A New Class of N-Hydroxycinnamoyltransferases

Agmatine coumaroyltransferase (ACT), which catalyzes the first step in the biosynthesis of antifungal hydroxycinnamoylagmatine derivatives, was purified to apparent homogeneity from 3-day-old etiolated barley (Hordeum vulgare L. ) seedlings. The enzyme was highly specific for agmatine as acyl acceptor and had the highest specificity forp-coumaroyl-CoA among various acyl donors with a specific activity of 29. 7 nanokatal × mg−1 protein. Barley ACT was found to be a single polypeptide chain of 48 kDa with a pI of 5. 20 as determined by isoelectric focusing. The 15 N-terminal amino acid residues were identified by micro-sequencing of the native protein and were used to clone a full-length barley ACT cDNA that predicted a protein of 439 amino acid residues. The sequence was devoid of N-terminal signal peptide, suggesting a cytosolic localization of barley ACT. Recombinant ACT produced and affinity-purified fromEscherichia coli had a specific activity of 189 nanokatal × mg−1 protein, thus confirming the identity of the purified native protein. A partial cDNA sequence for ACT was obtained from wheat that predicted a protein of 353 amino acid residues and had 95% sequence identity to barley ACT. Two motifs in the amino acid sequence reveal that barley ACT represents a new class of N-hydroxycinnamoyltransferases belonging to the transferase superfamily. The barley ACT is unique in producing the precursor of hordatine, a proven antifungal factor that may be directed toward Blumeria graminis. Agmatine coumaroyltransferase (ACT), which catalyzes the first step in the biosynthesis of antifungal hydroxycinnamoylagmatine derivatives, was purified to apparent homogeneity from 3-day-old etiolated barley (Hordeum vulgare L. ) seedlings. The enzyme was highly specific for agmatine as acyl acceptor and had the highest specificity forp-coumaroyl-CoA among various acyl donors with a specific activity of 29. 7 nanokatal × mg−1 protein. Barley ACT was found to be a single polypeptide chain of 48 kDa with a pI of 5. 20 as determined by isoelectric focusing. The 15 N-terminal amino acid residues were identified by micro-sequencing of the native protein and were used to clone a full-length barley ACT cDNA that predicted a protein of 439 amino acid residues. The sequence was devoid of N-terminal signal peptide, suggesting a cytosolic localization of barley ACT. Recombinant ACT produced and affinity-purified fromEscherichia coli had a specific activity of 189 nanokatal × mg−1 protein, thus confirming the identity of the purified native protein. A partial cDNA sequence for ACT was obtained from wheat that predicted a protein of 353 amino acid residues and had 95% sequence identity to barley ACT. Two motifs in the amino acid sequence reveal that barley ACT represents a new class of N-hydroxycinnamoyltransferases belonging to the transferase superfamily. The barley ACT is unique in producing the precursor of hordatine, a proven antifungal factor that may be directed toward Blumeria graminis. Agmatine coumaroyltransferase (ACT, 1The abbreviations used are: ACTagmatine coumaroyltransferaseCVcolumn volumesDEPCdiethyl pyrocarbonateESTexpressed sequence tagHCBTN-hydroxycinnamoyl/benzoyltransferaseRACErapid amplification of cDNA endsTHTtyramineN-hydroxycinnamoyltransferaseMOPS4-morpholinepropanesulfonic acidMES4-morpholineethanesulfonic acidBis-Tris2-bis (2-hydroxyethyl) amino-2- (hydroxymethyl) propane-1, 3-diolTES2-2-hydroxy-1, 1-bis (hydroxymethyl) ethylaminoethanesulfonic acidCAPS3- (cyclohexylamino) propanesulfonic acidcontiggroup of overlapping clones EC 2. 3. 1. 64) was the first amine N-hydroxycinnamoyltransferase characterized from plants (1Bird C. R. Smith T. A. Phytochemistry. 1981; 20: 2345-2346Crossref Scopus (30) Google Scholar, 2Bird C. R. Smith T. A. Phytochemistry. 1983; 22: 2401-2403Crossref Scopus (27) Google Scholar). The enzyme catalyzes the synthesis of hydroxycinnamoylagmatines from agmatine and hydroxycinnamoyl-CoA thiolesters in barley (Hordeum vulgare). Hydroxycinnamoylagmatines are direct precursor of hordatines (Fig. 1), which are antifungal compounds found to be highly abundant in the young barley seedling (3Stoessl A. Can. J. Chem. 1967; 45: 1745-1760Crossref Google Scholar). The hordatines seem to be confined to the genus Hordeum as preformed infection inhibitors (4Smith T. A. Best G. R. Phytochemistry. 