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ER Microsome Preparation and Subsequent IAA Quantification in Maize Coleoptile and Primary Root Tissue
玉米胚芽鞘和主根组织中的内质网(ER)微粒体制备和IAA的定量测定   

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Abstract

Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied (Darwin and Darwin, 1880). The auxin molecule in plants was first identified as indole-3-acetic acid (IAA) by Kögl et al. (1934). Active research over nearly a decade has shed light on many of the molecular mechanisms of its action but the complexity and redundancy of the auxin biosynthetic network raises questions about control of this system. We have shown that some enzymes involved in the YUCCA-route of auxin biosynthesis are not cytosolic but localised to the endoplasmic reticulum (ER) in both Arabidopsis thaliana (YUCCA4.2) (Kriechbaumer et al., 2012) as well as Zea mays (ZmTAR1 and ZmSPI) (Kriechbaumer et al., 2015). This is raising the intriguing possibility of subcellular compartmentation of auxin biosynthesis. To show that maize auxin biosynthesis indeed can take place in microsomal as well as cytosolic cellular fractions from maize seedlings we applied the protocol described here: Microsomes are being isolated from maize coleoptile and primary root tissue, enzyme assays with microsomal and cytosolic fractions using either tryptophan (Trp) or indole- -3pyruvic acid (IPyA) as a substrate are carried out and the auxin IAA is extracted and quantified.

Keywords: Maize(玉米), Microsomes(微粒体), Endoplasmic reticulum(内质网), Auxin(生长素), IAA(IAA)

Materials and Reagents

  1. Pipettes (1 ml, 200 µl and 10 µl) (Gilson Scientific Ltd., catalog number: F123602 , F123601 and F144802 )
  2. Eppendorf tubes (2 ml) (Eppendorf AG, catalog number: 0030120094 )
  3. Eppendorf conical tubes (50 ml) (Eppendorf AG, catalog number: 0030122178 )
  4. 0.45 µm syringe filters (Sigma-Aldrich, catalog number: F8273 )
  5. Cheese cloth (DUTSCHER SCIENTIFIC, catalog number: 789056 )
  6. Ice box and ice
  7. Liquid nitrogen
  8. Triethanolamine-acetic acid (TEA-HOAc) (Sigma-Aldrich, catalog number: 90278 )
  9. Potassium acetate (KOAc) (Sigma-Aldrich, catalog number: P1190 )
  10. Magnesium acetate [Mg(OAc)2] (Sigma-Aldrich, catalog number: M5661 )
  11. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  12. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
  13. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5391 )
  14. Tris-Hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: 252859 )
  15. β-Nicotinamide adenine dinucleotide phosphate disodium salt (NADPH) (Sigma-Aldrich, catalog number: 93205 )
  16. Flavin adenine dinucleotide (FAD) (Sigma-Aldrich, catalog number: F6625 )
  17. Indole-3-pyruvic acid (IPyA) (Sigma-Aldrich, catalog number: I7017 )
  18. Tryptophan (Sigma-Aldrich, catalog number: T0254 )
  19. Ethyl acetate (Sigma-Aldrich, catalog number: 270989 )
  20. Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795 )
  21. Distilled water
  22. Ethyl acetate for HPLC (Sigma-Aldrich, catalog number: 650528 )
  23. Acetic acid for HPLC (Sigma-Aldrich, catalog number: A6283 )
  24. Methanol for HPLC (Sigma-Aldrich, catalog number: 34860 )
  25. Stock solutions (see Recipes)
  26. Buffers (see Recipes)
    1. Buffers for microsome extraction (Buffer 1, Buffer 2, Buffer 3 and Buffer 4)
    2. Buffers for HPLC analysis

