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Quantification of Salicylic Acid (SA) and SA-glucosides in Arabidopsis thaliana
定量测定拟南芥中的水杨酸(SA)和SA糖苷   

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John V. Dean John V. Dean
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本实验方案简略版
Journal of Experimental Botany
Apr 2016

Abstract

Homeostasis between the cytoplasmic plant hormone salicylic acid (SA) and its’ inactive, vacuolar storage forms, SA-2-O-β-D-glucoside (SAG) and SA-β-D-Glucose Ester (SGE), regulates the fine-tuning of defense responses to biotrophic pathogens in Arabidopsis thaliana. This protocol describes a simplified, optimized procedure to extract and quantify free SA and total hydrolyzable SA in plant tissues using a classical HPLC-based method.

Keywords: Salicylic acid (水杨酸), SA-glucoside (SA糖苷), Defense hormone (防御激素), Arabidopsis thaliana (拟南芥), HPLC (HPLC)

Background

SA (2-hydroxybenzoic acid) is a plant hormone, which is synthesized in the chloroplast in response to pathogen attack. It is then exported to the cytoplasm, where it establishes both local and systemic-acquired resistance (SAR). In a generalized scheme, plant resistance to biotrophic pathogens is thought to be mediated through SA signaling, whereas resistance to necrotrophic pathogens is controlled by jasmonic acid (JA) and ethylene (ET). SA and JA/ET signaling pathways interact antagonistically. SA accumulation to high concentrations is toxic and leads to cell- and tissue damage. Most pathogen-induced SA is thus glycosylated by UDP-glucosyltransferases (UGTs) to form hydrophilic, non-toxic SAG and SGE (Noutoshi et al., 2012; George Thompson et al., 2017). SAG and SGE are then sequestered in vacuoles, where they form reusable sources for hydrolysis to active SA. Increasing amounts of total SA (SA + SAG/SGE) in plant tissues thus reflect SA synthesis as a response to biotrophic pathogen attack. However, the amplitude of defense responses in infected plant tissues is determined by the amount of available cytoplasmic, unconjugated SA. To evaluate both the onset of SA-dependent defense responses and their amplitude, it is essential to quantify free and conjugated SA, respectively. This article describes a method for measuring conjugated and unconjugated SA levels in phase-partitioned extracts from A. thaliana seedlings. It is based on a protocol established for SA analysis in cucumber leaves (Meuwly and Métraux, 1993), which we optimized and downscaled for convenient, routine use.

Materials and Reagents

  1. Pipette tips
  2. Nitrile gloves
  3. Microcentrifuge tubes (2 ml) (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 69720 )
  4. Centrifuge tubes (15 ml) (e.g., Corning, catalog number: 430791 )
  5. Microcentrifuge Tube Locks (LidLocksTM, VWR, catalog number: 14229-941)
    Manufacturer: Sorenson Bioscience, catalog number: 11870 .
  6. Glass wool (e.g., Sigma-Aldrich, catalog number: 18421 )
  7. Ten-day-old Arabidopsis thaliana seedlings (e.g., different genetic wild-type or mutant backgrounds, inoculated with pathogen or otherwise treated)
  8. Liquid nitrogen
  9. Ethanol (EtOH; e.g., Sigma-Aldrich, catalog number: 24103 )
  10. 70% aqueous EtOH (v/v)
  11. Methanol HPLC grade (MeOH; e.g., CARLO ERBA Reagents, catalog number: 412383 )
  12. 90% aqueous MeOH (v/v)
  13. Trichloracetic acid (TCA; e.g., Sigma-Aldrich, catalog number: T9159 )
  14. 20% aqueous TCA (w/v)
  15. Ethyl acetate, analytical grade (e.g., CARLO ERBA Reagents, catalog number: 448256 )
  16. Cyclohexane, analytical grade (e.g., CARLO ERBA Reagents, catalog number: 436903 )
  17. A mixture of ethyl acetate and cyclohexane (1:1, v:v)
  18. Trifluoracetic acid (TFA; e.g., Sigma-Aldrich, catalog number: T62200 )
  19. 10% aqueous MeOH (v/v) with 0.1% TFA (v/v)
  20. 82% aqueous MeOH (v/v) with 0.1% TFA (v/v)
  21. Concentrated hydrochloric acid (HCl; 37%, 12 M; e.g., Sigma-Aldrich, catalog number: 30721 )
  22. Ultra-pure water
  23. 2-Methoxybenzoic acid, o-Anisic acid (OAA; e.g., Sigma-Aldrich, catalog number: 169978 )
  24. Sodium salicylate (e.g., Sigma-Aldrich, catalog number: S3007 )
  25. o-Anisic acid 50x stock solution (see Recipes)