1978; 17: 1093-1098Crossref Scopus (65) Google Scholar, 5Stoessl A. Recent Adv. Phytochem. 1970; 3: 143-180Google Scholar), and recent studies indicate that the synthesis of hydroxycinnamoylagmatine derivatives are induced in response to fungal infection of the leaves (6Peipp H. Maier W. Schmidt J. Wray V. Strack D. Phytochemistry. 1997; 44: 581-587Crossref Scopus (96) Google Scholar, 7von Röpenack E. Parr A. Schulze-Lefert P. J. Biol. Chem. 1998; 273: 9013-9022Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Additionally, hydroxycinnamoylagmatine derivatives have been found in wheat (8Jin S. Yoshida M. Biosci. Biotechnol. Biochem. 2000; 64: 1614-1617Crossref PubMed Scopus (27) Google Scholar), and histochemical staining of epidermal leaf tissue confirms that these compounds might accumulate in cereals in general as a response to fungal infection (9Wei Y. D. de Neergaard E. Thordahl-Christensen H. Collinge D. B. Smedegaard-Petersen V. Physiol. Mol. Plant Pathol. 1994; 45: 469-484Crossref Scopus (23) Google Scholar). The function (s) of hydroxycinnamoylagmatine derivatives in plants is not known but may include cell wall fortification, restriction of pathogen ingress, and cytotoxicity to the invading pathogen (7von Röpenack E. Parr A. Schulze-Lefert P. J. Biol. Chem. 1998; 273: 9013-9022Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 9Wei Y. D. de Neergaard E. Thordahl-Christensen H. Collinge D. B. Smedegaard-Petersen V. Physiol. Mol. Plant Pathol. 1994; 45: 469-484Crossref Scopus (23) Google Scholar, 10Stoessl A. Unwin C. H. Can. J. Bot. 1970; 48: 465-470Crossref Google Scholar). Related hydroxycinnamic acid amides are found throughout the plant kingdom, and three plantN-hydroxycinnamoyltransferases have been purified and characterized: tyramine N-hydroxycinnamoyltransferase (THT, EC 2. 3. 1. 110) (11Hohlfeld H. Scheel D. Strack D. Planta. 1996; 199: 166-168Crossref Scopus (32) Google Scholar, 12Negrel J. Javelle F. Eur. J. Biochem. 1997; 247: 1127-1135Crossref PubMed Scopus (44) Google Scholar, 13Yu M. Facchini P. J. Planta. 1999; 209: 33-44Crossref PubMed Scopus (33) Google Scholar), putrescineN-hydroxycinnamoyltransferase (EC 2. 3. 1. 138) (14Negrel J. Paynot M. Javelle F. Plant Physiol. 1992; 98: 1264-1269Crossref PubMed Scopus (32) Google Scholar), and anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT, EC2. 3. 1. 144) (15Yang Q. Reinhard K. Schiltz E. Matern U. Plant Mol. Biol. 1997; 35: 777-789Crossref PubMed Scopus (126) Google Scholar). In general, the role of hydroxycinnamic acid amides remains speculative, but conjugation compounds of tyramine have been repeatedly reported to be present in cell wall fractions from several plant species, and they are believed to produce a phenolic barrier against pathogens by reducing cell wall digestibility (16Grandmaison J. Olah G. M. Van Calsteren M. R. Furlan V. Mycorrhiza. 1993; 3: 155-164Crossref Scopus (72) Google Scholar, 17McLusky S. R. Bennett M. H. Beale M. H. Lewis M. J. Gaskin P. Mansfield J. W. Plant J. 1999; 17: 523-534Crossref Scopus (207) Google Scholar, 18Negrel J. Pollet B. Lapierre C. Phytochemistry. 1996; 43: 1195-1199Crossref Scopus (92) Google Scholar, 19Pearce G. Marchand P. A. Griswold J. Lewis N. G. Ryan C. A. Phytochemistry. 1998; 47: 659-664Crossref Scopus (88) Google Scholar) and forming the phenolic domain of suberin (18Negrel J. Pollet B. Lapierre C. Phytochemistry. 1996; 43: 1195-1199Crossref Scopus (92) Google Scholar, 20Bernards M. A. Can. J. Bot. 2002; 80: 227-240Crossref Scopus (428) Google Scholar, 21Bernards M. A. Lewis N. G. Phytochemistry. 1998; 47: 915-933Crossref PubMed Scopus (177) Google Scholar). However, anthranilic acid amides have been predominantly detected as soluble compounds, and their synthesis can be induced in response to pathogen attack (22Niemann G. J. Phytochemistry. 1993; 34: 319-328Crossref Scopus (25) Google Scholar). The purified transferases are all cytosolic proteins. Putrescine N-hydroxycinnamoyltransferase and HCBT appeared as monomers with native molecular masses of about 44 to 50 kDa, whereas the all appeared as (11Hohlfeld H. Scheel D. Strack D. Planta. 1996; 199: 166-168Crossref Scopus (32) Google Scholar, 12Negrel J. Javelle F. Eur. J. Biochem. 1997; 247: 1127-1135Crossref PubMed Scopus (44) Google Scholar, 13Yu M. Facchini P. J. Planta. 