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Cytoplasmic calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in multiple signal transduction cascades (Allen et al., 1999). In plant cells, a dramatic and readily assayed response to stimulus is the change of stomatal aperture. Changes in [Ca2+]cyt of stomatal guard cells were involved in stomatal movement in response to various stimuli and cellular processes. In general, there are two available ways to measure [Ca2+]cyt in guard cells, i.e., loading of calcium-sensitive fluorescence dyes such as fluo-3 AM and fura-2 or expressing genetically encoded calcium indicators such as yellow cameleon (Krebs et al., 2012). In this protocol, we aim at describing the experimental procedure to record [Ca2+]cyt fluctuation in guard cells with loading of fluo-3 AM upon ABA or PA treatment combining with fluorescence imaging performed with confocal laser scanning microscope.

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A Protocol to Measure the Cytoplasmic Calcium in Arabidopsis Guard Cells
一种测定拟南芥保卫细胞中胞质钙离子的方法

植物科学 > 植物生理学 > 离子分析
作者: Li Li
Li LiAffiliation: College of Life Sciences, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China
Bio-protocol author page: a2175
Feng Lin
Feng LinAffiliation: College of Life Sciences, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China
Bio-protocol author page: a2176
Yana Qu
Yana QuAffiliation: College of Life Sciences, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China
Bio-protocol author page: a2177
 and Qun Zhang
Qun ZhangAffiliation: College of Life Sciences, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China
For correspondence: zhangqun@njau.edu.cn
Bio-protocol author page: a2178
Vol 5, Iss 9, 5/5/2015, 2402 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1462

[Abstract] Cytoplasmic calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in multiple signal transduction cascades (Allen et al., 1999). In plant cells, a dramatic and readily assayed response to stimulus is the change of stomatal aperture. Changes in [Ca2+]cyt of stomatal guard cells were involved in stomatal movement in response to various stimuli and cellular processes. In general, there are two available ways to measure [Ca2+]cyt in guard cells, i.e., loading of calcium-sensitive fluorescence dyes such as fluo-3 AM and fura-2 or expressing genetically encoded calcium indicators such as yellow cameleon (Krebs et al., 2012). In this protocol, we aim at describing the experimental procedure to record [Ca2+]cyt fluctuation in guard cells with loading of fluo-3 AM upon ABA or PA treatment combining with fluorescence imaging performed with confocal laser scanning microscope.

[Abstract]

Materials and Reagents

  1. Plant materials: Arabidopsis thaliana ecotype Columbia-0 (wild type) was obtained from ABRC at Ohio State University (Columbus)
  2. Glycine,N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxyethyl]-, (acetyloxy) methyl ester (Fluo-3, AM) (Life Technologies, InvitrogenTM, catalog number: F-14218 )
  3. Potassium chloride (KCl) (Sangon Biotech, catalog number: PB0440 )
  4. Calcium chloride dihydrate (CaCl2·2H2O) (Sangon Biotech, catalog number: C0556 )
  5. 2-(N-morpholino) ethanesulfonic acid (MES) (free acid, monohydrate) (Sigma-Aldrich, catalog number: M3671 )
  6. Potassium hydroxide (KOH) (Sangon Biotech, catalog number: PT1159 )
  7. Egtazic acid, Glycol ether diamine tetraacetic acid (EGTA) (Sangon Biotech, catalog number: ED007 )
  8. Chloroform (Sinopharm Chemical Reagent, catalog number: 10006818 )
  9. Sodium hypochlorite solution (Sangon Biotech, catalog number: S1944 )
  10. Agar (Sangon Biotech, catalog number: AJ637 )
  11. Murashige & Skoog (MS) Basal Medium w/Vitamins (PhytoTechnology Laboratories®, catalog number: M519 )
  12. Abscisic acid (ABA) (Sigma-Aldrich, catalog number: A1049 )
  13. Phosphatidic acid (PA) (Avanti Polar Lipid, catalog number: 840858C )
  14. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  15. Ethanol anhydrous (Sangon Biotech, catalog number: ET0737 )
  16. Tris (hydroxymethyl) aminomethane (Tris) (Sigma-Aldrich, catalog number: 252859 )
  17. Epidermal buffer (see Recipes)
  18. Fluo-3 AM stock solution (see Recipes)
  19. Loading buffer (see Recipes)
  20. ABA stock solution (see Recipes)
  21. ABA working solution (see Recipes)
  22. PA working solution (see Recipes)

