The biosynthesis of the plant hormones strigolactone and abscisic acid is, in part, controlled by a family of enzymes known as the carotenoid cleavage dioxygenases (CCDs), which perform an oxidative cleavage reaction on a carotenoid substrate (9’-cis-neoxanthin for abscisic acid and 9-cis-β-carotene and 9-cis-β-apo-10’-carotenal for strigolactone) to form apocarotenoids, which are metabolised to the functional phytohormone. Phenotypic effects on seed dormancy and shoot branching have been observed in Arabidopsis thaliana and Zea mays on the application of a selection of hydroxamic acids based inhibitors, designed to inhibit CCDs, whereby application of the inhibitor to the plant result in a decrease in the time take for germination (abscisic acid mediated) or an increase in the number of lateral shoot branches (strigolactone mediated). However, the biochemical basis of these phenotypes is not understood. In the present thesis, carotenoid cleavage dioxygenases from the abscisic acid biosynthesis pathway (9’-cis-epoxycarotenoid cleavage dioxygenases (NCED)) and strigolactone biosynthesis pathway (CCD7 and CCD8) were produced in vitro and assayed for inhibition against the hydroxamic acid inhibitors. The results show that Z. mays NCED is indeed inhibited by the hydroxamic acid inhibitors (D2: greater than 95% inhibition at 100 μM) in a time dependent fashion, indicating that inhibition of NCED is the basis of the seed germination phenotype. On the strigolactone biosynthesis pathway, recombinant A. thaliana CCD7 is not inhibited by the hydroxamic acids. However, recombinant A. thaliana CCD8 is inhibited by hydroxamic acids that show shoot branching phenotypes (D6 53% inhibition at 10 μM), suggesting that inhibition of CCD8 is the basis of the shoot branching phenotype. Structure activity relationships have also been performed to identify the key features of the hydroxamic acids required to inhibit each enzyme. The biochemistry of several CCDs has also been investigated, along with that of the enzyme Dwarf27, an isomerase enzyme required for the isomerisation of all-trans-β-carotene to 9-cis-β-carotene on the strigolactone biosynthesis pathway. Investigations indicate that Dwarf27 from Oryza sativa could be a novel iron-sulfur protein which isomerises β-carotene via a one electron transfer to or from the β-carotene substrate. D27 is also inhibited to some extent by certain hydroxamic acids (e.g. 41% by D30 at 100 μM). Biochemical characterisation of A. thaliana CCD8 provides evidence for a two-step mechanism involving acid-base catalysis, and evidence is obtained for an active site cysteine residue. A possible mechanism for the double oxidative cleavage reaction catalysed by CCD8 is proposed. These results provide an insight into the biochemistry of the biosynthesis of the phytohormones abscisic acid and strigolactone and demonstrate that enzymes on the biosynthesis pathways of these hormones can be selectively inhibited using a chemical genetics approach. This has potential to aid the development of novel agrochemical compounds which could influence these processes to improve plant architecture and crop yield.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:640961 |
| Date | January 2014 |
| Creators | Harrison, Peter J. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/66554/ |
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