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An Assay to Test Manganese Tolerance in Arabidopsis
拟南芥的耐锰实验分析   

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Abstract

Manganese (Mn) is an essential nutrient required for the catalytic or regulatory function of several cellular enzymes. However, excessive Mn concentrations in plant tissues are toxic to plant cells as they negatively affect enzymatic activities, lead to oxidative stress and disturb the uptake and distribution of other essential mineral elements (Ca, P, Mg or Fe). Plants have developed multiple mechanisms to avoid heavy metals (including Mn) toxicity, including transport across the plasma membrane or tonoplast. The genes encoding transporters involved in Mn detoxification are now being identified in different plant species, and functional characterization of genes isolated from species can be easily carried out in Arabidopsis.
Here we provide a method to evaluate the tolerance to excess Mn of Arabidopsis lines transformed with empty vector pMDC43 or the same vector carrying cucumber gene CsMTP8 encoding putative manganese transporter localized in the vacuolar membrane. We analyzed the growth and developmental phenotypes of plants grown in controlled conditions (phytotrone) on sterile plates containing different concentrations of MnSO4 during a 16 days period. Mn accumulation was measured in the same plants grown in liquid medium supplemented or not (control) with toxic Mn concentration.

Keywords: Heavy metals(重金属), Arabidopsis thaliana(拟南芥), Manganese(锰), Atomic Absorption Spectrometry(原子吸收光谱法)

Materials and Reagents

  1. The seeds of Arabidopsis thaliana ecotype Columbia (Col-0) transformed with empty vector (pMDC43) or with vector pMDC43 carrying the coding sequence of CsMTP8 gene under 35S CaM promoter (35S::CsMTP8)
  2. 96% ethanol
  3. 5% sodium hypochlorite (NaClO) (commercial detergent ACE produced by Procter and Gamble)
  4. Sterile water
  5. 1 M KOH (for pH establishment)
  6. Murashige & Skoog Basal Medium (MS) (Sigma-Aldrich, catalog number: M5519 )
  7. Phytagel (Sigma-Aldrich, catalog number: P8169 )
  8. Salts for media preparation:
    Ca(NO3)2 (POCH, catalog number: 874582797 )
    MgSO4.7H2O (POCH, catalog number: 613780111 )
    KH2PO4 (POCH, catalog number: 742020112 )
    K2HPO4 (POCH, catalog number: 742100117 )
    HNO3 (POCH, catalog number: 5296041 )
    KNO3 (POCH, catalog number: 738910115 )
    FeSO4.7H2O (POCH, catalog number: 902840115 )
    MnSO4.H2O (POCH, Poland, catalog number: 616940119 )
    H3BO3 (POCH, catalog number: 531360115 )
    CuSO4.5H2O (POCH, catalog number: ACRS42361 )
    ZnSO4.7H2O (POCH, catalog number: Ph. Eur. 6-265762730 )
    (NH4)6Mo7O24.4H2O (POCH, catalog number: 139000115 )
    Na2EDTA.2H2O (BioShop, catalog number: OM19432 )
  9. Medium composition for growing plants on plates (see Recipes)
  10. Medium composition for growing plants in liquid solution (see Recipes)
  11. 10 mM Na2EDTA (see Recipes)
  12. 65% HNO3 (see Recipes)

Equipment

  1. Square (120 x 120 mm) petri dishes polystyrene sterile
  2. Growth chamber or phytotrone (16/8 h photoperiod at 250 μmol·m−2·s−1 and 23 °C during the day and 22 °C during the night)
  3. Fume hood
  4. Laminar flow cabinet
  5. Autoclave
  6. Shaker
  7. Forceps
  8. Tubes 1.5 ml (Axygen)
  9. Heating digester with closed vessels (mineralizator) (Kiejdal Digestion Unit DK-20, VELP Scientifica, catalog number: F30100350 )
  10. Atomic absorption spectrophotometer (Perkin Elmer, model: AAS 3300 )
  11. Image capturing device (regular digital SLR-single less reflex camera, e.g. Nikon D40 camera)
  12. Precision balance (± 0.0001)

