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Staining of Callose Depositions in Root and Leaf Tissues
根部和叶片组织中的胼胝体染色   

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Abstract

The plant cell wall is a physical barrier, which fulfills a plethora of functions, for example it can efficiently prevent pathogen’s entry into the cell. In addition, its changing composition contributes to plants inducible defense mechanisms. This layer of defense includes pathogen perception and is followed by the activation of defense responses resulting, among others, in a modification and remodeling of the cell wall. This relatively late defense response (hours or days after contact with pathogen) comprises the accumulation of polysaccharides, such as the 1,3-ß-glucan callose, phenolic compounds and reactive oxygen species. Callose depositions occur during normal plant growth (e.g. in the phloem), they can be also a response to different stress stimuli. During the response to pathogen attack, callose depositions are essential part of cell wall reinforcement and are important for successful plant defense. Here, we describe a method to stain callose apposition spots, which can be used to quantify this defense response.

Materials and Reagents

  1. Plant material
  2. Flg22 (a flagellin-derived, 22 amino acid-long peptide) (QRLSTGSRINSAKDDAAGLQIA)
  3. 100% ethanol
  4. 100% acetic acid (Carl Roth, catalog number: 3738.5 )
  5. K2HPO4 (Carl Roth, catalog number: P749.2 )
  6. Aniline blue or water blue (Fluka Chemika, catalog number: 95290)
  7. Glycerol (Carl Roth, catalog number: 7530.4 )
  8. Filter paper (Munktell, catalog number: 3.00343.080)
  9. Destaining solution (see Recipes)
  10. Wash solution (see Recipes)
  11. Staining solution (see Recipes)

Equipment

  1. Plastic equipment allowing to float plant material in staining solution (Grainer Falcon 50 ml tubes, catalog number: 227261 ; Grainer 6-well culture plates, catalog number: 657160 )
  2. Vacuum pump (Savant Systems LLC, catalog number: SC110 : 25-30 Hg)
  3. Microscope (Zeiss, model: Axioplan 2ie) with UV lamp (FluoArc/001.26B) and ‘DAPI’ filter (excitation filter 390 nm; dichroic mirror 420 nm; emission filter 460 nm)

Procedure

  1. Plant material:
    We used either detached leaves from Arabidopsis plants or seedlings, depending on the experimental setup and the overall aim of the experiment. Plants and seedlings were grown in two different systems:
    1. Arabidopsis plants were grown on soil under short day conditions (8/16 h day/night photoperiod) at 21 °C for 5 weeks. Before the elicitor challenge, detached leaves were floated on plant growth media (½ MS) for 3 days for the appropriate pretreatment (Figure 1A).
    2. Arabidopsis seeds were surface-disinfected, germinated, and grown for 2 weeks on ½ MS plant growth media. Thereafter, seedlings were transferred into liquid plant growth media in 6-well plates for 3 days for the appropriate pretreatment (Figure 1B). The growth conditions were set to 22 °C with 150 µmol/m2/s light in 8/16 h day/night photoperiod and 65 % relative humidity.


      Figure 1. Plant material, staining with aniline blue and destaining. A. Leaves detached from 5-week-old, soil-grown Arabidopsis plants were floated on plant growth medium. B. Two-week-old Arabidopsis seedlings were floated on plant growth medium in 6-well plates. C. Translucent Arabidopsis seedlings after destaining. D. Arabidopsis seedlings transferred into aniline blue staining solution. E-F. Microscopic visualization of callose depositions.

  2. Plants were challenged with flagellin (flg22) to activate their defense responses. In the case of seedlings, flg22 was added to the medium at a final concentration of 100 nM for 24 h. For detached leaves, 1 µM flg22 solution was sprayed and then briefly vacuum infiltrated (Petri dishes with floating leaves were placed in a closed box until a pressure reached 0.84 bar), leaves were incubated on wet filter paper for 24 h.
  3. After 24 h incubation, plants or leaves were fixated and destained in 1:3 acetic acid/ethanol until the material was transparent (usually over night), the saturated destaining solution was replaced, if necessary (Figure 1C).
  4. Fixated and destained leaves or seedlings were washed in 150 mM K2HPO4 for 30 min.
  5. Plant material was incubated for at least 2 h in 150 mM K2HPO4 and 0.01% aniline blue (staining solution) in a Falcon tube wrapped in aluminium foil for light protection (Figure 1D).
  6. In order to facilitate the observations samples were embedded in 50% glycerol before further analysis. The embedment in 50% glycerol allows a prolonged observation time and reduces the disturbances caused by air bubbles.
  7. Callose depositions were quantified in a fluorescence microscope using a DAPI filter (Figure 1E-F). The optimal excitation wavelength for aniline blue is 370 nm, and the emission maximum is by 509 nm. For documentation we used the Zeiss AxioVision 3.0 software. The images were acquired with 1,300 x 1,030 resolution and left untreated except for the adjustment of brightness. Generally we used 300 ms exposure time.

