Glycosyltransferases from oat (Avena) implicated in the acylation of avenacins.

Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid.

Transgenesis and yield: what are our targets?

Plant metabolic engineering has been used to generate a wide range of transgenic lines in which specific metabolic steps have been targeted. Attempts to increase yield of some agronomically important crops using this approach have highlighted the inherent complexities of modulating plant metabolism. In light of the recent findings by Regierer et al. this article addresses the major challenges faced with respect to enhancing yield through transgenesis.

Sad3 and sad4 are required for saponin biosynthesis and root development in oat.

Avenacins are antimicrobial triterpene glycosides that are produced by oat (Avena) roots. These compounds confer broad-spectrum resistance to soil pathogens. Avenacin A-1, the major avenacin produced by oats, is strongly UV fluorescent and accumulates in root epidermal cells. We previously defined nine loci required for avenacin synthesis, eight of which are clustered. Mutants affected at seven of these (including Saponin-deficient1 [Sad1], the gene for the first committed enzyme in the pathway) have normal root morphology but reduced root fluorescence. In this study, we focus on mutations at the other two loci, Sad3 (also within the gene cluster) and Sad4 (unlinked), which result in stunted root growth, membrane trafficking defects in the root epidermis, and root hair deficiency. While sad3 and sad4 mutants both accumulate the same intermediate, monodeglucosyl avenacin A-1, the effect on avenacin A-1 glucosylation in sad4 mutants is only partial. sad1/sad1 sad3/sad3 and sad1/sad1 sad4/sad4 double mutants have normal root morphology, implying that the accumulation of incompletely glucosylated avenacin A-1 disrupts membrane trafficking and causes degeneration of the epidermis, with consequential effects on root hair formation. Various lines of evidence indicate that these effects are dosage-dependent. The significance of these data for the evolution and maintenance of the avenacin gene cluster is discussed.

Characterization of the genes encoding the cytosolic and plastidial forms of ADP-glucose pyrophosphorylase in wheat endosperm.

In most species, the synthesis of ADP-glucose (Glc) by the enzyme ADP-Glc pyrophosphorylase (AGPase) occurs entirely within the plastids in all tissues so far examined. However, in the endosperm of many, if not all grasses, a second form of AGPase synthesizes ADP-Glc outside the plastid, presumably in the cytosol. In this paper, we show that in the endosperm of wheat (Triticum aestivum), the cytosolic form accounts for most of the AGPase activity. Using a combination of molecular and biochemical approaches to identify the cytosolic and plastidial protein components of wheat endosperm AGPase we show that the large and small subunits of the cytosolic enzyme are encoded by genes previously thought to encode plastidial subunits, and that a gene, Ta.AGP.S.1, which encodes the small subunit of the cytosolic form of AGPase, also gives rise to a second transcript by the use of an alternate first exon. This second transcript encodes an AGPase small subunit with a transit peptide. However, we could not find a plastidial small subunit protein corresponding to this transcript. The protein sequence of the purified plastidial small subunit does not match precisely to that encoded by Ta.AGP.S.1 or to the predicted sequences of any other known gene from wheat or barley (Hordeum vulgare). Instead, the protein sequence is most similar to those of the plastidial small subunits from chickpea (Cicer arietinum) and maize (Zea mays) and rice (Oryza sativa) seeds. These data suggest that the gene encoding the major plastidial small subunit of AGPase in wheat endosperm has yet to be identified.

Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity.

Two mutant lines of barley, Risø 17 and Notch-2, were found to accumulate phytoglycogen in the grain. Like the sugary mutants of maize and rice, these phytoglycogen-accumulating mutants of barley lack isoamylase activity in the developing endosperm. The mutants were shown to be allelic, and to have lesions in the isoamylase gene, isa1 that account for the absence of this enzyme. As well as causing a reduction in endosperm starch content, the mutations have a profound effect on the structure, number and timing of initiation of starch granules. There are no normal A-type or B-type granules in the mutants. The mutants have a greater number of starch granules per plastid than the wild-type and, particularly in Risø 17, this leads to the appearance of compound starch granules. These results suggest that, as well as suppressing phytoglycogen synthesis, isoamylase in the wild-type endosperm plays a role in determining the number, and hence the form, of starch granules.