Procedure

  1. Sterilization and germination of seeds
    1. Seeds (10-15 mg) were surface sterilized in 1.5 ml tubes in a laminar flow cabinet by washing in 3% sodium hypochlorite dissolved in 96% ethanol for 5 min, and then by washing five to six times in 96% ethanol. After sterilization, the seeds were dried in laminar for at least two hours.
    2. Sterilized seeds were sown on Petri dishes (20 seeds per Petri dish: 10 seeds of control seeds with empty vector and 10 seeds carrying CsMTP8) containing 0.5 MS solid medium supplemented or not (control) with 2 mM MnSO4 in laminar flow cabinet (Figure 1A-C). The dishes with seeds were then kept in the dark at 4 °C for 2 days for stratification.


      Figure 1. View of seed sowing on petri dishes. The seeds were sterilized in 1.5 ml tubes A. The sterile plate was placed on a squared paper to facilitate the precise sowing and the seeds were placed along the line marked on the paper. B. The space between seeds was 0.5 cm. C. The view of the petri dish after sowing.

  2. Growth tests of A. thaliana plants in manganese excess
    1. Following stratification, the dishes with seeds were transferred to the culture chamber or phytotrone (22 °C under 16/8 h light/dark photoperiod) and grown for 16 days in vertical position (Figure 2A-B). Then, the plant images were captured using a Nikon D40 camera and the weight of the plants was measured. The experiment was repeated three times with three replicates made for each treatment.


      Figure 2. Example of measured effects of Mn excess on Arabidopsis growth in lines transformed with empty vector or vector carrying CsMTP8 (Migocka et al., 2014). Plants were grown in control 0.5 MS (A) or in 0.5 MS supplemented with 2 mM MnSO4 (B) for 16 days in phytotrone (22 °C under 16/8 h light/dark photoperiod).

    2. Measurement of plant fresh weight. Whole 16 day-old-plants grown in MS medium or MS medium supplemented with 2 mM MnSO4 were collected from a Petri dish using forceps and weighed using a precision balance (Figure 3A-B). Beside the estimation of plant weight, other phenotypes could be assessed in this assay, e.g. root length, leaf number and area or chlorophyll quantity.
      3. The obtained data were statistically analyzed using student's t tests and ANOVA (Excel) (P<0.05) (Figure 3C).


      Figure 3. The estimation of plant fresh weight. 16-day-old plants were carefully taken from petri dish and weighed with a precision balance (A-B). Data are expressed as means ± standard deviations of three independent experiments with at least 30 plants each (C). Different letters indicate significant differences between control and stress conditions (a) or between the line transformed with empty vector and two lines carrying CsMTP8 (b) (P<0.05; ANOVA Student-t tests).

  3. Measurement of manganese accumulation in plants
    1. To assess Mn accumulation in plants, the 16 day-old-plants grown on petri dishes containing control 0.5 MS were carefully transferred into liquid media of the composition described earlier (Morel et al., 2009), supplemented or not with 50 µM MnSO4 and further grown for five days (Figure 4A-B). Briefly, five plants were gently wrapped with a cotton wool and placed into a 15- ml Falcon containing liquid medium. After five days, plants were carefully harvested, washed in 10 mM Na2EDTA (incubated in a Na2EDTA solution for 5 min on bench, Figure 4C) and dried at 50 °C.


      Figure 4. Details of plant preparation for measurement of Mn accumulation. 16-day-old plants were carefully taken from petri dish, wrapped with a cotton wool (A) and transferred into 15-ml Falcon covered with aluminum foil (B; 5 plants per Falcon) containing liquid medium (Morel et al., 2009). After 5 days in phytotrone, the plants were washed in 10 mM Na2EDTA (C) for 5 min and dried.

