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本实验方案简略版
Aug 2018

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Quantification of Blumenol Derivatives as Leaf Biomarkers for Plant-AMF Association
蓝甲醇衍生物作为丛枝菌根真菌与植物共生标志物的定量研究   

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Abstract

Symbiotic interactions between arbuscular mycorrhizal fungi (AMF) and plants are widespread among land plants and can be beneficial for both partners. The plant is provided with mineral nutrients such as nitrogen and phosphorous, whereas it provides carbon resources for the fungus in return. Due to the large economic and environmental impact, efficient characterization methods are required to monitor and quantify plant-AMF colonization. Existing methods, based on destructive sampling and elaborate root tissue analysis, are of limited value for high-throughput (HTP) screening. Here we describe a detailed protocol for the HTP quantification of blumenol derivatives in leaves by a simple extraction procedure and sensitive liquid chromatography mass spectrometry (LC/MS) analysis as accurate proxies of root AMF-associations in both model plants and economically relevant crops.

Keywords: Arbuscular mycorrhizal fungi (丛枝菌根真菌), AMF (AMF), Fungus-plant symbiosis (真菌-植物共生), Leaf biomarker (叶片生物标志物), Shoot biomarker (根生物标志物), Blumenol derivatives (蓝甲醇衍生物), UHPLC-MS/MS technique (UHPLC-MS/MS技术)

Background

The widespread mutualistic relationship between AMF and plants involves not only the beneficial exchange of nutrients between the involved partners; phosphorous and nitrogen are supplied by the fungus and carbon is supplied by the plant in exchange; but is also thought to regulate plant growth and tolerance to various biotic and abiotic stresses (Maier et al., 1995; Barin et al., 2013; Aliferis et al., 2015; Wang et al., 2018). These interactions have fueled vast research programs and, in conjunction with dwindling natural phosphorous supplies, are of high interest for sustainable agriculture (Basu et al., 2018). Until now, the available approaches to measure and quantify AMF-plant associations require excavation of the roots followed by microscopic analysis, transcript analysis or quantification of fungal fatty acids (Barin et al., 2013). However, these methods are impractical for HTP screening due to the damage that results from root sampling, as well as being laborious (Barin et al., 2013; Wang et al., 2018). Hence, an HTP screening technique is needed to empower research and development in breeding programs for improved AMF-plant associations. Even though AMF colonization leads to systemic responses throughout the plant, until recently no AMF-specific metabolic response has been detected in plant parts other than in roots (Aliferis et al., 2015; Hill et al., 2018). The described protocol is based on a MeOH extraction of leaf tissue followed by Ultra High Performance Liquid Chromatography Mass Spectrometry (UHPLC-MS) analysis as described by Wang et al. (2018). The concentrations of foliar 11-hydroxy- and 11-carboxyblumenol C derivatives are not detectable in non-mycorrhized plants and are positively and quantitatively correlated with AMF root colonization and are transported from roots to the leaves after the formation of root-AMF associations (Wang et al., 2018). This protocol facilitates an HTP, non-destructive and quantitative characterization of AMF associations in various model and agricultural crop plant species.

