Detection of Alternative Oxidase Expression in Arabidopsis thaliana Protoplasts Treated with Aluminium

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Journal of Experimental Botany
Aug 2014



Aluminium (Al), a non-essential metal widespread in the environment that is known to be toxic to humans as well as to plants, can cause damage not only to the roots but also to the aerial parts of plants. Its toxicity has been recognized as one of the major factors that limit crop production on acid soil. Alternative oxidase, the respiratory terminal oxidase in plants, which contributes to maintain the electron flux and reduce mitochondrial ROS levels, is often dramatically induced to make plants to adapt better to stress conditions like Al stress. In this protocol, the expression of alternative oxidase induced by Al treatment was detected in Arabidopsis protoplasts using an adaptation of previous methods (Yamamoto et al., 2002; Li et al., 2011; Liu et al., 2014), which contribute to research on the mechanism of alternative oxidase in Al treatment.

Materials and Reagents

  1. Rosette leaves of Arabidopsis (Columbia, 3 weeks)
  2. AlCl3 (Sigma-Aldrich, catalog number: 237051 )
  3. CaCl2 (Sigma-Aldrich, catalog number: C7902 )
  4. HCl (Sigma-Aldrich, catalog number: 258148 )
  5. FDA (Sigma-Aldrich, catalog number: 596-09-8 )
  6. TRI reagent (Sigma-Aldrich, catalog number: 93289 )
  7. Cellulase R10 (Yakult Honsha, catalog number: C6260 )
  8. Macerozyme R10 (Yakult Honsha, catalog number: 8032-75-1 )
  9. Mannitol (Sigma-Aldrich, catalog number: M4125 )
  10. MES (Sigma-Aldrich, catalog number: M8250 )
  11. KCl (Sigma-Aldrich, catalog number: P3911 )
  12. Bovine serum albumin (Sigma-Aldrich, catalog number: A-6793 )
  13. NaCl (Sigma-Aldrich, catalog number: S6150 )
  14. Glucose (Sigma-Aldrich, catalog number: G7528 )
  15. Trition X-100 (Sigma-Aldrich, catalog number: T-8787 )
  16. Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: P7626 )
  17. Tris (Sigma-Aldrich, catalog number: T1378 )
  18. SDS (Sigma-Aldrich, catalog number: L6026 )
  19. (NH4)2S2O8 (Sigma-Aldrich, catalog number: A3426 )
  20. TEMED (Sigma-Aldrich, catalog number: T9281 )
  21. Tween-20 (Sigma-Aldrich, catalog number: P9416 )
  22. SuperScript II first-strand synthesis system (Life Technologies, InvitrogenTM, catalog number: 11904-018 )
  23. SYBR Premix Ex Taq (Takara, catalog number: RR420A )
  24. Bio-Rad protein assay kit (Bio-Rad Laboratories, catalog number: 500-0001 )
  25. Anti-AOX antibody (Agrisera, catalog number: AS10699 )
  26. Anti-Rubisco antibody (Agrisera, catalog number: AS03037 )
  27. Anti-rabbit IgG (DylightTM 800 4x PEG Conjugate) secondary antibody (CST, catalog number: 5151 )
  28. Anti-mouse IgG (DylightTM 800 Conjugate) secondary antibody (CST, catalog number: 5257 )
  29. Enzyme solution (see Recipes)
  30. W5 solution (see Recipes)
  31. 5 mM AlCl3 solution in Ca medium
  32. Real time PCR reaction solution (see Recipes)
  33. Lysis buffer (see Recipes)
  34. 10 ml separating gel (see Recipes)
  35. 6 ml stacking gels (see Recipes)
  36. TBST (see Recipes)


