Phytochemistry Reviews 3: 127–140, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Peroxidases and the metabolism of hydroxycinnamic acid amides in Poaceae Brian Kaare Kristensen∗ , Kim Burhenne & Søren Kjærsgaard Rasmussen RISØ National Laboratory, Plant Research Department, Plant Quality, Frederiksborgvej 399, PO Box 49, DK4000 Roskilde, Denmark; ∗ Author for correspondence (Phone +45 4677 4158; Fax +45 4677 4122; E-mail [email protected])

Key words: barley, cell wall, hydroxycinnamic acid amide, pathogenic fungi, defence

Abstract The arsenal of plants to fight off microorganisms and herbivores include hydroxycinnamic acid amides (HCAA) and their oxidation products. Hydroxycinnamic acid amides are widespread in the plant kingdom and in the recent years our knowledge of their biosynthesis and catabolism has increased substantially. Peroxidases are the primary candidates as the oxidative enzymes responsible for the turnover of hydroxycinnamic acid amide monomers. In barley, hydroxycinnamoylagmatine derivatives accumulate in young seedlings and in tissues infected with fungi. Hydroxycinnamoylagmatine is found as anti-fungal soluble dimers, called hordatines, and it is also a likely constituent of cell walls. Current evidence suggest that peroxidases are involved in the crosslinking of hydroxycinnamoylagmatine with cell wall components and possibly also in the synthesis of hordatines. Epidermal cell walls of barley respond to infection by the powdery mildew fungus with the deposition of polyphenolic material, that apparently contains hydroxycinnamic acid amides, at the site of attempted penetration. Accumulation of these compounds lowers the successful penetration by the fungus. The recent characterization of agmatine coumaroyl transferase (ACT), the N-hydroxycinnamoyltransferase responsible for the synthesis of hydroxycinnamoylagmatine in barley, has indicated that the production of these metabolites is widespread in the plant body and suggests multiple physiological functions for HCAA derivatives. The cloning of ACT has enabled the revelation of homologues genes in several monocots and the presence of a range of structurally diverse HCAAs in cereals suggests that their peroxidase-mediated metabolism is a common theme. The prospects for metabolic engineering of these pathways into other crops are discussed. Abbreviations: HCAA – hydroxycinnamic acid amide; HRPC – horseradish peroxidase C; ACT – agmatine coumaroyl transferase; THT – tyramine hydroxycinnamoyl transferase; HCBT – hydroxycinnamoyl/benzoylCoA:anthranilate N-hydroxycinnamoyl/benzoyl transferase; PHT – putrescine hydroxycinnamoyl transferase; SHT – spermidine/spermine hydroxycinnamoyl transferase; HHT – hydroxyanthranilate hydroxycinnamoyl transferase; p-CHA – p-coumaroyl hydroxyagmatine; p-CHDA – p-coumaroyl hydroxydehydroagmatine; PAL – phenylalanine ammonia lyase.

Hydroxycinnamic acid amides as peroxidase substrates in plants Secretory plant peroxidases (class III; E. C. 1.11.1.7) form a large multigene family with 73 members characterized in the model plant Arabidopsis thaliana (Welinder et al., 2002). Their multiplicity probably covers a large functional diversity of which we only

know little. Horseradish peroxidase C (HRPC), the model peroxidase, is capable of oxidising a wide variety of substrates under the consumption of hydrogenperoxide or under acidic conditions using oxygen and NADH (Dunford, 1991). The enzymatic properties of HRPC cannot be extrapolated to all other plant peroxidases as exemplified by the study of barley grain

128 peroxidase BP1 (Rasmussen et al., 1993) and the wide range of substrates identified by in vitro studies is probably not relevant to the in planta situation where temporal and spatial restrictions (in expression) are laid over substrate preferences of the individual enzymes. Expression profiling by RT-PCR and cDNA arrays of peroxidases in rice and Arabidopsis has already given better insight in peroxidase regulation and function on the organ and tissue level (Hiraga et al., 2000; Tognolli et al., 2002; Welinder et al., 2002). These studies suggest that only structurally different peroxidases (<70% amino acid sequence identity) are co-expressed in the same organs in Arabidopsis and rice (Welinder et al., 2002; Hiraga et al., 2000). This alone suggests that the structural diversity among peroxidases cover a spatial and biochemical functional diversity. However, expression analyses should be interpreted with caution since translational control may regulate the protein synthesis rate from peroxidase mRNAs (Østergaard et al., 1998) and consequently the accumulation levels of the various peroxidases should also be studied at the protein level. The most physiologically important substrates appear to be aromatic alcohols, such as monolignols, hydroxycinamic acids, hydroxycinnamic acid amides, indole acetic acid and tyrosine residues in structural proteins. The first hydroxycinnamic acid amides (HCAAs) characterized from plants were feruloylputrescine (Wheaton and Stewart, 1965) and p-coumaroylagmatine and its optically active dimer, hordatine (Stoessl, 1965, 1966). Following that, a wide range of structurally diverse HCAAs was characterized from a number of plant species spanning several genera (Martin-Tanguy et al., 1978 and references therein; Mayama et al., 1981). A selection of HCAAs is shown in Figure 1. The hydroxycinnamic acid moieties of the HCAAs are derived from the phenylpropanoid pathway before it branches of into the monolignol-specific part (Boerjan et al., 2003) and this part of the conjugate forms a conserved structure which can be used as a peroxidase substrate. The oxidation mechanism of phenolic compounds by peroxidase is known (reviewed by Smith and Veitch, 1998), and it liberates radicals, which can undergo hydrogen migration to generate a number of mesomeric forms that can dimerize and polymerise as described for lignin by Freudenberg (1965). The accumulation of soluble or cell wall bound HCAAs has been described as a stress response in a number of plant species, e.g. barley, wheat, oat, onion, potato, tobacco, tomato and carnation (Koshimizu