Materials and Reagents

  1. Pipette tips
  2. 96-well microplates with full skirt (Sapphire, Greiner Bio-One, catalog number: 652270)
  3. (Optional) Individual tubes
    Note: Individual tubes can be used instead of 96-well BioTubesTM for small batches of samples.
    1. 2 ml Eppendorf Safe-Lock tubes (Eppendorf, catalog number: 0030120094)
    2. 1.5 ml screw neck vials N9 (Macherey-Nagel, catalog number: 702282)
    3. N9 PP screw caps (Macherey-Nagel, catalog number: 702287.1)
    4. Steel balls Ø 4 mm (ASKUBAL, G100-1.4034, catalog number: 503012)
  4. Sealing film for 96-well microplates (Zone-freeTM, EXCEL Scientific, catalog number: ZAF-PE-50)
  5. 96-well PCR Plate (µltraAmp, SorensonTM BioScience Inc, catalog number: 21970)
  6. Domed 8-strip PCR caps (Eppendorf, catalog number: 0030124839)
  7. Steel balls Ø 3 mm (ASKUBAL, G100-1.4034, catalog number: 505001)
  8. Leaf material
  9. Liquid nitrogen
  10. MilliQ water
  11. Deuterated internal standard: D6-ABA (HPC Standards GmbH, 10 µg ml-1 in MeOH)
  12. Acetonitrile (VWR International, HiPerSolv CHROMANORM® for LC-MS, catalog number: BDH83640.100E)
  13. Formic acid (Fluka, for mass spectrometry, catalog number: 94318)
  14. Methanol (Merck, Gradient grade for LC LiChrosolv®, catalog number: 1060072500)
  15. Roseoside (Wuhan ChemFaces Biochemical Co., Ltd., catalog number: CFN98916)
  16. Corchoionoside C (Wuhan ChemFaces Biochemical Co., Ltd., catalog number: CFN99859)
  17. Blumenol C glucoside (Wuhan ChemFaces Biochemical Co., Ltd., catalog number: CFN99424)
  18. Byzantionoside B (Wuhan ChemFaces Biochemical Co., Ltd., catalog number: CFN99871)
  19. Extraction buffer with deuterated internal standard D6-ABA (see Recipes)

Equipment

  1. Stainless steel spatula
  2. Stainless steel tweezers
  3. 96-well tube racks (BioTubeTM, Simport® Scientific, catalog number: T101-1 and T100-20)
  4. Sealing mats for 96-well tube racks (ArctiSealTM, Arctic White LLC, catalog number: AWSM-2002RB)
  5. Cooling containers (Heathrow Scientific, True North®)
  6. Centrifuge (Eppendorf, model: 5415 R)
  7. Multipipette (Multipette® Xstream, Eppendorf, catalog number: 4986000025)
  8. Mortar and pestle (HaldenwangerTM, Fisher Scientific)
  9. Analytical balance (Sartorius, model: BP121S)
  10. 8-channel electronic pipette (Eppendorf, Xplorer®, 50-1,200 µl, catalog number: 4861000163)
  11. Tissue homogenizer (Geno/Grinder® 2000, SPEX SamplePrep)
  12. Cooled centrifuge equipped with 96-well plate rotor (Eppendorf, model: 5804 R, rotor A-2-DWP)
  13. UHPLC triple quadrupole MS instrument [Ultimate 3000 RSLC (Thermo Fisher Scientific); EVO-Q EliteTM (Bruker)]
  14. UHPLC column (ZORBAX Eclipse XDB-C18, 50 x 3.0 mm, 1.8 µm, Agilent, catalog number: 981757-302)
  15. -80 °C freezer

Software

  1. MS Data Review Version 8.2.1 (MS Workstation, Bruker Daltonics)

Procedure

Notes:

  1. In order to test the applicability of the method for the analyzed plant/AMF species, it is advised to perform an initial test screen with root tissue as the abundance of blumenol derivatives in root tissue is higher than in leaves.
  2. Blumenol levels can vary in different shoot tissues (Wang et al., 2018). Harvesting tissue samples from leaves at comparable developmental stages will reduce variation and allow better comparisons between plants.
  3. Blumenol levels reliably indicate AMF colonization 3 weeks after inoculation (Wang et al., 2018).

  1. Collection and preparation of leaf material
    1. Harvest leaves and immediately freeze in liquid nitrogen using stainless steel tweezers. Store at -80 °C until processing the samples.
    2. Grind the frozen leaf material with mortar and pestle under liquid nitrogen.
    3. Aliquot approximately 100 mg leaf material with a pre-cooled stainless steel spatula into liquid nitrogen-precooled and pre-weighted 96-well BioTubeTM racks containing two steel balls (Ø 3 mm). Record the exact mass and leave the samples on liquid nitrogen for extraction or store at -80 °C.
      Note: Instead of 96-well BioTubesTM, 2 ml Eppendorf tubes equipped with two steel balls (Ø 4 mm) can be utilized.

