ER Microsome Preparation and Subsequent IAA Quantification in Maize Coleoptile and Primary Root Tissue.
ER Microsome Preparation and Subsequent IAA Quantification in Maize Coleoptile and Primary Root Tissue.
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DOI：
10.21769/bioprotoc.1805
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发表时间：
2016-05-05
期刊：
影响因子：
0.8
通讯作者：
Kriechbaumer, Verena
中科院分区：
文献类型：
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作者：
Kriechbaumer, Verena
生长素是植物中主要的生长激素，是最早被发现和研究的植物激素（Darwin and Darwin, 1880）。Kogl et al.（1934）首先确定植物中的生长素分子为吲哚-3-乙酸（IAA）。近十年来的积极研究已经揭示了生长素作用的许多分子机制，但生长素生物合成网络的复杂性和冗余性提出了对该系统控制的问题。我们已经证明，在拟南芥（YUCCA4.2） （Kriechbaumer等人，2012）和玉米（ZmTAR1和ZmSPI） （Kriechbaumer等人，2015）中，一些参与生长素生物合成的yucca途径的酶不是细胞质的，而是定位于内质网（ER）。这引起了生长素生物合成亚细胞区隔的有趣可能性。为了证明玉米生长素确实可以在玉米幼苗的微粒体和细胞质细胞组分中进行生物合成，我们采用了这里描述的方案：从玉米胚芽包皮和初代根组织中分离微粒体，使用色氨酸（Trp）或吲哚- -3丙酮酸（IPyA）作为底物对微粒体和细胞质组分进行酶分析，提取和定量生长素IAA。
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 Kogl 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.