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
发布: 2016年05月05日第6卷第9期 DOI: 10.21769/BioProtoc.1805 浏览次数: 11584
评审: Samik BhattacharyaLaura ZaninAgnieszka Zienkiewicz
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参见作者原研究论文
The authors used this protocol in:
Sep 2015
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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
- Pipettes (1 ml, 200 µl and 10 µl) (Gilson Scientific Ltd., catalog number: F123602 , F123601 and F144802 )
- Eppendorf tubes (2 ml) (Eppendorf AG, catalog number: 0030120094 )
- Eppendorf conical tubes (50 ml) (Eppendorf AG, catalog number: 0030122178 )
- 0.45 µm syringe filters (Sigma-Aldrich, catalog number: F8273 )
- Cheese cloth (DUTSCHER SCIENTIFIC, catalog number: 789056 )
- Ice box and ice
- Liquid nitrogen
- Triethanolamine-acetic acid (TEA-HOAc) (Sigma-Aldrich, catalog number: 90278 )
- Potassium acetate (KOAc) (Sigma-Aldrich, catalog number: P1190 )
- Magnesium acetate [Mg(OAc)2] (Sigma-Aldrich, catalog number: M5661 )
- Sucrose (Sigma-Aldrich, catalog number: S0389 )
- DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
- Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5391 )
- Tris-Hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: 252859 )
- β-Nicotinamide adenine dinucleotide phosphate disodium salt (NADPH) (Sigma-Aldrich, catalog number: 93205 )
- Flavin adenine dinucleotide (FAD) (Sigma-Aldrich, catalog number: F6625 )
- Indole-3-pyruvic acid (IPyA) (Sigma-Aldrich, catalog number: I7017 )
- Tryptophan (Sigma-Aldrich, catalog number: T0254 )
- Ethyl acetate (Sigma-Aldrich, catalog number: 270989 )
- Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795 )
- Distilled water
- Ethyl acetate for HPLC (Sigma-Aldrich, catalog number: 650528 )
- Acetic acid for HPLC (Sigma-Aldrich, catalog number: A6283 )
- Methanol for HPLC (Sigma-Aldrich, catalog number: 34860 )
- Stock solutions (see Recipes)
- Buffers (see Recipes)
- Buffers for microsome extraction (Buffer 1, Buffer 2, Buffer 3 and Buffer 4)
- Buffers for HPLC analysis
- Buffers for microsome extraction (Buffer 1, Buffer 2, Buffer 3 and Buffer 4)
Equipment
- Porcelain mortar & pestle (Sigma-Aldrich, catalog number: Z247464 and Z247502 )
- Glass bottles for buffers (100 ml, 250 ml and 500 ml) (Sigma-Aldrich, catalog number: Z305170 , Z305189 and Z305197 )
- Refrigerated table centrifuge (Eppendorf AG, model: 5430R )
- Ultracentrifuge with swing-out rotor SW41 (Beckman Coulter, catalog number: 331362 )
- Ultracentrifuge corex tubes (Beckman Coulter, catalog number: 331372 )
- Glass rod and a 2 ml Potter–Elvehjem homogenizer (Sigma-Aldrich, catalog number: P7734-1EA )
- water bath (37 °C) (Thermo Fisher Scientific, model: TSGP02 )
- Speed-vac (LABCONCO, catalog number: 7810016 )
- Nanodrop ND-2000 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 13400504 )
- High-performance liquid chromatography (HPLC) (600E multisolvent delivery system) (WATERS, catalog number: 720000699EN )
- Reverse phase column (Apollo C18) (250 mm x 4.6 mm, 5 μm) (Thermo Fisher Scientific, GraceTM, catalog number: 5126767 )
- UV monitor 486 (WATERS) and a fluorescence monitor 470 (WATERS)
Procedure
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文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
植物科学 > 植物细胞生物学 > 细胞器分离
细胞生物学 > 细胞器分离 > 微粒体
植物科学 > 植物生物化学 > 植物激素
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