  2. Extraction
    1. Add 800 µl of ice-cold extraction buffer containing the internal standard D6-ABA to each tube with an 8-channel pipette. Replace the tube caps with a rubber sealing mat.
      Note: Samples should be kept on ice during the extraction procedure.
    2. Homogenize the samples in a Geno/Grinder® for 60 s at 1,000 strokes per minute (Geno/Grinder® 2000 setting: 1x at 000).
    3. Centrifuge the samples at 2,000 x g for 20 min at 4 °C, transfer the supernatant to a new 96-well BioTubeTM rack or Eppendorf tubes without steel balls and centrifuge again under the same conditions.
    4. Transfer 100 µl of the supernatant into skirted 96-well microplates and close wells with sealing film for LC-MS/MS analysis.
    5. As the sealing film is not suitable for long-term freezer storage, transfer 190 µl of the supernatant into 96-well PCR plates and seal with 8-strip caps as freezer backup.
      Note: In case Eppendorf tubes are used, transfer 700 µl of the supernatant to 1.5 ml screw neck vials (vials are stored in the freezer for re-analysis).
    6. Prepare a mixed quality control (QC) sample for each 96-well plate by combining 10 µl aliquots of each sample of the plate in a 1.5 ml screw neck vial.
    7. Use the extraction buffer as blank and for signal background calculations.

  3. UHPLC-MS/MS
    For the chromatographic separation, utilize an Agilent ZORBAX Eclipse XDB-C18 column. The mobile phase consists of 0.1% (v/v) acetonitrile and 0.05% (v/v) formic acid in MilliQ H2O for solvent A and 100% methanol as solvent B. The mobile phase gradient of the UHPLC method is shown in Table 1. The UHPLC instrument parameters comprise:
    Flow rate
    0.5 ml min-1
    Sample tray temperature
    10 °C
    Sample injection volume
    5 µl
    Column temperature
    42 °C

    Table 1. Mobile phase gradient of the UHPLC run


    The Bruker EVO-Q EliteTM triple quadrupole MS system is used in multiple reaction monitoring (MRM) mode. The heated electrospray ionization (HESI) source settings consist of:
    HESI spray voltage
    ± 4,500 V
    Cone temperature
    350 °C
    Probe temperature
    300 °C
    Cone gas flow
    35
    Nebulizer gas flow
    60
    Probe gas flow
    55
      System performance and general ESI parameters can be evaluated by injecting a standard solution of related blumenol glycoside compounds: Roseoside (Wuhan ChemFaces Biochemical Co., Ltd.; catalog number: CFN98916), Corchoionoside C (CFN99859), Blumenol C glucoside (CFN99424), Byzantionoside B (CFN99871). Standards for the 11-hydroxy- and 11-carboxyblumenol C derivatives are not commercially available.
      The MRM settings for the detection of specific blumenol derivatives are shown in Table 2 and a recording window of 1 min is set at the expected retention time (RT). The displayed compound table has been tested and found to be widely applicable. Additional markers can be identified in order to extend the method beyond the current list of plant species that have been investigated (Wang et al., 2018):
    Barley
    Hordeum vulgare
    Barrel clover
    Medicago truncatula
    Common rice
    Oryza sativa
    Common wheat
    Triticum aestivum
    Potato
    Solanum tuberosum
    Stiff brome
    Brachypodium distachyon
    Tomato
    Solanum lycopersicum
    Wild tobacco
    Nicotiana attenuate

    Table 2. Quantifier and Qualifier m/z fragments used to detect blumenol derivatives in plant leaves

    § Ionization polarity is indicated in parentheses.
    * Verified by NMR.
    ** Verified with authentic standard.
    & The fragmentation of the m/z 241.2 aglycon precursor [M+H-Glc]+ allows for a sensitive MRM detection in addition to the MRM of the m/z 403.2 molecular ion [M+H]+.
    ǂ Blumenol A and abscisic acid are not induced by AMF (Wang et al., 2018) and can be used as internal standards to evaluate the overall functionality of the carotenoid biosynthesis in the analyzed plant as well as providing valuable information about instrument performance.
    The identity of 11-carboxyblumenol C-Glc-Glc and 11-carboxyblumenol C-Mal-Glc detected in rice has not been confirmed.
    Internal Standard (typically showing 20-30% relative standard deviation after full extraction/analysis procedure).