  1. pH meter
  2. Thermal Cycler (Roche, model: Light cycler 2.0 )
  3. Confocal laser-scanning microscope (Carl-Zeiss, model: LSM510/ConfoCor2 )
  4. Auto microplate reader (Tecan Trading AG, model: infinite M200 )
  5. Two-color infrared imaging system (Odyssey, model: 9120 )
  6. Centrifuge
  7. Orbital shaker
  8. PVDF membranes (Bio-Rad Laboratories, catalog number: 162-0177 )
  9. Nylon mesh
  10. 96-well plates
  11. 1.5 ml microcentrifuge tube
  12. Generic razor blade
  13. Vacuum pump (Vacuubrand, model: MZ 2C )
  14. BioPhotometer (Eppendorf, model: AG 22331 )


  1. Image J 1.43 software


  1. Prepare for Arabidopsis protoplasts
    1. Healthy rosette leaves from Arabidopsis thaliana (3 weeks) were sliced with a razor blade into small leaf strips (0.5-1 mm).
    2. The leaves were covered with enzyme solution in a petri dish and placed in a vacuum chamber connected to a vacuum pump, and approx. 100 to 400 mmHg of vacuum was applied for 30 min. The leaves were then incubated in the dark for 3 h without shaking at room temperature.
    3. The digested sample was filtrated through a 75 µm nylon mesh, the crude protoplast filtrates were sedimented by centrifugation for 3 min at 100 x g at room temperature.
    4. The purified protoplasts were suspended in W5 solution.

  2. Al treatment for Arabidopsis protoplasts
    1. 10 μl AlCl3 solution in Ca medium was added to 90 µl of protoplast solution in 96-well plates.
    2. The protoplasts were incubated for 1 h at room temperature in darkness.
    3. The protoplasts were incubated with 50 µM FDA for 5 min and Al-induced protoplast death was observed by confocal microscopy (Figure 1).

      Figure 1. Al-induced protoplast death. Viability of wild-type (WT) Arabidopsis protoplasts after 0.5 mM Al treatment. Protoplasts (2 x 105/ml) were incubated with 50 µM FDA and observed with a confocal laser-scanning microscope.

  3. RNA extraction and quantitative RT-PCR
    1. Total RNA was extracted from Arabidopsis protoplasts according to the manufacturer’s instructions using TRI reagent.
    2. The concentration of RNA was determined by measuring A260 using BioPhotometer. 4 μg RNA was used for reverse transcription PCR.
    3. First strand cDNA was synthesized with SuperScript II First strand synthesis system for qRT-PCR. Eppendorf BioPhotometer was used to determine the concentrations of cDNA.
    4. The transcript of AOX genes were analyzed by quantitative RT-PCR using ACTIN2 as an endogenous control. The AOX1a gene was amplified with the primers 5’-ATGATGATAACTCGCGGTGGAGC-3’ and 5’-GCAACATTCAAAGAAAG CCGAATC-3'. PCR was carried out using 50 ng of cDNA and SYBR PCR Master Mix following the manufacturer’s protocol. For quantitative RT-PCR analyses, the Light Cycler 2.0 instrument was used to run the two-step program. PCR cycling conditions for amplification were 95 °C for 30 sec followed by 40 cycles of 95 °C for 5 sec, 55 °C for 30 sec.
    5. Relative expression levels were calculated using the 2(-∆∆Ct) analysis method.

      Figure 2. qRT-PCR analysis of the gene expression levels of AOX1a gene in Al-treated WT protoplasts

  4. Protein extraction and western blot
    1. The treated protoplasts were re-suspended in lysis buffer and incubated on ice with gentle shaking on a level shaker for 30 min.
    2. The samples were centrifuged for 5 min at 1,2000 x g at 4 °C, and the supernatants were transferred to new 1.5 ml tubes.
    3. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard (Bio-Rad protein assay kit).
    4. Proteins extracts were separated by 12% sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE).
    5. The gel was transferred to PVDF membranes and then electrophoresis for 45 min.
    6. The membrane was blocked with TBST containing 5% non-fat milk for 1 h.
    7. The membrane was incubated with anti-AOX antibody or anti-Rubisco antibody at 4 °C overnight.
    8. The membrane was washed with TBST three times (about 10 min each time), and then incubated with secondary antibody [AOX: Anti-rabbit IgG (DylightTM800 4x PEG Conjugate) secondary antibody; Rubisco: Anti-mouse IgG (DylightTM800 Conjugate) secondary antibody] at room temperature for 2 h.
    9. When the membrane was dried, detected by using Odyssey two-color infrared imaging system.

