Saponin glycosylation in cereals

Belinda Townsend1, Helen Jenner1 & Anne Osbourn1,2,*1The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich, Norfolk, NR4 7UH, United Kingdom;2Department of Metabolic Biology, John Innes Centre, Colney Lane, Norwich, Norfolk, NR4 7UH, UnitedKingdom; *Author for correspondence (Tel: +44-0-1603-450407; Fax: +44-0-1603-450014; E-mail:anne.osbourn@bbsrc.ac.uk)

Key words: avenacin, disease resistance, expressed sequence tag, glucosyltransferase, monocotyledon,triterpene

Abstract

Triterpene saponins are glycosylated plant secondary metabolites that are common in dicotyledonousspecies but rare in monocots. These compounds are antimicrobial and are important for plant defence.They also have a variety of pharmaceutical and industrial applications. Oat (Avena spp.) produces triter-pene saponins called avenacins, specialised metabolites that are not represented in other cereals. Theavenacins are synthesised and stored in roots and provide protection against soil-borne fungal pathogens.Glycosylation is often critical for bioactivity of saponins. We are using a combination of mutant analysis,database mining, expression profiling and functional characterisation to identify glycosyltransferasesrequired for addition of sugar units to avenacins. By identifying the molecular machinery required foravenacin biosynthesis we hope to understand the broader elements of the evolution of specialised metabolicpathways.

Saponins are glycosylated triterpenes, steroids, orsteroidal alkaloids (Hostettmann and Marston,1995). Research into these experimentally chal-lenging natural metabolites has been stimulated bytheir natural role in plant defence and their phar-maceutical applications. Triterpene saponins arefound in a wide variety of dicotyledonous plantspecies but are rare in monocots. Oats (Avena spp.)are unique amongst the cereals in that they pro-duce a group of defence-related triterpene sapo-nins called the avenacins (Osbourn et al., 1994;Hostettmann and Marston, 1995; Trojanowskaet al., 2000; Haralampidis et al., 2001). Avenacinsare synthesised and stored within the epidermalcells of the root tip, and so provide an effectivebarrier to soil-borne fungal pathogens. Dissectionof the biosynthetic pathway for these compoundsis providing important insights into the evolutionof natural product pathways in plants.

Avenacins consist of a triterpene core derivedfrom a modified b-amyrin backbone, an N-methy-lanthranilate or benzoate group attached at theC-21 position, and a branched sugar chain attachedat the C-3 position (Crombie and Crombie, 1986).There are four structurally related avenacins in oatroots (A-1, A-2, B-1 and B-2). These differ inoxidation status and in the nature of the acyl group.The avenacin composition varies during develop-ment but avenacin A-1 is the most abundant of thefour (Crombie andCrombie, 1986). The synthesis ofavenacin A-1 is predicted to involve a series ofmodifications in common with many terpene andother secondary metabolic pathways, in both orderand mechanism (Figure 1). The pentacyclic triter-pene backbone is derived from the cytosolicmevalonate pathway which supplies isopentenyldiphosphate for conversion to farnesyl diphosphateand then to 2,3-oxidosqualene, biosynthetic steps

Phytochemistry Reviews (2006) 5: 109–114 � Springer 2006DOI 10.1007/s11101-005-3852-3

that are shared with the sterol biosynthetic path-way. The triterpene and sterol pathways diverge atthe point of cyclisation of 2,3-oxidosqualene. Inavenacin biosynthesis the first committed step ismediated by the triterpene cyclase enzyme b-amyrinsynthase (AsbAS1), which catalyses the cyclisationof 2,3-oxidosqualene to b-amyrin, an oleanane-typetriterpene skeleton (Haralampidis et al., 2001).b-Amyrin then undergoes a series of hydroxylationsthat are most likely mediated by cytochrome P450monooxygenases. The aldehyde may be introducedby subsequent oxidation catalysed by a dehydro-genase, as has been shown to be the case in sesqui-terpene synthesis (de Kraker et al., 2001). The

N-methylanthranilate group, which is presumablyadded by an acyltransferase, renders avenacin A-1fluorescent under ultraviolet light, a property that isvery useful for experimental purposes. Finally,addition of the branched sugar chain onto theaglycone portion will involve glycosyltransferases(GTs) (Haralampidis et al., 2002).