1999; 209: 33-44Crossref PubMed Scopus (33) Google Scholar, J. Paynot M. Javelle F. Plant Physiol. 1992; 98: 1264-1269Crossref PubMed Scopus (32) Google Scholar, Q. Reinhard K. Schiltz E. Matern U. Plant Mol. Biol. 1997; 35: 777-789Crossref PubMed Scopus (126) Google Scholar). The HCBT cDNA (15Yang Q. Reinhard K. Schiltz E. Matern U. Plant Mol. Biol. 1997; 35: 777-789Crossref PubMed Scopus (126) Google Scholar) and from and A. Schmidt J. Scheel D. Strack D. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. P. A. J. Eur. J. Biochem. 1999; PubMed Scopus Google Scholar, K. Schmidt A. Strack D. Plant Physiol. PubMed Scopus Google Scholar) have been The amino acid sequence identity is the characterized but the HCBT of sequence to the M. J. P. A. J. Eur. J. Biochem. 1999; PubMed Scopus Google Scholar). these have a of the of the agmatine coumaroyltransferase sequence N-hydroxycinnamoyl/benzoyltransferase amplification of cDNA acid acid acid acid of overlapping clones of the hydroxycinnamoylagmatine derivatives and their synthesis and in plant a was to molecular of ACT. In enzyme and the of a barley ACT to apparent new of the protein and the of the 15 N-terminal amino ACT were and the identity of the purified protein was by of ACT cDNA coli in ACT protein sequence of the and that ACT is a new class of the belonging to a transferase Barley vulgare was of of and with a of The were in and in a of were in to a with a and a and for protein The hydroxycinnamoyl-CoA thiolesters were A The E. coli the was used to E. coli protein as D. Plant Physiol. 1996; PubMed Scopus Google Scholar). The A were to and The was as H. 1997; 44: Scopus Google Scholar), and 15 of derivatives were purified as H. 1997; 44: Scopus Google Scholar) that was by in all The hydroxycinnamoyl-CoA derivatives were in a to and The molecular of compounds were determined by ACT was × with in were with of acid and with of The was a A of and was to the for the were as 50 and was the by as the of were in from to and to The were used in the enzyme 50 and 50 and 50 as but of 50 as but as but of 50 and 50 used to the of the enzyme were and The 15 ACT and were by the of ACT activity was determined by the C. R. Smith T. A. Phytochemistry. 1983; 22: 2401-2403Crossref Scopus (27) Google Scholar). was in the of were and the were from for the the agmatine were by were The for hydroxycinnamoyl-CoA derivatives J. M. H. PubMed Scopus Google Scholar) were used for ACT activity were with were and was a protein from and used for ACT were The protein was used for of protein throughout the as the barley were with A in a and with a for 15 The was of and × for The was a and as The protein was in a in B. the was in of a step of and of of was a of of were and for ACT activity ACT activity from the were and to The was for and for The was in of 50 a in a in D. The was in of protein was with a in of a of of were and for ACT activity protein fractions from the of were and for against B. The protein was in of 50 a in B. The was in of a in 50 of was a of of were and for ACT activity protein fractions from the of were and to in and The ACT was a in F. was with a of and fractions of were and for ACT activity was in a was 50 50 and The isoelectric of ACT was determined with a in the were in and were to staining the reported in Biochem. 1981; PubMed Scopus Google Scholar) that and was The molecular of native ACT was by the a molecular The molecular of ACT was by by of molecular The ACT purified to apparent homogeneity was with a molecular protein was to and to the and was used for for The protein was with and for and in acid and for several the was several in and to The protein was and in was by a protein Two of of were A J. W. 1997; PubMed Scopus Google Scholar) the 15 N-terminal amino acid residues obtained from purified ACT identified clones Two of were for and for and for were used in with for amplification from cDNA of leaf from barley and wheat in and were used as in the amplification was with of cDNA as the as by the was by of by of for for and for was by a were in and and was the were and were by were used to the of were the of and was by E. coli the was in to a cell of The was to of which was for the of to were and in 15 50 and by with for 15 by and × The was to and by and soluble ACT was purified by as by the the was with and and the protein was with and were were for ACT and was by of ACT was in the of were in enzyme were The affinity-purified enzyme was with various of of ACT. a was with a of the ACT the ACT was for with the of the of amine N-hydroxycinnamoyltransferases in plants was (1Bird C. R. Smith T. A. Phytochemistry. 