Equipment

  1. Porcelain mortar & pestle (Sigma-Aldrich, catalog number: Z247464 and Z247502 )
  2. Glass bottles for buffers (100 ml, 250 ml and 500 ml) (Sigma-Aldrich, catalog number: Z305170 , Z305189 and Z305197 )
  3. Refrigerated table centrifuge (Eppendorf AG, model: 5430R )
  4. Ultracentrifuge with swing-out rotor SW41 (Beckman Coulter, catalog number: 331362 )
  5. Ultracentrifuge corex tubes (Beckman Coulter, catalog number: 331372 )
  6. Glass rod and a 2 ml Potter–Elvehjem homogenizer (Sigma-Aldrich, catalog number: P7734-1EA )
  7. water bath (37 °C) (Thermo Fisher Scientific, model: TSGP02 )
  8. Speed-vac (LABCONCO, catalog number: 7810016 )
  9. Nanodrop ND-2000 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 13400504 )
  10. High-performance liquid chromatography (HPLC) (600E multisolvent delivery system) (WATERS, catalog number: 720000699EN )
  11. Reverse phase column (Apollo C18) (250 mm x 4.6 mm, 5 μm) (Thermo Fisher Scientific, GraceTM, catalog number: 5126767 )
  12. UV monitor 486 (WATERS) and a fluorescence monitor 470 (WATERS)

Procedure

  1. Endoplasmic reticulum (ER) microsome preparation
    1. All steps are carried out on ice or at 4 °C unless indicated otherwise.
    2. Pre-cool all buffers, tubes etc. used in the procedure.
    3. 5 g of maize coleoptile or primary root tissue (4 d after germination) are ground to fine powder in liquid nitrogen using a pre-cooled mortar and pestle.
    4. The powder is then homogenized in 4 ml of ice-cold buffer 1 and transferred into a 50 ml falcon tube.
    5. 4 ml of ice-cold buffer 2 are added to the tube and the suspension was incubated on ice for 10 min. Then the homogenate is centrifuged at 1,000 x g for 10 min at 4 °C. The resulting supernatant is poured over 4 layers of cheese cloth into a fresh falcon tube. At this step, the resulting extract is considered total plant extract for later enzymatic assays. The extract is centrifuged again at 4,500 x g for 25 min at 4 °C.
    6. Prepare ultracentrifuge corex tubes with a 4 ml sucrose cushion (buffer 3) on the bottom.
    7. The 8 ml of plant suspension is layered on top of the sucrose cushion by slightly angling the tube and carefully and slowly pipetting the suspension at the side of the tube. The tube is centrifuged for 90 min at 93,000 x g (ultracentrifuge with swing-out rotor SW41).
    8. The resulting pellet is removed from the ultracentrifuge tube if necessary with 20 μl of buffer 4 and transferred to a 2 ml Potter–Elvehjem homogenizer. The supernatant is kept and used as cytosolic extract in later IAA quantifications.
    9. The final pellet was resuspended in 200 μl of buffer 4 using a glass rod and a 2 ml Potter-Elvehjem homogenizer. Protein content was measured using a Nanodrop. Freshly prepared microsomes were used for enzymatic assays straight away.
    10. This part of the procedure will take between 3 and 4 h depending on sample size.

  2. Microsomal enzymatic tests
    1. The YUCCA-route of auxin biosynthesis is a two-step process (Figure 1): In maize TAR-proteins [Tryptophan Aminotransferase of Arabidopsis-related proteins; ZmTAR1 (Chourey et al., 2010), ZmVT2 (Phillips et al., 2011), and ZmTAR3 (ZmAlliin1; Bernardi et al., 2012)] are converting tryptophan (Trp) to indole-3-pyruvic acid (IPyA) which is then converted by YUCCA proteins to IAA [ZmYUC1 and ZmSPI1 (Phillips et al., 2011)]. It was shown that ZmYUC1 and ZmVT2 are localized in the cytosol, whereas ZmSPI1, ZmTAR1 and ZmTAR3 are localized to the ER (Kriechbaumer et al., 2015).


      Figure1. YUCCA-route of auxin biosynthesis in maize. Cytosolic enzymes are labeled in green, ER-localised enzymes in blue.

    2. Enzymatic activity tests (100 μl total volume) with microsomal and cytosolic fractions were carried out in the following manner:
      20 μl of microsomal or cytosolic extract or total plant extract (see step A5)
      1 mM NADPH
      100 μM FAD
      100 μM IPyA or Trp (depending on experimental interest)
      100 mM Tris-HCl (pH 8.0) up to a total volume of 100 μl
      Mixed carefully in 2 ml Eppendorf tubes
      Note: For this the 100 mM Tris-HCl should be pipetted first, followed by the plant extract and then FAD. The reaction time is started with the addition of the substrate (IPyA or Trp).
      The assays were incubated for 1 h in a 37 °C water bath and snap-frozen in liquid nitrogen straight after the incubation time.
    3. For comparison enzymatic tests were also performed with the cytosolic fraction and total plant extract.
    4. At this stage the assays can be stored at -20 °C before the IAA analysis is carried out.