Equipment

  1. Graduated centrifuge tubes (1.5 ml)
  2. Petri dish (6 cm diameter)
  3. Cover glass (20 mm * 20 mm, 0.13-0.17 μm thickness)
  4. Slides (76 mm * 26 mm, 1-2 mm thickness)
  5. Forceps
  6. Pipettor and matched tips
  7. Growth chamber (Percival, model: I-41LL )
  8. Zeiss LSM780 confocal laser scanning microscope
  9. Microprocessor pH meter (HANNA® Instruments, model: HI-2211 )
  10. Ultrasonic cell crusher (Scientz, model: JY92Ⅱ )

Software

  1. Zeiss confocal software ZEN 2012 (blue edition)
  2. Microsoft Excel

Procedure

  1. Sample preparation
    1. Seeds of Arabidopsis thaliana were surface-sterilized by soaking in 75% ethanol for 3 min and 2% sodium hypochlorite for 10 min. The seeds were washed for 3 to 5 times with sterile distilled water, and then were placed on MS medium (1% agar). Arabidopsis seedlings were grown in a growth chamber at 160 μmol/m2/s light intensity and 14/10 h (24/21 °C) day/night regimes. The cotyledons of 7-day-old seedlings were cut from the plants and used for experiments.
    2. The cotyledons were incubated with epidermal buffer at 24 °C for 2 h in growth chamber (160 μmol/m2/s light intensity) to induce stomatal opening prior to various treatments.
    3. The samples were then incubated in 10 μM fluo-3 AM loading buffer (diluted from 5 mM stock solution in 10 mM MES-KOH pH 6.1) for 2 h at 4 °C in darkness. Higher concentration of fluo-3 AM at room temperature for incubation may result in false positive signals from other ions, for example Mn2+, Zn2+ and Pb2+.
    4. The samples were rinsed with loading buffer for 3 times to remove fluo-3 AM and were kept in growth chamber (24 °C) for 1 h before ABA or PA addition.
    5. For ABA or PA treatment, the cotyledons were incubated with 10 μM ABA or 50 μM PA working solution, and observed by the confocal laser scanning microscope.

  2. [Ca2+]cyt imaging
    1. [Ca2+]cyt imaging was performed with a 488 nm excitation and a 510-545 nm emission filter to record [Ca2+]cyt images. Increases in [Ca2+]cyt occurred after ABA or PA addition. Fluorescence intensity of Fluo-3 dye increases when bound to [Ca2+] in the cytosol. The Kd of Fluo-3 for calcium is 325 nM. Fluo-3 can also detect calcium near the chloroplasts membrane and plasma membrane, and display higher fluorescence in cytosol because of higher concentration of calcium. Changes in [Ca2+]cyt were visualized in pseudocolour as indicated by the color bars. The color bar showed the pixel intensity as the arrow indicated (Figure 1).
    2. The imaging parameters were as follows: Plan-Apochromat 10×/0.45 M27 objective, image dimension 1,024 x 1,024, pinhole 3 Airy unit, scanning speed 1936.28 ms. The images were acquired at the time indicated. Fluorescence pixel intensity values in guard cells were measured using the Zeiss confocal software ZEN 2012 (blue edition). The pixel intensity value of background is about 4.5 for subtraction.
      Please note that the laser power, gain, offset, zoom, scan speed, etc. should not be changed across experimental conditions if using intensity to distinguish phenotype. Set values at the brightest sample and continue imaging with same conditions to get an appropriate dataset. If the fluorescence is high enough, it can be performed with a fluorescence scope. The confocal scanning will be more sensitive and reproducible.