Equipment

  1. Micropipettes (e.g., Gilson, model: P2 , P20 , P200 , P1000 )
  2. Mortar (40 ml content) with pestle
  3. Fume hood
  4. Water purification system (Merck, EMD Millipore, catalog number: SYNS0HFWW )
  5. Vortex (e.g., IKA, model: MS 1 minishaker )
  6. Dry bath heating block for 2 ml microcentrifuge tubes (e.g., Major Science, model: EL-02 )
  7. Vacuum concentrator (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: Savant SpeedVac Concentrator )
  8. Vacuum pump (e.g., BÜCHI Labortechnik, model: V-300 )
  9. Microcentrifuge (e.g., Eppendorf, model: 5415 D with Rotor: Eppendorf, model: F45-24-11 )
  10. C18 HPLC column (e.g., Inertsil 5 ODS3, 5 µm, 250 x 4.6 mm i.d., Interchim, France) (GL Sciences, model: Inertsil® ODS3 )
  11. HPLC System (Shimadzu Prominence LC System) equipped with:
    2 solvent delivery units (Shimadzu Scientific, model: LC-20AD )
    A system controller (Shimadzu Scientific, model: CBM-20A )
    An autosampler (Shimadzu Scientific, model: SIL-20AC )
    A column oven (Shimadzu Scientific, model: CTO-20A )
    A diode array detector (Shimadzu Scientific, model: SPD-M20A )
    A fluorescence detector (Shimadzu Scientific, model: RF-10AXL )

Software

  1. Chromatography data system software (e.g., WATERS, Empower 3 Pro Chromatography Data Software)
  2. Microsoft Excel

Procedure

  1. Sample preparation
    1. Harvest and weigh 10 day-old Arabidopsis plantlets with fully developed cotyledons, leaf primordia, and hypocotyls, as shown in Figure 1. Transfer 400 mg fresh material (corresponding to ~250 plantlets) to a mortar, which was pre-cooled with liquid nitrogen, and grind to a fine powder under liquid nitrogen. Transfer this powder to a 2 ml microcentrifuge tube. Add 1.6 ml of 70% aqueous EtOH (v/v) and 32 µl o-anisic acid (OAA) stock solution (15.25 ng/µl; Figure 1).


      Figure 1. Flowchart for the preparation of SA-containing samples

    2. Vortex for 1 min.
    3. Centrifuge in a microcentrifuge for 10 min at 10,000 x g at room temperature.
    4. Transfer supernatant to a 15 ml centrifuge tube.
    5. From now on, use a fume hood. Add 1.6 ml of 90% aqueous MeOH (v/v) to the remaining pellet.
    6. Vortex for 1 min for re-extraction.
    7. Centrifuge in a microcentrifuge for 10 min at 10,000 x g at room temperature.
    8. Add this supernatant to the previous supernatant in the 15 ml centrifuge tube from Step A4.
    9. The pooled, clear supernatants contain free SA and SA-glucosides (Figure 1).

