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Extraction and Measurement of Abscisic Acid in a Unicellular Red Alga Cyanidioschyzon merolae
单细胞红藻Cyanidioschyzon merolae中脱落酸的提取和测定   

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Abstract

Abscisic acid (ABA) has been known as a phytohormone of land plants, which is synthesized in response to abiotic stresses and induces various physiological responses, but is also found from eukaryotic algae. Recently, we reported that a unicellular red alga Cyanidioschyzon merolae produced ABA, which prevented cell growth and enhanced salt stress tolerance (Kobayashi et al., 2016). This report describes the detailed method for the extraction and quantification of ABA in the model red alga C. merolae.

Keywords: Abscisic acid(脱落酸), Cyanidioschyzon merolae(温泉红藻纲), Algae(藻类), HPLC(HPLC), Phytohormone(植物激素)

Background

The phytohormone ABA has been found in divergent photosynthetic eukaryotes, but the function in unicellular algae remained unclear. In a recent study, we showed that a unicellular red alga C. melorae accumulates ABA in response to salt stress by the present protocol. This is the detail of the first published protocol for the extraction and quantification of ABA from C. merolae. This protocol is optimized for C. merolae based on the land plant protocol.

Materials and Reagents

  1. 500 ml centrifuge bottle (Hitachi, model: S305830A )
  2. Membrane filters Millex-GV syringe filter unit 0.22 μm (EMD Millipore, catalog number: SLGV033RS )
  3. Wild type C. merolae 10D cells (National Institute for Environmental Studies, Japan)
  4. Abscisic acid (Sigma-Aldrich, catalog number: A4906-250UG )
  5. NaCl (Wako Pure Chemical Industries, catalog number: 195-15975 )
  6. Liquid nitrogen
  7. Acetic acid (Wako Pure Chemical Industries, catalog number: 017-00256 )
  8. Diethyl ether (Wako Pure Chemical Industries, catalog number: 052-01165 )
  9. Methanol (HPLC grade) (Wako Pure Chemical Industries, catalog number: 132-06471 )
  10. Boric acid (H3BO3) (Wako Pure Chemical Industries, catalog number: 021-15645 )
  11. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Wako Pure Chemical Industries, catalog number: 133-00725 )
  12. Zinc sulfate heptahydrate (ZnSO4) (Wako Pure Chemical Industries, catalog number: 265-00415 )
  13. Sodium molybdate dehydrate (Na2MoO4·2H2O) (Wako Pure Chemical Industries, catalog number: 514-30001 )
  14. Copper(II) sulfate pentahydrate (CuSO4) (Wako Pure Chemical Industries, catalog number: 034-20065 )
  15. Cobalt(II) nitrate hexahydrate (Co[NO3]2·6H2O) (Wako Pure Chemical Industries, catalog number: 031-03752 )
  16. Ammonium sulfate ([NH4]2SO4) (Wako Pure Chemical Industries, catalog number: 016-03445 )
  17. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Wako Pure Chemical Industries, catalog number: 138-00415 )
  18. Sulfuric acid (H2SO4) (Wako Pure Chemical Industries, catalog number: 195-04706 )
  19. Potassium dihydrogen phosphate (KH2PO4) (Wako Pure Chemical Industries, catalog number: 166-04255 )
  20. Calcium chloride (CaCl2) (Wako Pure Chemical Industries, catalog number: 036-00485 )
  21. Iron(III) chloride hexahydrate (FeCl3) (Wako Pure Chemical Industries, catalog number: 090-02802 )
  22. Na2EDTA (Wako Pure Chemical Industries, catalog number: 345-01865 )
  23. Polyvinylpyrrolidone K-30 (Nacalai tesque, catalog number: 28314-82 )
  24. 2,6-di-tert-butyl-p-cresol (Tokyo chemical industry, catalog number: D0228 )
  25. MA2 medium (see Recipes)
  26. Extraction solution (see Recipes)