    The prepared QC samples will be analyzed repeatedly after every 15 to 20 samples with the identical UHPLC-MS/MS method. Comparisons of the QC runs will allow monitoring instrument performance and detecting retention time shifts or changes in mass spectrometer sensitivity in larger sample batches.

Data analysis

EXAMPLES of the blumenol derivative signals detected in leaves of barley (Hordeum vulgare) and tomato (Solanum lycopersicum) plants with and without AMF colonization are shown in Figure 1.


Figure 1. Chromatographic output for blumenol derivatives in different crop plants. Blumenol derivatives were extracted from leaf tissue of control plants (no AMF) and plants inoculated with Rhizophagus irregularis. 11-carboxy- and 11-hydroxyblumenol C-Glc were detected in AMF-colonized tomato (Solanum lycopersicum) and barley (Hordeum vulgare) plants, respectively. Details of the inoculation procedure can be found in Wang et al. (2018).

Peak area integration for the targeted compounds and the internal standard is carried out via the software MS Data Review Version 8.2.1 (MS Workstation, Bruker Daltonics). The analyte peak area is normalized to the internal standard D6-ABA and concentrations of blumenol derivatives are calculated as D6-ABA equivalents (ng mg-1 fresh mass) using the following equation:



and  represent the peak areas (in counts) of the target analyte and internal standard, respectively.
is the amount of internal standard (in ng) that is introduced to the sample via the extraction buffer.
  corresponds to the fresh mass (in mg) of the leaf tissue sample.

Recipes

  1. Extraction buffer including internal standard D6-ABA
    200 ml MilliQ H2O
    800 ml MeOH (gradient grade for LC)
    1.25 ml of 10 ng µl-1 D6-ABA (final concentration of 10 ng per 800 µl extraction buffer)

Acknowledgments

The work was funded by the Max Planck Society and ERC Advanced Grant ‘ClockworkGreen’ (293926). This protocol was adapted from the methods described in Wang et al. (2018) and Schäfer et al. (2016).

Competing interests

The procedure has been filed under PCT patent application PCT/EP2019/054738 with the European Patent Office.

References

  1. Aliferis, K. A., Chamoun, R. and Jabaji, S. (2015). Metabolic responses of willow (Salix purpurea L.) leaves to mycorrhization as revealed by mass spectrometry and 1H NMR spectroscopy metabolite profiling. Front Plant Sci 6: 344.
  2. Barin, M., Aliasgharzad, N., Olsson, P. A., Rasouli-Sadaghiani, M. H. and Moghddam, M. (2013). Abundance of arbuscular mycorrhizal fungi in relation to soil salinity around Lake Urmia in northern Iran analyzed by use of lipid biomarkers and microscopy. Pedobiologia 56(4-6): 225-232.
  3. Basu, S., Rabara, R. C. and Negi, S. (2018). AMF: The future prospect for sustainable agriculture. Physiol Mol Plant Pathol 102: 36-45.
  4. Hill, E. M., Robinson, L. A., Abdul-Sada, A., Vanbergen, A. J., Hodge, A. and Hartley, S. E. (2018). Arbuscular mycorrhizal fungi and plant chemical defence: effects of colonisation on aboveground and belowground metabolomes. J Chem Ecol 44(2): 198-208.
  5. Maier, W., Peipp, H., Schmidt, J., Wray, V. and Strack, D. (1995). Levels of a terpenoid glycoside (blumenin) and cell wall-bound phenolics in some cereal mycorrhizas. Plant Physiol 109(2): 465-470.
  6. Schäfer, M., Brutting, C., Baldwin, I. T. and Kallenbach, M. (2016). High-throughput quantification of more than 100 primary- and secondary-metabolites, and phytohormones by a single solid-phase extraction based sample preparation with analysis by UHPLC-HESI-MS/MS. Plant Methods 12: 30.
  7. Wang, M., Schäfer, M., Li, D., Halitschke, R., Dong, C., McGale, E., Paetz, C., Song, Y., Li, S., Dong, J., Heiling, S., Groten, K., Franken, P., Bitterlich, M., Harrison, M. J., Paszkowski, U., Baldwin, I. T. (2018). Blumenols as shoot markers of root symbiosis with arbuscular mycorrhizal fungi. eLife 7: e37093.

