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Quantification of Plant Cell Death by Electrolyte Leakage Assay

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The EMBO Journal
Sep 2017



We describe a protocol to measure the electrolyte leakage from plant tissues, resulting from loss of cell membrane integrity, which is a common definition of cell death. This simple protocol is designed to measure the electrolyte leakage from a tissue sample over a time course, so that the extent of cell death in the tissue can be monitored dynamically. In addition, it is easy to handle many tissue samples in parallel, which allows a high level of biological replication. Although the protocol is exemplified by cell death in Arabidopsis in response to pathogen challenge, it is easily applicable to other types of plant cell death.

Keywords: Plants (植物), Cell death (细胞死亡), Electrolyte leakage (电解质渗漏), Electrolytic conductivity (电解电导率)


When a cell dies and loses the integrity of the cell membrane, electrolytes, such as K+ ions, leak out of the cell. Thus, we can use the amount of electrolytes leaked from a tissue as a proxy for the extent of cell death in the tissue. A simple way to quantify such electrolytes leaked from a tissue is to measure the increase in electrolytic conductivity of water that contains the tissue with dying cells. This electrolyte leakage assay has been applied to plant tissues to assess the relative quantity of cells that died in response to biotic and abiotic stresses, such as pathogen challenge, insect herbivory, wounding, UV radiation, oxidative stress, salinity, drought, cold and heat stress (Demidchik et al., 2014).

The original method was designed to measure the conductivity of the aqueous bathing solution containing plant tissues before and after boiling it, in which the conductivity after boiling was used to normalize tissue size differences (Whitlow et al., 1992). Here we describe a procedure to dynamically monitor electrolyte leakage from leaf disks by measuring at multiple time points the electrolytic conductivity of water on which the leaf disks float in a 12-well plate. It is reasonable to assume that the total amount of electrolytes from tissue samples of the same size, such as disks of the same area punched out from leaves of a similar developmental stage, is comparable and that it is not necessary to measure the electrolytic conductivity after boiling the tissues. We inoculated leaves with the bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 pVSP61-avrRpt2 (Pto DC3000 avrRpt2) in order to trigger a type of programmed cell death, known as hypersensitive cell death. The protocol presented here has been applied to our studies (Igarashi et al., 2013; Bethke et al., 2016; Hatsugai et al., 2016; Hatsugai et al., 2017) and could also be used to quantify plant cell death triggered by any other stimuli. If detailed comparisons of the time courses of the electrolyte leakage are needed, fitting polynomial regression to the time courses is possible (Van Poecke et al., 2007; Qi et al., 2010), as it is easy to obtain electrolytic conductivity measurements at many time points with many replicates.

Materials and Reagents

  1. 2 ml microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-138 )
  2. Sterilized tubes for liquid bacterial cultures (Evergreen Scientific, catalog number: 222-2094-050 )
  3. Disposable 1-ml needleless syringes for bacterial inoculation (BD, catalog number: 309659 )
  4. 12-well cell culture plates (flat bottom with lid) (Corning, Costar®, catalog number: 3513 )
  5. 1-200 μl Pipette tips (Sorenson BioScience, catalog number: 3211 )
  6. 50-1,250 μl pipette tips (Sorenson BioScience, catalog number: 3205 )
  7. Arabidopsis thaliana accession Col-0 (Figure 1A)
    Note: Arabidopsis thaliana accession Col-0 carries the R gene RPS2, which confers resistance to Pto DC3000 avrRpt2 (Bent et al., 1994; Mindrinos et al., 1994).
  8. Pseudomonas syringae pv. tomato DC3000 pVSP61-avrRpt2 (Pto DC3000 avrRpt2)
    Note: Pto DC3000 avrRpt2 delivers the AvrRpt2 effector into plant cells, thereby inducing hypersensitive cell death in Arabidopsis Col-0.
  9. Sterilized ultrapure water (e.g., Milli-Q)
  10. Conductivity standard solution 1.41 mS/cm (HORIBA, model: Y071L, catalog number: 514-22 )
  11. Antibiotics
    1. Kanamycin sulfate (Thermo Fisher Scientific, GibcoTM, catalog number: 11815032 )
    2. Rifampicin (Sigma-Aldrich, catalog number: R3501 )
  12. Bacto proteose peptone No. 3 (BD, catalog number: 211693 )
  13. Glycerol (Fisher Scientific, catalog number: G33-500 )
  14. Dibasic potassium phosphate (K2HPO4) (Fisher Scientific, catalog number: BP363-500 )
  15. Bacto agar (BD, catalog number: 214010 )
  16. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A508-P500 )
  17. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 230391 )
  18. King’s B liquid medium (see Recipes)


