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Histochemical Staining of Silica Body in Rice Leaf Blades
对水稻叶片中硅质小体进行组织化学染色   

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参见作者原研究论文

本实验方案简略版
Plant & Cell Physiology
Feb 2015

Abstract

Silicon (Si) is a biologically important element for plants in the order Poales (Yamamoto et al., 2011; Kido et al., 2015). In rice, Si is mainly deposited in the motor cells and the cell walls of the leaf epidermis. However, the molecular basis of this overall process has not been elucidated. Thus, we propose a protocol for the histochemical staining of the silica body based on specific hydrogen bonding between silanol group and the carboxylate group of crystal violet lactone (Ichimura et al., 2008), as described by Isa et al. (2010), but with minor modifications. This modified protocol can be used for observing Si accumulation during rice development.

Keywords: Rice (大米), Cell wall (细胞壁), Silica body (硅体), Crystal violet lactone (结晶紫内酯), Motor cell (运动细胞)

Materials and Reagents

  1. Rice (Oryza sativa L. cv. Nipponbare) plants were grown in a liquid medium supplemented with 1.5 mM SiO32- in a growth chamber at 28 °C under a 15/9 h light/dark cycle (light at 150 μ mol-2 s-1). This protocol is performed with the fifth leaf blade at foliar age 5.2 (Kido et al., 2015). The foliar age “m.n” is defined as those in which the mth leaf is fully expanded and the (m+1)th leaf is under development with a length of n/10 of the fully expanded-length
  2. Paraformaldehyde (Wako Pure Chemical Industries, catalog number: 162-16065 )
  3. Sodium cacodylate buffer (Nacalai Tesque, catalog number: 37238-25 )
  4. Agar powder (Nacalai Tesque, catalog number: 01028-85 )
  5. Phosphate Buffered Saline (PBS) (Takara Bio, catalog number: T900 )
  6. Ethanol (Wako Pure Chemical Industries, catalog number: 057-00456 )
  7. Molecular sieves (Sigma-Aldrich, catalog number: M6141 )
  8. Benzene (Nacalai Tesque, catalog number: 04017-35 )
  9. Crystal violet lactone (Tokyo Chemical Industry UK Ltd, catalog number: C0741 )
  10. PBS tablets (Takara Bio, catalog number: T900)
  11. Phosphate-buffered saline (PBS) (see Recipes)
  12. 100% ethanol solution (see Recipes)
  13. Crystal violet lactone solution (see Recipes)

Equipment

  1. Growth chamber (Nippon Medical & Chemical Instruments, model: LH220S )
  2. Stirrer Hotplate (Thermo Fisher Scientific, model: Fisher Scientific Isotemp )
  3. Diaphragm vacuum pump (Leybold-Heraeus, model: Divac 2.2L )
  4. Desiccator (SANPLATEC, model: PC-250K )
  5. Microwave oven (Sharp Electronics, model: RE-T12 )
  6. Paraffin dish (Greiner Bio-One GmbH, catalog number: 908177 )
  7. Leica VT1200S vibrating blade microtome (Leica Microsystems, model: VT1200S )
  8. Microscope slide (Matsunami Glass, catalog number: S-2123 )
  9. Cover slip (Matsunami Glass, catalog number: C024361 )
  10. Optical microscope (Leica Microsystems, model: DMRPX )
  11. CCD camera (QImaging, model: Retiga EXi )

Procedure

  1. Prepare the fixative by dissolving 0.4 g of paraformaldehyde powder in 10 ml of 20 mM sodium cacodylate buffer (pH 7.4). Heat this solution in a fume hood on the hotplate/stirrer to approximately 70 °C until the solution clears completely, and allow it to cool. The fixative mixture should be prepared immediately before use.
  2. Cut the rice leaf blade into pieces measuring approximately 2-3 mm in length (Figure 1A) and immediately immerse it in the fixative (the cut tissue will float on the fixative).
  3. The tissue/fixative is placed in a desiccator, which is connected to a diaphragm vacuum pump. The tissue/fixative is vacuum degasified for 5 min at room temperature using the vacuum pump before releasing the vacuum very slowly. Pull and release the vacuum again until the tissue sinks.
  4. Incubate overnight at 4 °C without vacuum.
  5. Dissolve 5.0 g agar powder in 100 ml of PBS using a microwave oven. Place the molten agar medium on the hotplate/stirrer, which must be preheated to 60 °C.
  6. Remove the fixative. Rinse the tissue three times with PBS. Pour the tissue into a paraffin dish.
  7. Cover the paraffin dish with molten agar medium. Arrange the leaf blade tissue in a regular array (Figure 1B and Figure 2A). Allow the agar to solidify for at least 15 min (Figure 1C).
  8. Glue the agar block, in which pieces of cut leaf blade tissues were placed upright, onto the stage of a Leica VT1200S vibrating blade microtome with a drop of instant glue (Konishi Aron Alpha) (Figure 1D-E and Figure 2B).
  9. Cut the agar-embedded tissue transversely, set upright in the agar block, with a thickness of 70 μm with the vibroslicer (Figure 1F-H and Figure 2C). Collect the transverse sections in PBS by tweezers.