简介

丛枝菌根真菌(AMF)与植物之间的共生相互作用在陆地植物中广泛存在,并且对于两个伙伴而言可以是蜂窝状的。 基于破坏性取样和精细的根组织分析的存在方法对于高通量具有有限的价值(需要螺旋活性,环境影响,有效的表征方法来监测和量化植物-AMF定殖。 通过简单的提取程序和灵敏的液相色谱质谱(LC / MS)分析两种活动中根AMF-缔合的准确代理的叶片中的blumenol衍生物中HTP定量的特定方案
【背景】AMF与植物之间广泛的共生关系不仅涉及相关合作伙伴之间有益的营养交换。各种生物和非生物胁迫(Maier et al。,1995; Barin et al。,2013; Aliferis et al。,2015; Wang et al。, 2018)。这些相互作用推动了大量的研究项目,并与减少的天然磷光供应相结合,对可持续农业具有高度兴趣(Basu et al。 到目前为止,测量和量化AMF植物关联的现有方法需要通过显微镜分析,转录分析或真菌脂肪酸定量进行根分析(Barin et al。,2013)然而,由于r导致的损伤,这些方法对于HTP筛选是不切实际的抽样,以及费力(Barin et al。,2013; Wang et al。,2018)。因此,需要HTP筛选技术来增强电力研究和尽管AMF植物协会。即使AMF定植导致通过植物的系统反应,直到最近在植物部分中没有检测到AMF特异性代谢反应而不是根(Aliferis al。 Hill et al。,2018)。所描述的方案基于叶组织的MeOH提取,然后如所述的超高效液相色谱质谱(UHPLC-MS)分析。 Wang et al。(2018)。叶片中的11-羟基和11-羧酸丁内酯C衍生物的浓度在非菌根化植物中检测不到,并且与AMF根定植呈正相关和定量相关,并且是在根 - AMF关联形成后从根部运输到叶子(Wang et al。该协议促进了各种模型和农作物种类中AMF关联的HTP,非破坏性和定量表征。

关键字:丛枝菌根真菌, AMF, 真菌-植物共生, 叶片生物标志物, 根生物标志物, 蓝甲醇衍生物, UHPLC-MS/MS技术

材料和试剂

  1. 移液器吸头
  2. 96孔微孔板,带全裙(Sapphire,Greiner Bio-One,目录号:652270)
  3. (可选)单个管
    注意事项:单独的管可被用来代替96-孔BioTubes TM 用于样品的小批量
    1. 2毫升Eppendorf Safe-Lock管(Eppendorf,目录号:0030120094)
    2. 1.5毫升螺口瓶N9(Macherey-Nagel,目录号:702282)
    3. N9 PP螺旋盖(Macherey-Nagel,目录号:702287.1)
    4. 钢球Ø4mm(ASKUBAL,G100-1.4034,目录号:503012)
  4. 用于96孔微孔板的密封膜(Zone-freeTM,EXCEL Scientific,目录号:ZAF-PE-50)
  5. 96孔PCR板(μltraAmp,SorensonTMTM BioScience Inc,目录号:21970)
  6. 圆顶8条带PCR盖(Eppendorf,目录号:0030124839)
  7. 钢球Ø3mm(ASKUBAL,G100-1.4034,目录号:505001)
  8. 叶材料
  9. 液氮
  10. MilliQ水
  11. 氘代内标:D 6 -ABA(HPC Standards GmbH,10μgml -1 在MeOH中)
  12. 乙腈(VWR国际HiPerSolv CHROMANORM ®用于LC-MS,目录号:BDH83640.100E)
  13. 甲酸(Fluka,用于质谱,目录号:94318)
  14. 甲醇(Merck,LC LiChrosolv ®的梯度等级,目录号:1060072500)
  15. 罗索苷(武汉ChemFaces Biochemical Co.,Ltd。,目录号:CFN 98916)
  16. Corchoionoside C(武汉ChemFaces Biochemical Co.,Ltd。,目录号:CFN 99859)
  17. Blumenol C glucoside(武汉ChemFaces Biochemical Co.,Ltd。,目录号:CFN 99424)
  18. Byzantionoside B(武汉ChemFaces Biochemical Co.,Ltd。,目录号:CFN99871)
  19. 内标D 6 -ABA的提取缓冲液(见食谱)