      Figure 3. Western blot analysis of the level of total AOX proteins in Al-treated WT protoplasts. Blots were probed with monoclonal anti-AOX antibody.

      Quantitative analysis was carried out using Image J 1.43 software (Figure 4). A step-by-step guide on how to use Image J for this purpose is shown in Supplementary data 1.

      Figure 4. Quantitative analysis of western blot. Quantitative analysis was carried out using Image J software.


  1. Enzyme solution
    1%-1.5% (w/v) cellulose R10
    0.2%-0.4% (w/v) macerozyme R10
    0.4 M mannitol
    20 mM MES
    20 mM KCl
    10 mM CaCl2
    0.1% (w/v) bovine serum albumin
    pH 5.7
  2. W5 solution
    154 mM NaCl
    125 mM CaCl2
    5 mM KCl
    5 mM Glc
    15 mM MES-KOH (pH 5.6)
  3. 5 mM AlCl3 solution in Ca medium
    5 mM AlCl3
    30 mM CaCl2
    pH 4.5
  4. Real time PCR reaction solution
    SYBR premix Ex Taq
    10.0 µl
    PCR forward primer (10 µM)
    0.4 µl
    PCR reverse primer (10 µM)
    0.4 µl
    2.0 µl
    7.2 µl
  5. Lysis buffer
    15 mM NaCl
    1% Triton X-100
    100 mg/ml phenylmethylsulfonyl fluoride
    50 mM Tris-HCl (pH 8.0)
  6. 10 ml separating gel
    30% polyacrylamide
    4.0 ml
    1.5 M Tris-HCl (pH 8.8)
    2.5 ml
    10% SDS
    100 µl
    10% (NH4)2S2O8
    100 µl
    10 µl
    3.29 ml
  7. 6 ml stacking gels
    30% polyacrylamide
    1.0 ml
    1 M Tris-HCl (pH 6.8)
    0.75 ml
    10% SDS
    60 µl
    10% (NH4)2S2O8
    60 µl
    6 µl
    4.124 ml
  8. TBST
    10 mM Tris-HCl (pH 7.4)
    150 mM NaCl
    0.1% Tween-20


This protocol was supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT0829), the Key Program of NSFC-Guangdong Joint Funds of China (U0931005) and the National High Technology Research and Development Program of China (863 Program) (2007AA10Z204). This protocol was adapted from previous work (Yamamoto et al., 2002; Ye et al., 2013; Sun et al., 2012).


  1. Liu, J., Li, Z., Wang, Y. and Xing, D. (2014). Overexpression of ALTERNATIVE OXIDASE1a alleviates mitochondria-dependent programmed cell death induced by aluminium phytotoxicity in Arabidopsis. J Exp Bot 65(15): 4465-4478.
  2. Li, Z. and Xing, D. (2011). Mechanistic study of mitochondria-dependent programmed cell death induced by aluminium phytotoxicity using fluorescence techniques. J Exp Bot 62(1): 331-343.
  3. Sun, A., Nie, S. and Xing, D. (2012). Nitric oxide-mediated maintenance of redox homeostasis contributes to NPR1-dependent plant innate immunity triggered by lipopolysaccharides. Plant Physiol 160(2): 1081-1096.
  4. Yamamoto, Y., Kobayashi, Y., Devi, S. R., Rikiishi, S. and Matsumoto, H. (2002). Aluminum toxicity is associated with mitochondrial dysfunction and the production of reactive oxygen species in plant cells. Plant Physiol 128(1): 63-72.
  5. Ye, Y., Li, Z. and Xing, D. (2013). Nitric oxide promotes MPK6-mediated caspase-3-like activation in cadmium-induced Arabidopsis thaliana programmed cell death. Plant Cell Environ 36(1): 1-15.