Representative data

  1. For representative data see Schenk et al. (2014).
  2. For reproducibility see Notes.

Notes

Callose depositions can be easily quantified by counting all stained spots in the defined leaf area(s). In addition, the amount of deposits can be categorized in groups, ranked from the lowest to the highest density of spots per leaf or leaf area. Quantification of the intensity of callose with image analysis software is also possible, however focusing of the image to visualize all callose depositions in a particular leaf area may be difficult. To achieve high reproducibility of results, it is important to perform the experiments with leaves at equal age and to prevent injuries during the transfer of plants or leaves to the different solutions during sample preparation.

Recipes

  1. Destaining solution
    1:3 acetic acid/ethanol
  2. Wash solution
    5.2 g of K2HPO4 in 200 ml H2O
  3. Staining solution
    1.3 g of K2HPO4 with 5 mg of aniline blue in 50 ml H2O

Acknowledgments

The protocol was modified after Clay et al. (2009). This work was supported by the Federal Office for Agriculture and Food (BLE), Germany.

References

  1. Clay, N. K., Adio, A. M., Denoux, C., Jander, G. and Ausubel, F. M. (2009). Glucosinolate metabolites required for an Arabidopsis innate immune response. Science 323(5910): 95-101.
  2. Schenk, S. T., Hernandez-Reyes, C., Samans, B., Stein, E., Neumann, C., Schikora, M., Reichelt, M., Mithofer, A., Becker, A., Kogel, K. H. and Schikora, A. (2014). N-Acyl-Homoserine lactone primes plants for cell wall reinforcement and induces resistance to bacterial pathogens via the salicylic acid/oxylipin pathway. Plant Cell 26(6): 2708-2723.

简介

植物细胞壁是物理屏障,其实现过多的功能,例如它可以有效地防止病原体进入细胞。此外,其变化的组成有助于植物诱导防御机制。该防御层包括病原体感知,并且随后是防御反应的激活,导致尤其是细胞壁的修饰和重塑。这种相对较晚的防御反应(与病原体接触后的几小时或几天)包括多糖例如1,3-β-葡聚糖胼cal质,酚类化合物和活性氧簇的积累。 Callose沉积在正常植物生长期间(例如在韧皮部中)发生,它们也可以是对不同应激刺激的反应。在对病原体攻击的反应期间,胼lose质沉积是细胞壁增强的必要部分,并且对于成功的植物防御是重要的。在这里,我们描述了染色胼lose体位置斑点的方法,其可以用于量化该防御反应。

材料和试剂

  1. 植物材料
  2. Flg22(鞭毛蛋白衍生的,22个氨基酸长的肽)(QRLSTGSRINSAKDDAAGLQIA)
  3. 100%乙醇
  4. 100%乙酸(Carl Roth,目录号:3738.5)

  5. HPO 4(Carl Roth,目录号:P749.2)
  6. 苯胺蓝或水蓝(Fluka Chemika,目录号:95290)
  7. 甘油(Carl Roth,目录号:7530.4)
  8. 滤纸(Munktell,目录号:3.00343.080)
  9. 解决方案(参见配方)
  10. 洗涤溶液(见配方)
  11. 染色溶液(见配方)

设备

  1. 允许在染色溶液中漂浮植物材料的塑料设备(Grainer Falcon 50ml管,目录号:227261; Grainer6孔培养板,目录号:657160)
  2. 真空泵(Savant Systems LLC,目录号:SC110:25-30Hg)
  3. 使用UV灯(FluoArc/001.26B)和'DAPI'滤光片(激发滤光片390nm;分色镜420nm;发射滤光片460nm)的显微镜(Zeiss,型号:Axioplan 2ie)