  1. Walk-in Arabidopsis growth chamber (22 °C, 70% relative humidity, and 12-h/12-h day/night photoperiod) (Conviron, model: BDR40 )
  2. Tissue culture roller rotator drum for bacterial culture at 28 °C (New Brunswick Scientific, model TC-7 )
  3. Centrifuge (Eppendorf, model: 5415 D )
  4. Spectrophotometer to determine the density of bacterial culture (Beckman Coulter, model: DU-800 )
  5. Cork borer (size 4, diameter = 7.5 mm)
  6. Electrolytic conductivity meter (HORIBA, model: B-173 )
  7. Single-channel micropipettor (Eppendorf, 20-200 μl and 100-1,000 μl)
  8. Autoclave


  1. Microsoft Excel


  1. Inoculation of Arabidopsis leaves with Pto DC3000 avrRpt2
    1. Culture the bacterial strain in 2 ml King’s B liquid medium supplemented with 50 µg/ml kanamycin and 50 µg/ml rifampicin at 28 °C for 16-18 h.
      Note: The bacterial culture used should be in a late log phase of growth (OD600 = 1.5 - 2.0).
    2. Transfer 1 ml of the liquid culture to a 2-ml microcentrifuge tube.
    3. Harvest bacteria by centrifugation at 3,000 x g for 5 min at room temperature.
    4. Remove supernatant and then suspend bacteria in 1 ml sterilized ultrapure water.
    5. Repeat Steps A3-A4 one more time.
    6. Adjust the optical density of the bacterial suspension with sterilized ultrapure water to OD600 = 0.1 (measured by a spectrophotometer).
    7. Pressure-infiltrate the density-adjusted bacterial suspension (Katagiri et al., 2002) into two leaves per plant in a walk-in Arabidopsis growth chamber.
      Note: Gently pressure-infiltrate the bacterial suspension through the stomatal openings on the abaxial side of the leaf using a 1-ml needleless syringe. The infiltrated area of a leaf is readily visible as the appearance of the infiltrated area turns dark green due to flooding of the intercellular space of the leaf with bacterial suspension.

  2. Measurement of electrolytic conductivity
    1. Cut two leaf disks (7.5 mm diameter) from one plant (one disk per leaf) using a cork borer on paper towels (Figure 1B). Leaf disks should be cut out before the infiltrated bacterial suspension from Step A7 dries out from the intercellular space of the leaves, which occurs approximately 20 min after infiltration.
      Note: We typically use 4-weeks old plants grown under the growth conditions described in Equipment section and select the 7th and 8th leaves of each plant to obtain leaves of comparable developmental stages. Selecting leaves in similar developmental stages is important not only to have a similar amount of tissue for each leaf disk but also to have a similar response to the pathogen.
    2. Float two leaf disks (adaxial surface down) from a single plant on 2 ml of sterilized ultrapure water in one well of a 12-well plate, immediately after the leaf disks are cut out in Step B1 (Figure 1C). Use as many wells and 12-well plates as needed according to the number of plants to be assayed.
    3. Cover the plate with the lid and incubate it for 30 min in a walk-in Arabidopsis growth chamber.
    4. Replace the water in the well with 2 ml of fresh sterilized ultrapure water.
      Note: Steps B3 and B4 are to remove electrolytes initially leaked from the damaged cells on the edges of the leaf disks.
    5. Incubate the plate in a walk-in Arabidopsis growth chamber.
    6. Calibrate the electrolytic conductivity meter before the first use with the conductivity standard solution to 1.41 mS/cm.
    7. Wash the sensor of the electrolytic conductivity meter with sterilized ultrapure water.
    8. Sample 100 µl of the water per well at the determined time point and measure its conductivity using the sensor of an electrical conductivity meter (Figure 1D).

