 96% ethanol can be replaced, if necessary.
    3. Rehydrate the samples in sodium phosphate buffer for 30 min.
    4. Discard the phosphate buffer and cover the sample with 0.05% Methyl blue solution.
      Optional: Add 1/5 of fluorescent brightener solution (1 ml per each 4 ml of methyl blue solution used in the previous step).
    5. Incubate for 30 min and discard the solution.
    6. For Arabidopsis cotyledons go to step A8. Callose can be observed after 3-4 h of incubation with 0.05% Methyl blue as previously described by Luna et al. (2011).
    7. Cover the sample with 0.5% Methyl blue solution and incubate for 24 h in darkness. (Time may vary depending on the sample. Thought samples, such as citrus leaves, may require a vacuum stroke of 2 min and up to 7 days of staining.)
    8. For microscope observation, mount the samples on slides with the adaxial surface up using freshly prepared 0.05% Methyl blue.
    9. Observe the samples with an epifluorescence microscope with UV filter. Stained callose appears as fluorescent yellow spots (Figure 1).
      Note: Be aware to use all the time the same augmentation.


      Figure 1. Representative picture of callose deposition in tomato leaves upon Botrytis cinerea infection with fluorescent brightener solution (A) and without fluorescent brightener solution (B). The arrow highlights callose. Representative picture of control leaves (C).

  2. Image processing
    1. Open the images with the photography software (Figure 2).


      Figure 2. Yellow spots corresponding to stained callose analyzed for the number of pixels using A. GIMP (GNU Image Manipulation Program) or B. Adobe Photoshop

    2. Select the brilliant yellow pixels that correspond to callose using a Magic wand tool or select by color range.
    3. Use the histogram window to determine the number of pixels.
    4. Represent callose intensity as average number of bright pixels/number of total pixels.
      In the case of cotyledons it is possible to calculate the yellow pixels per leaf area.
      See Video for more details.

      Video 1. Quantification process using Gimp

      Video 2. Quantification process with photoshop

Recipes

  1. Sodium phosphate buffer (0.07 M, pH=9)
    Dissolve 12.46 g of Na2HPO4.2H2O in 1 L of distilled water
    Dissolve 0.966 g of NaH2PO4.2H2O in 100 ml of distilled water
    Adjust the pH of Na2HPO4.2H2O solution to 9, using the solution of NaH2PO4.2H2O
  2. Methyl blue solution freshly prepared
    For 0.5% solution: Dissolve 0.5 g of Methyl Blue in 100 ml of sodium phosphate buffer
    For 0.05% solution: Dissolve 0.05 g of Methyl Blue in 100 ml of sodium phosphate buffer

Optional solutions

  1. Tris.HCl buffer (0.1 M, pH =8.5)
    Dissolve 0.6057 g of Tris base in 50 ml of distilled water and adjust the pH at 8.5 with HCl
  2. Fluorescent brightener solution
    Dissolve 0.0125 g of Fluorescent Brightener 28 in 50 ml of Tris buffer
    Stored the solution in dark at room temperature

Acknowledgments

Authors thank Universitat Jaume I and the National R&D Plan (AGL2010-22300-C03-02, Spain for funding support.

References

  1. Chen, X. Y. and Kim, J. Y. (2009). Callose synthesis in higher plants. Plant Signal Behav 4(6): 489-492.
  2. Luna, E., Pastor, V., Robert, J., Flors, V., Mauch-Mani, B. and Ton, J. (2011). Callose deposition: a multifaceted plant defense response. Mol Plant Microbe Interact 24(2): 183-193.

简介

Callose是由β-1,3-葡聚糖组成的无定形均聚物,其广泛存在于高等植物中。 Callose涉及植物生长和发育的多个方面。其在细胞分裂期间在细胞板处的植物中合成,在花粉发育期间的几个阶段中,并且沉积在浆细胞上以调节分子的细胞至细胞运动。此外,它是响应多种生物和非生物胁迫产生的(Chen和Kim,2009)。考虑通过加强植物细胞以缓慢病原体感染并有助于植物的先天免疫来考虑Callose作为物理屏障。因此,胼lose质染色方法可用于定量植物免疫的活性。此外,这种染色可用于显现植物组织中的结构,其中胼cal质可以暗示在植物发育期间或对病原体感染的反应。该方法基于使用与(1→3)-β-葡聚糖反应以在UV光中产生明亮的黄色荧光的甲基蓝。此外,calcofluor对存在于真菌细胞膜中的壳多糖染色,并且在角质层损伤的位置结合纤维素。

关键字:胼胝质, 乳头, 植物抗性, 1,3-beta-glucans, 贯入阻力

材料和试剂

  1. 植物材料:拟南芥子叶和叶子,番茄叶子或柑橘叶
  2. 96%乙醇
  3. 2
  4. NaH 2 2 PO 4 4 。 2H 2
  5. 甲基蓝(Sigma-Aldrich,目录号:M6900)
  6. 磷酸钠缓冲液(见配方)
  7. 甲基蓝溶液0.5%和0.05%(见配方)

    可选
  1. (Sigma-Aldrich,目录号:T1503)
  2. 荧光增白剂28(同义词Calcofluor白)(Sigma-Aldrich,目录号:F3543)
  3. HCl
  4. Tris HCl缓冲液(参见配方)
  5. 荧光增白剂溶液0.01%(参见配方)

