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Histochemical Detection of Zn in Plant Tissues
植物组织中锌的组织化学检测

Ilya SereginIlya Seregin*Anna KozhevnikovaAnna Kozhevnikova* Henk SchatHenk Schat (*contributed equally to this work)
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Abstract

Accumulation of metals in plant tissues, and occasionally, different cells of the same tissue, may be highly non-uniform (Seregin and Kozhevnikova, 2008). Easy-to-use histochemical methods may greatly help to investigate the distribution and accumulation of metals within and among plant tissues, and also provide information on their subcellular localization (Seregin and Kozhevnikova, 2011). The histochemical techniques of zinc (Zn) visualization are based on the formation of the blue-colored complex of Zn with the metallochrome indicator Zincon (C20H15N4NaO6S), or the green-fluorescent complex with Zinpyr-1 (C46H36Cl2N6O5) (Seregin et al., 2011; Seregin and Kozhevnikova, 2011). A method for histochemical Zn detection in plant tissues using Zinpyr-1 was first proposed by Sinclair et al. (2007), and later modified by Seregin et al. (2011), and Seregin and Kozhevnikova (2011). Histochemical data supplement the results of quantitative analysis, thus allowing a detailed study of the distribution, accumulation, and translocation pathways of Zn within the plant, which are important topics in modern plant physiology. These histochemical techniques have been successfully applied in different plant species, for example Zea mays (Seregin et al., 2011), Noccaea caerulescens and Thlaspi arvense (Kozhevnikova et al., 2014a), Capsella bursa-pastoris and Lepidium ruderale (Kozhevnikova et al., 2014b), in which Zn was detected in different root and shoot tissues. Here, we present the full staining protocols for these methods, developed or modified in our lab (Seregin and Kozhevnikova, 2011; Kozhevnikova et al., 2014a; Kozhevnikova et al., 2014b).

Keywords: Zinc localization(锌的定位), Zincon(锌试剂), Zinpyr-1(zinpyr-1), Histochemical techniques(组织化学技术), Plant tissues(植物组织)

Materials and Reagents

  1. Staining with Zinpyr-1 (see Recipes)
    1. Zinpyr-1 or 4′, 5′-Bis[bis(2-pyridylmethyl)aminomethyl]-2′,7′-dichlorofluorescein (C46H36Cl2N6O5, Mr= 823.72) (Sigma-Aldrich, catalog number: 40667 or Millitech, catalog number: ZP1 )
    2. Dimethyl sulfoxide or DMSO (C2H6OS)
    3. Ethylenediaminetetraacetic acid (disodium salt) (Na2EDTA, C10H14O8N2Na2⋅2H2O, Mr = 372.24)
    4. Super demineralized water

  2. Staining with Zincon (see Recipes)
    1. Zincon sodium salt (C20H15N4NaO6S, Mr = 462.4) (Sigma-Aldrich, catalog number: 201332 )
    2. Borax (Na2B4O7⋅10 H2O)
    3. Sodium hydroxide (NaOH)
    4. Ethylenediaminetetraacetic acid, disodium salt (Na2EDTA, C10H14O8N2Na2⋅2H2O, Mr = 372.24)
    5. Super demineralized water

Equipment

  1. Light microscope with a color digital camera attachment (staining with Zincon); confocal microscope or fluorescence microscope with appropriate filters and digital camera attachment (staining with Zinpyr-1; see below for spectral characteristics of the dye)
  2. Micro pipettes (100-1,000 µl) and pipette tips
  3. Vortex
  4. Precision balances
  5. Razor blades
  6. 50 ml, 100 ml and 1 L flasks
  7. 2 ml microtubes
  8. 5 ml or 15 ml centrifuge tubes
  9. Heat-resistant 20 ml flask
  10. Microscope slides and cover glasses
  11. Tweezers
  12. Dissecting needles
  13. Magnetic stirrer with heating
  14. Axio Imager Z2 microscope (ZEISS)

Procedure

  1. Preparation of plant materials
    To get rid of the metal absorbed on the root surface, the roots should be incubated in Na2-EDTA (20 mM) for 10 min and then rinsed in demineralized water prior to the analysis. To prepare 20 mM Na2-EDTA solution, dissolve 7.44 g of Na2-EDTA in 1 L of demineralized water.