et al., 1963; Stoessl, 1965; Samborski and Rohringer, 1970; Mayama et al., 1981; Bouilliant et al., 1983; Negrel and Martin, 1984; Negrel and Lherminier, 1987; Grandmaison et al., 1993; Negrel et al., 1993; Wei et al., 1994; Pearce et al., 1998; Von Röpenack et al., 1998; McLusky et al., 1999; Newman et al., 2001; Von Roepenack-Lahaye et al., 2003). There is now a common agreement that HCAAs are important plant cell wall constituents that make the cell wall more resistant to degradation by hydrolytic enzymes and thereby provide physical defence towards pathogens (Von Röpenack et al., 1998; Facchini et al., 1999; McLusky et al., 1999; Bernards, 2002; Facchini, et al., 2002). The relation between peroxidases and HCAAs is very clear in the context of wound healing or pathogen challenged potato tubers (Negrel and Jeandet, 1987; Negrel et al., 1993, 1996; Bernards et al., 1999; Bernards and Razem, 2001; Razem and Bernards, 2002) where current evidence suggests that peroxidase-polymerised HCAAs, hydroxycinnamic acids and lignols form the phenolic domain of suberin in natural and wound periderms (reviewed by Bernards and Lewis, 1998; Bernards, 2002). However the first notion of peroxidase involvement in the metabolism of HCAAs was the demonstration by Stoessl (1967) that HRPC can form hordatine from hydroxycinnamoylagmatine. The biochemistry and physiology of the HCAAs with emphasis on HCAAs from tyramine in dicots was recently reviewed (Facchini et al., 2002). In the following paragraphs we will review the litterature on the biosynthesis and peroxidasemediated metabolism of HCAAs with emphasis on barley and other cereals where these compounds have been studied for the last forty years.

Biosynthesis and accumulation of HCAAs The HCAAs are synthesised from decarboxylated amino acids, di- and polyamines or anthranilate as acyl acceptors and coenzyme A-activated hydroxycinnamic acids from the phenylpropanoid pathway as acyl donors. Their biosynthesis is often stress-induced and they accumulate in roots, tubers, stems, leaves and reproductive organs where they may be metabolised by peroxidases. In the biosynthetic pathway to hydroxycinnamoylagmatine and hydroxycinnamoyltyramine, arginine and tyrosine are decarboxylated enzymatically (Smith and Best, 1978; Facchini et al., 1999) to form agmatine and tyramine, respectively.

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Figure 1. Structures of selected hydroxycinnamic acid amides.

Agmatine and tyramine are subsequently conjugated to the hydroxycinnamoyl CoA esters by ACT and THT, respectively (Bird and Smith, 1981, 1983; Hohlfeld et al., 1995, 1996; Farmer et al., 1999; Schmidt et al., 1999; Burhenne et al., 2003). The biosynthesis of p-coumaroylagmatine is shown in Figure 2. The acyltransferases (EC 2.3.1) responsible for the conjugation belongs to two distinct and highly diverse transferase families of which three members (ACT, THT and HCBT) have been characterized at the molecular level (Yang et al., 1997; Farmer et al., 1999; Schmidt et al., 1999; Burhenne et al., 2003; Von-Roepenack-Lahaye et al., 2003). ACT is a 48-kDa monomeric protein with very high specificity for agmatine as the acyl acceptor and preference for p-coumaroyl-CoA over feruloylCoA followed by caffeoyl-CoA as acyl donor for the cloned enzyme (Burhenne et al., 2003). The kinetics of the cloned recombinant enzyme corresponds closely to one of three isoforms purified by Burhenne et al. (2003). ACT is distantly related to HCBT, hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase (monomer of 53 kDa) that has been purified and cloned from Di-

anthus (Yang et al., 1997). The low sequence similarity and differences in substrate specificities strongly support the suggestion made (Burhenne et al., 2003) that ACT and HCBT belong to distinct functional groups. Accumulation of hydroxycinnamoylagmatines has been described in response to abiotic and biotic factors. Both synergistic (Lee et al., 1997; Peipp et al., 1997) and antagonistic microorganisms (Smith and Best, 1978; Von Röpenack et al., 1998), nutrient stress (Smith and Best, 1978) and signalling compounds such as jasmonic acid and abcisic acid (Ogura et al., 2001) induce accumulation of hydroxycinnamoylagmatines in barley. A clone from wheat closely related to barley ACT (Genbank Acc. No. AY228552) was obtained from a cDNA library of stressed wheat leaves (Burhenne et al., 2003) and the fact that HCAAs of agmatine accumulate in winter wheat leaves during cold hardening (Jin and Yoshida, 2000) and in mycorrhized roots (Fester et al., 1999) suggests similar functions for ACT in wheat and barley. Currently the sequence databases contain only ACT-homologous entries from wheat and rice as evidenced by recent BLAST searches (Altschul et al., 1990, GenBank