  2. Extraction of free SA
    1. Transfer 2 ml of the cleared supernatants to 2 ml microcentrifuge tubes (Figure 1).
    2. Evaporate alcohol (EtOH and MeOH) in a vacuum concentrator (without heating) for ~1.5 h.
    3. Transfer the remaining supernatant from the 15 ml centrifuge tube to the same 2 ml tube as used in Step A1.
    4. Continue evaporating the alcohol (EtOH and MeOH) in the vacuum concentrator for ~1.5 h.
    5. To the remaining aqueous solution (~600 µl) in the 2 ml microcentrifuge tube, add 65 µl of 20% aqueous TCA (w/v).
    6. Add 650 µl of a 1:1 (v/v) mixture of ethyl acetate and cyclohexane.
    7. Vortex for 30 sec.
    8. Centrifuge in a microcentrifuge for 2 min at 10,000 x g at room temperature for phase separation.
    9. Transfer the upper organic phase to a new 2 ml microcentrifuge tube.
    10. Re-extract the aqueous phase with 650 µl of the ethyl acetate-cyclohexane mixture.
    11. Centrifuge in a microcentrifuge for 2 min at 10,000 x g at room temperature for phase separation.
    12. Pool the organic phases in the new 2 ml centrifuge tube. They contain free, unconjugated SA. Store the aqueous phase for the treatment of SA-glucosides at 4 °C.
    13. Evaporate the solvents to dryness in a vacuum concentrator for ~30-45 min.
    14. Solubilize the dry residue in 100 µl of 10% aqueous MeOH (v/v) containing 0.1% aqueous TFA (v/v), vortex for 1 min. This fraction is ready for HPLC analysis (= free, unconjugated SA) (Figure 1).

  3. Extraction of hydrolyzable SA
    1. To the aqueous phase (B12; ~0.6 ml), add 0.5 volumes (~0.3 ml) of HCl (37%, 12 M) to obtain a final HCl concentration of 4 M (Figure 1).
    2. Secure the tubes with microcentrifuge tube locks and heat them for 1 h at 80 °C in a dry bath heating block. Let cool down to room temperature for 20 min.
    3. Add 0.02 volumes (18 µl) of 50x OAA stock solution.
    4. Add 1 volume (0.9 ml) of the 1:1 ethyl acetate–cyclohexane (v/v) mixture.
    5. Vortex for 30 sec.
    6. Centrifuge in a microcentrifuge for 2 min at 10,000 x g at room temperature for phase separation.
    7. Transfer the upper phase to a new 2 ml microcentrifuge tube.
    8. Re-extract the aqueous phase with 1 volume (0.9 ml) of the 1:1 (v/v) mixture of ethyl acetate and cyclohexane.
    9. Centrifuge in a microcentrifuge for 2 min at 10,000 x g at room temperature for phase separation.
    10. Pool the organic phases in the 2 ml centrifuge tube. They contain SA, which results from the acidic hydrolysis of SA-conjugates.
    11. Evaporate to dryness in a vacuum concentrator for ~30 min.
    12. Solubilize the dry residue in 100 µl of 10% aqueous MeOH (v/v) containing 0.1% aqueous TFA (v/v), vortex for 1 min. This fraction is ready for HPLC analysis (= total hydrolyzable SA) (Figure 1).

  4. SA quantification by HPLC
    1. Separations by HPLC are performed on a C18 column (250 x 4.6 mm, 5 µm) using a linear aqueous MeOH gradient from 10% to 82% (v/v), at a flow rate of 1 ml/min, over 30.4 min. Solvents contain 0.1% TFA (v/v) to maintain the protonated form of carboxylic acids. The column is maintained in an oven set at 30 °C. Eluates pass first the diode array detector, then the fluorescence detector.
    2. Inject 20 µl samples (B14 and C12) into the HPLC system. SA elutes at 25 min, OAA at 20 min. Determine the presence of SA and the internal standard with peak validation according to typical UV spectra (Figures 2A and 2B). Quantify with fluorimetric detection (excitation at 305 nm; emission at 407 nm) and determine areas under the corresponding peaks (Figures 2C and 2D).
    3. Using the peak area, apply the standard curve and linear equation to determine the amount (ng) of SA and of OAA in the injected 20 µl. Determine the correction factor by comparing the theoretic amounts of OAA in the samples with the experimentally measured ones. Multiply the measured SA amounts with this correction factor, calculate SA amounts (before and after hydrolysis), and express them as ng (SA and SAG/SGE, respectively; Note 5) per gram of cotyledon fresh weight as shown by Quentin et al. (2016).