  3. IAA extraction and quantification
    1. IAA is extracted by ethyl acetate phase separation (Park et al., 2003; Kriechbaumer et al., 2007) (Figure 2) from the enzyme assays prepared in section B. The whole sample of each assay is used for this procedure.
      1. For this the pH of sample is adjusted higher than 9.5 with 1 M Na2CO3, and the sample is then extracted with 400 μl of ethyl acetate.
      2. The aqueous lower phase is recovered and 200 μl of water are added.
      3. The partitioning procedure is repeated recover the aqueous phase again.
      4. The aqueous phases from both partitioning steps are combined.
      5. The collected aqueous phase is acidified with acetic acid to a pH below 2.5 and partitioned twice with addition of 400 μl of ethyl acetate for each step.
      6. The organic phases are collected and the liquid evaporated using a speed-vac (pre-set settings with room temperature). Evaporation will take around two hours but the sample should be checked from time to time.
      7. The dried substances are re-dissolved in 100% methanol and can be stored at -20 °C before further analysis if necessary.
    2. The IAA extraction can take up to 5 h depending on sample size and evaporation time in the speed-vac.


      Figure 2. Schematic representation of IAA extraction using ethyl acetate phase separation. The steps for IAA extraction by ethyl acetate phase separation are shown in this diagram. The Water phase is shown in blue, the organic phase in red. pH adjustments are indicated as well as changes in Eppendorf tubes.

    3. IAA is then analysed via high-performance liquid chromatography (HPLC) with a reverse phase column.
    4. For this the HPLC system is run in an isocratic flow of 0.8 ml min-1 with a 40:60 mixture of buffer A (10% methanol, 0.3% acetate) and buffer B (90% methanol, 0.3% acetate). Peaks were identified by comparison with the standard substances with respect to retention time and UV spectrum using both a UV monitor and a fluorescence monitor.
    5. The amount of IAA in each sample was calculated using the volume of the IAA-peak and compare it with the volume of IAA peaks resulting from IAA standards with known amounts. This will give the amount of IAA per sample. The protein amount in microsomal, cytosolic and total protein fractions is determined by measuring absorbance at 280 nm by an UV-Vis Spectrophotometer (e.g., by Nanodrop Instrument ND-2000). With this data the amount of IAA per µg protein can be calculated (Figure 4).

Representative data

To check for the purity of the microsomal fraction both the microsomal as well as the cytosolic fraction were probed with an anti-ZmNIT antibody detecting cytosolic nitrilases (Figure 3). This antibody was shown to detect the maize nitrilases 1 and 2 protein very specifically in various tissues in the range of ng mg-1 of total protein (Park et al., 2003; Kriechbaumer et al., 2007). The nitrilase band was only detectable in the cytosolic fraction indicating a rather pure microsomal fraction.


Figure 3. Immunoblot analysis for purity of microsomal fractions. Microsomal (M) and cytosolic (C) fractions were tested for nitrilase proteins using immunoblot analysis. 100 μg of protein from each fraction were probed with anti-ZmNIT1 antibodies (1:400) recognizing the cytosolic maize nitrilases 1 and 2. (Kriechbaumer et al., 2015).

Representative data for the conversion of tryptophan to IAA in maize coleoptiles and primary root is shown in Figure 4 [data from Kriechbaumer et al. (2015)].


Figure 4. Auxin biosynthetic capacity in maize. Enzymatic conversion of tryptophan to IAA by microsomal (Microsomes) fractions, cytosolic (Cytosol) fractions, or total plant extract from maize coleoptiles (Col, white bars) and primary roots (PR, black bars) 4 d after germination. Standard errors are indicated. n = 2 (two biological samples with three technical replicates each).

Notes

  1. Collect plant material straight into a chilled falcon tube that is kept on ice and try to collect the material as quickly as possible.
  2. Pre-chill tubes, glassware, buffers, centrifuges etc and keep your samples on ice as much as possible.
  3. Where possible timewise freshly prepared microsomes should be used for enzyme assays. Freezing in 20% glycerol for later assays is possible but not recommended.
  4. Assays can be performed with any HPLC system (e.g., Dionex, Beckman…)
  5. It is advisable to at least confirm some data points using mass spectrometry to be sure that the correct molecule is being quantified.