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Figure 2. Suggested biosynthesis and metabolism of hydroxycinnamoylagmatine. In the monomeric forms are shown R1 = H, p-coumaroyl-CoA and p-coumaroylagmatine and R2 = OMe, feruloyl-CoA and feruloylagmatine. For hordatine A, R1 = R2 = H. Hordatine B, R1 = OMe, R2 = H. Hordatine M, R1 = OMe/H, R2 = D-glucopyranosyl.

release 138.0). A survey of expressed sequence tag (EST) databases show that ACT or highly homologues genes are expressed throughout the vegetative parts of barley, wheat and rice and are responsive to biotic and abiotic stress. In cereals, a hydroxycinnamoyl transferase enzyme with preference for putrescine (PHT) is also found. Hydroxycinnamoylputrescines and diferuloylputrescine has been found in wheat and maize reproductive tissues (Martin-Tanguy et al., 1978, 1979). Hydroxycinnamoylputrescines accumulate in response to methyljasmonate treatment and mycorrhiza infection of barley roots (Lee et al., 1997; Peipp

et al., 1997) and in rice endosperm (Bonneau et al., 1994), and hydroxycinnamoyl hydroxyputrescines accumulates in wheat leaves in response to rust infection (Samborski and Rohringer, 1970). HCAAs of spermidine and spermine have been found in rice endosperm (Bonneau et al., 1994) and in maize anthers and kernels (Martin-Tanguy et al., 1979) suggesting the presence of spermidine/spermine hydroxycinnamoyl transferases (SHT) in these species. To our knowledge the presence of HCAAs of spermine or spermidine in other cereals have not been investigated further. HCAAs of tyramine, putrescine and spermidine accumulating in rice endosperm were

131 suggested to function as a nitrogen pool for germination (Bonneau et al., 1994). Martin-Tanguy et al. (1979) showed that fertile maize anthers accumulate HCAAs of putrescine, spermine and spermidine but not those carring cytoplasmic male sterility and it was later suggested that the accumulation of these HCAAs is functionally linked with reproduction capability (Smith et al., 1983). Like ACT and HCBT, PHT is a monomeric enzyme (Negrel et al., 1992) and it is tempting to speculate that PHT and SHT are strongly related to ACT because of the structural resemblance of the substrates. The avenanthramides (Figure 1) are a highly diversified group of HCAAs found in oat and these soluble HCAAs accumulate in the leaves in response to biotic stress. Mayama et al. (1982) showed that avenanthramides accumulate in the apoplast of oat leaves infected by the rust fungus Puccinia coronata at levels far exceeding the inhibitory concentration for rust spore germination. Their accumulation results from de novo synthesis of avenanthramides through the phenylpropanoid pathway (Ishihara et al., 1999a, b). Elicitors (chitin oligomers) mimicking fungal cell wall breakdown products (Ishihara et al., 1998, 1999b) also induce both the biosynthesis of avenanthramides and increase the activity of hydroxyanthranilate hydroxycinnamoyl transferase (HHT). HHT has been characterized biochemically and the enzyme has similar, but still distinct, characteristics from HCBT (Ishihara et al., 1998, 1999a,b; Matsukawa et al., 2000). It will be very interesting to see if HHT is more highly related to ACT, THT or HCBT. The potato, tomato and tobacco THTs are dimeric and highly related enzymes of ca. 25 kDa per subunit and are responsible for the synthesis of HCAAs of hydroxycinnamoyltyramines, hydroxycinnamoyloctopamines and hydroxycinnamoylnoradrenaline found in these species (Negrel et al., 1993, 1996; Hohlfeld et al., 1995, 1996; Von Roepenack-Lahaye et al., 2003). These THTs vary slightly in their specificity for the acyl acceptor and display broad, but different, substrate specificity towards the acyl donor. The HCAAs of tyramine observed to accumulate in intact organs in these species are most likely the product of several THT isoforms like in tobacco leaves (Fleurence and Negrel, 1989). Within monocots, THT activity has been detected in wheat and barley roots and leaves (Louis and Negrel, 1991), maize leaves (Ishihara et al., 2000) and onion epidermis (Allium cepa) (McLusky et al., 1999). In 3-day old barley seedlings the activity of THT was

approximately 5 times higher in roots compared to the shoot (Louis and Negrel, 1991). THT from maize was partially purified and the native molecular weight determined by gel filtration to ca. 40 kDa (Ishihara et al., 2000). The maize THT has distinct enzymatic properties compared to the THTs known from dicots as it has a broader specificity for the acyl acceptor. In barley and wheat THT activity has also been detected and the specificity resembles (Louis and Negrel, 1991) more the maize than the Solanaceae THT activities (Farmer et al., 1999; Schmidt et al., 1999). We have investigated the presence of Solanaceae THT homologs in monocots by BLAST searches on sequence databases and we found rice, maize and barley cDNAs and ESTs encoding proteins with 34–35% amino acid sequence identity. Rice accumulates HCAAs of tyramine in the endosperm (Bonneau et al., 1994), but it is currently unknown if these compounds are products of the THT homologs. These rice THT homologs might be functional THTs, but the distinctive substrate specificity and the concomitant low similarity to the known THTs makes such an annotation highly speculative. Experiments are needed to conclude if these cDNAs codes for THTs and if the monocot or cereals enzymes are related to the known THTs. Phenylalanine ammonia lyase (PAL) performs the first committed step in the phenylpropanoid pathway as reviewed by Boerjan et al. (2003) and PAL induction may be a prerequisite for biosynthesis of HCAAs (Ishihara et al., 1999a, b). Attack by the powdery mildew fungus Blumeria graminis on wheat, barley and oat induces PAL expression and activity in the leaves (Carver et al., 1992; Clark et al., 1994; Boyd et al., 1995). The fungus attempts to penetrate through the epidermal cell wall and at the sites of attack localised cell wall appositions, termed papillae, are formed (reviewed by Thordal-Christensen et al., 2000). The autofluorescence of the papillae and surrounding area (halo) show that polyphenolic materials are components of the papillae. The materials are not extractable and presumably cross-linked to the cell wall by peroxidases present in the papillae (Scott-Craig et al., 1995; Thordal-Christensen et al., 1997; Von Röpenack et al., 1998). These phenolics are derived from the phenylpropanoid pathway and are involved in the penetration resistance of these cereals since treatment with PAL inhibitor results in increased penetration frequency by the powdery mildew fungus and in reduced autofluorescence from haloes and papillae (Carver et al., 1992, 1994; Zeyen et al., 1995). The nature of the polyphenolics in barley papillae has for many years been a