      Figure 1. Experimental procedure to measure the electrolytic leakage from Arabidopsis leaf disks. A. 4-weeks old Arabidopsis thaliana accession Col-0 grown under the growth conditions described in Equipment section. B. Cut two leaf disks (7.5 mm diameter) from one plant (one disk per leaf) using a cork borer on paper towels. C. Float two leaf disks (adaxial surface down) on 2 ml of sterilized ultrapure water in one well of a 12-well cell culture plates. D. Drop 100 µl of the water from one well with a pipette onto the sensor of an electrical conductivity meter and measure its conductivity.

    9. Return the sampled water to the well to maintain the water at constant volume during the time course.
    10. Continue incubating the plate and repeating Steps B8-B9 at additional time points.
      Note: The sensor of the electrical conductivity meter should be rinsed with sterilized ultrapure water between one sample and the other.

Data analysis

  1. For data analysis, directly use the values of the conductivity value obtained from the sensor of the electrical conductivity meter. We recommend inclusion of uninoculated leaf disks in the experiment as a negative control.
    Note: Data analysis is done using Microsoft Excel.
  2. The experiments were performed at least three independent times, each of which had four to six biological replicates. For statistical analysis, Student’s t-test was used for comparison between two genotypes and a one-way analysis of variance (ANOVA) was used for comparison of more than two genotypes. The increased electrolytic conductivity is interpreted as the extent of cell death (Figure 2).

    Figure 2. Electrolyte leakage from leaf disks at 2 to 12 h after bacterial inoculation. Representative data of electrolyte leakage from leaf disks. Pto DC3000 avrRpt2 was infiltrated into Arabidopsis leaves of wild-type and rps2 mutant plants. Electrolytic conductivity dramatically increased with wild-type plants after bacterial inoculation, whereas the increase was limited with rps2 mutant plants. Error bars indicate standard errors of three independent experiments, each with four biological replicates. Asterisks indicate significant differences compared with rps2 mutant plants (**, P < 0.01, two-tailed t-tests).


  1. King’s B liquid medium
    20 g of Bacto proteose peptone No. 3
    15 ml of glycerol
    1.5 g of dibasic potassium phosphate (K2HPO4)
    15 g of Bacto agar
    Dissolve the ingredients in distilled water (1 L), adjust the pH of the solution to 7-6.8 with hydrochloric acid (HCl), and then autoclave it
    After autoclaving, add 6 ml of 1 M filter-sterilized magnesium sulfate heptahydrate (MgSO4·7H2O) to the 1-L medium
    Note: MgSO4·7H2O is added after autoclaving to avoid precipitation. If storing liquid KB medium, do not add MgSO4 until just before use.


We thank Jane Glazebrook for critical reading of the manuscript. This work was funded by grants (grant No. IOS-1121425 and MCB-1518058 to F.K) from the National Science Foundation, U.S.A. Many laboratories used similar protocols in the past (Imanifard et al., 2018; Bach-Pages and Preston, 2018). We have adapted this protocol from (Mackey et al., 2002). The authors have no conflicts of interest or competing interests.