Recipes

  1. Stock solutions
    1 M TEA-HOAc (pH 7.5)
    1 M KOAc (pH 7.5)
    0.1 M Mg(OAc)2
    1 M sucrose
    1 M DTT
    0.5 M EDTA
    1 M Tris-HCl (pH 8.0)
    1 M Na2CO3
    0.1 M NADPH
    0.1 M FAD
    0.1 M IPyA
    0.1 M Trp

  2. Buffers
    Note: Filter buffers 1-4 through 0.45 µm syringe filters. Add DTT from 1M stock fresh prior to use.
    1. Buffers for microsome extraction
      1. Buffer 1
        25 mM TEA-HOAc (pH 7.5)
        50 mM KOAc (pH 7.5)
        5 mM Mg(OAc)2
        0.25 M sucrose
        4 mM DTT
      2. Buffer 2
        100 mM TEA-HOAc (pH 7.5)
        20 mM EDTA
      3. Buffer 3
        25 mM TEA-HOAc (pH 7.5)
        25 mM KOAc (pH 7.5)
        2 mM Mg(OAc)2
        0.5 M sucrose
        4 mM DTT
      4. Buffer 4
        25 mM TEA-HOAc (pH 7.5)
        0.25 M sucrose
        1 mM DTT
    2. Buffers for HPLC analysis
      Buffer A: 10% methanol, 0.3% acetate
      Buffer B: 90% methanol, 0.3% acetate

Acknowledgments

This protocol was adapted from Kriechbaumer et al. (2015). This work was supported by a research scholarship from the Korean Federation of Science and Technology Societies (KOFST) awarded to Dr Verena Kriechbaumer and the British Biotechnology and Biological Sciences Research Council (grant No. BB/J004987/1 research grant awarded to Prof Chris Hawes.

References

  1. Abell, B. M., Holbrook, L. A., Abenes, M., Murphy, D. J., Hills, M. J. and Moloney, M. M. (1997). Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil body targeting. Plant Cell 9(8): 1481-1493.
  2. Bernardi, J., Lanubile, A., Li, Q. B., Kumar, D., Kladnik, A., Cook, S. D., Ross, J. J., Marocco, A. and Chourey, P. S. (2012). Impaired auxin biosynthesis in the defective endosperm18 mutant is due to mutational loss of expression in the ZmYuc1 gene encoding endosperm-specific YUCCA1 protein in maize. Plant Physiol 160(3): 1318-1328.
  3. Chourey, P. S., Li, Q. B. and Kumar, D. (2010). Sugar-hormone cross-talk in seed development: two redundant pathways of IAA biosynthesis are regulated differentially in the invertase-deficient miniature1 (mn1) seed mutant in maize. Mol Plant 3(6): 1026-1036.
  4. Darwin, C. and Darwin, F. (1880). The power of movement in plants. John Murray.
  5. Kögl, F., Haagen-Smit, A. J. and Erxleben, H. (1934). Über ein neues Auxin (Heteroauxin) aus Harn. 11. Mitteilung über pflanzliche Wachstumsstoffe. Hoppe-Seyler’s Zeitschrift für Physiologisch Chemie 228: 90-103.
  6. Kriechbaumer, V., Park, W. J., Piotrowski, M., Meeley, R. B., Gierl, A. and Glawischnig, E. (2007). Maize nitrilases have a dual role in auxin homeostasis and beta-cyanoalanine hydrolysis. J Exp Bot 58(15-16): 4225-4233.
  7. Kriechbaumer, V., Seo, H., Park, W. J. and Hawes, C. (2015). Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes. J Exp Bot 66(19): 6009-6020.
  8. Park, W. J., Kriechbaumer, V., Moller, A., Piotrowski, M., Meeley, R. B., Gierl, A. and Glawischnig, E. (2003). The Nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid. Plant Physiol 133(2): 794-802.
  9. Phillips, K. A., Skirpan, A. L., Liu, X., Christensen, A., Slewinski, T. L., Hudson, C., Barazesh, S., Cohen, J. D., Malcomber, S. and McSteen, P. (2011). Vanishing tassel2 encodes a grass-specific tryptophan aminotransferase required for vegetative and reproductive development in maize. Plant Cell 23(2): 550-566.