1981; 20: 2345-2346Crossref Scopus (30) Google Scholar), ACT was purified from young etiolated barley C. R. Smith T. A. Phytochemistry. 1983; 22: 2401-2403Crossref Scopus (27) Google Scholar). hydroxycinnamoylagmatine derivatives have been detected and characterized in barley (6Peipp H. Maier W. Schmidt J. Wray V. Strack D. Phytochemistry. 1997; 44: 581-587Crossref Scopus (96) Google Scholar, 7von Röpenack E. Parr A. Schulze-Lefert P. J. Biol. Chem. 1998; 273: 9013-9022Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, J. Schmidt J. B. M. Phytochemistry. 1997; 44: Scopus Google Scholar, A. H. PubMed Scopus (27) Google Scholar). In 3-day-old barley were as the for the ACT the first plants are (4Smith T. A. Best G. R. Phytochemistry. 1978; 17: 1093-1098Crossref Scopus (65) Google Scholar, C. R. Smith T. A. Bot. Scopus Google Scholar). was used as the hydroxycinnamoyl-CoA to the enzyme activity was of E. coli A D. Plant Physiol. 1996; PubMed Scopus Google Scholar) and purified H. 1997; 44: Scopus Google Scholar). The of the purified and was as reported J. M. H. PubMed Scopus Google Scholar). Additionally, of a of the molecular of was of a of barley were for ACT were in ACT etiolated were as the enzyme to the of the activity be detected in the soluble protein the for ACT which The of ACT activity in the of young barley a of the ACT activity the first step to the was used for of the enzyme was obtained from the with was to ACT from the However, of were to the and a of with ACT were the of ACT activity from etiolated barley was from of etiolated barley The was from of etiolated barley seedlings. in a new was as the step the in ACT activity step was found to be for ACT ACT was and not was used as the The from the step of the used to the enzyme and the The step ACT activity a of and the as from a of 48 kDa, was The ACT activity were to molecular In the protein was but the first ACT from the was not whereas the ACT purified to apparent homogeneity as by The a with a of the in activity in the ACT in was for of of enzyme activity was The molecular of native ACT was determined by a with molecular ACT with apparent molecular of kDa, suggesting that the native barley ACT enzyme is a The isoelectric of the purified native enzyme was determined by isoelectric to be found ACT activity a C. R. Smith T. A. Phytochemistry. 1983; 22: 2401-2403Crossref Scopus (27) Google Scholar). that ACT activity in the to the purified protein not In a in enzyme activity was found 15 of The of a was found to be to ACT activity was for and was and a activity was by in the of and were for and activity A with activity was in with the reported C. R. Smith T. A. Phytochemistry. 1983; 22: 2401-2403Crossref Scopus (27) Google Scholar). activity was in the The purified ACT was not by to of ACT activity by and to not ACT The ACT activity was and 50 and the highest was found the of the apparent of was to be of the ACT purified to apparent homogeneity was to and to a The protein by staining was and by a of derivatives were in the first that the protein be The first amino were from and a was of ACT protein. the protein appeared to be but the first 15 amino were N-terminal found that the with ACT activity three molecular all three ACT apparent molecular masses of kDa, and in the of the specific and nanokatal × these the three ACT were by studies The activity to the single activity of the protein purified to homogeneity by and specificity of native and and from barley the three native ACT that as in The the sequence in which the were and from barley the three native ACT that as in The the sequence in which the were and from barley the three native ACT that as in The the sequence in which the were ACT in E. coli and and agmatine were used as the acyl and acyl specificity is for the of enzyme to × × × × × × and ACT × × specificity is for the of enzyme to × × × × × × and ACT × × specificity is for the of enzyme to × × × × × × and ACT × × specificity is for the of enzyme to × × × × × × and ACT × × and agmatine were used as the acyl and acyl and from barley the three native ACT that as in The the sequence in which the were Recombinant ACT in E. coli and and agmatine were