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Final Activity and Management Report Summary - IAOX (Dissection of the metabolic grid of IAOx, a precursor for natural plant products and the plant hormone auxin)

Glucosinolates (GSLs) are amino acid-derived natural product present in the plants of order Brassicales. Upon disruption of plant tissue by, for example, wounding or mastication, GSLs are hydrolysed by the thioglucosidases, myrosinases, which produce a range of breakdown products, primarily isothiocyanates and nitriles, with diverse biological activities. GSLs and their breakdown products act as antimicrobials against pathogens, feeding deterrents toward generalist herbivores. For humans, the well-studied sulforaphane, which is derived from 4-methylsulfinylbutyl (4-MSB) GSL are considered very potent cancer-preventive agents. Biosynthesis of methionine-derived methylsulfinylalkyl GSLs can be divided into three steps. First, methionine undergoes a varying number of chain-elongation cycles to produce chain-elongated methionine derivatives. Second is the synthesis of the core GSL structure. Finally, side-chain modification via S-oxygenation of Methylthioalkyl (MT) to Methylsulfinylalkyl (MS) GSL occurs.

The main achievement of the project is the identification and characterisation of a subclade of flavin monooxygenases as enzymes in the biosynthesis of aliphatic GSLs in Arabidopsis that catalyses the S-oxygenation of methylthioalkyl to methylsulfinylalkyl GSLs. Spatial expression pattern of FMOGS-OXs were primarily detected. In return phase of this project, based on previous work of the incoming phase, characterisation of spatial expression pattern of FMOGS-OXs genes were performed. Another two FMO genes were detected can alter aliphatic GSLs content while overexpressed in Arabidopsis.

Furthermore, a small RNA was identified involved in regulation of GSLs biosynthesis.

1) Localisation of FMOGS-OXs FMOGS-OX1-5 was expressed basically in vascular tissues, especially in phloem cells, like other GSLs biosynthetic genes. They were also found in endodermis-like cells in flower stalk and epidermal cells in leaf, which is a location that has not been reported for other GSLs biosynthetic genes. Interestingly, our results suggested that the spatial expression pattern of FMOGS-OX1-5 determines the access of enzymes to their substrate and therefore affects the glucosinolate profile. For subcellular level, FMOGS-OX1-YFP fusion protein analysis identified FMOGS-OX1 as a cytosolic protein. Together with the subcellular locations of the other biosynthetic enzymes, an integrated map of the multicompartmentalised aliphatic glucosinolate biosynthetic pathway (cytosol-choloroplast-cytosol-ER-cytosol) is established.

2) Indentification of another two FMO genes that can alter aliphatic glucosinolates profile while overexpressed in Arabidopsis. According to our previous phylogenic analysis (Hansen et al., 2006), a clade of genes from the phylogenetic tree of plant FMOs in rice, poplar and Arabidopsis which contains FMOGS-OXs subclade was supposed to involve in S-oxygenating reaction. Since all the five genes in FMOGS-OX subclade were confirmed to have the activity of S-oxygenation that can catalyse MT to MS GSLs. I extended the research to the neighbour subclade which contains another two FMO genes At1g12130 and At1g12160. Biochemical characterisation of the recombinant protein showed that these two FMO proteins cannot S-oxygenated MT GSLs to MS GSLs in vitro like FMOGS-OX1-5. Consistently, no GSLs profile alteration was detected in T-DNA knock-out mutants. However, the GSLs profile was significantly altered with a decrease of MT:(MT+MS) when over expressed At1g12130 and At1g12160 genes respectively in Arabidopsis, which suggested the overexpression of the two genes significantly converted MT to MS like the other FMOGS-OXs. As the S-oxygenating clade includes FMOs from poplar and rice, this clade may be involved in producing a diversity of oxidised sulphur compounds within plant primary and secondary metabolism.

Our results indicated that the proteins encoded by At1g12130 and At1g12160 have S-oxygenating activity, while at the normal expression level, the substrate in vivo might not be GSLs but some sulphur containing compounds in Arabidopsis.

3) Identification of a small RNA involved in regulation of glucosinolates biosynthesis Structural gene discovery in GSLs biosynthesis, greatly aided by a combination of molecular, genetic and genomic approaches in Arabidopsis has made big progress. Biosynthetic genes in the pathway have largely been identified and confirmed. Current research increasingly focuses on regulatory mechanism of GSLs biosynthesis.

MicroRNAs (miRNA) are small (20-25 nt) non-coding RNA molecules that regulate gene expression through interaction with mRNA and control a vast array of biological processes. MiRNAs regulate gene expression by complementary base pairing to target mRNA. In plants the base-pairing is almost perfect along the whole length of miRNA and it makes it relatively easy to do the computational predictions of both the targets for known miRNAs and the novel miRNA candidates for all known mRNAs. Lindow et al. identified about 1200 miRNA candidate genes by the method of intragenomic matching in conjunction with a supervised learning approach (Lindow et al. 2005, 2007). Several GSLs biosynthetic genes were predicted to be the target of miRNA. The expression of one candidate miRNA gene targeted to superroot1 gene was detected by stem-loop RT realtime PCR and northern blot. RACE analysis suggested that this small RNA can cleave mRNA of superroot1 at the predicted cite. Although these data did not satisfy the criteria of a miRNA, it is first time discovered that there is small RNA regulation in GSLs biosynthesis.

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