132 mystery, partly because they are not stained with lignin stains (e.g. Wei et al., 1994). Accumulating evidence now suggest that these polyphenolics are composed of HCAAs polymerised by peroxidases. This evidence and the involvement of specific peroxidases in this process will be discussed below.

Biosynthesis of hordatine In barley, the hydroxycinnamic acid amide pcoumaroylagmatine was described as a co-metabolite of hordatine A that is found in barley coleoptiles (Stoessl, 1966, 1967). The hordatines (Figure 2) are found in four molecular forms, hordatine A is the dimer of two p-coumaroylagmatines, hordatine B is the dimer of p-coumaroylagmatine and feruloylagmatine, while hordatine M is the mixture of the glycoconjugates of hordatine A and B (Stoessl, 1967). In vitro experiments showed that horseradish peroxidase or hydrogenperoxide dependent enzymes (likely to be peroxidases) present in crude extracts of barley seedlings could generate hordatine (Stoessl, 1967; Negrel and Smith, 1984), suggesting that a barley peroxidase was mediating the dimerisation. Very interestingly the hordatines isolated from coleoptiles are optically active (Stoessl, 1967) like the lignans (Davin et al., 1997). This shows that one of the two enantiomers is synthesised in molar excess, or exclusively, in vivo. The in vitro peroxidase-generated hordatines did not show optical rotation (Stoessl, 1967; Negrel and Smith, 1984), suggesting either that peroxidases are not involved in the dimerisation or that a protein, functionally similar to the dirigent protein involved in lignan biosynthesis (Davin et al., 1997), participates in the biosynthesis of hordatines. The subcellular locations of the production and storage of the hordatines are unknown, but the majority of known glycosylated secondary plant metabolites are stored in the vacuole (Boller and Wiemken, 1986) which is also true for certain phytoalexins (SorianoRichards et al., 1998). Hordatine M is likely to accumulate in barley epidermis cells since the metabolites is abundant in 2–4 day old seedlings, i.e., coleoptiles that predominantly consists of epidermal tissue. Hordatine M is probably resistant to further peroxidasemediated polymerisation due to the glycosyl-group present on the coumaric hydroxyl group (Stoessl and Unwin, 1970) as Bernards et al. (1999) demonstrated protection from peroxidase-mediated polymerisation by phenol O-glycosylation of hydroxycinnamic acids.

The protective group may make it possible for hordatine M to be stored in the same compartment as vacuolar peroxidases. If glycosidases are introduced to the storage compartment, e.g. during an encounter with a necrotrophic pathogen or herbivore, the released hordatines could be imagined to polymerise or be converted to toxic radicals intracellularly. In wheat cells attacked by stem rust, lignin formed intracellularly has been suggested to be the mediator of the hypersensitive response (HR) (Beardmore et al., 1983; Moerschbacher et al., 1990) or are at least connected to HR in wheat. Hordatines also accumulate, slowly but substantially, in the barley leaf in response to powdery mildew infection (Smith and Best, 1978). Three days after inoculation with powdery mildew fungus no increase in the content of either hordatines or p-coumaroylagmatine could be detected. However, 13 days after infection, the content of hordatine A and B had increased approximately six-fold and hordatine M had increased two-fold, while the content of p-coumaroylagmatine was unchanged (Smith and Best, 1978). It is not clear if the late accumulation was a result of a general stress in the infected tissue or if the accumulation was the result of reduced flux to the cell wall bound fraction in this compatible interaction (see also later). Studies on peroxidases in barley vegetative tissues have focussed on two isoperoxidases HvPrx7 (Genbank Acc. No. AJ003141) and HvPrx8 (Genbank Acc. No. X58396) (e.g. Kerby and Somerville, 1989; Thordal-Christensen et al., 1992; Kristensen et al., 1999). The accumulation of HvPrx7 in coleoptiles and in stressed barley leaves is correlated with the accumulation of the putative vacuolar hordatine M (Smith and Best, 1978; Kristensen et al., 1999). HvPrx7 is abundant in the coleoptile where hordatine M also accumulates, and the slow accumulation of HvPrx7 in leaves in response to inoculation with powdery mildew parallels the accumulation of hordatine. Likewise, HvPrx7 is present at low levels in non-inoculated lightgrown leaves where the accumulation of hordatine is low. Immunolocalization using monoclonal antibodies shows that HvPrx7 is present in the epidermal vacuole in leaves and coleoptile (Kristensen, Frøkier and Rasmussen, unpublished), while the distinct HvPrx8 (41% amino acid identity) seem to be localised in the cell walls predominantly around mesophyll cells in leaves (Scott-Craig et al., 1995; Gregersen et al., 1997; Kristensen et al., 1997). HvPrx7 was expressed in stressed barley roots and early induction was cor-