简介

生长素是植物中的主要生长激素和待发现和研究的第一种植物激素(Darwin和Darwin,1880)。植物中的植物生长素分子首先由Kögl等人确定为吲哚-3-乙酸(IAA)。 (1934)。近十年的积极研究揭示了其作用的许多分子机制,但是生长素生物合成网络的复杂性和冗余性提出了对该系统的控制的问题。我们已经显示,参与植物生长素生物合成的YUCCA途径的一些酶不是细胞质的,而是定位于拟南芥中的内质网(ER)(YUCCA4.2)(Kriechbaumer等人。( Zm TAR1和 Zm SPI)(Kriechbaumer /em>,2015)。这提高了生长素生物合成的亚细胞区室的有趣的可能性。为了显示玉米生长素生物合成确实可以在来自玉米幼苗的微粒体以及胞质细胞部分中发生,我们应用本文所述的方案:从玉米胚芽鞘和原代根组织分离微粒体,使用色氨酸的微粒体和胞浆部分的酶测定(Trp)或吲哚-3-丙酮酸(IPyA)作为底物,并提取并定量生长素IAA。

关键字:玉米, 微粒体, 内质网, 生长素, IAA

材料和试剂

  1. 移液管(1ml,200μl和10μl)(Gilson Scientific Ltd.,目录号:F123602,F123601和F144802)
  2. Eppendorf管(2ml)(Eppendorf AG,目录号:0030120094)
  3. Eppendorf锥形管(50ml)(Eppendorf AG,目录号:0030122178)
  4. 0.45μm注射器过滤器(Sigma-Aldrich,目录号:F8273)
  5. 奶酪布(DUTSCHER SCIENTIFIC,目录号:789056)
  6. 冰盒和冰
  7. 液氮
  8. 三乙醇胺 - 乙酸(TEA-HOAc)(Sigma-Aldrich,目录号:90278)
  9. 乙酸钾(KOAc)(Sigma-Aldrich,目录号:P1190)
  10. 乙酸镁[Mg(OAc)2](Sigma-Aldrich,目录号:M5661)
  11. 蔗糖(Sigma-Aldrich,目录号:SO389)
  12. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43815)
  13. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E5391)
  14. Tris-盐酸(Tris-HCl)(Sigma-Aldrich,目录号:252859)
  15. β-烟酰胺腺嘌呤二核苷酸磷酸二钠盐(NADPH)(Sigma-Aldrich,目录号:93205)
  16. 黄素腺嘌呤二核苷酸(FAD)(Sigma-Aldrich,目录号:F6625)
  17. 吲哚-3-丙酮酸(IPyA)(Sigma-Aldrich,目录号:I7017)
  18. 色氨酸(Sigma-Aldrich,目录号:T0254)
  19. 乙酸乙酯(Sigma-Aldrich,目录号:270989)
  20. 碳酸钠(Na 2 CO 3)(Sigma-Aldrich,目录号:S7795)
  21. 蒸馏水
  22. 用于HPLC的乙酸乙酯(Sigma-Aldrich,目录号:650528)
  23. HPLC的乙酸(Sigma-Aldrich,目录号:A6283)
  24. HPLC的甲醇(Sigma-Aldrich,目录号:34860)
  25. 库存解决方案(参见配方)
  26. 缓冲区(参见配方)
    1. 用于微粒体提取的缓冲液(缓冲液1,缓冲液2,缓冲液3和缓冲液4)
    2. 用于HPLC分析的缓冲液