    Figure 1. Section preparation. A. The fifth leaf blades cut with a razor blade. B-E. Embedding of pieces of rice blade tissues in agar. F-H. Sectioning with a vibroslicer equipped with a razor blade.


    Figure 2. Schematic drawing of section preparation. Rice leaf blade tissues are embedded in agar (A), and the agar block is glued on the stage of a Leica VT1200S vibrating blade microtome (B). The agar-embedded tissue is cut parallel to the surface of the stage (shown as dotted lines in C).

  10. Transfer the sections and incubate them in sequential dehydration treatments with 0.5 ml of 70%, 80%, 90%, and 100% ethanol for 30 min in each solution without shaking.
  11. The sections are then transferred and incubated for 30 min in each step in increasing concentrations of benzene in ethanol, ranging from 10% up to 100% with 10% stepwise increases, until the solvent has been replaced with benzene.
  12. The benzene-equilibrated sections are stained with 0.1% crystal violet lactone solution for 10 min to visualize the silicified cells.
  13. Place the stained sections on a microscope slide and cover it gently with a cover slip. Seal the cover slip with nail polish.
  14. The sections are observed using an optical microscope and images are recorded using a CCD camera (Figure 2).

Representative data


Figure 3. Crystal violet lactone staining of transverse section of the rice leaf blade at foliar age 5.2. cs: cortical sclerenchyma, pc: parenchyma. mc: motor cell. Scale bar = 50 µm

Notes

Rice (Oryza sativa L. cv Nipponbare) plants are grown in a growth chamber at 28 °C under a 15/9 h light/dark cycle (light at 150 μmol-2 s-1). This protocol is performed using the fifth leaf blades of rice plants grown in +Si conditions (Kido et al., 2015).

Recipes

  1. PBS
    Dissolve 10 PBS tablets in distilled water to make a total volume of 1,000 ml
  2. 100% ethanol solution
    To prepare the 100% ethanol solution, use 100% bulk ethanol with molecular sieves in the bottom of the bottle
  3. Crystal violet lactone solution
    Dissolve crystal violet lactone as a 0.1% solution in benzene

Acknowledgments

This protocol has been adapted or modified from a previous study by Isa et al. (2010). This study was supported by the Japan Society for Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan [a Grant-in-Aid for Scientific Research on Innovative Areas “Plant Cell Wall” (No. 24114001, 24114005) to K.N. and a Grant-in-Aid for Scientific Research (C) (25440124) to R.Y.].

References

  1. Ichimura, K., Funabiki, A., Aoki, K. and Akiyama, H. (2008). Solid phase adsorption of crystal violet lactone on silica nanoparticles to probe mechanochemical surface modification. Langmuir 24(13): 6470-6479.
  2. Isa, M., Bai, S., Yokoyama, T., Ma, J.F., Ishibashi, Y., Yuasa, T., Iwaya-Inoue, M. (2010). Silicon enhances growth independent of silica deposition in a low-silica rice mutant, lsi1. Plant Soil 331(1-2): 361-375.
  3. Kido, N., Yokoyama, R., Yamamoto, T., Furukawa, J., Iwai, H., Satoh, S. and Nishitani, K. (2015). The matrix polysaccharide (1;3,1;4)-beta-D-glucan is involved in silicon-dependent strengthening of rice cell wall. Plant Cell Physiol 56(2): 268-276.
  4. Yamamoto, T., Nakamura, A., Iwai, H., Ishii, T., Ma, J. F., Yokoyama, R., Nishitani, K., Satoh, S. and Furukawa, J. (2012). Effect of silicon deficiency on secondary cell wall synthesis in rice leaf. J Plant Res 125(6): 771-779.