  2. Staining procedure
    1. Make series of sections of the examined plant material on a glass slide using a safety razor blade. The optimal thickness is about 1-2 intact cells, but it depends on plant material. Leaf epidermis can be peeled with tweezers. It is important to use fresh living plant material. It is not useful to analyze the zinc distribution in fixed plant tissue owing to potential redistribution or wash-out of part of the cellular Zn during the procedure of chemical fixation. For example, large losses (up to 75-80%) of loosely bound Zn from roots to the fixative and dehydrating solutions during tissue preparation by conventional fixation for electron microscopy have been documented by Davies et al. (1991).
    2. Add 3-4 drops of analytical reagent.
    3. Cover the sections or peels with a cover glass. If needed, remove excessive reagent with filter paper. If during the examination the reagent dries out on the slide, it should be added under the cover glass.

  3. Microscopy

    Staining with Zinpyr-1
    During the incubation keep the preparations in the dark. Preparations can be examined under a confocal scanning fluorescent light microscope, or fluorescent light microscope after 15-60 min (depending on the size and amount of plant material). The excitation and emission maxima of Zinpyr-1 are both within the visible spectrum: 490 and 525 nm, respectively. We used filter set 38 for the Axio Imager Z2 microscope, with excitation wavelength range 450-490 nm and emission wavelength range 500-550 nm. In order to avoid background fluorescence, it is important to remove the Zinpyr-1 solution from the slides after the treatment using filter paper and substitute it with superdemineralized water. The location of Zn in plant tissues is indicated by the green fluorescence of the Zn-Zinpyr-1 complex (Figure 1 A-C).

    Staining with Zincon
    Preparations can be examined under the light microscope after 5-15 min (depending on the size and amount of plant material). Preparations cannot be stored longer than a couple of hours. The location of Zn in plant tissues is indicated by the blue color of the Zn-Zincon complex (Figure 1 D-G).

Representative data



Figure 1. Zn localization in plant tissues using Zinpyr-1 (A-C) and Zincon (D-G). A. Root section, B. Root, C. Leaf petiole section of Capsella bursa-pastoris exposed to 20 µM Zn for 8 weeks; D. Root section, E-F. Leaf sections, G. Leaf epidermal peel of Noccaea caerulescens exposed to 800 µM Zn for 8 weeks. Green fluorescence indicates the location of the Zn-Zinpyr-1 complex, blue colour indicates the location of the Zn-Zincon complex. Designations: C-cortex; E-endodermis; Ep-epidermis; GC-stomata guard cells; M-mesophyll; P-pericycle; Ph-phloem; R-rhizodermis; RH-root hairs; SC-subsidiary cell; VB-vascular bundle; WSC-water-storage epidermal cell; X-xylem. Bar: 10 µm (A, D-G); 50 µm (B, C)

Video 1. The procedure of sample preparation and staining with Zincon dye

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Notes

  1. To prove that green fluorescence observed after staining with Zinpyr-1 or blue coloring observed after staining with Zincon are Zn-dependent indeed, tissue sections can be incubated in a 1mM-solution of TPEN [N, N, N', N'-tetrakis (2-pyridylmethyl)ethane-1, 2-diamine] for 2 h at room temperature prior to staining with Zinpyr-1 or Zincon. TPEN is a chelator capable of penetrating cell membranes and showing high affinity and specificity toward Zn ions. Treatment of samples with TPEN should lead to almost complete absence of Zn-dependent green fluorescence after subsequent treatment with Zinpyr-1 or absence of blue staining after subsequent treatment with Zincon.
  2. The intensity of staining corresponds with the level of Zn accumulation in cells and tissues. Therefore, it is possible to semi-quantitatively estimate and compare the Zn contents within and among sections on a per unit area basis. The Zn contents of cells of different tissues can be compared only if the cell sizes in these tissues are similar. In particular, when Zn distribution in the growing parts of plants is analyzed, one should take into consideration that the reduced staining intensity of elongating cells, as compared with meristematic cells, may point to a decrease in the Zn content in elongating cells only on a per unit volume basis. The Zn content per cell may be the same or even higher as a result of ongoing Zn uptake during the elongation phase.
  3. In our hands, Zincon staining was visible at Zn concentrations of 10 μM or higher. The threshold Zn concentration of the fluorescence method (Zinpyr-1), as determined by analysis of the emission spectrum of Zinpyr-1 at various Zn concentrations, was 1 nM. One should bear in mind that the lack of coloration of certain tissues or organs only means that the Zn content in the tissues is below the detection limit of the histochemical method. However, plants can accumulate Zn at concentrations that are one or more orders of magnitude higher than in their nutrient or soil solution. Therefore, even when the Zn concentration in the medium is identical to or below the detection limit of these histochemical methods, the Zn content within the plants will most likely be sufficient for detection.