    2. The dried plants collected from liquid media supplemented or not with MnSO4 were ground to the powder in mortar with pestle.
    3. 100 mg of the ground tissue was placed in the glass vessel (Figure 5A) and then digested overnight with 10 ml of the concentrated (65%) HNO3 at room temperature under the fume hood.
    4. After that, plant samples were boiled in the same solution at 150 °C for 12 h under the fume hood (Figure 5A). The solution was then analyzed by atomic absorbtion spectrophotometry using Perkin Elmer A3300 (Figure 5B). This is a multielement spectrophotometer equipped with cationic lamps (e.g. Mn lamp) which are bought separately. The samples with metal are atomized in a flame and then absorb the specific wavelength emitted by the lamp. However, the elemental analysis by Atomic Absorption Spectrometry provides reliable and reproducible results when the concentration of metal in the samples is relatively high (higher than that met in natural conditions).
    5. The obtained data were statistically analyzed using student's t tests and ANOVA (Excel) (P<0.05) (Figure 5C).


      Figure 5. Details of the analysis of Mn accumulation in Arabidopsis plants. A. Dried plants were digested in closed glass vessels by heating in 10 ml of 65% HNO3 in heating digester at 150 for 12 h. B. Atomic absorbtion spectrophotometer was used to estimate Mn content in the solution following digestion (mineralization). C. The example data are present as means ± standard deviations of three independent experiments with at least 15 plants each. Asterisks indicate significant differences between the line transformed with empty vector and two lines carrying CsMTP8 (b) (P<0.05; ANOVA Student-t tests).

Recipes

  1. Medium composition for growing plants on plates
    1. For 1 L of 0.5 MS control medium
      2.2 g of Murashige & Skoog (MS) medium
      Adjust pH to 5.7 with KOH
      Add 5 g agar
      Sterilized for 20 min at 121 °C/1 atm using an autoclave
    2. For 1 plate of the medium MS supplemented with 2 mM MnSO4
      1 ml of 100 mM MnSO4 solution was added to 50 ml of medium
  2. Medium composition for growing plants in liquid solution
    1. For 1 L of control medium
      Macroelements:
      Ca(NO3)2.4H2O
      189 mg/L
      MgSO4.7H2O
      270 mg/L
      KH2PO4
      100 mg/L
      K2HPO4
      10 mg/L
      KNO3
      200 mg/L
      Microelements:
      FeSO4.7H2O
      5.54 mg/L
      MnSO4.5H2O
      0.59 mg/L
      H3BO3
      0.56 mg/L
      CuSO4.5H2O
      0.195 mg/L
      ZnSO4.7H2O
      0.86 mg/L
      (NH4)6Mo7O24.4H2O
      0.092 mg/L
      Na2EDTA.2H2O
      7.44 mg/L
    2. For 1 L of the medium supplemented with 50 µM MnSO4
      0.5 ml of 100 mM MnSO4 solution was added to the medium
  3. 10 mM Na2EDTA
    3.722 g disodium ethylenediaminetatraacetate-2H2O (Na2EDTA.2H2O, Mw = 372.24) was added to 800 ml of double distilled H2O and stirred vigorously
    The pH was adjusted to 8.0 with NaOH and the volume of the solution was adjusted to 1 L with double distilled water
    The solution was sterilized by autoclaving
    4. 65% HNO3
    65% HNO3 was purchased as a ready-to-use concentrated solution

Acknowledgments

We gratefully acknowledge Dr. Sophie Filleur and Dr. Sébastien Thomine (Institut des Sciences du Végétal (ISV), Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, France) for giving us the opportunity to transform of A. thaliana plants. This work was supported by the Polish Ministry of Science and Higher Education (grant no. IP2010 026470). This protocol was adapted from Migocka et al. (2014).