设备

  1. 不锈钢刮刀
  2. 不锈钢镊子
  3. 96孔管架(BioTube TM ,Simport ® Scientific,目录号:T101-1和T100-20)
  4. 用于96孔管架的密封垫(ArctiSealTM,Arctic White LLC,目录号:AWSM-2002RB)
  5. 冷却容器(Heathrow Scientific,True North ®)
  6. 离心机(Eppendorf,型号:5415 R)
  7. Multipipette(Multipette ® Xstream,Eppendorf,目录号:4986000025)
  8. 砂浆和杵(HaldenwangerTM,Fisher Scientific)
  9. 分析天平(赛多利斯,型号:BP121S)
  10. 8通道电子移液器(Eppendorf,Xplorer®,50-1,200μl,目录号:48610000163)
  11. 组织匀浆器(Geno / Grinder ® 2000,SPEX SamplePrep)
  12. 冷却离心机配备96孔板转子(Eppendorf,型号:5804 R,转子A-2-DWP)
  13. UHPLC三重四极杆MS仪器[Ultimate 3000 RSLC(赛默飞世尔科技); EVO-Q Elite (布鲁克)]
  14. UHPLC色谱柱(ZORBAX Eclipse XDB-C18,50 x 3.0 mm,1.8μm,Agilent,目录号:981517-302)
  15. -80°C冰柜

软件

  1. MS Data Review Version 8.2.1(MS Workstation,Bruker Daltonics)

程序

注意:

  1. 为了测试该方法对分析的植物/ AMF物种的适用性,建议对根组织进行初始测试筛选,因为根组织中蓝霉素衍生物的丰度高于叶片。
  2. 在可比较的发育阶段从叶子中收获组织样品将减少变异并允许更好的比较 Blumenol水平在不同的芽组织中可以变化(Wang 等 ,2018)。植物之间。
  3. Blumenol水平表明接种后3周AMF定植(Wang 等 ,2018)。

  1. 叶材料的收集和准备
    1. 储存在-80°C直至处理样品。收获叶子并使用不锈钢镊子立即冷冻液氮。
    2. 在液氮下用研钵和研杵研磨冷冻的叶子材料。
    3. 用预先冷却的不锈钢刮刀将约100 mg叶材料分装到液氮预冷和预加重的96孔BioTube TM 机架中,该机架包含两个钢球(Ø3mm)。记录准确质量并将样品留在液氮中进行提取或储存在-80°C。
      注意:可以使用配备两个钢球(Ø4mm)的2 ml Eppendorf管代替96孔BioTubesTM。

  2. 萃取
    1. 用橡胶密封垫替换管帽。
      用8通道移液器向每个管中加入800μl含有内标D 6 -ABA的冰冷提取缓冲液。 注意:在提取过程中,样品应保存在冰上。
    2. 将Geno /Grinder®中的样品均匀化为每分钟1000次冲程60次(Geno /Grinder®2000设置:1x at 000)。
    3. 在4℃下将样品在2,000 xg 离心20分钟,将上清液转移到新的96孔BioTube TM 支架或不带钢球的Eppendorf管中,再次离心。相同的条件。
    4. 将100μl上清液转移到带裙边的96孔微量培养板中,并用密封膜封闭孔用于LC-MS / MS分析。
    5. 由于密封膜不适合长期冷冻储存,将190μl上清液转移到96孔PCR板中,并用8条带盖作为冷冻备用密封。
      注意:如果使用Eppendorf试管,将700μl上清液转移至1.5 ml螺旋瓶小瓶(小瓶存放在冰箱中进行重新分析)。
    6. 通过将每个平板的10μl等分试样在1.5ml螺旋颈瓶中混合,为每个96孔板制备混合质量控制(QC)样品。
    7. 使用提取缓冲液作为空白并进行信号背景计算。