133 related with resistance against Dreschlera graminea (Valé et al., 1994). A HvPrx7 homologue is also present in wheat and its expression is induced in the epidermis of sheaths by fungi (Bishop, 2002). The phylogenetic survey by Duroux and Welinder (2003) and EGO-BLAST searches (Eukaryotic gene orthologs database, Version 6.0, released March 30, 2004 (http://www.tigr.org/tdb/tgi/ego/index.shtml)) suggest that orthologs of HvPrx7 are found in wheat, maize and rice. HvPrx7 is expressed in barley in stressed and un-stressed roots, powdery mildew fungus infected leaves, etiolated leaves, coleoptiles, in callus, and the embryo sac early after anthesis. The putative orthologs are expressed in non-stressed and abiotic stressed roots (salt or drought), water-logged roots, fungi infected leaves, and suspension cell culture. Clearly these peroxidases are present in organs where accumulation of HCAAs is also induced. The effects of peroxidase overexpression in a barley epidermis single-cell transient expression system was analysed by Kristensen et al. (2001). Surprisingly, HvPrx7 overexpression led to an increase in the susceptibility of epidermal cells towards the pathogen, suggesting that the peroxidase perturbed the defence responses of the cell (Kristensen et al., 2001). Apparently, the pertubation was connected to the targeting of the peroxidase to the vacuole, as deletion of the carboxy-terminal extension from HvPrx7 did not result in increased fungal susceptibility. This carboxyterminal extension was absent from the native peroxidase purified from barley tissue (Kristensen et al., 1999) and it was shown to function as a vacuolar targeting signal as it is necessary and sufficient for targeting of green fluorescent protein in the endoplasmatic reticulum to the vacuole (Kristensen, Frøkier and Rasmussen, unpublished). This suggests that the effect of HvPrx7 overexpression in transient studies was determined by its vacuolar localization. The time lap between transfection and fungal challenge (24 h) may permit redirection of biosynthetic pathways in the plant cell. A high flux in a pathway involving HvPrx7 may deplete competing pathways for substrates used in the defence response. The significance of this type of transient expression assays is currently difficult to assess, as there is no prior experience to gauge whether the results obtained by these methods can be transferred to the whole-plant level. A possible sequence of the final steps in the biosynthesis of hordatines could be as follows: In a prevacuolar compartment p-coumaroylagmatines are dimerised by HvPrx7 and a dirigent protein. Sub-

sequently hordatines are glycosylated and the biosynthetic vesicle containing hordatine M, HvPrx7 and the glycosyl-transferase are fused to the epidermal vacuole. Transfer into the vacuole could also be mediated by tonoplast located glutathione pumps, as has been demonstrated for transport of the anthocyanin precursor cyanidin 3-glucoside in maize (Alfenito et al., 1998). The phenylcoumaran structure of orantine, a macrobicyclic HCAA formed by cyclization of di-p-coumaroyl spermine (Hedberg et al., 1996; Nezbedová et al., 2001), is the same as that of hordatine (Yoshihara et al., 1983) and is also suggested to be formed by oxidative phenol coupling as the last stereo selective step in its biosynthesis (Nezbedová et al., 2001). Nezbedová et al. (2001) showed that an enzyme with properties similar to microsomal cytochrome P-450 would be a potential candidate for the last step. This may also be the case in the synthesis of hordatine giving an alternative hypothesis to the peroxidase-mediated dimerisation of the hydroxycinnamoylagmatines, as suggested above and indirectly by Stoessl (1967) and Negrel and Smith (1984). Dirigent protein homologues are present in barley (Gang et al., 1999). These proteins are secretory, but since the vacuole is also a secretory compartment it could be that vacuolar dirigent proteins has also evolved.

Peroxidase metabolism of HCAAs Peroxidases are mediating the polymerisation of HCAAs in the phenolic domain of suberin in potato tubers and tobacco leaves (Negrel and Lherminier, 1987; and reviewed by Bernards, 2002). To our knowledge, Bernards et al. (1999) are the first and the only ones to report on the activities of a specific peroxidase towards HCAAs by showing that HCAAs of tyramine are better than the monolignols as substrates for the suberization-associated anionic peroxidase of potato. This can be expected for several of the peroxidases present in plants, but currently the association of other specific peroxidases with the oxidation of HCAAs can only be based on correlative evidence from HCAA accumulation patterns and peroxidase expression profiles. The following will list a number of situations where peroxidases are implicated in the metabolism of apoplastic HCAAs and discuss specific peroxidases implicated in these processes. McLusky et al. (1999) provided the most detailed single study of peroxidase-mediated HCAA metabolism in a monocot so far. In onion epidermal cells