      Figure 2. UV spectra for SA (A) and OAA (B), and chromatograms of fluorescence-detected free SA (C) and SA-hydrolysates (D) in extracts from Arabidopsis seedlings, four days after inoculation with the Hyaloperonospora arabidopsidis isolate Waco 9

  5. Calibration curves for SA and OAA
    1. Prepare five solutions containing SA and OAA at different concentrations, ranging from 0.1 µg/ml to 5 µg/ml in 10% aqueous MeOH (v/v) containing 0.1% TFA (v/v).
    2. Inject 20 µl from each preparation, run the linear gradient and detect as described in Step D1. Repeat two times.
    3. Using the chromatography data system software, integrate the area under the peaks at 20 min (OAA) and 25 min (SA) for every concentration. Plot a graph of peak area versus ng of injected SA and OAA. Calculate the equation of the trend line using linear regression analysis (R2 must be > 0.98).

Data analysis

  1. At least three biological replicates should be used for the quantification of free and total hydrolyzable SA.
  2. Statistical analysis should be performed by calculating means and standard deviations across these replicates. The significance of differences between two samples (different Arabidopsis genotypes or different treatments) might be estimated with the non-parametric Kruskal-Wallis test by ranks. Analyses can be performed with Microsoft Excel or comparable software.

Notes

  1. Optional step after Step A8: If the pooled supernatants contain visible cellular debris, remove them by filtering through home-made glass wool-filled 1 ml pipet tips.
  2. Vacuum concentrator and vacuum pump should be used in a fume hood or in appropriate areas, when evaporating organic solvents.
  3. Fluorimetric detection with excitation at 305 nm and emission at 407 nm is optimal for SA, but not for OAA, which has maximum emission at 365 nm when excited at 305 nm. However, OAA gives quantifiable peaks at 407 nm (Figures 2C and 2D).
  4. In our HPLC set-up, eluates pass the diode array detector first and then the fluorescence detector. This leads to a lag of ~0.1 min for fluorescence detection, when compared to UV detection.
  5. It is established that SAG and SGE represent the large majority of stress-induced SA conjugates in wild-type Arabidopsis (Zhao et al., 2017). However, when transposing the protocol to other plants, or to Arabidopsis lines harboring mutations and transgenes that affect the plant secondary metabolism, SAG and SGE must first be confirmed as main SA conjugates. This might be achieved by enzymatic cleavage of SAG and SGE with β-glucosidase and short chain esterase, respectively (Enyedi et al., 1992). Obtaining comparable recovery of SA from double enzymatic digestion and acid hydrolysis is a prerequisite to express results as the amount of SA released from SAG + SGE.

Recipes

  1. 2-Methoxybenzoic acid (o-Anisic acid, OAA; internal standard) stock solution (50x, 0.1 µM)
    Dissolve 152 mg OAA in 10 ml 70% aqueous EtOH (v/v) and dilute 1:1,000 in ultra-pure water

Acknowledgments

This protocol was modified from previously published works (Meuwly and Métraux, 1993; Quentin et al., 2016). The work was supported by the French Government (National Research Agency, ANR) through the ‘Investments for the Future’ LABEX SIGNALIFE [program reference #ANR-11-LABX-0028-01]. The authors declare no conflicts of interest or competing interests.

References

  1. Enyedi, A. J., Yalpani, N., Silverman, P. and Raskin, I. (1992). Localization, conjugation, and function of salicylic acid in tobacco during the hypersensitive reaction to tobacco mosaic virus. Proc Natl Acad Sci U S A 89: 2480-2484.
  2. George Thompson, A. M., Iancu, C. V., Neet, K. E., Dean, J. V. and Choe, J. Y. (2017). Differences in salicylic acid glucose conjugations by UGT74F1 and UGT74F2 from Arabidopsis thaliana. Sci Rep 7: 46629.
  3. Meuwly, P. and Métraux, J. P. (1993). Ortho-anisic acid as internal standard for the simultaneous quantitation of salicylic acid and its putative biosynthetic precursors in cucumber leaves. Anal Biochem 214(2): 500-505.
  4. Noutoshi, Y., Okazaki, M., Kida, T., Nishina, Y., Morishita, Y., Ogawa, T., Suzuki, H., Shibata, D., Jikumaru, Y., Hanada, A., Kamiya, Y. and Shirasu, K. (2012). Novel plant immune-priming compounds identified via high-throughput chemical screening target salicylic acid glucosyltransferases in Arabidopsis. Plant Cell 24(9): 3795-3804.
  5. Quentin, M., Baurès, I., Hoefle, C., Caillaud, M. C., Allasia, V., Panabières, F., Abad, P., Hückelhoven, R., Keller, H. and Favery, B. (2016). The Arabidopsis microtubule-associated protein MAP65-3 supports infection by filamentous biotrophic pathogens by down-regulating salicylic acid-dependent defenses. J Exp Bot 67(6): 1731-1743.
  6. Zhao, P., Lu, G. H. and Yang, Y. H. (2017). Salicylic acid signaling and its role in responses to stresses in plants. In: Pandey, G. K. (Ed.). Mechanisms of Plant Hormone Signaling under Stress. John Wiley & Sons 413-441.

