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Seed Coat Ruthenium Red Staining Assay
种皮钌红染色试验   

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Abstract

The goal of this protocol is to assay for defects in synthesis/secretion/release of seed coat mucilage by ruthenium red staining of mature whole seeds.
The mucilage secretory cells of the Arabidopsis seed coat synthesize and secrete a large quantity of primarily pectinaceous mucilage to a ring-shaped apical domain during their differentiation. This makes them an excellent model system to identify genes involved in both cell wall synthesis and secretion (et al., 2000). When wild-type seeds are incubated in ruthenium red stain, hydrated mucilage is extruded from epidermal cells and a ‘halo’ of red-stained mucilage is observed surrounding the seed (Western et al., 2000). Reduced mucilage staining may result from defects in cell wall biosynthesis, secretion, or impaired release upon hydration.

Keywords: Arabidopsis(拟南芥), Seed coat(种子大衣), Mucilage(粘液), Ruthenium red(钌红), Pectin(果胶)

Materials and Reagents

  1. Seeds
  2. 0.01% ruthenium red solution (Sigma-Aldrich, catalog number: 11103-72-3 )
  3. 50 mM EDTA (optional)
  4. dH2O (pH 6.5-7.5) [buffer with 10 mM Tris (pH 7.6), if necessary]

Equipment

  1. Microfuge tubes
  2. Transfer pipet or aspirator
  3. Orbital shaker
  4. White porcelin depression plate or clean depression slides
  5. Dissecting or compound microscope

Procedure

  1. Place ~20 seeds in a clean (but not necessarily sterile) microfuge tube.
  2. Add 800 μl dH2O or 50 mM EDTA.
    Shake vigorously (~400 rpm) on an orbital shaker for 2 h at room temperature to hydrate, and therefore release the mucilage from the epidermal seed coat (see Note 6).
  3. Remove as much of the first solution as possible with a pipet or an aspirator and replace with 800 μl of 0.01% ruthenium red solution.
  4. Shake vigorously (~400 rpm) on an orbital shaker for 1 h at room temperature.
  5. Remove the ruthenium red solution and replace with dH2O.
  6. Observe the seeds, mounted in water, under a dissecting microscope in a white porcelin depression plate, or on a clean depression slide under a compound microscope.


    Figure 1. Mucilage staining of WT and echidna mutant seeds incubated in EDTA and ruthenium red solution (Gendre et al., 2013). Wild-type seeds hydrated and stained with ruthenium red display a ‘halo’ of stained mucilage around the seed. This halo was almost completely absent in echidna seeds indicating a putative defect in mucilage biosynthesis, secretion, or release.

Notes

  1. Ruthenium red is a non-specific chemical dye that binds to negative charges, therefore it will stain the acidic polysaccharides that make up pectin.
  2. EDTA treatment chelates Ca2+ ions from solution, thereby reducing pectin crosslinking. Thus, EDTA treatment can particularly be used to promote the release of mucilage both from mutants with reduced amounts of mucilage (e.g. Arsovski et al., 2009a) and those with chemically altered mucilage that has altered hydration and swelling (e.g. Arsovski et al., 2009b).
  3. If dH2O is significantly lower than pH 7, the dye won't work very well.


    Figure 2. Effect of the pH and EDTA on the ruthenium staining. WT seeds were incubated for 2 h in water with an acidic pH 5.8, a buffered Tris solution or EDTA, prior to the ruthenium red staining. The acidity is affecting the staining intensity. The EDTA treatment promote mucilage release giving rise to a thicker halo.

  4. Seeds should be completely desiccated (1-2 weeks after harvesting minimum) before staining.
  5. Environmental variations in plant growth can lead to differences in mucilage. If plants are under stress conditions (i.e. water stress, pathogen attack, deficiency or excess of nutriments) the seed quality, and therefore mucilage production, may be affected. It is best to compare seed mucilage from plants that were grown together, and/or to repeat the experiment several times on different batches of seeds.
  6. Seeds have two layers of mucilage: a loose outer layer that washed away with shaking, and a more tightly bound layer that remains associated with the seed even after shaking or washing. To observe both layers, seeds can be stained without shaking in individual wells of a tissue culture plate (e.g. 24-48 well plate) (Harpaz-Saad et al., 2011).

Recipes

  1. 0.01% ruthenium red
    0.1 g in 1 L dH2O (pH 6.5 -7.5)
    Stored at 4 ºC
    Discard solution if no longer bright pink (It may turn brownish, orange, purple.)
  2. 50 mM EDTA
    1,861 g in 100 ml dH2O
    Add EDTA to 80 ml of dH2O
    Bring pH to ~8-9 (until EDTA dissolves) with NaOH pellets or 10 N NaOH solution (~2 g of pellets)
    Bring volume to 100 ml

Acknowledgments

This protocol was developed by T. Western at the University of British Columbia while in the laboratory of Dr. George Haughn, funded through National Sciences and Engineering Research Council grants to G. Haughn. While the first use was shown in Western et al. (2000) referenced in the text, the protocol was first described in Western et al. (2001) and elaborated for details of EDTA pretreatment in Arsovski et al. (2009a), referenced in the text.