  1. Bach-Pages, M. and Preston, G. M. (2018). Methods to quantify biotic-induced stress in plants. Methods Mol Biol 1734: 241-255.
  2. Bent, A. F., Kunkel, B. N., Dahlbeck, D., Brown, K. L., Schmidt, R., Giraudat, J., Leung, J. and Staskawicz, B. J. (1994). RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes. Science 265(5180): 1856-1860.
  3. Bethke, G., Thao, A., Xiong, G., Li, B., Soltis, N. E., Hatsugai, N., Hillmer, R. A., Katagiri, F., Kliebenstein, D. J., Pauly, M. and Glazebrook, J. (2016). Pectin biosynthesis is critical for cell wall integrity and immunity in Arabidopsis thaliana. Plant Cell 28(2): 537-556.
  4. Demidchik, V., Straltsova, D., Medvedev, S. S., Pozhvanov, G. A., Sokolik, A. and Yurin, V. (2014). Stress-induced electrolyte leakage: the role of K+-permeable channels and involvement in programmed cell death and metabolic adjustment. J Exp Bot 65(5): 1259-1270.
  5. Hatsugai, N., Hillmer, R., Yamaoka, S., Hara-Nishimura, I. and Katagiri, F. (2016). The μ subunit of Arabidopsis adaptor Protein-2 is involved in effector-triggered immunity mediated by membrane-localized resistance proteins. Mol Plant Microbe Interact 29: 345-351.
  6. Hatsugai, N., Igarashi, D., Mase, K., Lu, Y., Tsuda, Y., Chakravarthy, S., Wei, H. L., Foley, J. W., Collmer, A., Glazebrook, J. and Kataqiri, F. (2017). A plant effector-triggered immunity signaling sector is inhibited by pattern-triggered immunity. EMBO J 36: 2758-2769.
  7. Igarashi, D., Bethke, G., Xu, Y., Tsuda, K., Glazebrook, J. and Katagiri, F. (2013). Pattern-triggered immunity suppresses programmed cell death triggered by fumonisin b1. PLoS One 8(4): e60769.
  8. Imanifard, Z. Vandelle, E. and Bellin, D. (2018). Measurement of hypersensitive cell death triggered by avirulent bacterial pathogens in Arabidopsis. Methods Mol Biol 1743: 39-50.
  9. Katagiri, F., Thilmony, R. and He, S. Y. (2002). The Arabidopsis thaliana-Pseudomonas syringae interaction. Arabidopsis Book 1: e0039.
  10. Mackey, D., Holt III, B. F., Wiig, A. and Dangl, J. L. (2002). RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 108(6): 743-754.
  11. Mindrinos, M., Katagiri, F., Yu, G. L. and Ausubel, F. M. (1994). The A. thaliana disease resistance gene RPS2 encodes a protein containing a nucleotide-binding site and leucine-rich repeats. Cell 78(6): 1089-1099.
  12. Qi, Y., Tsuda, K., Joe, A., Sato, M., Nguyen le, V., Glazebrook, J., Alfano, J. R., Cohen, J. D. and Katagiri, F. (2010). A putative RNA-binding protein positively regulates salicylic acid-mediated immunity in Arabidopsis. Mol Plant Microbe Interact 23(12): 1573-1583.
  13. Van Poecke, R. M., Sato, M., Lenarz-Wyatt, L., Weisberg, S. and Katagiri, F. (2007). Natural variation in RPS2-mediated resistance among Arabidopsis accessions: correlation between gene expression profiles and phenotypic responses. Plant Cell 19(12): 4046-4060.
  14. Whitlow, T. H., Bassuk, N. L., Ranney, T. G. and Reichert, D. L. (1992). An improved method for using electrolyte leakage to assess membrane competence in plant tissues. Plant Physiol 98(1): 198-205.


我们描述了测量植物组织中电解质渗漏的方案,其由细胞膜完整性的丧失导致,这是细胞死亡的常见定义。 这个简单的方案设计用于测量组织样本在一段时间内的电解质泄漏,从而可以动态监测组织中细胞死亡的程度。 另外,平行处理许多组织样品很容易,这允许高水平的生物复制。 尽管该方案以响应病原体攻击的拟南芥中的细胞死亡为例,但它很容易应用于其他类型的植物细胞死亡。

【背景】当细胞死亡并丧失细胞膜的完整性时,电解质如K +离子就会从细胞中渗出。因此,我们可以使用组织中泄漏的电解质的量作为组织中细胞死亡程度的代表。量化从组织泄漏的电解质的简单方法是测量含有将死细胞的组织的水的电解电导率的增加。这种电解质渗漏测定法已经应用于植物组织,以评估响应于生物和非生物胁迫而死亡的细胞的相对数量,所述细胞例如病原体攻击,昆虫食草,伤口,UV辐射,氧化应激,盐度,干旱,寒冷和热压力(Demidchik et al。,2014)。