简介

细胞质植物激素水杨酸(SA)与其无活性的液泡形式,SA-2-葡萄糖苷(SAG)和SA-β-D-葡萄糖酯之间的稳态 (SGE)规定了拟南芥中对营养性病原体防御反应的微调。 该协议描述了一种简化的优化程序,使用传统的基于HPLC的方法提取和定量植物组织中的游离SA和总可水解SA。

【背景】SA(2-羟基苯甲酸)是植物激素,其在叶绿体中响应于病原体攻击而合成。然后输出到细胞质中,在细胞质中建立局部和系统获得性抗性(SAR)。在广义方案中,植物对生物营养性病原体的抗性被认为是通过SA信号传导介导的,而对于坏死性病原体的抗性受茉莉酸(JA)和乙烯(ET)控制。 SA和JA / ET信号通路相互作用。 SA积累到高浓度是有毒的并导致细胞和组织损伤。因此,大多数病原体诱导的SA被UDP-葡糖基转移酶(UGT)糖基化以形成亲水的,无毒的SAG和SGE(Noutoshi等人,2012; George Thompson等人, 2017)。然后将SAG和SGE隔离在液泡中,在那里它们形成水解成活性SA的可重复使用的来源。因此,植物组织中增加的总SA(SA + SAG / SGE)量反映了SA合成作为对生物营养性病原体攻击的反应。然而,受感染植物组织中防御反应的幅度由可用的胞质未结合SA的量决定。为了评估SA依赖性防御反应的发作和它们的幅度,分别定量游离和共轭SA是必要的。本文介绍了一种测量A相分配提取物中共轭和非共轭SA水平的方法。 thaliana 幼苗。它基于为黄瓜叶片中的SA分析建立的协议(Meuwly和Métraux,1993),我们对其进行了优化和缩减以便于日常使用。

关键字:水杨酸, SA糖苷, 防御激素, 拟南芥, HPLC

材料和试剂

  1. 移液器吸头
  2. 丁腈手套
  3. 微量离心管(2ml)(例如,Thermo Fisher Scientific,Thermo Scientific TM,目录号:69720)
  4. 离心管(15ml)(如emning,Corning,目录号:430791)
  5. 微量离心管锁(LidLocks TM ,VWR,目录号:14229-941)
    制造商:Sorenson Bioscience,目录号:11870。
  6. 玻璃棉(,例如,Sigma-Aldrich,目录号:18421)
  7. 十日龄拟南芥幼苗(例如,不同的遗传野生型或突变体背景,接种病原体或以其他方式处理)
  8. 液氮
  9. 乙醇(EtOH;例如,Sigma-Aldrich,目录号:24103)
  10. 70%EtOH水溶液(v / v)
  11. 甲醇HPLC级(MeOH;例如,CARLO ERBA试剂,目录号:412383)
  12. 90%MeOH水溶液(v / v)
  13. 三氯乙酸(TCA;例如,Sigma-Aldrich,目录号:T9159)
  14. 20%含水TCA(w / v)
  15. 乙酸乙酯,分析等级(例如,CARLO ERBA试剂,目录号:448256)
  16. 环己烷,分析等级(例如,,CARLO ERBA试剂,目录号:436903)
  17. 乙酸乙酯和环己烷的混合物(1:1,v:v)
  18. 三氟乙酸(TFA;例如,Sigma-Aldrich,目录号:T62200)
  19. 10%含0.1%TFA(v / v)的MeOH水溶液(v / v)
  20. 含有0.1%TFA(v / v)的82%MeOH水溶液(v / v)
  21. 浓盐酸(HCl; 37%,12M;例如,Sigma-Aldrich,目录号:30721)
  22. 超纯水
  23. 2-甲氧基苯甲酸,肉桂酸(OAA;例如,Sigma-Aldrich,目录号:169978)
  24. 水杨酸钠(例如,Sigma-Aldrich,目录号:S3007)
  25. o - 肉豆蔻酸50x原液(见食谱)