设备

  1. 瓷砂浆 杵(Sigma-Aldrich,目录号:Z247464和Z247502)
  2. 缓冲液(100ml,250ml和500ml)的玻璃瓶(Sigma-Aldrich,目录号:Z305170,Z305189和Z305197)
  3. 冷冻台式离心机(Eppendorf AG,型号:5430R)
  4. 具有摆出转子SW41(Beckman Coulter,目录号:331362)的超速离心机
  5. 超速离心机corex管(Beckman Coulter,目录号:331372)
  6. 玻璃棒和2ml Potter-Elvehjem匀浆器(Sigma-Aldrich,目录号:P7734-1EA)
  7. 水浴(37℃)(Thermo Fisher Scientific,型号:TSGP02)
  8. Speed-vac(LABCONCO,目录号:7810016)
  9. Nanodrop ND-2000(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:13400504)
  10. 高效液相色谱(HPLC)(600E多溶剂递送系统)(WATERS,目录号:720000699EN)
  11. 反相柱(Apollo C18)(250mm×4.6mm,5μm)(Thermo Fisher Scientific,Grace TM,目录号:5126767)
  12. UV监视器486(WATERS)和荧光监视器470(WATERS)

程序

  1. 内质网(ER)微粒体制剂
    1. 除非另有说明,所有步骤均在冰上或4℃下进行
    2. 预冷所有在程序中使用的缓冲液,管等
    3. 5g玉米胚芽鞘或原代根组织(4 d后 发芽)在液氮中用a研磨成细粉 预冷砂浆和杵。
    4. 然后将粉末在4ml冰冷的缓冲液1中匀化,并转移到50ml falcon管中。
    5. 将4ml冰冷的缓冲液2加入管和悬浮液中 在冰上孵育10分钟。 然后将匀浆在37℃下离心 1,000×g/g,在4℃温育10分钟。 将所得上清液倒在4上   干酪布层进入新鲜的猎鹰管。 在这一步, 所得提取物被认为是用于以后酶促的总植物提取物 测定。 将提取物在4,500xg下再次离心25分钟,4℃ ℃。
    6. 准备超速离心机corex管与4毫升蔗糖垫(缓冲区3)在底部
    7. 将8ml植物悬浮液层叠在蔗糖的顶部 通过稍微倾斜管子并小心地和缓慢地移液来缓冲 悬浮液在管的侧面。 将管离心90分钟 min,93,000×g (带有转出转子SW41的超速离心机)。
    8. 将所得沉淀从超速离心管中取出 必需用20μl缓冲液4并转移到2ml Potter-Elvehjem匀浆器。 保持上清液并用作 胞质提取物在以后的IAA定量
    9. 最终沉淀 重悬于200μl缓冲液4中,使用玻璃棒和2ml Potter-Elvehjem匀浆器。 蛋白质含量使用a Nanodrop。 新鲜制备的微粒体用于酶测定 直接。
    10. 根据样本大小,这部分过程将需要3到4小时。

  2. 微粒体酶测试
    1. 生长素生物合成的YUCCA途径是两步过程(图1): 在玉米TAR-蛋白[拟南芥相关蛋白的色氨酸氨基转移酶; Zm TAR1(Chourey 等人,2010), Zm VT2 (Phillips等人,2011)和 Zm TAR3( Zm Alliin1; Bernardi )] 将色氨酸(Trp)转化为吲哚-3-丙酮酸(IPyA)  然后通过YUCCA蛋白转化成IAA [em] Zm YUC1和 Zm SPI1(Phillips等  al。,2011)]。已显示 Zm YUC1和 Zm VT2已本地化在 而 Zm SPI1, Zm TAR1和 Zm TAR3本地化到ER (Kriechbaumer et al。,2015)。


      图1。 YUCCA-玉米中生长素生物合成的途径。 胞质酶以绿色标记,ER定位的酶以蓝色标记。

    2. 酶活性试验(100μl总体积)与微粒体和胞质级分按如下方式进行:
      20微升的微粒体或胞质提取物或总植物提取物(参见步骤A5) 1mM NADPH 100μMFAD
      100μMIPyA或Trp(取决于实验兴趣)
      100mM Tris-HCl(pH8.0)直到总体积为100μl
      在2ml Eppendorf管中小心混合
      注意:为此,应首先移取100 mM Tris-HCl,然后 通过植物提取物然后FAD。 反应时间从开始   加入底物(IPyA或Trp)。
      将测定在37℃水浴中孵育1小时,并在孵育时间后直接在液氮中快速冷冻。
    3. 为了比较,还用胞质部分和总植物提取物进行酶测试
    4. 在该阶段,可以在进行IAA分析之前将测定储存在-20℃。