References

  1. Arsovski, A. A., Villota, M. M., Rowland, O., Subramaniam, R. and Western, T. L. (2009a). MUM ENHANCERS are important for seed coat mucilage production and mucilage secretory cell differentiation in Arabidopsis thaliana. J Exp Bot 60(9): 2601-2612.
  2. Arsovski, A. A., Popma, T. M., Haughn, G. W., Carpita, N. C., McCann, M. C. and Western, T. L. (2009b). AtBXL1 encodes a bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells. Plant Physiol 150(3): 1219-1234.
  3. Gendre, D., McFarlane, H. E., Johnson, E., Mouille, G., Sjodin, A., Oh, J., Levesque-Tremblay, G., Watanabe, Y., Samuels, L. and Bhalerao, R. P. (2013). Trans-Golgi network localized ECHIDNA/Ypt interacting protein complex is required for the secretion of cell wall polysaccharides in Arabidopsis. Plant Cell 25(7): 2633-2646.
  4. Harpaz-Saad, S., McFarlane, H. E., Xu, S., Divi, U. K., Forward, B., Western, T. L. and Kieber, J. J. (2011). Cellulose synthesis via the FEI2 RLK/SOS5 pathway and cellulose synthase 5 is required for the structure of seed coat mucilage in Arabidopsis. Plant J 68(6): 941-953.
  5. Western, T. L., Skinner, D. J. and Haughn, G. W. (2000). Differentiation of mucilage secretory cells of the Arabidopsis seed coat. Plant Physiol 122(2): 345-356.
  6. Western, T. L., Burn, J., Tan, W. L., Skinner, D. J., Martin-McCaffrey, L., Moffatt, B. A. and Haughn, G. W. (2001). Isolation and characterization of mutants defective in seed coat mucilage secretory cell development in Arabidopsis. Plant Physiol 127(3): 998-1011.
  7. Western, T. L. (2012). The sticky tale of seed coat mucilages: production, genetics, and role in seed germination and dispersal. Seed Sci Res 22(1): 1.

简介

该协议的目的是通过成熟全种子的钌红染色来测定种皮粘液合成/分泌/释放中的缺陷。拟南芥种皮的粘液分泌细胞合成 并在其分化期间将大量的主要果胶粘液分泌到环状顶端结构域。 这使得它们是鉴定参与细胞壁合成和分泌的基因的极好的模型系统(等人,2000)。 当野生型种子在钌红染色中孵育时,水合粘液从表皮细胞中挤出,并且在种子周围观察到红色染色的粘液的"晕"(Western等人,2000)。 减少粘液染色可能由细胞壁生物合成,分泌缺陷或水合作用时释放受损而导致。

关键字:拟南芥, 种子大衣, 粘液, 钌红, 果胶

材料和试剂

  1. 种子
  2. 0.01%钌红溶液(Sigma-Aldrich,目录号:11103-72-3)
  3. 50 mM EDTA(可选)
  4. dH 2 O(pH6.5-7.5)[如果需要,用10mM Tris(pH7.6)缓冲液]

设备

  1. 微透镜管
  2. 移液管或吸液器
  3. 轨道振动器
  4. 白色瓷器凹陷板或干净的凹陷滑道
  5. 解剖或复合显微镜

程序

  1. 将〜20粒种子放在干净(但不一定无菌)微量离心管中
  2. 加入800μldH 2 O或50mM EDTA 在定轨振荡器上在室温下剧烈摇动(〜400rpm)2小时以水合,并且因此从表皮种皮释放粘液(参见注释6)。
  3. 使用移液管或吸气器尽可能多地除去第一种溶液,并更换为800μl的0.01%钌红溶液。
  4. 在定轨振荡器上在室温下剧烈摇动(〜400rpm)1小时
  5. 除去钌红溶液,并用dH 2 O代替。
  6. 观察种子,安装在水中,在解剖显微镜下在白色瓷器凹陷板,或在复合显微镜下的清洁凹陷幻灯片上。


    图1.在EDTA和钌红溶液(Gendre 中孵育的野生型和兰花突变种子的Mucilage染色> ,2013)。野生型种子水合并用钌红染色显示种子周围染色粘液的"晕"。这种晕在种子中几乎完全不存在,表明在粘液生物合成,分泌或释放中的推定缺陷。

笔记

  1. 钌红是与负电荷结合的非特异性化学染料,因此它会染色构成果胶的酸性多糖。
  2. EDTA处理螯合来自溶液的Ca 2+离子,从而减少果胶交联。因此,EDTA处理可以特别地用于促进来自具有减少量的粘液的突变体(例如,Arsovski等人,2009a)的粘液的释放和那些具有化学改变的已经改变水合和肿胀的粘液(例如 Arsovski等人,2009b)。
  3. 如果dH 2 O明显低于pH 7,染料将不能很好地工作。


