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, Microsomes, Endoplasmic reticulum, Auxin, IAA
Materials and Reagents
Equipment
Procedure
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
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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
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