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ER Microsome Preparation and Subsequent IAA Quantification in Maize Coleoptile and Primary Root Tissue.

Bio-protocol (2017-02-10)
Verena Kriechbaumer
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Tris(hydroxymethyl)aminomethane, ACS reagent, ≥99.8%
Sigma-Aldrich
Flavin adenine dinucleotide disodium salt hydrate, ≥95% (HPLC), powder
Sigma-Aldrich
Acetic acid, glacial, ReagentPlus®, ≥99%
Sigma-Aldrich
Methanol, suitable for HPLC, ≥99.9%
Sigma-Aldrich
L-Tryptophan, reagent grade, ≥98% (HPLC)