Representative data



Figure 1. Elevation of cytoplasmic calcium induced by ABA and PA in Arabidopsis guard cells. The confocal images of [Ca2+]cyt in guard cells were monitored by fluo-3 AM dye before (A) or after treatment with10 μM ABA (B) or 50 μM PA (C) for 12 min. The color bars showed the intensity as the arrow indicated. D. Changes in the relative levels of [Ca2+]cyt in ABA- or PA-treated guard cells. Regions of interests used to measure the intensities were indicated by white rectangle with background subtraction. Values are the mean ± SD (n = 50-60 from not less than 10 cotyledons) from three independent experiments.

Recipes

  1. Epidermal buffer
    10 mM KCl
    0.2 mM CaCl2
    0.1 mM EGTA
    10 mM MES-KOH
    Adjust the pH to 6.15 with KOH (0.1 M)
  2. Fluo-3 AM stock solution
    5 mM Fluo-3 AM in DMSO
  3. Fluo-3 AM Loading buffer
    10 mM MES
    Adjust the pH to 6.1 with KOH (0.1 M)
    10 μM Fluo-3 AM
  4. ABA stock solution
    Dissolve 26.432 mg ABA powder in 1 ml ethanol to a concentration of 100 mM
  5. ABA working solution (for treatment)
    Add ABA stock solution (10,000x) to the epidermal buffer to a concentration of 10 μM ABA
  6. PA working solution (for treatment)
    PA in chloroform were dried under N2 and suspended in 1 ml of epidermal solution by sonication for preparing the stock solution (50 mM, 1,000x). The PA stock solution was diluted into 50 μM with epidermal solution before treatment.

Acknowledgments

The methods were adapted from (Jiang et al., 2014). Techniques were also adapted from all of the references cited. This work was supported by grants from National Basic Research Program of China (31100194 and 31470364) and the Fundamental Research Funds for the Central Universities (KYZ201423) to Q Zhang.

References

  1. Allen, G. J., Kwak, J. M., Chu, S. P., Llopis, J., Tsien, R. Y., Harper, J. F. and Schroeder, J. I. (1999). Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. Plant J 19(6): 735-747.
  2. Jiang, Y., Wu, K., Lin, F., Qu, Y., Liu, X. and Zhang, Q. (2014). Phosphatidic acid integrates calcium signaling and microtubule dynamics into regulating ABA-induced stomatal closure in Arabidopsis. Planta 239(3): 565-575.
  3. Krebs, M., Held, K., Binder, A., Hashimoto, K., Den Herder, G., Parniske, M., Kudla, J. and Schumacher, K. (2012). FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca(2)(+) dynamics. Plant J 69(1): 181-192.