简介

硅(Si)对于Poales(Yamamoto等人,2011; Kido等人,2015年)的植物是重要的生物元素。 在稻中,Si主要沉积在运动细胞和叶表皮的细胞壁中。 然而,该整个方法的分子基础尚未阐明。 因此,我们提出了用于基于硅烷醇基团和结晶紫内酯的羧酸酯基团之间的特异性氢键的二氧化硅体的组织化学染色的方案(Ichimura等人,2008),如Isa (2010),但稍作修改。 这个修改的协议可以用于观察水稻开发过程中的硅积累。

关键字:大米, 细胞壁, 硅体, 结晶紫内酯, 运动细胞

材料和试剂

  1. 在生长室中,在补充有1.5mM SiO 3 3+的液体培养基中培养水稻(Orysa sativa L.cv。Nipponbare)植物。在28℃,15/9h光/暗循环下(150μmol<-2μm -1 的光)。该方案在叶龄5.2(Kido等人,2015)下用第五叶叶进行。叶年龄"mn "定义为其中第m个叶完全扩展并且第(m + 1)叶是正在开发,其长度为完全展开长度的长度 n/ 10
  2. 多聚甲醛(Wako Pure Chemical Industries,目录号:162-16065)
  3. 二甲胂酸钠缓冲液(Nacalai Tesque,目录号:37238-25)
  4. 琼脂粉(Nacalai Tesque,目录号:01028-85)
  5. 磷酸盐缓冲盐水(PBS)(Takara Bio,目录号:T900)
  6. 乙醇(Wako Pure Chemical Industries,目录号:057-00456)
  7. 分子筛(Sigma-Aldrich,目录号:M6141)
  8. 苯(Nacalai Tesque,目录号:04017-35)
  9. 结晶紫内酯(Tokyo Chemical Industry UK Ltd,目录号:C0741)
  10. PBS片(Takara Bio,目录号:T900)
  11. 磷酸盐缓冲盐水(PBS)(见配方)
  12. 100%乙醇溶液(见配方)
  13. 水晶紫内酯溶液(见配方)

设备

  1. 生长室(Nippon Medical& Chemical Instruments,型号:LH220S)
  2. Stirrer热板(Thermo Fisher Scientific,型号:Fisher Scientific Isotemp)
  3. 膜片真空泵(Leybold-Heraeus,型号:Divac 2.2L)
  4. 干燥器(SANPLATEC,型号:PC-250K)
  5. 微波炉(Sharp Electronics,型号:RE-T12)
  6. 石蜡皿(Greiner Bio-One GmbH,目录号:908177)
  7. Leica VT1200S振动刀片切片机(Leica Microsystems,型号:VT1200S)
  8. 显微镜载片(Matsunami Glass,目录号:S-2123)
  9. 盖玻片(Matsunami Glass,目录号:C024361)
  10. 光学显微镜(Leica Microsystems,型号:DMRPX)
  11. CCD相机(QImaging,型号:Retiga EXi)

程序

  1. 通过将0.4g多聚甲醛粉末溶解在10ml的20mM的二甲胂酸钠缓冲液(pH 7.4)中制备固定剂。将该溶液在加热板/搅拌器上的通风橱中加热至约70℃,直到溶液完全澄清,并使其冷却。固定剂混合物应在使用前立即制备
  2. 将稻叶切成长约2-3mm的片(图1A),并立即将其浸入固定剂中(切割的组织将漂浮在固定剂上)。
  3. 将组织/固定剂置于干燥器中,其连接至隔膜真空泵。在非常缓慢地释放真空之前,使用真空泵在室温下将组织/固定剂真空脱气5分钟。再次拉出并释放真空,直到组织下沉。
  4. 在4℃,无真空下孵育过夜
  5. 使用微波炉将5.0g琼脂粉末溶解在100ml PBS中。将熔融的琼脂培养基置于加热板/搅拌器上,其必须预热至60℃。
  6. 删除固定剂。用PBS冲洗组织三次。将组织倒入石蜡皿中。
  7. 用熔化的琼脂培养基覆盖石蜡皿。将叶片组织排列成规则阵列(图1B和图2A)。让琼脂固化至少15分钟(图1C)。
  8. 使用一滴瞬间胶(Konishi Aron Alpha)(图1D-E和图2B)将其中切割的叶片组织块竖立放置的琼脂块胶化到Leica VT1200S振动刀片切片机的台上(图1D-E和图2B)。 />
  9. 切下琼脂嵌入组织横向,设置在琼脂块,与振动器70微米的厚度(图1F-H和图2C)。用镊子收集PBS中的横切面