The structural complexity and low abundanceof saponins coupled with the lack of availablestandards has proved to be problematic for sapo-nin research. However, advances in phytochemis-try, including chromatographic and spectroscopictechniques, have for the most part enabled thesedifficulties to be overcome (Marston et al., 2000;Huhman and Sumner, 2002; Joshi et al., 2002).Integrated approaches to understanding saponinbiosynthesis at the genome and metabolite levelsare underway in Panax ginseng (Jung et al., 2003;Choi et al., 2005) andMedicago truncatula (Suzukiet al., 2002; Achnine et al., 2005). Triterpenoidbiosynthesis is also being investigated inArabidopsis thaliana by a genome mining approach(Husselstein-Muller et al., 2001; Ebizuka et al.,2003; Fazio et al., 2004). To understand saponinbiosynthesis in oats, we are using a combination ofgenetics, metabolite profiling, expressed sequencetag (EST) mining, expression analysis, and bio-chemical characterisation.

Clustering of avenacin biosynthetic genes

The fluorescent property of avenacin A-1 underultraviolet light has been exploited to screen forsodium-azide generated mutants of diploid oat(Avena strigosa) that are defective in avenacinsynthesis. Saponin-deficient (sad) mutants wereisolated which display reduced root fluorescence.These mutants have enhanced susceptibility tofungal infection (Papadopoulou et al., 1999). Todate eight loci required for avenacin biosynthesishave been defined (Sad1–8). This mutant collec-tion represents a powerful tool for investigatingavenacin biosynthesis. A combination of metabo-lite profiling and biochemical analysis has revealedthat sad1 mutants accumulate 2,3-oxidosqualeneand are deficient in b-amyrin synthase activity(Trojanowska et al., 2001) (Figure 1). sad1mutants were subsequently shown to have under-gone point mutations within the b-amyrin syn-thase (AsbAS1) gene (Haralampidis et al., 2001).

O

2,3-Oxidosqualene

α-L-arabinose

β-D-glucose (1 2)

β-D-glucose (1 4)

NHCH3

O

O

O

OH

OH

O

CHO

AvenacinA-1

Sad3 Sad4

Glucosylation

OH

Sad7 Acylation

HO

CHO

β-Amyrin

Cyclisation Sad1 (AsbAS1)

Hydroxylation Sad2

Figure 1. The biosynthesis of avenacin A-1 proceeds via thecyclisation of 2,3-oxidosqualene to b-amyrin by b-amyrin syn-thase. Subsequent steps are under characterisation and arepredicted to involve enzymes such as cytochrome P450 hy-droxylases, O-methyltransferases, acyltransferases, dehydro-genases and glycosyltransferases. Biosynthetic lesions in sadmutants are indicated. A dashed line indicates that manyother modifications are likely to occur and the order of stepsindicated by arrows after sad2 are unconfirmed.

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Subsequent biochemical analysis has shown thatother sad mutants accumulate different triterpeneintermediates, suggestive of blocks in hydroxyl-ation (sad2), acylation (sad7), and glucosylation(sad3 and sad4) (Papadopoulou et al., 1999; Tro-janowska et al., 2001; Qi et al., 2004) (Figure 1).

Genetic analysis has revealed that five loci(Sad2, Sad3, Sad6, Sad7, and Sad8) are linked tothe Sad1 locus (Qi et al., 2004). This observation isstriking because it suggests that the genes for a setof biochemically distinct enzymes involved inavenacin biosynthesis are clustered within thegenome (Qi et al., 2004). Such biosynthetic geneclusters are common in fungi but rare in plants. Theonly other well-characterised example of clusteredgenes for a secondary metabolic pathway in plantsis that of the benzoxazinoids in maize. Benzoxazi-noids are cyclic hydroxamic acids that are struc-turally unrelated to avenacins (Frey et al., 1997,2003; von Rad et al., 2001). The significance ofgene clustering in plant secondary metabolism isunclear at present. Clustered genes may confer aselective advantage because they are inherited as adiscrete functional unit undergoing evolutionarypressures such as linkage disequilibrium orco-adaptation. Gene clustering may also facilitateco-ordinate regulation of gene expression (Gierland Frey, 2001; Qi et al., 2004).