最初的方法被设计成测量含有植物组织的水浴溶液在煮沸之前和之后的电导率,其中煮沸后的电导率被用于使组织尺寸差异标准化(Whitlow等人,1992年)。在这里我们描述了一个程序,通过在多个时间点测量叶片浮在12孔板上的水的电解电导率来动态监测叶盘中的电解质渗漏。可以合理地假设来自相同大小的组织样品的电解质的总量,例如从类似发育阶段的叶子冲出的相同面积的圆盘是相当的,并且没有必要测量电解质电导率之后煮沸组织。我们用细菌病原体,即丁香假单胞菌 pv接种叶子。为了触发已知的程序性细胞死亡类型,已知有番茄 DC3000 pVSP61- avrRpt2 ( pto DC3000 avrRpt2 作为过敏性细胞死亡。这里介绍的协议已经应用到我们的研究中(Igarashi et al。,2013; Bethke et al。,2016; Hatsugai et al。 ,2016; Hatsugai et al。,2017),也可用于量化任何其他刺激触发的植物细胞死亡。如果需要详细比较电解质泄漏的时间进程,则可以对时间进程进行拟合多项式回归(Van Poecke等人,2007; Qi等人, ,2010),因为很多重复试验很容易在许多时间点获得电解电导率测量结果。

关键字:植物, 细胞死亡, 电解质渗漏, 电解电导率


  1. 2 ml微量离心管(Fisher Scientific,目录号:05-408-138)
  2. 用于液体细菌培养的灭菌管(Evergreen Scientific,目录号:222-2094-050)

  3. 用于细菌接种的一次性1毫升无针注射器(BD,目录号:309659)
  4. 12孔细胞培养板(带盖的平底)(Corning,Costar ,产品目录号:3513)
  5. 1-200μl移液枪头(Sorenson BioScience,产品目录号:3211)

  6. 50-1,250μl移液枪头(Sorenson BioScience,产品目录号:3205)
  7. 拟南芥登录Col-0(图1A)
    注:拟南芥登录号Col-0携带R基因RPS2,其赋予对Pto DC3000 avrRpt2的抗性(Bent等,1994; Mindrinos等,1994)。
  8. 丁香假单胞菌 pv。 DC3000 pVSP61- avrRpt2 ( Pto DC3000 avrRpt2 )
    注意:Pto DC3000 avrRpt2将AvrRpt2效应子传递到植物细胞中,从而在拟南芥Col-0中诱导过敏性细胞死亡。
  9. 消毒的超纯水(如emi,Milli-Q)
  10. 电导率标准溶液1.41mS / cm(HORIBA,型号:Y071L,目录号:514-22)
  11. 抗生素
    1. 卡那霉素硫酸盐(Thermo Fisher Scientific,Gibco TM,目录号:11815032)
    2. 利福平(Sigma-Aldrich,目录号:R3501)
  12. 细菌蛋白胨蛋白胨3号(BD,目录号:211693)
  13. 甘油(Fisher Scientific,目录号:G33-500)
  14. 二元磷酸钾(KH 2 HPO 4)(Fisher Scientific,目录号:BP363-500)
  15. 细菌琼脂(BD,目录号:214010)
  16. 盐酸(HCl)(Fisher Scientific,目录号:A508-P500)
  17. 硫酸镁七水合物(MgSO 4•7H 2 O)(Sigma-Aldrich,目录号:230391)
  18. King's B液体培养基(见食谱)


  1. 步入植物拟南芥生长室(22℃,70%相对湿度和12小时/ 12小时昼/夜光周期)(Conviron,型号:BDR40)
  2. 组织培养滚筒转子滚筒用于28°C细菌培养(New Brunswick Scientific,TC-7型)
  3. 离心机(Eppendorf,型号:5415 D)
  4. 用分光光度计测定细菌培养物的密度(Beckman Coulter,型号:DU-800)
  5. 软木蛀虫(大小4,直径= 7.5毫米)
  6. 电解电导率计(HORIBA,型号:B-173)
  7. 单通道微量移液器(Eppendorf,20-200μl和100-1,000μl)
  8. 高压灭菌器