设备

  1. 装备有Leica DC300F照相机(Leica Microsystems,型号:Leica IRB)的UV滤光器(BP 340至380nm,LP 425nm)的Leica IRB落射荧光显微镜,

软件

  1. 数码照片分析软件(如GIMP( http://www.gimp.org )或Adobe Photoshop( adobe.com

程序

注意:使用荧光增白剂染色是可选的。这将使存在于样品中的甲壳质染色,并且可用于染色真菌。然而,使用这种染色可以掩盖胼fluorescence质发出的荧光。因此,如果实验的目的是量化胼cal质沉积,不推荐使用荧光增白剂。

  1. 染色程序
    1. 该程序可用于染色拟南芥子叶 和叶子,番茄叶子或柑橘叶子很少 根据组织韧性的变化
    2. 抽样整个叶 ?或来自植物的子叶,并将它们放置在50ml管中 含96%乙醇去除叶绿素。丢弃样品 96%乙醇,直到它们显示完全白色(时间可以变化 天的小叶,如拟南芥,直到一个星期 柑橘叶)。饱和蒸馏96%乙醇可以替换,如果 ?必要。
    3. 将样品在磷酸钠缓冲液中再水化30分钟
    4. 弃去磷酸盐缓冲液,并用0.05%甲基蓝溶液覆盖样品 可选:加入1/5的荧光增白剂溶液(每4ml上一步中使用的甲基蓝溶液1ml)。
    5. 孵育30分钟并丢弃溶液。
    6. 对于拟南芥子叶进入步骤A8。可以观察到Callose 如前所述用0.05%甲基蓝孵育3-4小时 ?由Luna等人(2011)。
    7. 用0.5%甲基蓝覆盖样品 溶液并在黑暗中孵育24小时。 (时间可能会有所不同 例子。思考样品,如柑橘叶,可能需要真空 ?行程2分钟,最多7天染色。)
    8. 显微镜 观察,将样品安装在具有向上表面的载玻片上 使用新制备的0.05%甲基蓝
    9. 观察样品 具有UV滤光器的落射荧光显微镜。染色胼。质 表现为荧光黄斑(图1) 注意:请注意始终使用相同的扩充。

      A                           ;                    B                    ;                         ;    C

      图1.番茄叶中胼cal质沉积的代表性图片 ?用荧光增白剂溶液感染灰葡萄孢菌 (A),不含荧光增白剂溶液(B)。箭头 突出胼。。对照叶的代表图(C)。

  2. 图像处理
    1. 使用摄影软件打开图像(图2)。

      A

      B

      图2.分析对应于染色的胼cal质的黄色斑点 使用A GIMP(GNU图像处理程序)或 B. Adob??e Photoshop
    2. 使用魔术棒工具选择对应于胼cal质的亮黄色像素,或者按颜色范围选择
    3. 使用直方图窗口确定像素数。
    4. 将胼lose质强度表示为亮像素的平均数量/总像素数量。
      在子叶的情况下,可以计算每叶面积的黄色像素。
      有关详情,请参阅视频。

      视频1.使用Gimp的量化过程
      <! - [if!IE]> - <! - <![endif] - >

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      获取Adobe Flash Player

      <! - [if!IE]> - >
      <! - <![endif] - >

      视频2.使用photoshop的量化过程
      <! - [if!IE]> - <! - <![endif] - >

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      获取Adobe Flash Player

      <! - [if!IE]> - >
      <! - <![endif] - >

食谱

  1. 磷酸钠缓冲液(0.07M,pH = 9)
    将溶于1L蒸馏水中的12.46g Na 2 HPO 4 Sub 2 .2H 2 O溶于溶剂中。
    在100ml蒸馏水中溶解0.966g NaH 2 PO 4 PO 4·2H 2 2H 2 O。 使用NaH的溶液将Na 2 HPO 4+,2H 2 2H 2 O溶液的pH调节至9, sub> 2 PO 4 2H <2> O
  2. 新鲜制备的甲基蓝溶液
    对于0.5%溶液:将0.5g甲基蓝溶解在100ml磷酸钠缓冲液中 对于0.05%的溶液:将0.05g甲基蓝溶解在100ml磷酸钠缓冲液中
    可选解决方案
  3. Tris.HCl缓冲液(0.1M,pH = 8.5) 将0.6057g Tris碱溶于50ml蒸馏水中,用HCl调节pH至8.5
  4. 荧光增白剂溶液
    将0.0125g荧光增白剂28溶解在50ml Tris缓冲液中
    将溶液在室温下避光保存

致谢

作者感谢Jaume I大学和国家R& D计划(AGL2010-22300-C03-02,西班牙资金支持。

参考文献

  1. Chen,X.Y。和Kim,J.Y。(2009)。 高等植物中的Callose合成。 植物信号分析 4 (6):489-492。
  2. Luna,E.,Pastor,V.,Robert,J.,Flors,V.,Mauch-Mani,B.and Ton,J。(2011)。 Callose沉积:多方面植物防御反应 Mol Plant Microbe Interact 24(2):183-193。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Scalschi, L., Llorens, E., Camañes, G., Pastor, V., Fernández-Crespo, E., Flors, V., García-Agustín, P. and Vicedo, B. (2015). Quantification of Callose Deposition in Plant Leaves. Bio-protocol 5(19): e1610. DOI: 10.21769/BioProtoc.1610.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。