134 attacked by Botrytis allii blue autofluorescent papillae form at the sites of attempted fungal penetration (McLusky et al., 1999). Peroxidases present in the papillae supposedly cross-linked the material to the cell wall, but the identification of the peroxidases was not pursued. The blue fluorescent material was also present as extractable granular deposits in the papilla matrix and it was identified as feruloyltyramine, feruloyl 3 -methoxytyramine and p-coumaroyltyramine (McLusky et al., 1999). The closely related grasses barley and wheat respond very similar to attack by the powdery mildew fungus. In barley cells attacked by the powdery mildew fungus vesicles containing peroxidase activity, hydrogenperoxide, and probably HCAAs, accumulate beneath the site of attempted fungal penetration. The vesicles fuse with the plasma membrane and deliver materials for the papillae formation (Hückelhoven et al., 1999; Collins et al., 2003). The papillae stain with the guanidine-specific Sakaguchi reagent which indicates that the polyphenolic therein contain HCAAs of agmatine (Wei et al., 1994). The presence of hydrogenperoxide and peroxidase enzyme and activity in papillae and barley epidermis inoculated with the fungus also indicates that the secreted HCAAs are cross-linked to the papilla matrix by peroxidases (Wei et al., 1994; Scott-Craig et al., 1995; Thordal-Christensen et al., 1997; Von Röpenack et al., 1998; Kristensen et al., 1999). This clearly suggests that the hydroxycinnamoylagmatine compounds accumulate as polymerisation products in the papillae and cell walls (Wei et al., 1994; Von Röpenack et al., 1998), but it is not known if the monomeric HCAAs accumulates selectively or if hordatines also are crosslinked to the cell wall. The later accumulation of soluble HCAAs (Smith and Best, 1978; Von Röpenack et al., 1998) could relate to reduced flux to peroxidasemediated cell wall cross-linking. Further studies on the barley peroxidases and on the metabolism of hydroxycinnamoylagmatines are necessary to clarify this sequence of events. The hypothesis presented here that hydroxycinnamoylagmatines and derived products are part of the polyphenolic material deposited in barley papillae by the action of peroxidases and is contributing to an inhibition of the pathogenic fungus, are substantiated by experimental evidence in several ways: (1) The biosynthesis of hydroxycinnamoylagmatines is sensitive to PAL inhibitors which have been shown to increase the quantitative susceptibility in barley and reduce the autofluorescence of the papillae halo (Carver et al., 1994; Zeyen et al., 1995); (2) Papil-

lae stain with the guanidine specific Sakaguchi reagent (Wei et al., 1994); (3) In penetration-resistant Mlo barley mutants, the biosynthesis of the hydroxylated p-coumaroylagmatines is enhanced compared to susceptible near isogenic Mlo plants (Von Röpenack et al., 1998); (4) Hydrogenperoxide and peroxidases are present in papillae (Thordal-Christensen et al., 1997; Scott-Craig et al., 1995) and several peroxidase isoforms accumulate in the epidermis in response to inoculation with fungal spores (Kristensen et al., 1999); (5) Proteins and phenolics are not extractable from papillae, even under relatively harsh conditions (Thordal-Christensen et al., 1997), suggesting that covalent linkages attach the phenolics. It appears as if the HCAAs of agmatine in barley leaves has taken over at least a part of the role of HCAAs of tyramine in defence and wound responses in Solanaceae and onion. The apoplastic HvPrx8 peroxidase is the major stress-induced isoenzyme in infected barley leaves (Kerby and Somerville, 1989, 1992; Kristensen et al., 1999) and its messenger RNA accumulate rapidly after inoculation (Thordal-Christensen et al., 1992; ScottCraig et al., 1995) but predominantly in mesophyll cells which are not in direct contact with the pathogen (Gregersen et al., 1997; Kristensen et al., 1999). Single cell overexpression of the stress-induced apoplastic HvPrx8 peroxidase in barley leaves (Kristensen et al., 2001) did not have any effects on the susceptibility towards colonisation by the powdery mildew fungus. This was in contrast to the effect of the homologous peroxidase in wheat (pPOX381/WIR3), which mediated increased resistance using a similar transient expression system (Schweizer et al., 1999). The differences between the effects of the HvPrx8-type peroxidase in wheat and barley may reflect genotypic differences. The timing of events in the cereal powdery mildew interaction apparently is crucial for the outcome of pathogen challenge (Boyd et al., 1995). In the experiments of Kristensen et al. (2001) a time lap of 24 h was allowed between transfection of peroxidase constructs and inoculation with fungal spores, allowing vector driven expression of HvPrx8 for many hours prior to the plant defence induction. This was in contrast to the experiments by Schweizer et al. (1999) where inoculation with fungal spores was done immediately after transfection of wheat leaves. In barley epidermis, HvPrx8 activity is not strongly induced following pathogen attack, while it is highly induced in the mesophyll cells and abundant in the apoplast surrounding them (Gregersen et al., 1997;