References

  1. Migocka, M., Papierniak, A., Maciaszczyk-Dziubinska, E., Pozdzik, P., Posyniak, E., Garbiec, A. and Filleur, S. (2014). Cucumber metal transport protein MTP8 confers increased tolerance to manganese when expressed in yeast and Arabidopsis thaliana. J Exp Bot 65(18): 5367-5384.
  2. Morel, M., Crouzet, J., Gravot, A., Auroy, P., Leonhardt, N., Vavasseur, A. and Richaud, P. (2009). AtHMA3, a P1B-ATPase allowing Cd/Zn/Co/Pb vacuolar storage in Arabidopsis. Plant Physiol 149(2): 894-904.

简介

锰(Mn)是几种细胞酶的催化或调节功能所需的必需营养素。然而,植物组织中过量的Mn浓度对植物细胞是有毒的,因为它们对酶活性有负面影响,导致氧化应激并扰乱其他必需矿物元素(Ca,P,Mg或Fe)的摄取和分布。植物已经开发了多种机制以避免重金属(包括Mn)毒性,包括跨质膜或质膜的转运。编码参与Mn解毒的转运蛋白的基因现在在不同的植物物种中被鉴定,并且从物种中分离的基因的功能表征可以在拟南芥中容易地进行。
在这里,我们提供了一种方法来评估用空载体pMDC43或携带黄瓜基因的相同载体 CsMTP8 编码推定的锰转运蛋白的拟南芥线的过量Mn的耐受性定位于液泡膜中。我们分析了在16天期间在含有不同浓度的MnSO 4的无菌平板上在受控条件下生长的植物(植物软体)的生长和发育表型。在相同的植物中测量Mn积累,所述植物在补充有或没有(对照)有毒的Mn浓度的液体培养基中生长

关键字:重金属, 拟南芥, 锰, 原子吸收光谱法

材料和试剂

  1. 用空载体(pMDC43)或携带CsMTP8的编码序列的载体pMDC43转化的拟南芥生态型哥伦比亚( Col-0 基因在35S CaM启动子(<35S :: CsMTP8 )下
  2. 96%乙醇
  3. 5%次氯酸钠(NaClO)(由Procter and Gamble生产的商业洗涤剂ACE)
  4. 无菌水
  5. 1 M KOH(用于pH建立)
  6. Murashige& Skoog基础培养基(MS)(Sigma-Aldrich,目录号:M5519)
  7. Phytagel(Sigma-Aldrich,目录号:P8169)
  8. 介质制备盐:
    (POCH,目录号:874582797)。
    MgSO 4 .7H 2 O(POCH,目录号:613780111)。

    (POCH,目录号:742020112)
    HPO <4>(POCH,目录号:742100117)
    HNO <3>(POCH,目录号:5296041)
    KNO 3 (POCH,目录号:738910115)

    O(POCH,目录号:902840115)

    (POCH,波兰,目录号:616940119)。



    (POCH,目录号:531360115) (POCH,目录号:ACRS42361)
    (POCH,目录号:Ph.Eur.6-265762730)
    (NH 4)6 Mo 7+ O 24+。在本发明的一个实施方案中, O(POCH,目录号:139000115)
    (BioShop,目录号:OM19432)的缓冲液洗涤
  9. 用于在板上种植植物的中等组成(参见配方)
  10. 用于在液体溶液中生长植物的中等组成(参见配方)
  11. 10mM Na 2 EDTA(参见Recipes)
  12. 65%HNO 3 (见配方)

设备

  1. 方形(120×120mm)培养皿聚苯乙烯无菌
  2. 生长室或植物光酮(白天期间在250μmol·m -2 -2℃/s和<23℃下的16/8小时光周期和夜间22℃)
  3. 通风橱
  4. 层流柜
  5. 高压灭菌器
  6. 振动器
  7. 镊子
  8. 管1.5 ml(Axygen)
  9. 具有封闭容器的加热消化器(矿化器)(Kiejdal Digestion Unit DK-20,VELP Scientifica,目录号:F30100350)
  10. 原子吸收分光光度计(Perkin Elmer,型号:AAS 3300)
  11. 图像捕获设备(常规数码单反单反相机,如 Nikon D40相机)
  12. 精密平衡(±0.0001)
  13. (对照)与2mM MnSO 4在层流柜(图1A-C)中。 的 然后将具有种子的培养皿在黑暗中在4℃下保持2天 分层。