    图2.pH和EDTA对钌染色的影响。在钌红染色前,将WT种子在酸性pH5.8,缓冲的Tris溶液或EDTA中在水中孵育2小时。酸度影响染色强度。 EDTA治疗促进粘液释放,导致更厚的晕
  4. 种子应完全干燥(收获后最少1-2周),然后染色
  5. 植物生长中的环境变化可导致粘液的差异。如果植物处于胁迫条件下(即水胁迫,病原体攻击,缺乏或过量的营养),种子质量和因此的粘液产生可能受到影响。最好比较种植在一起的植物的种子粘液,和/或在不同批种子上重复实验几次。
  6. 种子具有两层胶浆:用振动洗掉的松散外层,以及甚至在摇动或洗涤后仍与种子结合的更紧密结合的层。为了观察两层,可以在组织培养板(例如24-48孔板)的单个孔中不振动地将种子染色(Harpaz-Saad等人,2011 )。

食谱

  1. 0.01%钌红
    0.1g在1L dH 2 O(pH 6.5-7.5)中的溶液 储存在4ºC
    丢弃溶液,如果不再亮粉色(它可能变成棕色,橙色,紫色。)
  2. 50mM EDTA
    在100ml dH 2 O中的1,861g/dm 2 将EDTA加入到80ml dH 2 O 2中 用NaOH颗粒或10N NaOH溶液(〜2g颗粒)将pH调至〜8-9(直到EDTA溶解)。
    将体积设为100 ml

致谢

该协议由不列颠哥伦比亚大学的T.Western开发,在George Haughn博士的实验室,由国家科学和工程研究委员会资助G. Haughn。虽然在本文中引用的Western等人(2000)中显示了第一次使用,但是该方案首先在Western等人(2001)中描述并且详细阐述了细节的EDTA预处理在Arsovski等人(2009a),在文中引用。

参考文献

  1. Arsovski,A.A.,Villota,M.M.,Rowland,O.,Subramaniam,R。和Western,T.L。(2009a)。 MUM ENHANCERS对于种子衣壳粘液生产和粘液分泌细胞分化在拟南芥中是重要的。 60(9):2601-2612。
  2. Arsovski,A.A.,Popma,T.M.,Haughn,G.W.,Carpita,N.C.,McCann,M.C。和Western,T.L。(2009b)。 AtBXL1编码果胶阿拉伯聚糖修饰所需的双功能β-D-木糖苷酶/α-L-阿拉伯呋喃糖苷酶拟南芥粘液分泌细胞。植物生理 150(3):1219-1234。
  3. Gendre,D.,McFarlane,HE,Johnson,E.,Mouille,G.,Sjodin,A.,Oh,J.,Levesque-Tremblay,G.,Watanabe,Y.,Samuels,L.and Bhalerao,RP 2013)。 Trans-Golgi网络定位的ECHIDNA/Ypt相互作用蛋白复合物是细胞壁多糖分泌所需的拟南芥。植物细胞 25(7):2633-2646。
  4. Harpaz-Saad,S.,McFarlane,H.E.,Xu,S.,Divi,U.K.,Forward,B.,Western,T.L.and Kieber,J.J。(2011)。 通过FEI2 RLK/SOS5途径和纤维素合酶5的纤维素合成是种皮结构所必需的拟南芥中的粘液。 植物J 68(6):941-953。
  5. Western,T.L.,Skinner,D.J.and Haughn,G.W。(2000)。 拟南芥种皮的粘液分泌细胞分化。 Plant Physiol 122(2):345-356。
  6. Western,T.L.,Burn,J.,Tan,W.L.,Skinner,D.J.,Martin-McCaffrey,L.,Moffatt,B.A。和Haughn,G.W。(2001)。 拟南芥中种子衣壳粘液分泌细胞发育缺陷的突变体的分离和表征。 em> Plant Physiol 127(3):998-1011。
  7. Gendre,D.,McFarlane,HE,Johnson,E.,Mouille,G.,Sjodin,A.,Oh,J.,Levesque-Tremblay,G.,Watanabe,Y.,Samuels,L.and Bhalerao,RP 2013)。 Trans-Golgi网络定位的ECHIDNA/Ypt相互作用蛋白复合物是细胞壁多糖分泌所需的拟南芥。植物细胞 25(7):2633-2646。
  8. Harpaz-Saad,S.,McFarlane,H.E.,Xu,S.,Divi,U.K.,Forward,B.,Western,T.L.and Kieber,J.J。(2011)。 通过FEI2 RLK/SOS5途径和纤维素合酶5的纤维素合成是种皮结构所必需的拟南芥中的粘液。 植物J 68(6):941-953。
  9. Western,T.L.,Skinner,D.J.and Haughn,G.W。(2000)。 拟南芥种皮的粘液分泌细胞分化。 Plant Physiol 122(2):345-356。
  10. Western,T.L.,Burn,J.,Tan,W.L.,Skinner,D.J.,Martin-McCaffrey,L.,Moffatt,B.A。和Haughn,G.W。(2001)。 拟南芥中种子衣壳粘液分泌细胞发育缺陷的突变体的分离和表征。 em> Plant Physiol 127(3):998-1011。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:McFarlane, H. E., Gendre, D. and Western, T. L. (2014). Seed Coat Ruthenium Red Staining Assay. Bio-protocol 4(7): e1096. DOI: 10.21769/BioProtoc.1096.
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