135 Kristensen et al., 1999). The HCAAs that seem to accumulate in the periclinal cell walls between epidermis cells and mesophyll cells (Wei et al., 1994) are potential substrates for HvPrx8. The apoplastic isoperoxidases P4.8, P6.3 and P9.6 are more strongly enhanced in the inoculated epidermis (Kristensen et al., 1999) and one, or all, of these peroxidases are likely to perform the cross-linking of the HCAAs to papillae. The presence of these isoforms in the non-inoculated epidermis and the rapid and abundant accumulation of these peroxidases in inoculated epidermis make them prime candidates as enzymes involved in papillae formation. Brown et al. (1998) showed by electron microscopical studies of the defence response of French bean against the bean pathogen Xanthomonas campestris that the bacteria was caught by an encapsulation process taking place in the free space in cell wall corners between bean cells. The material that contained the bacteria was rich in phenolics indicated by increased electron density and peroxidase as revealed by immuno-gold labelling (Brown et al., 1998). The same mechanism is probably also active against vascular fungal pathogens. Keller et al. (1996) showed that excess HCAAs that are not cross-linked in cell wall appositions are secreted and thus are available for such reactions. The abundant stress-induced peroxidases are probably utilizing these substrates in the encapsulation process. Hydroxycinnamoylputrescines and feruloylagmatine accumulated transiently in the soluble fraction of extracts from mycorrhized barley roots (Peipp et al., 1997). The prescence of cell wall bound HCAAs was not investigated, but it is known that in onions feruloyltyramine accumulates in the cell wall fraction of mycorrhized roots and presumably the HCAA is cross-linked to the cell wall by peroxidases (Grandmaison et al., 1993). Opposed to p-coumaroyltyramine the HCAAs of putrescine may not be cross-linked to cell walls by peroxidases since inhibition of H2 O2 production does not change their amounts in the soluble fraction of wound suberizing potato tubers (Razem and Bernards, 2002). In this context it is puzzling that the suberization-associated anionic peroxidase from potato has optimum activity towards feruloylputrescine (Bernards et al., 1999) but is apparently not utilizing this substrate in vivo. Spatial separation of feruloylputrescine from the peroxidase activity may explain this contradictory observation. THT activity has been found in wounded maize leaves, and wheat and barley leaves (Louis and Negrel, 1991; Ishihara et al., 2000). But until three days after germination

the THT activity is higher in roots compared to the leaves in wheat and barley (Louis and Negrel 1991). The absence of soluble HCAAs of tyramine in the roots of wheat and barley despite the relative high activity of THT (up to 33 nkat/mg protein in wheat) (Louis and Negrel, 1991) can be taken as an implication of rapid cross-linking of the HCAAs to the cell wall. This suggests a similar function for hydroxycinnamoyltyramine in the suberization of cereal root tissues as in potatoes. This hypothesis should be supported by chemical analysis of wheat and barley roots and a profiling of the cell-wall peroxidases present in root tissues. Duroux and Welinder (2003) showed that most of the phylogenetic groups of peroxidases in grasses (Poaceae) are structurally distinct from and have evolved independently from the peroxidases in the dicots. This makes it hard to identify peroxidase orthologs between the plant classes showing the need for functional analysis of the grass peroxidases separately. However the monocot peroxidases clearly clustered into 14 speciesindependent similarity groups (Duroux and Welinder, 2003). One of these clusters contains apoplastic wound, stress- and pathogen-induced peroxidases. Some of the members of this potential orthology group are: from barley, HvPrx8 and homologues, wheat (pPOX381, Genbank Acc. No. X56011) (Rebmann et al., 1991), oat (PXC2 and PXC6, Genbank Acc. No. AAC31550 and AAC31551), and rice (POC1, Genbank Acc. No. AF247700), (POX8.1, POX 22.3, POXgX9) (Chittoor et al., 1997), (RICPERX, Genbank Acc. No. BAA03911) (Hiraga et al., 2000). The rice peroxidases in this group are the best studied with respect to their global expression patterns (Chittor et al., 1997; Hiraga et al., 2000). Their results may suggest that there is functional redundancy among these homologs i.e., more than one of the highly similar peroxidases is expressed in an organ in response to a specific stimulus. However these studies are not addressing tissue-specific expression or protein accumulation patterns, and an additional level of complexity can be found here (Gregersen et al., 1997; Kristensen et al., 1999). These peroxidases probably have common functions and more functional analyses of peroxidases and HCAAs would bring new exciting understanding of the stress-physiology of grasses. The functional characterization of monocot peroxidases will be a challenging task. This task will need to embrace expression patterns, accumulation patterns and the enzymology of the peroxidases to be solved. The availability of recombinant transferases (Farmer

136 et al., 1999; Schmidt et al., 1999; Burhenne et al., 2003) to supply the peroxidase substrates for kinetic investigations will hopefully further our understanding of the role of HCAAs as peroxidase substrates.

Bioactivity of HCAA’s and derivatives The term phytoalexin describes anti-microbial, low molecular weight compounds produced in response to pathogen attack, while the term phytoanticipin covers anti-microbial, low molecular weight compounds produced before pathogenic aggression (VanEtten et al., 1994). Phytoalexins and phytoanticipins cover a large variation in biological activity. The flavonoid phytoalexins are in general strongly inhibitory in the 50–100 micromolar range depending on the compound and fungal species in question (VanEtten and Pueppke, 1976). Activation of HCAAs by peroxidase oxidation may result in an even higher potency than that described below. This was originally proposed for monolignols by Ride (1978) but should be tested with appropriate mixtures of pathogens, HCAAs, H2 O2 and peroxidases. Feruloyltyramine in onion (Allium cepa) roots strongly reduced growth of the symbiotic endomycorrhizal fungus Glomus intraradix when added in vitro at twice the concentration (44 µg ml−1 ≈ 150 µM) naturally occuring in 5-week-old mycorrhised roots (Grandmaison et al., 1993). However, solutions of 400 µg ml−1 of feruloyltyramine or pcoumaroyltyramine did not show any antifungal activity when tested against conidia and germ tubes of Botrytis allii or B. cinerea (McLusky et al., 1999). Both compounds were found to be bactericidal when added to liquid cultures of Xanthomonas campestris at a concentration of 250 µM (Newman et al., 2001). The presence of hordatines (1 mmol/kg fresh weight (Smith and Best, 1978) prior to inoculation was correlated to seedling resistance against Cochliobulus sativus and it was shown that the epidermis-rich coleoptile was the major tissue of hordatine accumulation (Ludwig et al., 1960; Stoessl, 1967; Stoessl and Unwin, 1970). Stoessl and Unwin (1970) performed in vitro bioassays to analyse the effects of the hordatines on the spore germination on a number of fungal species and as expected non-pathogens of barley were found to be more sensitive than pathogens. Monilinia fructicola was the most sensitive, with spore germination completely inhibited at concentrations of 7 µM of either hordatine A, B or M. Spores of the barley pathogen Cochliobulus sativus were the most