  3. IAA提取和量化
    1. 通过乙酸乙酯相分离提取IAA(Park等人,2003; Kriechbaumer等人,2007)(图2)   每个测定的全部样品用于该程序。
      1. 为此,用1M调节样品的pH高于9.5 Na 2 CO 3 3,然后用400μl乙酸乙酯萃取样品。
      2. 回收水相下层相并加入200μl水
      3. 重复分配过程以再次回收水相。
      4. 将来自两个分配步骤的水相合并。
      5. 将收集的水相用乙酸酸化至pH 低于2.5,并加入400μl乙酸乙酯分配两次   为每个步骤。
      6. 收集有机相和液体 使用speed-vac(预设置为室温)蒸发。 蒸发将需要大约两个小时,但样品应检查 时。
      7. 将干燥的物质重新溶解在100%甲醇中,如果需要,可以在进一步分析之前储存在-20℃。
    2. 根据样品量和蒸发时间的不同,IAA提取可能需要长达5小时

      图2.使用乙基的IAA提取的示意图 乙酸酯相分离。 用乙酸乙酯萃取IAA的步骤 相分离如图所示。水相显示 蓝色,有机相为红色。表明pH调节以及 在Eppendorf管中的变化
    3. 然后通过具有反相柱的高效液相色谱(HPLC)分析IAA
    4. 为此,HPLC系统以0.8ml min -1的等梯度流与缓冲液A(10%甲醇,0.3%乙酸盐)的40:60混合物和 缓冲液B(90%甲醇,0.3%乙酸盐)。鉴定峰 与标准物质的保留时间比较和使用UV监测器和荧光监测器的UV光谱
    5. 使用的体积计算每个样品中IAA的量 IAA峰,并将其与得自的IAA峰的体积进行比较 IAA标准。 这将给出IAA的数量 样品。 微粒体,胞质和总蛋白中的蛋白量 通过用UV-Vis测量280nm处的吸光度来测定级分 分光光度计(例如通过Nanodrop Instrument ND-2000)。 有了这个数据 可以计算每μg蛋白质的IAA量(图4)。

代表数据

为了检查微粒体部分的纯度,用检测胞质腈水解酶的抗ZmNIT抗体探测微粒体以及胞质部分(图3)。 该抗体显示检测玉米腈水解酶1和2蛋白质在各种组织中在总蛋白质的ng mg -1 sup范围内非常特异性(Park等人,2003; Kriechbaumer等人。,2007)。腈水解酶条带仅在胞质级分中可检测到,表明相当纯的微粒体部分

图3.微粒体级分的纯度的免疫印迹分析使用免疫印迹分析测试微粒体(M)和胞质(C)级分的腈水解酶蛋白。用识别胞质玉米腈水解酶1和2的抗-Zm NIT1抗体(1:400)探测来自每个级分的100μg蛋白质。(Kriechbaumer等人,2015 )。

玉米胚芽鞘和原生根中色氨酸转化为IAA的代表性数据显示在图4中[来自Kriechbaumer等人(2015)的数据]。


图4.玉米中的生长素生物合成能力。 发芽后4天,通过微粒体(微粒体)级分,胞质(Cytosol)级分或来自玉米胚芽鞘(Col,白色条)和主根(PR,黑色条)的总植物提取物将色氨酸酶促转化为IAA。显示标准错误。 n = 2(两个生物样品,每个具有三个技术重复)。

笔记

  1. 将植物材料直接收集到保持在冰上的冷冻猎鹰管中,尽可能快地收集材料。
  2. 预冷管,玻璃器皿,缓冲液,离心机等,并尽可能保持您的样品在冰上
  3. 在可能的情况下,应当使用时间上新鲜制备的微粒体用于酶测定。 在20%甘油中冷冻用于以后的测定是可能的,但不推荐
  4. 可以使用任何HPLC系统(例如Dionex,Beckman ...)进行测定。
  5. 建议至少使用质谱法确认一些数据点,以确保正确的分子被定量。

食谱

  1. 库存解决方案
    1M TEA-HOAc(pH7.5) 1M KOAc(pH7.5)
    0.1M Mg(OAc)2
    1 M蔗糖
    1 M DTT
    0.5 M EDTA
    1 M Tris-HCl(pH 8.0)
    1 M Na 2 CO 3 sub
    0.1 M NADPH
    0.1 M FAD
    0.1 M IPyA
    0.1 M Trp