used as the acyl and acyl The specificity is for the of enzyme to × × × × × × and ACT × × in a new The N-terminal sequence from purified barley ACT was found to be highly to predicted N-terminal from barley and wheat clones and Two were to the and used with for cDNA from Blumeria leaves from barley and Two of and were by from the barley of the in and that they in the they partial but that were amplification the to the sequence and the to the cDNA a that the of ACT. clone sequence identity to and sequence identity to in the The full-length cDNA a protein of 439 amino acid residues with a molecular of and isoelectric which is with the determined for native barley ACT. the amino acid sequence of and with the N-terminal sequence of the native barley protein, the ACT N-terminal signal peptide, suggesting a cytosolic in with from A partial sequence was obtained from wheat for amino acid residues in barley ACT a protein of the amino acid sequence in a protein A. E. S. R. S. M. 2002; PubMed Scopus Google Scholar) that ACT to a highly transferase for acyl plant transferases have been the HCBT from (15Yang Q. Reinhard K. Schiltz E. Matern U. Plant Mol. Biol. 1997; 35: 777-789Crossref PubMed Scopus (126) Google Scholar), the (EC B. P. V. Plant J. 1998; PubMed Scopus Google Scholar), and the (EC from H. K. M. M. M. Plant J. 1998; PubMed Google Scholar). The barley ACT a is to the highly found in transferases belonging to is a sequence identity to ACT among plant A the B. P. V. Plant J. 1998; PubMed Scopus Google Scholar), can be found in barley ACT but to be in of the barley the amino acid of the highest obtained in a J. W. 1997; PubMed Scopus Google Scholar), the wheat and the amino acid sequence from a wheat clone are in A of characterized plant the of S. F. P. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A that to from the of and are in that a of the barley ACT and the with highest from the were in a a to be might to from the acid amides of and are in the plant T. A. J. C. R. Adv. 1983; Scholar). The amino acid sequence of and identity to the that ACT with in in A identity and the transferases of the The the barley cDNA in the and ACT with amino a and a the of ACT. The and the were and of soluble ACT was first the of for a of ACT activity was found in the soluble of protein the of protein in the induced and not in the in the to the molecular of for the ACT the and the the to by the of soluble enzyme The ACT was affinity-purified and used for to ACT purified from The three ACT purified from barley had and toward the all the highest specificity for in that the specificity for and was that detected for and The for the acyl acceptor was the native and highly specific for In to tyramine and were as for but activity be are in to found for the purified ACT from barley C. R. Smith T. A. Phytochemistry. 1983; 22: 2401-2403Crossref Scopus (27) Google Scholar). The of the were to the native ACT in with of The specific activity nanokatal × mg−1 of the affinity-purified enzyme that detected for the native ACT purified to apparent homogeneity but with The of activity the ACT was by and The of a of not to the molecular of The of the not was to a of (7von Röpenack E. Parr A. Schulze-Lefert P. J. Biol. Chem. 1998; 273: 9013-9022Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), thus the identity of the purified protein. is known to be a for thus of in the of the ACT about a with of activity be a of but the in ACT activity was ACT was for with of the with suggesting the of residues in the of ACT. ACT catalyzes the first step in the synthesis of hydroxycinnamoylagmatine step the and the in that seem to be in the of barley to fungal the to the biosynthesis of these molecular ACT was purified by and agmatine as the C. R. Smith T. A. Phytochemistry. 1983; 22: 2401-2403Crossref Scopus (27) Google Scholar). In a new was a in ACT to apparent homogeneity and the of three ACT in young barley seedlings. The of the purified enzyme were in with the detected for purified barley ACT C. R. Smith T. A. Phytochemistry. 1983; 22: 2401-2403Crossref Scopus (27) Google Scholar). The molecular native of kDa was as The three characterized ACT all the highest specificity for as the acyl and activity was acyl specificity for the acyl acceptor been reported for the purified which activity in the of anthranilate (15Yang Q. Reinhard K. Schiltz E. Matern U. Plant Mol. Biol. 1997; 35: 777-789Crossref PubMed Scopus (126) Google Scholar). In purified putrescineN-hydroxycinnamoyltransferase and have been reported to be in specificity with to acyl J. Javelle F. Eur. J. Biochem. 