Equipment

  1. Spectrophotometer (Beckman Coulter, model: DU730 )
  2. Refrigerated centrifuge (Hitachi, model: CF16RXII )
  3. Angle rotor (Hitachi, model: R10A3 )
  4. Mortar and pestle
  5. Vortex mixer (M & S Instruments, model: VORTEX-GENIE 2 Mixer )
  6. Microcentrifuge (TOMY DIGITAL BIOLOGY, model: MX150 )
  7. Vacuum centrifugal evaporator with low temperature trapper (TOMY DIGITAL BIOLOGY, model: CC-105 system )
  8. pH meter (As One, model: KR5E )
  9. HPLC system (Shimadzu, model: X2 HPLC system ) equipped with a photodiode array detector (PDA) and column (5 μm, 4.6 x 250 mm) (Senshu Scientific, model: ODS SP100 )

Procedure

  1. Extraction
    1. The optical density (OD) of C. merolae liquid culture is measured by spectrophotometer at 750 nm. When the OD750 reaches 10, cells are diluted to yield an OD750 of approximately 0.5 in 350 ml MA2 medium (Kobayashi et al., 2010). Grow the cells under illumination with fluorescent white light (50 μM photons m-2 s-1) at 42 °C, bubbled with air supplemented with 2% CO2. After incubation for 16 h, measure the OD750, transfer the culture to 500 ml centrifuge bottle, and collect the cells by centrifuging with angle rotor at 3,000 x g for 3 min at room temperature. Gently resuspend the pellet in 350 ml MA2 medium containing 500 mM NaCl and further cultivate for 3 h under the same condition.
    2. Harvest the cells (OD750 = 0.8, containing about 2 x 107 cells/ml) by centrifugation at 3,000 x g for 3 min at 4 °C, discard the medium by decantation.
    3. Remove the remaining medium by pipetting. Dissolve the pellet in MA2 (1-2 ml) and flash freeze in liquid nitrogen (Video 1).
    4. Grind the frozen cell suspension to powder by a mortar and pestle (Video 1). 

      Video 1. Video for ABA extraction steps A3 and A4. This video supports the cell harvesting and grinding.

    1. Homogenize the powdered sample in 20 ml extraction solution by vortexing for 5 min and centrifuge at 10,000 x g for 15 min at 4 °C (Video 2).
    2. Filter the supernatant with 0.22 μm membrane filter (Video 2). 

      Video 2. Video for ABA extraction steps A5 and A6. This video supports the cell extraction and filtration.

    1. Concentrate the aqueous phase by vacuum centrifugal evaporator at room temperature (collect about 4 ml aqueous phase) (Video 3).
    2. Measure the pH of aqueous phase by pH meter. Adjust the aqueous phase to pH 2.8 by 0.5 M acetic acid and filter with 0.22 μm membrane (Video 3).

      Video 3. Video for ABA extraction steps A7 and A8. This video supports the purification of extract.

    1. Extract the ABA from the aqueous phase by three partitions with 5 ml diethyl ether (Video 4).
    2. Dry up the collected ether layer by vacuum centrifugal evaporator at room temperature and dissolve the pellet in the 300 μl methanol (Video 4).

      Video 4. Video for ABA extraction steps A9 and A10. This video supports the continuation of purification of extract.

  2. HPLC analysis
    1. Chromatography is conducted on an HPLC system equipped with a PDA using the full visible spectrum with monitoring employed at 254 nm. Column is a tandem jointed ODS SP100, 250 x 4.6 mm, S = 5 µm.
    2. Perform isocratic separation by 50% methanol. Flow rate is 0.5 ml/min at 40 °C, and apply the samples in 10-50 μl. The peak of ABA was detected at retention time 7.1 min (Figure 1).
    3. Standard calibration curves were generated at 254 nm with ABA reference.
    4. Standard ABAs of concentration of 0, 10, 50, 100, 200, 500, 1000 pmol/μl are prepared and applied to HPLC in 10 μl. HPLC analysis should be performed in triplicates.


      Figure 1. HPLC separation of the C. merolae extract with ABA standard reference. Cells were treated with 500 mM NaCl for 3 h. The ABA standard reference and 500 mM NaCl treated cell extract were separated by HPLC with PDA. The absorption spectra of 0.2 mM ABA standard (left panel), 500 mM NaCl treated cell extract (center panel) and merged (right panel) are shown.