    图1.种子在培养皿上播种的视图。 种子在1.5ml试管A中灭菌。将无菌平板置于其上   一个方形纸,以方便精确播种和种子 沿着纸上标记的线放置。 B.种子之间的空间 0.5cm。 C.播种后培养皿的视图。

  • A的生长测试。 thaliana 植物中的锰过量
    1. 分层后,将具有种子的碟转移至 培养室或植物甾醇(22℃,16/8h光照/黑暗) 光周期),并在垂直位置生长16天(图2A-B)。 然后,使用Nikon D40照相机捕获植物图像 测量植物的重量。实验重复三次 次,每次处理重复三次。


      图2。 Mn过量对线的增长的测量效应的例子 拟南芥 用空载体或载有  2014)。植物在对照0.5MS(A)或补充0.5MS中生长 与2mM MnSO 4(B)在植物甾醇中培养16天(22℃,16/8小时 亮/暗光周期)。

    2. 测量植物鲜重。  整个16日龄植物生长在MS培养基或MS培养基中补充 使用镊子从培养皿中收集2mM MnSO 4 使用精密天平称重(图3A-B)。除了估计 的植物重量,可以在该测定中评估其它表型,例如根长度,叶数和面积或叶绿素量。 使用学生t检验和ANOVA(Excel)(P <0.05)(图3C)对获得的数据进行统计分析。


      图3.植物鲜重的估计。16天龄的植物 小心地从培养皿中取出并用精密天平称重 (A-B)。数据表示为平均值±三的标准偏差 每个至少30株植物的独立实验(C)。不同的字母 表明控制和胁迫条件之间的显着差异 (a)或用空载体转化的线和两条线之间 携带CsMTP8(b)(P <0.05; ANOVA Student-t检验)。

  • 植物中锰积累的测量
    1. 为了评估植物中的Mn积累,16天龄植物生长 将含有对照0.5MS的陪替氏培养皿小心地转移入 (Morel等人,2009)的液体培养基, 补充或不补充50μMMnSO 4并进一步生长5天 (图4A-B)。简言之,用棉花轻轻包裹5株植物 羊毛并置于含15-ml Falcon的液体培养基中。五后  天,小心收获植物,在10mM Na 2 EDTA中洗涤 (在Na 2 EDTA溶液中在台上孵育5分钟,图4C)和 在50℃下干燥。


      图4.植物准备的细节 测量Mn积累。仔细采集16天的植物 (A),并转移到用铝箔(B;每个Falcon 5植物)覆盖的15-ml Falcon中,所述培养基含有 液体培养基(Morel等人,2009)。植物甾醇5天后, 将植物在10mM Na 2 EDTA(C)中洗涤5分钟并干燥
    2. 干燥的植物从补充或不补充的液体培养基收集 MnSO 4在研钵中用杵研磨成粉末。
    3. 100 mg   将研磨的组织放置在玻璃容器中(图5A),然后 在室温下用10ml浓缩(65%)HNO 3消化过夜 温度下通风橱。
    4. 之后,植物样品 在通风橱中在150℃下在相同溶液中煮沸12小时 (图5A)。 然后通过原子吸收分析该溶液 使用Perkin Elmer A3300分光光度法(图5B)。 这是一个 装备有阳离子灯的多元件分光光度计(例如Mn 灯)。 具有金属的样品被雾化 然后吸收由灯发射的特定波长。 然而,通过原子吸收光谱法的元素分析 提供可靠和可重复的结果时的浓度 金属在样品中相对较高(高于在自然界遇到的  条件)。
    5. 使用学生的测试和ANOVA(Excel)(P <0.05)(图5C)对所获得的数据进行统计分析。