Recipes

  1. Staining with Zinpyr-1
    1. To prepare 1 mM Zinpyr-1 stock solution dissolve 1 mg of Zinpyr-1 in 500 µl of DMSO, stir it well using a vortex mixer, pipette the solution into a 2 ml microtube and add 714 µl of DMSO. Vortex it well again. Make 50 µl aliquots of Zinpyr-1 stock solution and store them in a freezer at -20 °C.
    2. Directly prior to the analysis, dilute an aliquot of Zinpyr-1 DMSO stock solution (1 mM) to a final concentration of 5 or 10 μM with superdemineralized water. To prepare 5 ml of 10 μM Zinpyr-1 working solution dissolve 50 µl of Zinpyr-1 DMSO stock solution in 4.95 ml of superdemineralized water. Alternatively, to prepare 5 ml of 5 μM Zinpyr-1 working solution dissolve 25 µl of Zinpyr-1 DMSO stock solution in 4.975 ml of superdemineralized water.
  2. Staining with Zincon
    1. To prepare 50 ml of 1 M NaOH stock solution dissolve 2 g of NaOH in 50 ml of superdemineralized water.
    2. To prepare 10 ml of working solution dissolve 0.0065 g of Zincon and 0.1906 g of borax in 9 ml of superdemineralized water. Add 0.2 ml of 1 M NaOH and adjust the volume with superdemineralized water to 10 ml.
    3. Heat up the solution to 80-90 °C on a magnetic stirrer with heating and then cool down to room temperature. The solution may be stored for a week in darkness at room temperature.

Acknowledgments

This work was supported by the Russian Foundation for Basic Research (RFBR, # 11-04-00513, 15-04-02236). We thank Prof. V. B. Ivanov and Dr. A. Voronkov for fruitful discussions. The protocol for Zn staining with Zinpyr-1 was modified from the work of Dr. S. A. Sinclair and co-authors (2007).

References

  1. Davies, K. L., Davies, M. S. and Francis, D. (1991). Zinc-induced vacuolation in root meristematic cells of Festuca rubra L. Plant Cell Environ 14(4): 399-406.
  2. Kozhevnikova, A. D., Seregin, I. V., Erlikh, N. T., Shevyreva, T. A., Andreev, I. M., Verweij, R. and Schat, H. (2014a). Histidine-mediated xylem loading of zinc is a species-wide character in Noccaea caerulescens. New Phytol 203(2): 508-519.
  3. Kozhevnikova, A. D., Erlikh, N. T., Zhukovskaya, N. V., Obroucheva, N. V., Ivanov, V. B., Belinskaya, A. A., Khutoryanskaya, M. Y. and Seregin, I. V. (2014b). Nickel and zinc effects, accumulation and distribution in ruderal plants Lepidium ruderale and Capsella bursa-pastoris. Acta Physiologiae Plantarum 36(12): 3291-3305.
  4. Seregin, I. and Kozhevnikova, A. (2008). Roles of root and shoot tissues in transport and accumulation of cadmium, lead, nickel, and strontium. Russ J Plant Physiol 55(1): 1-22.
  5. Seregin, I. and Kozhevnikova, A. (2011). Histochemical methods for detection of heavy metals and strontium in the tissues of higher plants. Russ J Plant Physiol 58(4): 721-727.
  6. Seregin, I., Kozhevnikova, A., Gracheva, V., Bystrova, E. and Ivanov, V. (2011). Tissue zinc distribution in maize seedling roots and its action on growth. Russ J Plant Physiol 58(1): 109-117.
  7. Sinclair, S. A., Sherson, S. M., Jarvis, R., Camakaris, J. and Cobbett, C. S. (2007). The use of the zinc‐fluorophore, Zinpyr‐1, in the study of zinc homeostasis in Arabidopsis roots. New Phytol 174(1): 39-45.