Data analysis

  1. The retention time of the single peak areas is recorded and calculate the average of each peak.
  2. Create a calibration curve using the average of each peak. Calculate the ABA amount using a specific standard calibration curve (An example is shown in Figure 2).


    Figure 2. ABA standard curve. Standard curve was made based on peak value in HPLC analysis. Example: 300 μl extract was obtained starting from salt stressed cell culture (350 ml, OD750 = 0.8), and 10 μl of the 300 μl was subjected to HPLC analysis, which resulted in the read of 133.533 AU. Based on the calibration curve, the total amount of ABA contained in the starting material was calculated as 68487.5 pmole.

Notes

ABA extraction and HPLC method was modified from Kojima method (Kojima et al., 1995). ABA is detected from C. merolae cells only under the salt stressed condition.

Recipes

  1. MA2 medium
    Mix solutions I to III and mess up to 1 L. Sterilize by autoclaving. Solution IV should be sterilized by filtration through a 0.22-micron filter. Add solution IV to the mix after autoclave.

    Solution I


    Solution II


    Solution III


    Solution IV


    A6 minor salts


  2. Extraction solution
    0.6 g/L polyvinylpyrrolidone
    0.22 g/L 2,6-di-tert-butyl-p-cresol
    Dissolve in 80% (v/v) methanol

Acknowledgments

This protocol was adapted from Kobayashi et al. (2016). The authors thank Dr. Tadao Asami for technical help in ABA detection. This study was supported by MEXT/JSPS KAKENHI (Grant numbers: 21370015, 23120505, 2424806, 15K14539 to K.T., 13274350, 15621958 to Y.K.)

References

  1. Kobayashi, Y., Ando, H., Hanaoka, M. and Tanaka, K. (2016). Abscisic acid participates in the control of cell-cycle initiation through heme homeostasis in the unicellular red alga Cyanidioschyzon merolae. Plant Cell Physiol 57(5), 953-960.
  2. Kobayashi, Y., Ohnuma, M., Kuroiwa, T., Tanaka, K. and Hanaoka, M. (2010). The basics of cultivation and molecular genetic analysis of the unicellular red alga Cyanidioschyzon merolae. Endocytobiosis Cell Res 20: 53-61.
  3. Kojima, K., Yamada, Y. and Yamamoto, M. (1995). Effects of abscisic acid injection on sugar and organic acid contents of citrus fruit. J Japan Soc Hort Sci 64(1): 17-21.

简介

脱落酸(ABA)已知为陆生植物的植物激素,其响应于非生物胁迫合成并诱导各种生理反应,但也可从真核藻类中发现。最近,我们报道了单细胞红藻(Cyanidioschyzon merolae)产生ABA,其阻止细胞生长和增强盐胁迫耐受性(Kobayashi等人,2016)。该报告描述了在红海藻模型中提取和定量ABA的详细方法。 。
关键字:脱落酸,藻类, Cyanidioschyzon merolae ,HPLC,植物激素

] 植物激素ABA已在发散光合真核生物中发现,但单细胞藻类的功能仍不清楚。在最近的研究中,我们显示单细胞红藻。 melorae 通过本方案累积ABA以应答盐胁迫。这是用于从C中提取和定量ABA的第一已公布方案的细节。 merolae 。此协议针对 C进行了优化。基于陆地植物协议的。