tolerant, requiring 28 µM hordatine A for 100% inhibition of spore germination. The other tested pathogens were all intermediary in their response to hordatines (Stoessl and Unwin, 1970). The hordatine precursor p-coumaroylagmatine was only slightly inhibitory to germination of M. fructicola even at 220 µM (Stoessl and Unwin, 1970). In barley coleoptiles the concentration of p-coumaroylagmatine is ca 350 µmol/kg fresh weight (Smith and Best, 1978). Feruloylagmatine was shown to inhibit the snowmold fungus (Microdocium nivale) (Jin and Yoshida, 2000), while p-coumaroylagmatine did not inhibit powdery mildew appressoria formation (Von Röpenack et al., 1998). Besides p-coumaroylagmatine, Von Röpenack et al. (1998) also found that the closely related pcoumaroyl hydroxyagmatine (p-CHA) is present in barley epidermis. The accumulation of p-CHA is faster and more abundant in epidermis of barley mloresistant plants after inoculation with powdery mildew conidia. In an in vitro powdery mildew spore germination assay, p-CHA at a concentration of 100 µM was found to reduce the appressoria formation rate by nearly 30%. The p-CHA derivative p-coumaroyl hydroxydehydroagmatine (p-CHDA) was more potent. Using 0.1 µM a 35% reduction in appressoria formation was registered. The p-CHDA is naturally present in barley, and appears to be a non-enzymatic oxidative product of p-CHA, but this is not clear. Von Röpenack et al. (1998) also showed that infiltration of 200 µM p-CHA into leaves reduced the powdery mildew haustoria formation by 20% after application of spores. Application of p-CHDA was only slightly more efficient in this assay. The effect of the antifungal hydroxycinnamic acid amide family of compounds appears to be greater on non-pathogens compared to pathogens on barley. This tendency appears to be general and has previously been characterised for the flavonoid phytoalexin pisatin from pea, and pea pathogens (reviewed by VanEtten and Pueppke (1976) and VanEtten et al. (1989)). From this evidence it is clear that various HCAAs have strong antimicrobial effects and most likely contribute to the constitutive and induced defences of cereals both as soluble and structural compounds.

Prospects for metabolic engineering The picture of stress-related HCAA metabolism in barley may be valid throughout the grass family. How-

137 ever it must be emphazised that species differences clearly exist. Wei et al. (1994) used the guanidinespecific histochemical Sakaguchi reagent to indicate that p-coumaroylagmatines are present in barley and rye papillae, and at low level in papillae of wheat epidermal cells. In contrast, wheat papillae stained with the lignin-indicating phloroglucinol-HCl reagent, while the barley papillae did not react to this stain (Wei et al., 1994). This finding suggests speciesspecific differences in the metabolism of HCAAs and lignols even between these closely related cereals. In wheat, HCAAs may not be produced or sequestered into the cell walls to any great extent compared to lignin. Given that this is the case, metabolic engineering to increase the production of HCAAs in wheat may be feasible. The transfer of a stilbene synthase from peanut to tobacco, increasing the resistance in the transgenic plants (Hain et al., 1993) has already exemplified such interspecific transfer of foreign phytoalexins. The strategy has been predicted to be a powerful way to improve pathogen resistance in plants, because it exposes an adapted pathogen to a compound to which it has had no evolutionary experience (Lamb et al., 1992). The biosynthesis of HCAAs requires the action of two separate pathways, the phenylpropanoid and the polyamine pathway or an amino acid decarboxylation, merged by the action of the ACT, THT or HHT. Overexpression of an oat arginine decarboxylase in transgenic tobacco resulted in a 10–20-fold accumulation of agmatine over the level in wild type without any adverse effects on the plant phenotype and without inducing biosynthesis of HCAAs of putrescine (Burtin and Michael, 1997), although tobacco clearly contains these compounds (Negrel, 1989). However, overexpression of a poppy tyrosine decarboxylase in transgenic Brassica napus led to an increase in THT activity and in cell wall bound tyramine, suggesting that tyramine was converted to HCAAs before peroxidase-mediated polymerisation to the cell wall (Facchini et al., 1999). This may suggest a difference in the readiness for flux in HCAA metabolism between the two pathways, or perhaps differences between the plant species. Overexpression of an anionic peroxidase in transgenic tomato led to increased accumulation of cell-wall bound polyphenolics (Lagrimini et al., 1993), but the nature of these phenolics was not analysed. The studies on single cell overexpression of barley and wheat peroxidases hints that temporal and spatial expression is crucial for the concerted action of the enzymes in the pathways. A strategy to increase the flux of HCAAs into a desired

fraction (soluble or cell wall bound) must take these differences into account. The availability of characterised decarboxylases, transferases and peroxidases provides the tools for exploring these differences and ultimately for metabolic engineering of these traits into crop plants.

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