简介

植物组织中金属的累积,以及偶尔,相同组织的不同细胞,可能是高度不均匀的(Seregin和Kozhevnikova,2008)。易于使用的组织化学方法可以极大地帮助调查金属在植物组织内和之间的分布和积累,并提供其亚细胞定位的信息(Seregin和Kozhevnikova,2011)。锌(Zn)可视化的组织化学技术基于Zn与金属色素指示剂Zincon(C 20 H 15 SO 3)的蓝色络合物的形成, 4 NaO 6 S)或具有Zincpyr-1(C 46 NH 36)的绿色 - 荧光复合物, (Seregin等人,2011; Seregin和Kozhevnikova,2011)。使用Zinpyr-1的植物组织中组织化学Zn检测的方法首先由Sinclair等人(2007)提出,并且稍后由Seregin等人(2011)修改。 ,和Seregin和Kozhevnikova(2011)。组织化学数据补充定量分析的结果,从而允许详细研究植物中Zn的分布,积累和易位途径,这是现代植物生理学中的重要议题。这些组织化学技术已经成功地应用于不同的植物物种中,例如玉米(Seregin等人,2011),玉米螟(Noccaea caerulescens)和 Thlaspi arvense (Kozhevnikova ,2014a), bursa-pastoris 和 Reperale (Kozhevnikova et al。,2014b),其中在不同的根和芽组织中检测到Zn。在这里,我们提出这些方法的全染色方案,在我们的实验室中开发或修改(Seregin和Kozhevnikova,2011; Kozhevnikova等人,2014a; Kozhevnikova等人 ,2014b)。

关键字:锌的定位, 锌试剂, zinpyr-1, 组织化学技术, 植物组织

材料和试剂

  1. 用Zinpyr-1染色(参见配方)
    1. 金属吡啶-I或4' 5'-双[双(2-吡啶基甲基)氨基甲基] -2',7'-二氯荧光素 (C 46 H 36 N 6 Cl 2 N 6 6 O 5,Mr = 823.72 )(Sigma-Aldrich,目录号:40667或 Millitech,目录号:ZP1)
    2. 二甲基亚砜或DMSO(C 2 H 6 OS)
    3. 乙二胺四乙酸(二钠盐)(Na 2 EDTA,C 10 H 14 O 8 S N 2 2 2h <2 O,Mr = 372.24)
    4. 超级软化水

  2. 用Zincon染色(见配方)
    1. 锌指钠盐(C 20 H 15 N 15 Si 4 NaO 6 S,Mr = 462.4)(Sigma-Aldrich ,目录号:201332)
    2. 硼砂(Na 2 B 4 B 4 O 7·10 H 2 O)
    3. 氢氧化钠(NaOH)
    4. 乙二胺四乙酸二钠盐(Na 2 EDTA,C 10 H 14 O 8 S N 2,/sub> 2 sub 2 H sub 2 O,Mr = 372.24)
    5. 超级软化水

设备

  1. 光学显微镜与彩色数码相机附件(用Zincon染色); 共聚焦显微镜或荧光显微镜,具有适当的过滤器和数码相机附件(用Zinpyr-1染色;关于染料的光谱特性参见下文)
  2. 微量移液器(100-1,000μl)和移液器吸头
  3. 涡流
  4. 精确余额
  5. 剃刀刀片
  6. 50ml,100ml和1L烧瓶中
  7. 2 ml微量管
  8. 5ml或15ml离心管
  9. 耐热20ml烧瓶
  10. 显微镜载玻片和盖玻璃
  11. 镊子
  12. 解剖针
  13. 带加热的磁力搅拌器
  14. Axio Imager Z2显微镜(ZEISS)

程序

  1. 植物材料的制备
    为了去除在根表面上吸收的金属,根应该在Na 2 EDTA-EDTA(20mM)中温育10分钟,然后在分析前在软化水中漂洗。 为了制备20mM Na 2 EDTA溶液,将7.44g Na 2 EDTA溶解在1L软化水中。