材料和试剂

  1. 植物材料:拟南芥生态型Columbia-0(野生型)获自俄亥俄州立大学(哥伦布)的ABRC
  2. 甘氨酸N- [4- [6 - [(乙酰氧基)甲氧基] -2,7-二氯-3-氧代-3H-呫吨-9-基] -2- [2- [2- [双[2- [ (乙酰氧基)甲氧基] -2-氧乙基]氨基] -5-甲基苯氧基]乙氧基]苯基] -N- [2 - [(乙酰氧基)甲氧基] -2-氧乙基] - ,(乙酰氧基)甲酯(Fluo- AM)(Life Technologies,Invitrogen TM,目录号:F-14218)
  3. 氯化钾(KCl)(Sangon Biotech,目录号:PB0440)
  4. 氯化钙二水合物(CaCl 2·2H 2 O)(Sangon Biotech,目录号:C0556)
  5. 2-(N-吗啉代)乙磺酸(MES)(游离酸,一水合物)(Sigma-Aldrich,目录号:M3671)
  6. 氢氧化钾(KOH)(Sangon Biotech,目录号:PT1159)
  7. 乙酸,乙二醇四乙酸四乙酸(EGTA)(Sangon Biotech,目录号:ED007)
  8. 氯仿(国药化学试剂,目录号:10006818)
  9. 次氯酸钠溶液(Sangon Biotech,目录号:S1944)
  10. 琼脂(Sangon Biotech,目录号:AJ637)
  11. Murashige& Skoog(MS)基础培养基维生素( Phyto 技术实验室®,目录号:M519)
  12. 脱落酸(ABA)(Sigma-Aldrich,目录号:A1049)
  13. 磷脂酸(PA)(Avanti Polar Lipid,目录号:840858C)
  14. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  15. 无水乙醇(Sangon Biotech,目录号:ET0737)
  16. 三(羟甲基)氨基甲烷(Tris)(Sigma-Aldrich,目录号:252859)
  17. 表皮缓冲液(见配方)
  18. Fluo-3 AM储备溶液(见配方)
  19. 加载缓冲区(见配方)
  20. ABA储备溶液(参见食谱)
  21. ABA工作解决方案(见配方)
  22. PA工作解决方案(见配方)

设备

  1. 刻度离心管(1.5 ml)
  2. 培养皿(直径6厘米)
  3. 覆盖玻璃(20mm×20mm,厚度0.13-0.17μm)
  4. 幻灯片(76 mm * 26 mm,1-2 mm厚)
  5. 镊子
  6. 移液器和匹配的提示
  7. 生长室(Percival,型号:I-41LL)
  8. 蔡司LSM780共焦激光扫描显微镜
  9. 微处理器pH计(HANNA ®仪器,型号:HI-2211)
  10. 超声波破碎机(Scientz,型号:JY92Ⅱ)

软件

  1. 蔡司共焦软件ZEN 2012(蓝版)
  2. Microsoft Excel

程序

  1. 样品制备
    1. 将拟南芥的种子通过浸泡在75% 乙醇3分钟和2%次氯酸钠10分钟。种子是 用无菌蒸馏水洗涤3至5次,然后进行洗涤 置于MS培养基(1%琼脂)上。将拟南芥种植在a中 生长室为160μmol/ m 2 / s光强度和14/10h(24/21℃) 日夜制度。从7日龄幼苗的子叶上切下来 植物用于实验。
    2. 子叶是 与表皮缓冲液在生长室(160℃)中于24℃孵育2小时 μmol/ m 2 / s光强度)以诱导各种气孔开放 治疗。
    3. 然后将样品在10μM氟-3AM中孵育 加载缓冲液(从10mM MES-KOH pH的5mM储备溶液中稀释 6.1)在4℃下在黑暗中2小时。较高浓度的fluo-3 AM在 孵化的室温可能导致假阳性信号 来自其他离子,例如Mn 2 + ,Zn 2 + 和Pb 2 + 。
    4. 样品 用加载缓冲液冲洗3次以除去fluo-3AM并进行  在ABA或PA加入前,保持在生长室(24℃)中1小时。
    5. 对于ABA或PA处理,子叶与10μMABA孵育  或50μMPA工作溶液,并通过共焦激光观察 扫描显微镜。