铝(Al),一种已知对人类以及对植物有毒的环境中普遍存在的非必需金属,不仅对根而且对植物的地上部分都造成损害。 其毒性已被认为是限制作物在酸性土壤上生产的主要因素之一。 替代氧化酶,植物中的呼吸末端氧化酶,有助于维持电子通量和降低线粒体ROS水平,经常被显着诱导,使植物更好地适应应力条件,如铝胁迫。 在该方案中,使用先前方法的改编,在拟南芥原生质体中检测由A1处理诱导的替代氧化酶的表达(Yamamoto等人,2002; et al。,2011; Liu et al。,2014),它们有助于研究Al处理中替代氧化酶的机制。


  1. 拟南芥的玫瑰花叶片(哥伦比亚,3周)
  2. AlCl 3(Sigma-Aldrich,目录号:237051)
  3. CaCl 2(Sigma-Aldrich,目录号:C7902)
  4. HCl(Sigma-Aldrich,目录号:258148)
  5. FDA(Sigma-Aldrich,目录号:596-09-8)
  6. 材料和试剂

    1. 拟南芥的玫瑰花叶片(哥伦比亚,3周)
    2. AlCl 3(Sigma-Aldrich,目录号:237051)
    3. CaCl 2(Sigma-Aldrich,目录号:C7902)
    4. HCl(Sigma-Aldrich,目录号:258148)
    5. FDA(Sigma-Aldrich,目录号:596-09-8)
    6. ... Bovine serum albumin (Sigma-Aldrich, catalog number: A-6793)
    7. NaCl (Sigma-Aldrich, catalog number: S6150)
    8. Glucose (Sigma-Aldrich, catalog number: G7528)
    9. Trition X-100 (Sigma-Aldrich, catalog number: T-8787)
    10. Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: P7626)
    11. Tris (Sigma-Aldrich, catalog number: T1378)
    12. SDS (Sigma-Aldrich, catalog number: L6026)
    13. (Sigma-Aldrich,目录号:A3426),br /(NH 4)2 SubS 2 O 8 >
    14. TEMED(Sigma-Aldrich,目录号:T9281)
    15. Tween-20(Sigma-Aldrich,目录号:P9416)
    16. SuperScript II第一链合成系统(Life Technologies,Invitrogen TM ,目录号:11904-018)
    17. SYBR Premix Ex Taq(Takara,目录号:RR420A)
    18. Bio-Rad蛋白测定试剂盒(Bio-Rad Laboratories,目录号:500-0001)
    19. 抗AOX抗体(Agrisera,目录号:AS10699)
    20. 抗Rubisco抗体(Agrisera,目录号:AS03037)
    21. 抗兔IgG(Dylight?sup?800 4x PEG缀合物)二抗(CST,目录号:5151)
    22. 抗小鼠IgG(Dylight TM sup TM 800 Conjugate)二抗(CST,目录号:5257)
    23. 酶溶液(见配方)
    24. W5解决方案(参见配方)
    25. 5mM AlCl 3在Ca培养基中的溶液
    26. 实时PCR反应溶液(参见配方)
    27. 裂解缓冲液(见配方)
    28. 10ml分离凝胶(见配方)
    29. 6 ml堆叠凝胶(见配方)
    30. TBST(参见配方)