1997; 247: 1127-1135Crossref PubMed Scopus (44) Google Scholar, J. Paynot M. Javelle F. Plant Physiol. 1992; 98: 1264-1269Crossref PubMed Scopus (32) Google Scholar, H. W. Scheel D. Strack D. Plant Physiol. PubMed Scopus Google Scholar). the of barley soluble in to the a in was detected in the leaves of a barley C. R. Smith T. A. Phytochemistry. 1983; 22: 2401-2403Crossref Scopus (27) Google Scholar). be to of the purified can the as a ACT activity be detected as acyl C. R. Smith T. A. Phytochemistry. 1983; 22: 2401-2403Crossref Scopus (27) Google Scholar). The ACT had a specific activity that of the native protein. indicate that ACT activity the purified protein is that ACT is The specific activity of purified N-hydroxycinnamoyltransferases was in the nanokatal × mg−1 protein (11Hohlfeld H. Scheel D. Strack D. Planta. 1996; 199: 166-168Crossref Scopus (32) Google Scholar, 12Negrel J. Javelle F. Eur. J. Biochem. 1997; 247: 1127-1135Crossref PubMed Scopus (44) Google Scholar, 13Yu M. Facchini P. J. Planta. 1999; 209: 33-44Crossref PubMed Scopus (33) Google Scholar, J. Paynot M. Javelle F. Plant Physiol. 1992; 98: 1264-1269Crossref PubMed Scopus (32) Google Scholar, Q. Reinhard K. Schiltz E. Matern U. Plant Mol. Biol. 1997; 35: 777-789Crossref PubMed Scopus (126) Google Scholar), which is in to for native and barley ACT. The activity of was by H. W. Scheel D. Strack D. Plant Physiol. PubMed Scopus Google Scholar), and activity was by in the J. Javelle F. Eur. J. Biochem. 1997; 247: 1127-1135Crossref PubMed Scopus (44) Google Scholar). of these ACT However, the of the enzyme the is in the of these of ACT that the protein was A acid sequence of the was but the of the amino acid residues is to that of the were to In a several barley clones from etiolated barley to the N-terminal sequence of barley ACT. of the ACT the N-terminal sequence The of ACT the of several Two partial clones were and highly The sequence identity the full-length and the partial that they The of the clone in E. coli in synthesis of highly ACT protein, suggesting that ACT Barley ACT a new class of belonging to a of with plant transferases have been characterized and seem to be highly The can be found throughout of the that is a of the of the J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). of barley ACT the that a is for the of which is by the of ACT that the of The is in the of the but been to is that in the is by residues in the in ACT the that not to acyl The of the hydroxycinnamoylagmatine derivatives is not and ACT activity been detected in the young of is of that the ACT clone and ACT clones were from from plants with fungal The ACT clones obtained in were from a cDNA from barley leaves with of B. clone was from of leaves barley with of B. clone was from wheat with that ACT is in of barley ACT clones have been from and that ACT is in the barley The hordatines have been known to be antifungal compounds in the young barley and they seem to be to the (4Smith T. A. Best G. R. Phytochemistry. 1978; 17: 1093-1098Crossref Scopus (65) Google Scholar, 5Stoessl A. Recent Adv. Phytochem. 1970; 3: 143-180Google Scholar). The in the synthesis of the hordatines is not known but been that the can be by in the of of a of the A. Scopus Google Scholar). the hydroxycinnamoylagmatines are to in cereals that hydroxycinnamoylagmatine derivatives have been detected in wheat (8Jin S. Yoshida M. Biosci. Biotechnol. Biochem. 2000; 64: 1614-1617Crossref PubMed Scopus (27) Google Scholar) and a clone to barley ACT been from wheat The of the ACT cDNA clones to the of ACT and in response to a pathogen In be to ACT in cereals a as barley and of leaf indicate that compounds are highly abundant in the of several cereals infection (9Wei Y. D. de Neergaard E. Thordahl-Christensen H. Collinge D. B. Smedegaard-Petersen V. Physiol. Mol. Plant Pathol. 1994; 45: 469-484Crossref Scopus (23) Google Scholar). The is a cell wall of produced in the cell wall to the be to the of by the synthesis of hydroxycinnamoylagmatine derivatives barley a Additionally, of the ACT of protein to and J. S. and P. for cDNA C. J. for the E. coli the A S. for in of the H. for the to H. for of the