设备

  1. Micropipettes( ,Gilson,型号:P2,P20,P200,P1000)
  2. 杵(含40毫升)杵
  3. 通风橱
  4. 水净化系统(Merck,EMD Millipore,目录号:SYNS0HFWW)
  5. 涡流(,例如,IKA,型号:MS 1 minishaker)
  6. 用于2ml微量离心管的干浴加热块(,例如,Major Science,型号:EL-02)
  7. 真空浓缩器(例如,Thermo Fisher Scientific,Thermo Scientific TM,型号:Savant SpeedVac浓缩器)
  8. 真空泵(例如,BÜCHILabortechnik,型号:V-300)
  9. 微量离心机(例如Eppendorf,型号:5415 D,带转子:Eppendorf,型号:F45-24-11)
  10. C18 HPLC柱(例如,Inertsil 5ODS3,5μm,250x4.6mm内径,Interchim,法国)(GL Sciences,型号:InertsilODS3) >
  11. 配备有:
    的HPLC系统(Shimadzu Prominence液相色谱系统) 2个溶剂输送装置(岛津科学,型号:LC-20AD)
    系统控制器(Shimadzu Scientific,型号:CBM-20A)
    自动进样器(Shimadzu Scientific,型号:SIL-20AC)
    柱温箱(Shimadzu Scientific,型号:CTO-20A)
    二极管阵列检测器(Shimadzu Scientific,型号:SPD-M20A)
    荧光检测器(Shimadzu Scientific,型号:RF-10A XL )

软件

  1. 色谱数据系统软件(,例如,WATERS,Empower 3 Pro色谱数据软件)
  2. Microsoft Excel

程序

  1. 样品制备
    1. 如图1所示,收获并称量具有完全发育的子叶,叶原基和下胚轴的10日龄拟南芥幼苗。将400mg新鲜材料(对应于〜250个植株)转移到研钵中,其用液氮预冷,在液氮下研磨成细粉。将此粉末转移到2 ml微量离心管中。加入1.6ml 70%EtOH水溶液(v / v)和32μl茴香酸(OAA)原液(15.25ng /μl;图1)。


      图1.制备含有SA的样品的流程图

    2. 涡旋1分钟。
    3. 在室温下以10000×g的密度在微量离心机中离心10分钟。
    4. 将上清转移到15 ml离心管中。
    5. 从现在开始,使用通风橱。
      加入1.6 ml的90%甲醇水溶液(v / v)至剩余的颗粒
    6. 涡旋1分钟以重新提取。
    7. 在室温下以10000×g的密度在微量离心机中离心10分钟。
    8. 将上清液加入到步骤A4的15ml离心管中的上清液中。
    9. 汇集的清澈上清液含有游离的SA和SA葡萄糖苷(图1)。

  2. 提取游离SA
    1. 将2毫升澄清的上清液转移至2毫升微量离心管中(图1)。

    2. 在真空浓缩器中(不加热)蒸发酒精(EtOH和MeOH)约1.5小时。
    3. 将剩余的上清液从15ml离心管中转移到步骤A1中使用的相同的2ml管中。

    4. 在真空浓缩器中继续蒸发酒精(EtOH和MeOH)约1.5小时。
    5. 向2 ml微量离心管中剩余的水溶液(〜600μl)中加入65μl20%TCA(w / v)。
    6. 加入650μl乙酸乙酯和环己烷的1:1(v / v)混合物。
    7. 涡旋30秒。