    1. pH计
    2. 热循环仪(Roche,型号:Light Cycler 2.0)
    3. 共聚焦激光扫描显微镜(Carl-Zeiss,型号:LSM510/ConfoCor2)
    4. 自动酶标仪(Tecan Trading AG,型号:无限M200)
    5. 双色红外成像系统(Odyssey,型号:9120)
    6. 离心机
    7. 轨道振动器
    8. PVDF膜(Bio-Rad Laboratories,目录号:162-0177)
    9. 尼龙网
    10. 96孔板
    11. 1.5 ml微量离心管
    12. 通用剃刀刀片
    13. 真空泵(Vacuubrand,型号:MZ 2C)
    14. BioPhotometer(Eppendorf,型号:AG 22331)


    1. Image J 1.43软件


    1. 准备拟南芥原生质体
      1. 将来自拟南芥(Arabidopsis thaliana)(3周)的健康莲座叶片用刀片切成小叶片(0.5-1mm)。
      2. 在培养皿中用酶溶液覆盖叶子 放置在连接真空泵的真空室中, 100 至400mmHg真空30分钟。 然后叶子 在黑暗中孵育3小时,在室温下不摇动
      3. 将消化的样品通过75μm尼龙网过滤 通过离心3分钟沉淀粗制原生质体滤液 在100×g下在室温下进行
      4. 将纯化的原生质体悬浮在W5溶液中。

    2. 拟南芥原生质体的治疗
      1. 将10μl在Ca培养基中的AlCl 3溶液加入96孔板中的90μl原生质体溶液中。
      2. 将原生质体在室温下在黑暗中孵育1小时
      3. 将原生质体与50μM的FDA温育5分钟, 通过共聚焦显微镜观察Al诱导的原生质体死亡(图 1)。

        图1.Al诱导的原生质体死亡。野生型的存活力 (WT)拟南芥原生质体。 原生质体(2×   10 /ml)与50μMFDA孵育并用共焦观察 激光扫描显微镜。

    3. RNA提取和定量RT-PCR
      1. 根据制造商的说明书使用TRI试剂从拟南芥原生质体提取总RNA。
      2. 通过使用测量A 260来测定RNA的浓度 生物光度计。 4μgRNA用于逆转录PCR
      3. 用SuperScript II第一链合成第一链cDNA qRT-PCR的合成系统。 使用Eppendorf BioPhotometer 确定cDNA的浓度
      4. 基因的转录物   通过使用ACTIN2作为内源的定量RT-PCR进行分析 控制。 使用引物扩增AOX1a 基因 5'-ATGATGATAACTCGCGGTGGAGC-3'和5'-GCAACATTCAAAGAAAG CCGAATC-3'。 PCR 使用50ng cDNA和SYBR PCR Master Mix进行 制造商的协议。 对于定量RT-PCR分析,光   Cycler 2.0仪器用于运行两步程序。 PCR循环   扩增条件为95℃30秒,随后40℃ 95℃5秒,55℃30秒的循环
      5. 使用2 ( - ΔΔCt)分析方法计算相对表达水平

        图2.A1处理的WT原生质体中 AOX1a基因的基因表达水平的qRT-PCR分析

    4. 蛋白质提取和蛋白质印迹
      1. 将处理的原生质体重悬于裂解缓冲液中,并在冰上温和振荡在水平振荡器上孵育30分钟。
      2. 将样品在4℃下以1,2000×g离心5分钟,将上清液转移到新的1.5ml管中。
      3. 蛋白质浓度通过Bradford方法使用测定 牛血清白蛋白作为标准(Bio-Rad蛋白测定试剂盒)
      4. 蛋白质提取物通过12%十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳(SDS-PAGE)分离
      5. 将凝胶转移到PVDF膜上,然后电泳45分钟
      6. 用含有5%脱脂乳的TBST封闭膜1小时
      7. 将膜与抗AOX抗体或抗Rubisco抗体在4℃下温育过夜
      8. 将膜用TBST洗涤三次(每次约10分钟) 时间),然后与第二抗体[AOX:抗兔IgG (Dylight TM TM 800 4x PEG缀合物)二抗; Rubisco:抗老鼠 IgG(Dylight?sup?800 Conjugate)二抗]   2小时。
      9. 当膜干燥时,通过使用Odyssey双色红外成像系统检测