  3. UHPLC-MS / MS
    流动相由0.1%(v / v)乙腈和0.05%(v / v)甲酸在MilliQ H 2 O中进行色谱分离,使用Agilent ZORBAX Eclipse XDB-C18色谱柱。 UHPLC方法的流动相梯度如表1所示.UHPLC仪器参数:
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:450px;”border =“0”cellspacing =“0”cellpadding =“2”>流量
    0.5 ml min -1
    样品盘温度
    10°C
    样品注射量
    5μl
    柱温
    42°C

    表1. UHPLC运行的流动相梯度


    Bruker EVO-Q EliteTM三重四极杆质谱系统用于多反应监测(MRM)模式。加热电喷雾电离(HESI)源设置一致:
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:450px;”border =“0”cellspacing =“0”cellpadding =“2”>HESI喷涂电压
    ±4,500 V
    锥温
    350°C
    探头温度
    300°C
    锥形气流
    35
    雾化器气流
    60
    探测气体流量
    55 ;&NBSP系统性能和一般ESI参数可以通过注入相关blumenol糖苷化合物的标准溶液进行评价:Roseoside(武汉ChemFaces生化有限公司;目录号:CFN98916),Corchoionoside C(CFN99859),Blumenol C糖苷(CFN99424 ,Byzantionoside B(CFN 99871).11-羟基 - 和11-羧酸联苯胺C衍生物的标准品尚未商业化。
    显示的化合物表已经过测试,用于检测特定特定衍生物的MRM设置如表2所示,并且在预期保留时间(R T )的设定值为1分钟的记录窗口。可以鉴定其他标记以将该方法扩展到已经研究的当前植物物种列表之外(Wang et al。,2018): class =“ke-zeroborder”bordercolor =“#000000”style =“width:450px;”border =“0”cellspacing =“0”cellpadding =“2”>大麦
    Hordeum vulgare
    桶三叶草
    Medicago truncatula
    普通大米
    Oryza sativa
    普通小麦
    Triticum aestivum
    马铃薯
    Solanum tuberosum
    僵硬的雀斑
    Brachypodium distachyon
    番茄
    Solanum lycopersicum
    野生烟草
    Nicotiana followntuate

    表2.用于检测植物叶片中的blumenol衍生物的Quantifier和Qualifier m / z片段

    §电离极性用括号表示。
    * 经核磁共振验证。
    ** 用真实标准验证。
    & m / z 241.2 aglycon前体[M + H-Glc] + 的碎裂除了m / z的MRM之外还允许灵敏的MRM检测403.2分子离子[M + H] +。
    Blumenol A和脱落酸不是由AMF诱导的(Wang et al。,2018),可以作为内标来评估类胡萝卜素生物合成的总体功能。分析的工厂以及提供有关仪器性能的有价值信息。
    †在水稻中检测到的11-碳基丁内酯C-Glc-Glc和11-羧基苄基醇C-Mal-Glc的特性尚未得到证实。
    ¶内标(完全提取/分析程序后通常显示20-30%的相对标准偏差)。

    使用相同的UHPLC-MS / MS方法,每15至20个样品后,将重复分析制备的QC样品。质量控制运行的比较将允许监测仪器性能并检测保留时间的变化或质谱的变化

数据分析

在大麦叶检测的blumenol衍生物信号的实施例(实施大麦)和番茄(番茄)有和没有AMF定植植物在图1中示出


图1.不同作物植物中不同衍生物的色谱输出。从对照植物(无AMF)的叶组织和接种 Rhizophagus irregularis 的植物中提取Blumenol衍生物。羧基和11-hydroxyblumenol C-GLC在AMF定殖的番茄中检测到(番茄)和大麦(大麦)植物,接种过程的分别。细节可以可以在Wang et al。(2018)中找到。