    8. 在室温下在10,000×g g离心2分钟以离心分离。
    9. 将上层有机相转移到新的2 ml离心管中。

    10. 用650μl乙酸乙酯 - 环己烷混合物重新提取水相

    11. 在室温下在10,000×g g离心2分钟以离心分离。
    12. 在新的2 ml离心管中合并有机相。它们包含免费的,非共轭的SA。
      在4°C储存SA-葡萄糖苷水相。

    13. 在真空浓缩器中蒸发溶剂至干燥约30-45分钟。
    14. 将干燥的残余物溶于100μl含有0.1%TFA水溶液(v / v)的10%MeOH水溶液(v / v)中,涡旋1分钟。这部分可用于HPLC分析(=游离的,未结合的SA)(图1)。

  3. 可水解SA的提取
    1. 向水相(B12;〜0.6ml)中加入0.5体积(〜0.3ml)HCl(37%,12M)以获得4M的最终HCl浓度(图1)。
    2. 用微量离心管锁固定管,并在80℃的干浴加热块中加热1小时。冷却至室温20分钟。

    3. 添加0.02体积(18μl)的50x OAA原液
    4. 加入1体积(0.9ml)1:1乙酸乙酯 - 环己烷(v / v)混合物。
    5. 涡旋30秒。

    6. 在室温下在10,000×g g离心2分钟以离心分离。
    7. 将上层相转移到新的2毫升微量离心管中。
    8. 用1体积(0.9ml)1:1(v / v)乙酸乙酯和环己烷的混合物重新萃取水相。

    9. 在室温下在10,000×g g离心2分钟以离心分离。
    10. 将有机相在2 ml离心管中合并。它们含有SA,这是SA偶联物酸性水解的结果。

    11. 在真空浓缩器中蒸发至干燥约30分钟。
    12. 将干燥的残余物溶于100μl含有0.1%TFA水溶液(v / v)的10%MeOH水溶液(v / v)中,涡旋1分钟。这部分已准备好用于HPLC分析(=全部可水解的SA)(图1)。

  4. SA通过HPLC定量
    1. 通过HPLC在C18柱(250×4.6mm,5μm)上使用10%至82%(v / v)的线性MeOH水溶液梯度以1ml / min的流速在30.4分钟内进行分离。溶剂含有0.1%TFA(v / v)以维持羧酸的质子化形式。色谱柱保持在30℃的烘箱中。洗脱物先通过二极管阵列检测器,然后通过荧光检测器。
    2. 将20μl样品(B14和C12)注入HPLC系统。 SA在25分钟时洗脱,OAA在20分钟时洗脱。根据典型的紫外光谱(图2A和2B)确定SA和内标物的存在以及峰验证。用荧光检测(305nm激发; 407nm发射)量化并确定相应峰下的面积(图2C和2D)。
    3. 使用峰面积,应用标准曲线和线性方程来确定注入的20μl中SA和OAA的量(ng)。通过比较样品中的OAA的理论量和实验测量的量来确定校正因子。将测量的SA量乘以该校正因子,计算SA量(水解前后),并将其表示为ng(分别为SA和SAG / SGE;注5)每克子叶鲜重,如Quentin em所示(2016)。


      图2. SA(A)和OAA(B)的紫外光谱以及来自拟南芥幼苗的提取物中荧光检测的游离SA(C)和SA-水解产物(D)的色谱图,接种后用透明霜霉分离物Waco 9
      接种4天
  5. SA和OAA的校准曲线
    1. 在含有0.1%TFA(v / v)的10%甲醇水溶液(v / v)中制备含有不同浓度的SA和OAA的五种溶液,范围从0.1μg/ ml到5μg/ ml。
    2. 从每个制备物中注入20μl,运行线性梯度并按照步骤D1中所述进行检测。重复两次。
    3. 使用色谱数据系统软件,在每个浓度20分钟(OAA)和25分钟(SA)的峰下面积积分。绘制注射的SA和OAA的峰面积对ng的图。使用线性回归分析计算趋势线的方程(R 2必须> 0.98)。

数据分析


  1. 至少有三个生物学复制品可用于定量游离和总可水解的SA
  2. 统计分析应通过计算平均值和标准偏差进行。两个样品(不同的拟南芥基因型或不同的处理)之间差异的显着性可以用非参数Kruskal-Wallis检验按等级估算。分析可以使用Microsoft Excel或类似软件执行。