  2. [Ca 2 + ] > cyt 成像
    1. 使用488nm激发和510-545nm激光进行[Ca 2 + ] cyt 成像  发射滤波器记录[Ca 2 + ] cyt 图像。在ABA或PA添加后发生[Ca 2 + ] cyt 的增加。 Fluo-3染料的荧光强度 当与细胞质中的[Ca 2 + ]结合时增加。 Fluo-3的Kd 钙是325nM。 Fluo-3还可以检测叶绿体附近的钙 膜和质膜,并在细胞质中显示更高的荧光  因为钙浓度较高。 [Ca 2 + ] cyt 变化 以彩色条指示的虚拟色调可视化。彩条  显示像素强度为箭头所示(图1)
    2. 成像参数如下:Plan-Apochromat 10×/ 0.45 M27 客观,图像尺寸1,024 x 1,024,针孔3通风单元,扫描 速度1936.28 ms。在指定的时间拍摄图像。 使用保护细胞中的荧光像素强度值进行测量 蔡司共焦软件ZEN 2012(蓝版)。像素 减法的背景强度值约为4.5 请 请注意,激光功率,增益,偏移,变焦,扫描速度等 如果使用强度,不能在实验条件下改变 区分表型将值设置在最亮的样本并继续 在相同条件下进行成像以获得适当的数据集。如果 荧光足够高,可以用荧光进行 范围。共焦扫描将更加敏感和可重复

代表性资料



图1:拟南芥中ABA和PA诱导的细胞质钙的保护细胞保守细胞 在(A)之前或用10μMABA(B)或50μMPA(C)处理12分钟后,通过荧光-3AM染料监测保卫细胞中的cyt 。彩条显示强度如箭头所示。 D.在ABA或PA-处理的保卫细胞中[Ca 2+ / 2]亚细胞的相对水平的变化。用于测量强度的兴趣区域由背景扣除的白色矩形表示。值是来自三个独立实验的平均值±SD(不少于10个子叶的50-60)。

食谱

  1. 表皮缓冲液
    10 mM KCl
    0.2mM CaCl 2
    0.1 mM EGTA
    10 mM MES-KOH
    用KOH(0.1M)
    调节pH至6.15
  2. Fluo-3 AM库存解决方案
    5 mM Fluo-3 AM in DMSO
  3. Fluo-3 AM加载缓冲区
    10 mM MES
    用KOH(0.1M)
    调节pH至6.1 10μMFluo-3 AM
  4. ABA库存解决方案
    将26.432 mg ABA粉末溶解在1 ml乙醇中,浓度为100 mM
  5. ABA工作解决方案(治疗)
    将ABA储备溶液(10,000x)加入到表皮缓冲液中至浓度为10μMABA
  6. PA工作方案(待处理)
    PA在氯仿中在N 2下干燥,并通过超声处理悬浮于1ml表皮溶液中以制备储备溶液(50mM,1,000x)。 PA处理前用表皮溶液将PA储备溶液稀释至50μM

致谢

这些方法从(Jiang等人,2014年)改编而来。所有引用的参考文献也改编了技术。这项工作得到了中国国家基础研究计划(31100194和31470364)以及中央大学基础研究基金(KYZ201423)给Q张的资助。

参考文献

  1. Allen,G.J.,Kwak,J.M.,Chu,S.P.,Llopis,J.,Tsien,R.Y.,Harper,J.F.and Schroeder,J.I。(1999)。 Cameleon钙指示剂报告拟南芥保卫细胞中的细胞质钙动力学。 a>植物J 19(6):735-747。
  2. Jiang,Y.,Wu,K.,Lin,F.,Qu,Y.,Liu,X. and Zhang,Q.(2014)。 磷脂酸将钙信号传导和微管动力学整合到调节ABA诱导的拟南芥气孔闭合中/ ;。 Planta 239(3):565-575。
  3. Krebs,M.,Held,K.,Binder,A.,Hashimoto,K.,Den Herder,G.,Parniske,M.,Kudla,J.and Schumacher,K。(2012)。 基于FRET的遗传编码传感器允许Ca(2)(+)的高分辨率活细胞成像动植物。植物J 69(1):181-192。
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How to cite this protocol: Li, L., Lin, F., Qu, Y. and Zhang, Q. (2015). A Protocol to Measure the Cytoplasmic Calcium in Arabidopsis Guard Cells. Bio-protocol 5(9): e1462. DOI: 10.21769/BioProtoc.1462; Full Text



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