MS数据审查版本8.2.1(MS Workstation,Bruker Daltonics)。分析物峰面积归一化为内标D 6 > -ABA和blumenol衍生物的浓度使用以下等式计算为D 6 -ABA当量(ng mg -1 新鲜质量):



和  代表目标分析和内部标准的峰值区域(计数),值得尊重。
是通过萃取缓冲液引入样品的内标量(以ng计)。
  对应于叶组织样品的新鲜质量(以mg计)。

食谱

  1. 提取缓冲液包括内标D 6 -ABA
    200毫升MilliQ H 2 O
    800毫升MeOH(梯度等级用于LC)
    1.25ml10ngμl-1 D 6 -ABA(最终浓度为10ng /800μl提取缓冲液)

致谢

该方案改编自Wang et al。(2018)和Schäfer等人所述的方法。该工作由Max Planck Society和ERC Advanced Grant'TlockworkGreen'(293926)资助。 (2016)。

竞争利益

该程序已在欧洲专利局的PCT专利申请PCT / EP2019 / 054738下提交。

参考

  1. Aliferis,KA,Chamoun,R。和Jabaji,S。(2015)。柳树的代谢反应( Salix purpurea L.)通过质谱和1 H NMR光谱代谢物分析释放出来进行菌根化。 Front Plant Sci 6:344。
  2. Barin,M.,Aliasgharzad,N.,Olsson,PA,Rasouli-Sadaghiani,MH和Moghddam,M。(2013)。 “目标=”_空白“>使用脂质生物标志物和显微镜分析了伊朗北部乌尔米亚湖周围土壤盐度相关的丛枝菌根真菌的丰度。 Pedobiologia 56(4-6) :225-232。
  3. Basu,S.,Rabara,RC和Negi,S。(2018)。 AMF:未来可持续农业的前景。 Physiol Mol Plant Pathol 102:36-45。
  4. Hill,EM,Robinson,LA,Abdul-Sada,A.,Vanbergen,AJ,Hodge,A。和Hartley,SE(2018)。 J Chem Ecol 44(2):198-208。
  5. Maier,W.,Peipp,H.,Schmidt,J.,Wray,V。和Strack,D。(1995)。一些谷类菌根中萜类糖苷(blumenin)和细胞壁结合酚类的水平。 植物生理学 109(2):465-470。
  6. Schäfer,M.,Brutting,C.,Baldwin,IT和Kallenbach,M。(2016)。通过UHPLC-HESI-MS / MS分析,通过基于单一固相萃取的样品制备,对100多种一级和二级代谢物以及植物激素进行高通量定量分析。 植物方法他们> 12:30。
  7. Wang,M.,Schäfer,M.,Li,D.,Halitschke,R.,Dong,C.,McGale,E.,Paetz,C.,Song,Y.,Li,S.,Dong,J., Heiling,S.,Groten,K.,Franken,P.,Bitterlich,M.,Harrison,MJ,Paszkowski,U.,Baldwin,IT(2018)。 Blumenols作为与丛枝菌根真菌共生的根系共生标记。 eLife 7:e37093。
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Copyright Mindt et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mindt, E., Wang, M., Schäfer, M., Halitschke, R. and Baldwin, I. T. (2019). Quantification of Blumenol Derivatives as Leaf Biomarkers for Plant-AMF Association. Bio-protocol 9(14): e3301. DOI: 10.21769/BioProtoc.3301.
  2. Wang, M., Schäfer, M., Li, D., Halitschke, R., Dong, C., McGale, E., Paetz, C., Song, Y., Li, S., Dong, J., Heiling, S., Groten, K., Franken, P., Bitterlich, M., Harrison, M. J., Paszkowski, U., Baldwin, I. T. (2018). Blumenols as shoot markers of root symbiosis with arbuscular mycorrhizal fungi. eLife 7: e37093.
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