关键字:脱落酸, 温泉红藻纲, 藻类, HPLC, 植物激素

材料和试剂

  1. 500ml离心瓶(日立,型号:S305830A)
  2. 膜过滤器Millex-GV注射器过滤单元0.22μm(EMD Millipore,目录号:SLGV033RS)
  3. 野生型。 merolae 10D细胞(国家环境研究所,日本
  4. 脱落酸(Sigma-Aldrich,目录号:A4906-250UG)
  5. NaCl(Wako Pure Chemical Industries,目录号:195-15975)
  6. 液氮
  7. 乙酸(Wako Pure Chemical Industries,目录号:017-00256)
  8. 二乙醚(Wako Pure Chemical Industries,目录号:052-01165)
  9. 甲醇(HPLC级)(Wako Pure Chemical Industries,目录号:132-06471)
  10. 硼酸(H 3 BO 3)(Wako Pure Chemical Industries,目录号:021-15645)
  11. 氯化锰(II)四水合物(MnCl 2·4H 2 O)(Wako Pure Chemical Industries,目录号:133-00725)
  12. 硫酸锌七水合物(ZnSO 4)(Wako Pure Chemical Industries,目录号:265-00415)
  13. 无水钼酸钠(Na 2 MoO 4·2H 2 O)(Wako Pure Chemical Industries,目录号:514-30001)
  14. 硫酸铜(II)五水合物(CuSO 4)(Wako Pure Chemical Industries,目录号:034-20065)
  15. 将硝酸钴(II)六水合物(Co [NO 3] sub 2·6H 2 O)(Wako Pure Chemical Industries,目录号:031- 03752)
  16. 硫酸铵([NH 4] 2 Sub SO 4)(Wako Pure Chemical Industries,目录号:016-03445)
  17. 硫酸镁七水合物(MgSO 4·7H 2 O)(Wako Pure Chemical Industries,目录号:138-00415)
  18. 硫酸(H 2 SO 4)(Wako Pure Chemical Industries,目录号:195-04706)
  19. 磷酸二氢钾(KH 2 PO 4)(Wako Pure Chemical Industries,目录号:166-04255)
  20. 氯化钙(CaCl 2)(Wako Pure Chemical Industries,目录号:036-00485)
  21. 氯化铁(III)六水合物(FeCl 3)(Wako Pure Chemical Industries,目录号:090-02802)
  22. Na 2 EDTA(Wako Pure Chemical Industries,目录号:345-01865)
  23. 聚乙烯吡咯烷酮K-30(Nacalai tesque,目录号:28314-82)
  24. 2,6-二叔丁基对甲酚(东京化成工业,目录号:D0228)
  25. MA2介质(参见配方)
  26. 提取溶液(参见配方)

设备

  1. 分光光度计(Beckman Coulter,型号:DU730)
  2. 冷冻离心机(日立,型号:CF16RXII)
  3. 角转子(日立,型号:R10A3)
  4. 砂浆和杵
  5. 涡旋混合器(M& S Instruments,型号:VORTEX-GENIE 2混合器)
  6. 微量离心机(TOMY DIGITAL BIOLOGY,型号:MX150)
  7. 带有低温捕集器的真空离心蒸发器(TOMY DIGITAL BIOLOGY,型号:CC-105系统)
  8. pH计(As One,型号:KR5E)
  9. 装备有光电二极管阵列检测器(PDA)和柱(5μm,4.6×250mm)(Senshu Scientific,型号:ODS SP100)的HPLC系统(Shimadzu,型号:X2 HPLC系统)

程序

  1. 萃取
    1. C的光密度(OD)。 merolae液体培养物通过分光光度计在750nm测量。当OD 750达到10时,在350ml MA2培养基中稀释细胞以产生约0.5的OD 750值(Kobayashi等人, 2010)。在42℃下用荧光白光(50μM光子m -1 -2s -1 s -1)照射下培养细胞,用补充有2%CO 2的空气鼓泡, 2 。在孵育16小时后,测量OD 750,将培养物转移至500ml离心瓶中,并通过用角转子以3,000xg离心3分钟收集细胞室内温度。轻轻地将沉淀重悬于350ml含有500mM NaCl的MA2培养基中,并在相同条件下进一步培养3小时。
    2. 通过在4℃以3,000xg离心3分钟,收获细胞(OD 750 = 0.8,含有约2×10 7个细胞/ml) ℃,通过倾析弃去介质。
    3. 通过移液除去剩余的培养基。将颗粒溶解在MA2(1-2ml)中并在液氮中快速冷冻(视频1)
    4. 用研钵和研杵将冷冻的细胞悬浮液研磨成粉末(视频1)。

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    1. 通过涡旋5分钟并在4℃下以10,000×g离心15分钟,使粉末样品在20ml提取溶液中均质化(视频2)。
    2. 用0.22μm膜滤器过滤上清液(视频2)。