  2. 染色程序
    1. 在玻璃载玻片上制作系列检查植物材料的切片 使用安全剃刀刀片。 最佳厚度约为1-2完整 细胞,但它取决于植物材料。 叶片表皮可以剥离 用镊子。 重要的是使用新鲜的活植物材料。 它是   不利于分析固定植物组织中锌的分布   到部分细胞Zn的潜在再分布或洗出 在化学固定的过程中。 例如,大损失(up   至75-80%)从根到固定剂的松散结合的锌 脱水溶液在组织制备期间通过常规固定   对于电子显微镜已经由Davies等人(1991)记录。
    2. 加入3-4滴分析试剂。
    3. 用盖玻片覆盖这些部分或皮。 如果需要,请删除 过量试剂与滤纸。 如果在考试期间 试剂在载玻片上干燥,应将其加到盖子下面 玻璃。

  3. 显微镜

    使用Zinpyr-1染色
    在孵化期间保持在黑暗中的制剂。可以在共聚焦扫描荧光显微镜或荧光显微镜下在15-60分钟后检查制剂(取决于植物材料的大小和量)。 Zinpyr-1的激发和发射最大值都在可见光谱内:490和525nm。我们使用Axio Imager Z2显微镜的滤光片组38,激发波长范围为450-490nm,发射波长范围为500-550nm。为了避免背景荧光,重要的是在使用滤纸处理后用载玻片除去Zinpyr-1溶液,并用超级矿物质的水代替。植物组织中Zn的位置由Zn-Zinpyr-1复合物的绿色荧光指示(图1A-C)。

    使用Zincon染色
    可以在光学显微镜下在5-15分钟后检查制剂(取决于植物材料的大小和量)。准备时间不能超过几个小时。植物组织中Zn的位置由Zn-Zincon复合物的蓝色指示(图1D-G)。

代表数据



图1.使用Zincpyr-1(AC)和Zincon(DG)的Zn在植物组织中的定位。 A.根部,B.根,C.叶片的叶柄部分暴露于20μMZn 8周; D.根部,
使用Zinpyr-1染色
在孵化期间保持在黑暗中的制剂。可以在共聚焦扫描荧光显微镜或荧光显微镜下在15-60分钟后检查制剂(取决于植物材料的大小和量)。 Zinpyr-1的激发和发射最大值都在可见光谱内:490和525nm。我们使用Axio Imager Z2显微镜的滤光片组38,激发波长范围为450-490nm,发射波长范围为500-550nm。为了避免背景荧光,重要的是在使用滤纸处理后用载玻片除去Zinpyr-1溶液,并用超级矿物质的水代替。植物组织中Zn的位置由Zn-Zinpyr-1复合物的绿色荧光指示(图1A-C)。

使用Zincon染色
可以在光学显微镜下在5-15分钟后检查制剂(取决于植物材料的大小和量)。准备时间不能超过几个小时。植物组织中Zn的位置由Zn-Zincon复合物的蓝色指示(图1D-G)。

代表数据



图1.使用Zincpyr-1(AC)和Zincon(DG)的Zn在植物组织中的定位。 A.根部,B.根,C.叶片的叶柄部分暴露于20μMZn 8周; D.根部,...