  1. Microsoft Excel


  1. 接种拟南芥与 Pto DC3000 avrRpt2
    1. 培养细菌菌株在2毫升King's B液体培养基中,培养基中添加50μg/ ml卡那霉素和50μg/ ml利福平,时间为16-18小时。
      注意:所使用的细菌培养物应该处于生长后期(OD 600 = em = 1.5-2.0)的晚期阶段。
    2. 将1毫升液体培养液转移到2毫升微量离心管中。

    3. 在室温下3000×g离心5分钟收获细菌。
    4. 除去上清液,然后将细菌悬浮在1ml无菌超纯水中。

    5. 重复步骤A3-A4。
    6. 用灭菌的超纯水调节细菌悬浮液的光密度至OD 600 = 0.1(用分光光度计测量)。

    7. 在步入式拟南芥生长室中,将密度调整的细菌悬浮液(Katagiri等,2002)压入渗透到每株植物的两片叶子中。

  2. 电解电导率的测量
    1. 使用纸巾上的软木钻(图1B),从一个植物(每片叶片一片)切下两片叶片(直径7.5毫米)。在步骤A7的浸润的细菌悬浮液从叶片的细胞间隙中干燥之前应该切掉叶盘,其在渗透约20分钟后发生。
    2. 在步骤B1(图1C)中切下叶盘后立即在12孔板的一个孔中的2ml灭菌的超纯水上漂浮两片叶片(正面朝下)。根据需要测定的植物数量,根据需要使用尽可能多的孔和12孔板。
    3. 用盖子覆盖平板并在步入式拟南芥生长室中孵育30分钟。
    4. 用2毫升新鲜无菌超纯水替换孔中的水。

    5. 在步入式的拟南芥生长室中孵育平板
    6. 在第一次使用电导率标准溶液之前,校准电解电导率计至1.41 mS / cm。

    7. 用无菌超纯水清洗电解电导率仪的传感器。
    8. 在确定的时间点每孔取100μl水,并使用电导率仪的传感器测量其电导率(图1D)。

      图1.测量来自拟南芥叶片的电解质渗漏的实验程序。 :一种。在设备部分中描述的生长条件下生长4周大的拟南芥属种Col-0。 B.用纸巾上的软木钻将一张植物(每片一片一片)的两片叶片(7.5毫米直径)切下。 C.在12孔细胞培养板的一个孔中,在2ml无菌超纯水上漂浮两片叶片(正面向下)。 D.用移液管将100μl水从一个孔中滴入电导率仪的传感器并测量其电导率。

    9. 。将采样水返回井中,以便在时间过程中保持水量恒定。
    10. 继续孵育平板并在更多时间点重复步骤B8-B9。


  1. 对于数据分析,直接使用从电导率仪的传感器获得的电导率值。我们建议在实验中包含未接种的叶盘作为阴性对照。
    注意:数据分析是使用Microsoft Excel完成的。
  2. 实验进行至少三次独立的时间,其中每一次都有四到六次生物学复制。为了进行统计分析,使用Student's检验用于两种基因型之间的比较,并且使用单因素方差分析(ANOVA)来比较两种以上的基因型。电解质电导率的提高被解释为细胞死亡的程度(图2)。

    图2.细菌接种后2至12小时叶盘中的电解质泄漏。有代表性的叶片电解质泄漏数据。将pto DC3000
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hatsugai, N. and Katagiri, F. (2018). Quantification of Plant Cell Death by Electrolyte Leakage Assay. Bio-protocol 8(5): e2758. DOI: 10.21769/BioProtoc.2758.



Babak Pakdaman Sardrood
Assistant Professor at Ramin Agriculture and Natural Resources University of Khuzestan
Dear Scientist
Hoping that You are fine and appreciating your efforts, I have a question that I will be more thankful if You kindly answer to it: I put leaf discs exposed to toxic extracts, then I omit the discs and measure their absorbance of 285-425 nm, and compared them with the control of each extract production medium without the added discs. I want to develop a simple method for the comparison of the phytotoxicity of different culture extracts. Is this possible? May You discuss and lead me? What range of spectra can be suitable for this purpose? Thanks, Babak
4/29/2019 8:50:24 AM Reply