程序

  1. 植物材料:
    我们使用来自拟南芥植物或幼苗的分离的叶子,这取决于实验设置和实验的总体目的。植物和幼苗在两种不同的系统中生长:
    1. 拟南芥植物在短日条件下(8/16小时)在土壤上生长  日/夜光周期)在21℃下5周。诱导者之前 挑战,将分离的叶漂浮在植物生长培养基(1/2MS)上  3天进行适当的预处理(图1A)。
    2. 拟南芥种子经表面消毒,发芽,生长2天 周在½MS植物生长培养基上。此后,转移幼苗  在6孔板中的液体植物生长培养基中培养3天 适当的预处理(图1B)。生长条件设定为 22℃,在8/16小时日/夜光周期中具有150μmol/m 2 s/s光照,65% 相对湿度

      图1.植物材料,用苯胺染色 蓝色和脱色。A.从5周龄土壤生长的拟南芥植物上分离的叶漂浮在植物生长培养基上。 B.将两周龄的拟南芥幼苗漂浮在6孔的植物生长培养基上 板。 C.脱色后的半透明拟南芥幼苗。 D.拟南芥幼苗转移到苯胺蓝染色溶液中。 E-F。胼lose质沉积的显微可视化

  2. 用鞭毛蛋白(flg22)攻击植物以激活其防御反应。在幼苗的情况下,将flg22以100nM的终浓度加入培养基中24小时。对于分离的叶,喷洒1μMflg22溶液,然后短暂地真空浸润(具有漂浮叶的培养皿置于密闭的盒中,直到压力达到0.84巴),将叶在湿滤纸上孵育24小时。
  3. 孵育24小时后,将植物或叶子固定并在1:3乙酸/乙醇中脱色,直到材料透明(通常过夜),如果需要,替换饱和脱色溶液(图1C)。
  4. 固定和脱色的叶或幼苗在150mM K 2 HPO 4中洗涤30分钟。
  5. 将植物材料在包裹在铝箔中的Falcon管中在150mM K 2 HPO 4和0.01%苯胺蓝(染色溶液)中温育至少2小时,用于光保护(图1D)。
  6. 为了便于观察,在进一步分析之前将样品包埋在50%甘油中。包埋在50%甘油中允许延长的观察时间,并减少由气泡引起的扰动
  7. 使用DAPI过滤器在荧光显微镜中定量Callose沉积(图1E-F)。苯胺蓝的最佳激发波长为370nm,发射最大值为509nm。对于文档,我们使用Zeiss AxioVision 3.0软件。用1,300×1,030分辨率获得图像,除了亮度的调节之外不进行处理。一般来说,我们使用300毫秒的曝光时间

代表数据

  1. 有代表性的数据参见Schenk等人。(2014)。
  2. 有关再现性,请参见注释

笔记

可以通过计数限定的叶面积中的所有染色斑点容易地定量Callose沉积。 此外,沉积物的量可以分组,从每个叶或叶面积的最低点到最高点的密度排列。 用图像分析软件对胼cal质的强度进行定量也是可能的,然而图像的聚焦以可视化特定叶面积中的所有胼cal质沉积可能是困难的。 为了实现结果的高再现性,重要的是在相等年龄的叶子上进行实验并且在样品制备期间在植物或叶子转移到不同溶液期间防止损伤。

食谱

  1. 脱色解决方案
    1:3乙酸/乙醇
  2. 洗液
    在200ml H 2 O中的5.2g的K 2 HPO 4子
  3. 染色溶液
    1.3克K 2 HPO 4与5毫克苯胺蓝在50毫升H 2 O中的混合物

致谢

该方案在Clay等人(2009)后修改。 这项工作得到了德国联邦农业和食品局(BLE)的支持。

参考文献

  1. Clay,N.K.,Adio,A.M.,Denoux,C.,Jander,G.and Ausubel,F.M。(2009)。 拟南芥先天免疫反应所需的葡萄糖苷酸代谢物 科学 323(5910):95-101。
  2. Schenk,ST,Hernandez-Reyes,C.,Samans,B.,Stein,E.,Neumann,C.,Schikora,M.,Reichelt,M.,Mithofer,A.,Becker,A.,Kogel,KH和 Schikora,A。(2014)。 N-酰基 - 高丝氨酸内酯引物植物用于细胞壁增强并通过水杨酸诱导对细菌病原体的抗性 酸/oxylipin途径。植物细胞26(6):2708-2723。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Schenk, S. T. and Schikora, A. (2015). Staining of Callose Depositions in Root and Leaf Tissues. Bio-protocol 5(6): e1429. DOI: 10.21769/BioProtoc.1429.
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