  2. 缓冲区
    注意:过滤缓冲区1-4到0.45μm注射器过滤器。 在使用之前,从1M新鲜的原液中加入DTT。
    1. 微粒子提取缓冲液
      1. 缓冲区1
        25mM TEA-HOAc(pH7.5) 50mM KOAc(pH7.5) 5mM Mg(OAc)2 0.25 M蔗糖 4 mM DTT
      2. 缓冲2
        100mM TEA-HOAc(pH7.5) 20 mM EDTA
      3. 缓冲区3
        25mM TEA-HOAc(pH7.5)25mM KOAc(pH7.5) 2mM Mg(OAc)2
        0.5 M蔗糖 4 mM DTT
      4. 缓冲区4
        25mM TEA-HOAc(pH7.5) 0.25 M蔗糖 1mM DTT
    2. 用于HPLC分析的缓冲液
      缓冲液A:10%甲醇,0.3%乙酸盐 缓冲液B:90%甲醇,0.3%乙酸

致谢

该协议改编自Kriechbaumer等人。 (2015)。这项工作得到了韩国科学技术协会(KOFST)授予Dr Verena Kriechbaumer和英国生物技术与生物科学研究委员会的研究奖学金(授予教授Chris Hawes的授予号BB/J004987/1研究资助)的支持。 。

参考文献

  1. Abell,B.M.,Holbrook,L.A.,Abenes,M.,Murphy,D.J.,Hills,M.J.and Moloney,M.M。(1997)。 脯氨酸结基序在油质蛋白内质网拓扑学和油体靶向中的作用。 em> Plant Cell 9(8):1481-1493。
  2. Bernardi,J.,Lanubile,A.,Li,Q.B.,Kumar,D.,Kladnik,A.,Cook,S.D.,Ross,J.J.,Marocco,A.and Chourey,P.S。(2012)。 生长素受损在缺陷型胚乳18突变体中的生物合成是由于在玉米中编码胚乳特异性YUCCA1蛋白的Zm Yuc1基因中的突变表达缺失。植物生理学160 ):1318-1328。
  3. Chourey,P.S.,Li,Q.B.and Kumar,D。(2010)。 种子发育中的糖激素交互作用:IAA生物合成的两个冗余途径在转化酶缺陷型miniature1(mn1)种子突变体在玉米中。 3(6):1026-1036。
  4. 达尔文,C。和达尔文,F.(1880)。 植物中的运动的力量 约翰Murray。
  5. Kögl,F.,Haagen-Smit,A.J.and Erxleben,H。(1934)。 Überein neues Auxin(Heteroauxin)aus Harn。 11. Mitteilungüberpflanzliche Wachstumsstoffe。 Hoppe-Seyler's ZeitschriftfürPhysiologisch Chemie 228:90-103。
  6. Kriechbaumer,V.,Park,W.J.,Piotrowski,M.,Meeley,R.B.,Gierl,A.and Glawischnig,E。(2007)。 玉米腈水解酶在生长素稳态和β-氰基丙氨酸水解中具有双重作用。 J Exp Bot 58(15-16):4225-4233。
  7. Kriechbaumer,V.,Seo,H.,Park,W.J.and Hawes,C。(2015)。 内质网定位和玉米生长素生物合成酶的活性。 J Exp Bot 66(19):6009-6020。
  8. Park,W.J.,Kriechbaumer,V.,Moller,A.,Piotrowski,M.,Meeley,R.B.,Gierl,A.and Glawischnig,E。(2003)。 Nitrilase ZmNIT2将吲哚-3-乙腈转化为吲哚-3-乙酸。 Plant Physiol 133(2):794-802。
  9. Phillips,K.A.,Skirpan,A.L.,Liu,X.,Christensen,A.,Slewinski,T.L.,Hudson,C.,Barazesh,S.,Cohen,J.D.,Malcomber,S.and McSteen, 消失的流苏2编码在玉米中营养和繁殖发育所需的草特异性色氨酸转氨酶。 植物细胞 23(2):550-566。
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引用:Kriechbaumer, V. (2016). ER Microsome Preparation and Subsequent IAA Quantification in Maize Coleoptile and Primary Root Tissue. Bio-protocol 6(9): e1805. DOI: 10.21769/BioProtoc.1805.
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