Glycosylation of avenacins

The branched sugar chain of avenacin A-1 is pre-dicted, by analogy to other glycosylated smallmolecules, to be synthesised by the sequentialaddition of sugar units onto the aglycone compo-nent, most probably by the activity of three differ-ent GTs. The first step in glycosylation involves theaddition of a-L-arabinose onto the C3 hydroxylgroup of the aglycone, mediated by an arabinosyl-transferase. This is followed by the addition of twob-D-glucose molecules, one at the C2 position of thearabinose and the other at the C4 position, medi-ated by one (or possibly two) glucosyltransferases.

The order in which the two glucose units areadded has been inferred from intermediates thataccumulate in certain sad mutants. Mutants rep-resenting two different loci (Sad3 and Sad4) bothaccumulate avenacin A-1 lacking one glucose unit(Papadopoulou et al., 1999), indicating that glu-cosylation is perturbed in some way. Sugar linkage

analysis indicates that for both sad3 and sad4mutants the intermediates that accumulate arelacking the b-D-glucose at the C4 position of thearabinoside, suggesting that this glucose is addedlast (B. Qin and A. Osbourn, unpublished data).

Glycosylation may be important for storageand appropriate subcellular compartmentalisationof the final product of the pathway. Unlike othersad mutants, sad3 and sad4 mutants both havealtered root morphologies; the roots are thicker,shorter and are deficient in root hairs (P. Mylonaand A. Osbourn, unpublished data). Whether thealtered root morphology of these mutants is acause or consequence of the mis-localisation andaccumulation of avenacin intermediates is not yetknown.

Database mining for GT genes

GTs belong to a large family of enzymes thattransfer saccharide units from activated donormolecules onto a wide spectrum of potentialacceptor molecules. The array of potential accep-tors includes proteins, lipids, polysaccharides andsmall molecules, which may be involved in diversecellular processes such as cell wall synthesis andsignalling (Coutinho et al., 2003). The GTs areclassified into families based on sequence similarity,predicted protein folds, reaction mechanisms andpresence of conserved regions (Coutinho et al.,2003). Of 77 GT families with representativesspanning all Kingdoms, the GT Family 1 is one ofthe largest (Carbohydrate active enzymes websitehttp://afmb.cnrs-mrs.fr/CAZY/; Coutinho andHenrissat, 1999). Family 1 consists of GTs thatoperate via an inverting catalytic mechanism ofsugar transfer, usually onto low molecular weightacceptor molecules (Vogt and Jones, 2000; Lim andBowles, 2004). There are several hundred plant GTsin Family 1 and the number is steadily increasingdue to the ongoing contributions from large-scalegenome sequencing projects. Arabidopsis has 121predicted Family 1 sequences (excluding pseudog-enes) and progress is being made in elucidating therole of each family member (Li et al., 2001; Limet al., 2003; Paquette et al., 2003). Since avenacinA-1 is a lowmolecular weight secondary metabolitethen the GTs involved in glycosylation of the cor-responding aglycone are likely to belong to GTFamily 1. Therefore, a sequence-based database

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mining approach was taken to identify Family 1GTs involved in avenacin glycosylation.