      图5.拟南芥植物中Mn积累分析的细节。A.干燥的植物在封闭的玻璃容器中通过加热消化  在10ml 65%HNO 3中的加热消化器中在150℃下加热12小时。 B.原子 使用吸收分光光度计估计Mn含量 溶液(矿化)。 C.示例数据为 作为三次独立实验的平均值±标准偏差 每个至少有15株植物。星号表示显着差异  (b)(P <0.05; ANOVA学生t检验)的两条线之间的差异。
  • 食谱

    1. 板材上生长植物的中等组成
      1. 对于1L的0.5MS对照培养基
        2.2g Murashige& Skoog(MS)介质
        用KOH调节pH至5.7 加入5克琼脂
        使用高压灭菌器在121℃/1atm下灭菌20分钟
      2. 对于补充有2mM MnSO 4·7H 2 O的1个平板的培养基MS 将1ml 100mM MnSO 4溶液加入50ml培养基中
    2. 用于在液体溶液中生长植物的培养基组合物
      1. 对于1L对照培养基
        宏元素:
        Ca(NO 3)2 sub 2 4H 2 O
        189 mg/L
        MgSO 4。 。 O
        270 mg/L
        KH 2 PO 4
        100 mg/L
        K 2 HPO 4
        10 mg/L
        KNO 3
        200 mg/L
        微元件:
        FeSO 4 7H <2> O
        5.54 mg/L
        MnSO 4 5H sub 2 O
        0.59 mg/L
        H 3 BO 3
        0.56 mg/L
        CuSO 4 5H sub 2 O
        0.195 mg/L
        ZnSO 4 。 7H O
        0.86 mg/L
        (NH 4)6 Mo 7+ O 24+。在本发明的一个实施方案中, O
        0.092mg/L
        Na EDTA 2H 2 7.44 mg/L
      2. 对于1L补充有50μMMnSO 4
        的培养基 将0.5ml 100mM MnSO 4溶液加入到培养基中
    3. 10mM Na 2 EDTA 3.722g乙二胺四乙酸二钠-2H 2 O(Na 2 EDTA),2H 2 O,Mw = 372.24)为 加入到800ml双蒸水H 2 O中并剧烈搅拌 用NaOH将pH调节至8.0,用双蒸水
      将溶液的体积调节至1L 通过高压灭菌将溶液灭菌
      65%HNO 3
      65%HNO 3作为即用型浓缩溶液购买

    致谢

    我们衷心感谢Sophie Filleur博士和SébastienThomine博士(法国国家科学技术中心(ISV),法国Gif-sur-Yvette中心),让我们有机会 变换。 thaliana 植物。 这项工作得到了波兰科学和高等教育部的支持(授权号IP2010 026470)。 该协议改编自Migocka等人(2014)。

    参考文献

    1. Migocka,M.,Papierniak,A.,Maciaszczyk-Dziubinska,E.,Pozdzik,P.,Posyniak,E.,Garbiec,A.and Filleur,S。(2014)。 黄瓜金属转运蛋白MTP8在酵母中表达时赋予锰增加的耐受性,并且 Arabidopsis thaliana 。 65(18):5367-5384。
    2. Morel,M.,Crouzet,J.,Gravot,A.,Auroy,P.,Leonhardt,N.,Vavasseur,A.and Richaud,P.(2009)。 AtHMA3,一种允许Cd/Zn/Co/Pb液泡储存在拟南芥中的P1B-ATP酶 。植物生理学 149(2):894-904。
    • 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:Migocka, M. and Biskup, R. (2015). An Assay to Test Manganese Tolerance in Arabidopsis. Bio-protocol 5(9): e1460. DOI: 10.21769/BioProtoc.1460.
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