    图1.剖面准备。 A.用刀片切割第五片叶片。是。将稻叶片组织片块嵌入琼脂中。 F-H.用装有刀片的振动器切片。


    图2.切片准备的示意图。将水稻叶片组织包埋在琼脂(A)中,将琼脂块胶合在Leica VT1200S振动刀片切片机(B)的台上。将琼脂包埋的组织平行于载物台的表面切割(如C中的虚线所示)
  10. 转移切片,并在连续脱水处理中用0.5ml 70%,80%,90%和100%乙醇孵育30分钟,每个溶液不摇动。
  11. 然后将切片转移并在每个步骤中以递增浓度的苯在乙醇中的温度孵育30分钟,范围从10%至100%,以10%逐步增加,直到溶剂被苯替代。
  12. 将苯平衡的切片用0.1%结晶紫内酯溶液染色10分钟以显现硅化细胞。
  13. 将染色的部分放在显微镜载玻片上,轻轻盖上盖玻片。用指甲油密封盖玻片。
  14. 使用光学显微镜观察切片并使用CCD照相机记录图像(图2)

代表数据


图3.在叶龄5.2时,水稻叶片横断面的结晶紫内酯染色。 cs:皮层鳞状细胞,pc:薄壁组织。 mc:运动细胞。比例尺=50μm

笔记

在28℃,在15/9h光/暗循环(150μmol-2μm)下,在生长室中生长水稻(水稻,日本晴> s -1 )。该方案使用在+ Si条件下生长的水稻植物的第五叶叶进行(Kido等人,2015)。

食谱

  1. PBS
    将10个PBS片溶于蒸馏水中,使总体积为1000ml
  2. 100%乙醇溶液
    为了制备100%乙醇溶液,在瓶子底部使用100%含有分子筛的大量乙醇
  3. 结晶紫内酯溶液
    将结晶紫内酯溶解在0.1%的苯溶液中

致谢

该协议已经由Isa等人(2010)的先前研究调整或修改。本研究由日本科学促进会(JSPS)和教育,文化,体育科学技术部(MEXT),日本[创新地区科学研究资助"植物细胞壁"(No.24114001,24114005)和用于科学研究的Grant-in-Aid(C)(25440124)。

参考文献

  1. Ichimura,K.,Funabiki,A.,Aoki,K.and Akiyama,H。(2008)。 结晶紫内酯对二氧化硅纳米粒子的固相吸附以探测机械化学表面改性。 em> Langmuir 24(13):6470-6479。
  2. Isa,M.,Bai,S.,Yokoyama,T.,Ma,J.F.,Ishibashi,Y.,Yuasa,T.,Iwaya-Inoue,M。(2010)。 硅在低二氧化硅水稻突变体中促进生长而与二氧化硅沉积无关, lsi1 。 植物土壤 331(1-2):361-375。
  3. Kido,N.,Yokoyama,R.,Yamamoto,T.,Furukawa,J.,Iwai,H.,Satoh,S.and Nishitani,K。 基质多糖(1; 3,1; 4)-β-D-葡聚糖参与硅依赖性强化稻细胞壁。植物细胞生理学56(2):268-276。
  4. Yamamoto,T.,Nakamura,A.,Iwai,H.,Ishii,T.,Ma,J.F.,Yokoyama,R.,Nishitani,K.,Satoh,S.and Furukawa,J.(2012)。 缺硅对水稻次生细胞壁合成的影响 Plant Res 125(6):771-779。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yokoyama, R., Kido, N., Yamamoto, T., Furukawa, J., Iwai, H., Satoh, S. and Nishitani, K. (2015). Histochemical Staining of Silica Body in Rice Leaf Blades. Bio-protocol 5(19): e1609. DOI: 10.21769/BioProtoc.1609.
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