笔记

  1. 步骤A8后的可选步骤:如果合并的上清液含有可见的细胞碎片,请通过使用自制玻璃棉填充1 ml移液器吸头进行过滤将其去除。

  2. 在蒸发有机溶剂时,应在通风橱或适当区域使用真空浓缩器和真空泵
  3. 在305nm激发和407nm发射的荧光检测对于SA是最佳的,但对于在305nm激发时在365nm具有最大发射的OAA不是。然而,OAA在407nm处给出可量化的峰(图2C和2D)。
  4. 在我们的HPLC设置中,洗脱液首先通过二极管阵列检测器,然后通过荧光检测器。与紫外检测相比,这导致荧光检测延迟约0.1分钟。
  5. 已经证实,SAG和SGE代表野生型拟南芥中大部分胁迫诱导的SA缀合物(Zhao等人,2017)。然而,将该方案转移到其他植物或含有影响植物次生代谢的突变和转基因的拟南芥属系中时,必须首先确认SAG和SGE为主要的SA缀合物。这可以通过分别用β-葡糖苷酶和短链酯酶酶促切割SAG和SGE来实现(Enyedi等人,1992)。从双酶消化和酸水解中获得可比较的SA回收率是表示SAAG + SGE释放SA量的结果的先决条件。

食谱

  1. 2-甲氧基苯甲酸( o -Anisic acid,OAA;内标)储备溶液(50×,0.1μM)
    将152毫克OAA溶解在10毫升70%EtOH水溶液(v / v)中并在超纯水中稀释1:1,000

致谢

该协议是从以前发表的作品(Meuwly和Métraux,1993; Quentin等人,2016年)进行修改的。法国政府(国家研究机构,ANR)通过“未来投资”LABEX SIGNALIFE [程序参考#ANR-11-LABX-0028-01]支持该项工作。作者声明不存在利益冲突或利益冲突。

参考

  1. Enyedi,A.J.,Yalpani,N.,Silverman,P.and Raskin,I。(1992)。 烟草花叶病毒超敏反应过程中水杨酸在烟草中的定位,结合和功能< Proc Natl Acad Sci USA 89:2480-2484。
  2. George Thompson,A.M.,Iancu,C.V.,Neet,K.E.,Dean,J.V。和Choe,J.Y。(2017)。 拟南芥UGT74F1和UGT74F2在水杨酸葡萄糖结合中的差异拟南芥。 Sci Rep 7:46629.
  3. Meuwly,P.和Métraux,J. P.(1993)。 邻茴香酸作为同时定量测定黄瓜中水杨酸及其推定的生物合成前体的内标叶子。 Anal Biochem 214(2):500-505。
  4. Noutoshi,Y.,Okazaki,M.,Kida,T.,Nishina,Y.,Morishita,Y.,Ogawa,T.,Suzuki,H.,Shibata,D.,Jikumaru,Y.,Hanada, Kamiya,Y.和Shirasu,K。(2012)。 通过高通量化学筛选目标水杨酸葡萄糖基转移酶鉴定的新型植物免疫启动化合物 Arabidopsis 。 Plant Cell 24(9):3795-3804。
  5. Quentin,M.,Baurès,I.,Hoefle,C.,Caillaud,MC,Allasia,V.,Panabières,F.,Abad,P.,Hückelhoven,R.,Keller,H.和Favery,B。(2016 )。 拟南芥微管相关蛋白MAP65-3支持丝状生物营养通过下调水杨酸依赖性防御的病原体。 J Exp Bot 67(6):1731-1743。
  6. Zhao,P.,Lu,G.H。和Yang,Y.H。(2017)。 水杨酸信号转导及其在植物逆境中的作用在: Pandey,GK(Ed。)。植物激素信号胁迫下的机制。 John Wiley&amp;儿子 413-441。
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引用:Allasia, V., Industri, B., Ponchet, M., Quentin, M., Favery, B. and Keller, H. (2018). Quantification of Salicylic Acid (SA) and SA-glucosides in Arabidopsis thaliana. Bio-protocol 8(10): e2844. DOI: 10.21769/BioProtoc.2844.
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