An EST resource was constructed using RNAisolated from oat root tips, the tissue in whichavenacin biosynthesis occurs (Trojanowska et al.,2000; Haralampidis et al., 2001). Around 16,000ESTs were searched for similarity to plant aminoacid sequences representative of the sequencediversity of GT Family 1, including a sterol gluco-syltransferase gene. A conserved signature motif of44 amino acids common to many plant GT Family1 members, the Putative Secondary Product Gly-cosyltransferase Box (PSPG Box), was also used inthe search algorithm. The PSPG box is located inthe C-terminal portion of the protein and is believedto be the binding site for UDP glucose (Hughes andHughes, 1994; Mackenzie et al., 1997; Kubo et al.,2004). This search highlighted 27 distinct sequenceswith similarity to GT Family 1 sequences, (B.Townsend and A. Osbourn, unpublished data),including the oat UDP-glucose:sterol glucosyl-transferase gene (Warnecke et al., 1997).

Establishing a function for candidate GTs

A large collection of plantGTFamily 1 sequences ispublicly available. However, until recently func-tional information has been sparse. Many GT en-zymes have been purified from plant proteinextracts by traditional methods and their activitiesstudied. They include the oat nuatigenin UDP-glucosyltransferase that glucosylates the aglyconeof avenacosides (Kalinowska and Wojciechowski,1988). The avenacosides are steroidal saponinsproduced in leaves as opposed to the avenacins thatoccur in roots (reviewed in Osbourn, 1996). A so-yasapogenolUDP-glucuronosyltransferase involvedin triterpenoid saponin production in Glycine maxhas also been purified (Kurosawa et al., 2002). Todate only a few genes for saponin GTs have beenidentified. One such saponinGT is solanidineUDP-glucosyltransferase which is involved in the steroi-dal glycoalkaloid pathway in potato (Solanumtuberosum) (Moehs et al., 1997). The recentlycharacterised UDP-glucosyltransferases from bar-rel medic (M. truncatula) utilise the triterpeneaglycones hederagenin, medicagenic acid or soyas-apogenols B and E as acceptors for saponinbiosynthesis (Achnine et al., 2005).

We have cloned full-length cDNAs for can-didate oat GTs into a heterologous expressionconstruct for production of N-terminal histidineprotein fusions in bacteria. Insolubility of theheterologously expressed proteins was a signifi-cant complication but was overcome by inductionof expression at 16 �C overnight with low inducerconcentrations (0.1 mM IPTG). Growth in smallculture volumes with the addition of sorbitol andbetaine as protein stabilising agents also served toimprove yield of soluble protein. Several proteinshave been successfully expressed and purified byaffinity chromatography. With these purifiedproteins we have set out to determine theirin vitro donor and acceptor profiles. To assay theacceptor profiles of our candidate GTs we areusing natural acceptors and synthetic analoguesto identify the GTs that specifically transfer thesugar units onto avenacin. Intermediates purifiedfrom sad3 and sad4 mutants (Papadopoulouet al., 1999; B. Qin and A. Osbourn, unpublisheddata) and/or the products of enzymatic digestionof avenacin A-1 with a microbial hydrolase canserve as acceptor substrates for GT enzyme as-says. In addition to natural substrates, syntheticsubstrates based on benzoic acid are also avail-able and are being used to study the regioselec-tivity of these GTs (Mukhopadhyay and Field,2004).

Avenacin biosynthesis occurs as part of nor-mal growth and development and does not ap-pear to be induced by external treatments, unlikethe situation in plants such as M. truncatula andPanax ginseng where triterpene glycoside synthe-sis can be induced by elicitors (Suzuki et al.,2002; Achnine et al., 2005; Choi et al., 2005; Junget al., 2005). Strategies for the identification oftarget GTs will include assessing transcriptabundance and expression patterns in variousplant tissues and correlating these with expressionof AsbAS1 and other genes in the pathway duringroot development. Mutants that are defective inavenacin glycosylation also represent a valuableresource for implicating candidate GT sequences.Whilst these approaches can provide compellingcircumstantial evidence to identify GTs requiredfor triterpene glycosylation, investigating of theproperties of the enzyme remains one of the mostdefinitive methods for confirming biological sig-nificance.

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Acknowledgements

The Sainsbury Laboratory is supported by theGatsby Charitable Foundation. Parts of this workhave been funded by the Biotechnology and Bio-logical Sciences Research Council (B.T.) and byDuPont (H.J.).

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