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Notes

  1. To prove that green fluorescence observed after staining with Zinpyr-1 or blue coloring observed after staining with Zincon are Zn-dependent indeed, tissue sections can be incubated in a 1mM-solution of TPEN [N, N, N', N'-tetrakis (2-pyridylmethyl)ethane-1, 2-diamine] for 2 h at room temperature prior to staining with Zinpyr-1 or Zincon. TPEN is a chelator capable of penetrating cell membranes and showing high affinity and specificity toward Zn ions. Treatment of samples with TPEN should lead to almost complete absence of Zn-dependent green fluorescence after subsequent treatment with Zinpyr-1 or absence of blue staining after subsequent treatment with Zincon.
  2. The intensity of staining corresponds with the level of Zn accumulation in cells and tissues. Therefore, it is possible to semi-quantitatively estimate and compare the Zn contents within and among sections on a per unit area basis. The Zn contents of cells of different tissues can be compared only if the cell sizes in these tissues are similar. In particular, when Zn distribution in the growing parts of plants is analyzed, one should take into consideration that the reduced staining intensity of elongating cells, as compared with meristematic cells, may point to a decrease in the Zn content in elongating cells only on a per unit volume basis. The Zn content per cell may be the same or even higher as a result of ongoing Zn uptake during the elongation phase.
  3. In our hands, Zincon staining was visible at Zn concentrations of 10 μM or higher. The threshold Zn concentration of the fluorescence method (Zinpyr-1), as determined by analysis of the emission spectrum of Zinpyr-1 at various Zn concentrations, was 1 nM. One should bear in mind that the lack of coloration of certain tissues or organs only means that the Zn content in the tissues is below the detection limit of the histochemical method. However, plants can accumulate Zn at concentrations that are one or more orders of magnitude higher than in their nutrient or soil solution. Therefore, even when the Zn concentration in the medium is identical to or below the detection limit of these histochemical methods, the Zn content within the plants will most likely be sufficient for detection.

食谱

  1. 用Zinpyr-1染色
    1. 为了制备1mM的Zincpyr-1储备溶液,将500mg的1mg的Zinpyr-1溶解   μl的DMSO,使用涡旋混合器充分搅拌,吸取溶液 加入2ml微量管中,加入714μlDMSO。 再次涡旋。 使   将50μl等份的Zinpyr-1储备溶液并储存在冷冻箱中 在-20℃
    2. 在分析之前,稀释等分试样 Zinpyr-1 DMSO储备溶液(1mM)至终浓度为5或10 μM的超级矿物水。 制备5ml10μM的Zincpyr-1 工作溶液溶解50μl在4.95中的Zinpyr-1 DMSO储备溶液 ml超顺磁水。 或者,制备5ml的5μM Zinpyr-1工作溶液溶解25μl的Zinpyr-1 DMSO储备溶液   在4.975ml超高温矿化水中。
  2. 用Zincon染色
    1. 为了制备50毫升1M NaOH储备溶液,将2克NaOH溶解在50毫升超纯矿泉水中。
    2. 为了制备10ml的工作溶液,溶解0.0065g的Zincon和   0.1906g硼砂在9ml超高温矿化水中的溶液。 加入0.2ml 1M   NaOH并用超纯水调节体积至10ml
    3. 在加热的磁力搅拌器上将溶液加热至80-90℃ 然后冷却至室温。 可以存储该解   周在黑暗中室温。

致谢

这项工作是由俄罗斯基础研究基金会(RFBR,#11-04-00513,15-04-02236)的支持。 我们感谢V. Ivanov教授和A. Voronkov博士进行富有成果的讨论。 使用Zinpyr-1的Zn染色的方案从S.A.Sincclair博士和合作者的工作(2007)修改。

参考文献

  1. Davies,K.L.,Davies,M.S.and Francis,D。(1991)。 锌诱导的根分生细胞中的空泡化 Festuca rubra L. Plant Cell Environ 14(4):399-406。
  2. Kozhevnikova,A.D.,Seregin,I.V.,Erlikh,N.T.,Shevyreva,T.A.,Andreev,I.M.,Verweij,R.and Schat,H。(2014a)。 组氨酸介导的木质部锌负载是一种物种范围的特征,在N藜(Noccaea caerulescens) New Phytol 203(2):508-519。
  3. Kozhevnikova,A.D.,Erlikh,N.T.,Zhukovskaya,N.V.,Obroucheva,N.V.,Ivanov,V.B.,Belinskaya,A.A.,Khutoryanskaya,M.Y.and Seregin,I.V。(2014b)。 镍和锌的影响,在ruderal植物中的积累和分布 Lepidium ruderale 36(12):3291-3305。
  4. Seregin,I。和Kozhevnikova,A。(2008)。 根和芽组织在镉,铅,镍和锶的运输和积累中的作用。 Russ J Plant Physiol 55(1):1-22。
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引用:Seregin, I., Kozhevnikova, A. and Schat, H. (2015). Histochemical Detection of Zn in Plant Tissues. Bio-protocol 5(10): e1470. DOI: 10.21769/BioProtoc.1470.
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