        图3.蛋白印迹分析总AOX蛋白水平 Al处理的WT原生质体 。用单克隆抗AOX 抗体
        使用Image J进行定量分析 1.43软件(图4)。 有关如何使用Image J的分步指南   此目的显示在补充数据1 中。

        图4.蛋白质印迹的定量分析 。使用Image J软件进行定量分析。


    1. 酶溶液
      0.2%-0.4%(w/v)macerozyme R10
      0.4M甘露醇 20 mM MES
      20 mM KCl
      10mM CaCl 2
      0.1%(w/v)牛血清白蛋白 pH5.5
    2. W5解决方案
      154 mM NaCl 125mM CaCl 2。 5 mM KCl
      5 mM Glc
      15mM MES-KOH(pH 5.6)
    3. 5mM AlCl 3在Ca培养基中的溶液
      5mM AlCl 3
      30mM CaCl 2
      pH 4.5
    4. 实时PCR反应溶液
      SYBR预混料Ex Taq
      ddH sub 2 O
    5. 裂解缓冲液
      15mM NaCl 1%Triton X-100 100mg/ml苯甲基磺酰氟
      50mM Tris-HCl(pH8.0)
    6. 10ml分离凝胶
      30%聚丙烯酰胺 4.0 ml
      1.5 M Tris-HCl(pH 8.8)
      2.5 ml
      10%(NH 4)2 SubS 2 O 8 sub
      ddH sub 2 O
      3.29 ml
    7. 6 ml堆积凝胶
      30%聚丙烯酰胺 1.0 ml
      1 M Tris-HCl(pH 6.8)
      0.75 ml
      10%(NH 4)2 SubS 2 O 8 sub
      ddH sub 2 O
    8. TBST
      10mM Tris-HCl(pH7.4) 150mM NaCl 0.1%Tween-20


    该协议得到了长江学者和大学创新研究团队(IRT0829),国家自然科学基金委 - 广东省联合基金重点项目(U0931005)和国家高技术研究与发展计划(863计划) 2007AA10Z204)。 该方案改编自以前的工作(Yamamoto等人,2002; Ye等人,2013; Sun 。,2012)。


    1. Liu,J.,Li,Z.,Wang,Y.and Xing,D。(2014)。 过表达 ALTERNATIVE OXIDASE1a 可缓解铝诱导的线粒体依赖性程序性细胞死亡拟南芥中的植物毒性。 J Exp Bot 65(15):4465-4478。
    2. Li,Z.和Xing,D。(2011)。 使用荧光技术的铝植物毒性诱导的线粒体依赖性程序性细胞死亡的机制研究。 J Exp Bot 62(1):331-343。
    3. Sun,A.,Nie,S.and Xing,D。(2012)。 一氧化氮介导的维持氧化还原稳态有助于NPR1依赖植物由脂多糖引发的先天免疫。/a> Plant Physiol 160(2):1081-1096
    4. Yamamoto,Y.,Kobayashi,Y.,Devi,S.R.,Rikiishi,S。和Matsumoto,H。(2002)。 铝毒性与线粒体功能障碍和植物细胞中活性氧的产生有关。 Plant Physiol 128(1):63-72。
    5. Ye,Y.,Li,Z.和Xing,D。(2013)。 一氧化氮促进镉诱导的拟南芥程序性细胞死亡中MPK6介导的半胱天冬酶3样激活 。植物细胞环境36(1):1-15。
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引用:Liu, J., Li, Z. and Xing, D. (2015). Detection of Alternative Oxidase Expression in Arabidopsis thaliana Protoplasts Treated with Aluminium. Bio-protocol 5(13): e1515. DOI: 10.21769/BioProtoc.1515.