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    1. 在室温下通过真空离心蒸发器浓缩水相(收集约4ml水相)(视频3)。
    2. 通过pH计测量水相的pH。用0.5M乙酸调节水相至pH2.8,用0.22μm膜过滤(视频3)
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    1. 通过用5ml二乙醚的三个分配从水相萃取ABA(视频4)。
    2. 在室温下通过真空离心蒸发器干燥收集的醚层,并将沉淀溶解在300μl甲醇中(视频4)。

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  2. HPLC分析
    1. 在配备有PDA的HPLC系统上进行色谱,使用完全可见光谱,在254nm处进行监测。柱是串联连接的ODS SP100,250×4.6mm,S =5μm。
    2. 用50%甲醇进行等度分离。在40℃下的流速为0.5ml/min,并将样品加入10-50μl。在保留时间7.1分钟检测到ABA的峰(图1)。
    3. 使用ABA参照在254nm下产生标准校准曲线。
    4. 制备浓度为0,10,50,100,200,500,1000pmol /μl的标准ABA,并以10μl应用于HPLC。 HPLC分析应进行三次。


      图1.E.C的HPLC分离。 merolae提取物与ABA标准参考。细胞用500mM NaCl处理3小时。通过具有PDA的HPLC分离ABA标准参考和500mM NaCl处理的细胞提取物。显示了0.2mM ABA标准品(左图),500mM NaCl处理的细胞提取物(中心图)和合并(右图)的吸收光谱。

数据分析

  1. 记录单个峰面积的保留时间并计算每个峰的平均值。
  2. 使用每个峰的平均值创建校准曲线。使用特定的标准校准曲线计算ABA量(示例如图2所示)

    图2. ABA标准曲线。 基于HPLC分析中的峰值进行标准曲线。实施例:从盐胁迫细胞培养物(350ml,OD 750 = 0.8)开始获得300μl提取物,并将10μl300μl进行HPLC分析,结果读取133.533 AU。基于校准曲线,计算起始材料中所含的ABA的总量为68487.5pmol。

笔记

ABA提取和HPLC方法从Kojima方法修改(Kojima等人,1995)。从C检测ABA。 merolae细胞仅在盐胁迫条件下。

食谱

  1. MA2介质
    混合溶液I到III和混乱到1 L.通过高压灭菌消毒。溶液IV应通过0.22微米过滤器过滤灭菌。高压灭菌后将溶液IV加入到混合物中。

    解决方案I


    解决方案II


    解决方案III


    解决方案IV


    A6次要盐


  2. 提取溶液
    0.6g/L聚乙烯吡咯烷酮 0.22g/L 2,6-二叔丁基对甲酚
    溶于80%(v/v)甲醇中

致谢

该协议改编自Kobayashi等人。 (2016年)。作者感谢Tadao Asami博士在ABA检测中的技术帮助。该研究由MEXT/JSPS KAKENHI(批准号:21370015,23120505,2424806,15K14539至K.T.,13274350,15621958至Y.K.)支持,

参考文献

  1. Kobayashi,Y.,Ando,H.,Hanaoka,M.and Tanaka,K。(2016)。 脱落酸参与细胞周期启动的控制在单细胞红藻(Cyanidioschyzon merolae)中的血红素稳态。


    植物细胞生理学57(5),953-960。
  2. Kobayashi,Y.,Ohnuma,M.,Kuroiwa,T.,Tanaka,K.and Hanaoka,M。(2010)。 单细胞红藻的培养和分子遗传分析的基础知识em> Cyanidioschyzon merolae 。  Endocytobiosis Cell Res 20:53-61。
  3. Kojima,K.,Yamada,Y.and Yamamoto,M。(1995)。 脱落酸注射对糖和有机酸含量。日本Soc Hort Sci 64(1):17-21。
  • English
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kobayashi, Y. and Tanaka, K. (2016). Extraction and Measurement of Abscisic Acid in a Unicellular Red Alga Cyanidioschyzon merolae. Bio-protocol 6(23): e2033. DOI: 10.21769/BioProtoc.2033.
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