Avenanthramide Concentrations and Hydroxycinnamoyl- Coa:Hydroxyanthranilate N-Hydroxycinnamoyltransferase Activities in Developing Oats$

ARTICLE IN PRESS

Journal of Cereal Science 47 (2008) 101–108 www.elsevier.com/locate/jcs

Avenanthramide concentrations and hydroxycinnamoyl- CoA:hydroxyanthranilate N-hydroxycinnamoyltransferase activities in developing oats$

David M. Petersona,b,Ã, Lena H. Dimbergc

aCereal Crops Research Unit, Agricultural Research Service, U.S. Department of Agriculture, 502 Walnut St., Madison, WI 53726, USA bDepartment of Agronomy, University of Wisconsin-Madison, USA cDepartment of Food Science, Swedish University of Agricultural Sciences, P.O. Box 7051, SE-750 07 Uppsala, Sweden

Received 5 October 2006; received in revised form 15 February 2007; accepted 23 February 2007

Abstract

Avenanthramides are unique components of oats (Avena sativa L.) that are described as phytoalexins and that have potential health promoting properties. The objectives of this study were to examine the avenanthramide contents and the activity of the avenanthramide biosynthetic enzyme hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) in spikelets and leaves of developing ﬁeld-grown oats and to examine the presence of avenanthramides in unelicited seedling leaves of oats raised in a growth chamber. Avenanthramides were evident in spikelets of ﬁeld-grown plants within 3–5 days after heading, and they generally increased in concentration throughout maturation. HHT activity was not detected until 21–22 days after heading, but the activity increased with age in most cultivars. In leaves, avenanthramides were evident before heading and generally increased in concentration until about 15 days after heading. At maturity, the concentrations of avenanthramides in spikelets were generally higher than in leaves. Seedling leaves from controlled environments that were not exposed to elicitors had low concentrations of avenanthramides at 7 days after planting, which increased in one cultivar, but not in another, over the next 14 days. These results indicate that unelicited seedling leaves contain avenanthramides, i.e. that avenanthramides are constitutively present in both grains and leaves. r 2007 Elsevier Ltd. All rights reserved.

Keywords: Avenanthramide; Development; Genotype; Oat

1. Introduction

Avenanthramides are novel compounds found in oat Abbreviations: 2c, N-(30,40-dihydroxy)-(E)-cinnamoyl-5-hydroxyanthranilic acid; 2f, N-(40-hydroxy-30-methoxy)-(E)-cinnamoyl-5-hydroxyan- (Avena sativa L.) grain (Collins, 1989; Dimberg et al., 1993) thranilic acid; 2p, N-(40-hydroxy)-(E)-cinnamoyl-5-hydroxyanthranilic that have antioxidant activity in vitro (Bratt et al., 2003; acid; API-ES, Atmospheric pressure ionization-electrospray; DAD, Diode Peterson et al., 2002) and in vivo (Chen et al., 2004; Ji et al., array detector; HHT, hydroxycinnamoyl-CoA:hydroxyanthranilate N- 2003). They also are potential anti-inﬂammatory and anti- hydroxycinnamoyl transferase; HPLC-MS, high performance liquid atherogenic agents (Liu et al., 2004; Nie et al., 2006a, b). chromatography-mass spectrometry; MOPS, 4-morpholinepropanesulfo- nic acid The avenanthramide molecules consist of an anthranilic $Names are necessary to report factually on available data; however, acid linked to a hydroxycinnamic acid with an amide bond the USDA neither guarantees nor warrants the standard of the product, (Collins, 1989). Although about 40 different avenanthra- and the use of the name by USDA implies no approval of the product to mide-like compounds were identiﬁed in oats by thin-layer the exclusion of others that may also be suitable. chromatography (Collins, 1989), three are most abundant ÃCorresponding author. Cereal Crops Research Unit, Agricultural Research Service, U.S. Department of Agriculture, 502 Walnut St., in oat grain (Bratt et al., 2003; Emmons and Peterson, Madison, WI 53726, USA. Tel.: +1 608 262 3355; fax: +1 608 890 0302. 1999). These are termed 2c, 2p and 2f, the 2 indicating 5- E-mail address: [email protected] (D.M. Peterson). hydroxyanthranilic acid (versus 1 for anthranilic acid), and

0733-5210/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2007.02.007 ARTICLE IN PRESS 102 D.M. Peterson, L.H. Dimberg / Journal of Cereal Science 47 (2008) 101–108 c, p or f indicating caffeic, p-coumaric or ferulic acid ice, where green leaf blades were excised and spikelets moieties, respectively (Bratt et al., 2003). Avenanthramides containing developing grain were stripped from the are of interest because of their putative health benefits for panicles. The samples were freeze-dried and stored at humans and animals and their possible role as a defense ambient temperature until analysis. mechanism against fungal attack in the oat plant (Mayama Seeds of cultivars Gem and Vista were planted in flats of et al., 1981, 1982). potting mix, irrigated with a complete nutrient solution, Avenanthramide concentrations in oat grain are influ- and allowed to germinate and grow in a growth chamber, enced by genotype (Bryngelsson et al., 2002; Dimberg programmed at 20 1C with a 16-h photoperiod. At 7, 14, et al., 1993, 1996; Emmons and Peterson, 1999, 2001). The 21, and 28 days after planting, seedling leaves were growing environment also has a profound effect on harvested and freeze dried for subsequent extraction and avenanthramide concentrations (Dimberg et al., 2005; analysis. The experiment had two replications. Emmons and Peterson, 2001; Peterson et al., 2005). For example, oat cultivars grown at Sturgeon Bay, Wisconsin 2.2. Avenanthramide extraction had considerably higher avenanthramide concentrations than the same cultivars grown at six other Wisconsin Freeze-dried samples of field-grown spikelets and leaves locations (Emmons and Peterson, 2001). Also, oats grown and growth chamber grown seedlings were ground in a in arid western regions of the USA and Canada generally Retsch ZM-1 mill (Brinkmann) to pass a 0.5-mm sieve. have lower concentrations than oats grown in the Midwest Duplicate 2.0-g aliquots of the ground samples were (Peterson, unpublished data). The growing conditions in extracted three times with 10 mL 80% ethanol in 25-mL different years are also of importance (Dimberg et al., screw-capped culture tubes at 50 1C for 20 min in a shaking 2005; Peterson et al., 2005). The reasons for the environ- water bath. After centrifugation, the supernatants were mental effect are unknown, but environmental stress may combined and dried under vacuum at 40 1C with a rotary play a role. evaporator. The residues were dissolved in 2 mL methanol Avenanthramides are elicited in oat seedling leaves by and stored at –20 1C until analyzed. spores of incompatible races of crown rust (Puccinia To eliminate chlorophyll from the leaf extracts, 0.5 mL- coronata Corda. var. avenae Fraser & Ledingham) or other aliquots were vacuum dried in a SpeedVac Concentrator elicitors (Mayama et al., 1981, 1982; Miyagawa et al., (ThermoSavant), and the residues were redissolved in 1995). In oat grain, however, their presence is believed to 0.5 mL 40% methanol. The solutions were applied to a be constitutive (Matsukawa et al., 2000). The activity of the 1-mL C18 Bond Elut column (Varian) that had been enzyme, hydroxycinnamoyl-CoA:hydroxyanthranilate N- equilibrated with 40% methanol, and the avenanthramides hydroxycinnamoyl transferase (HHT), which catalyzes the were quantitatively eluted with 5 mL 40% methanol. The final step in avenanthramide biosynthesis, is believed to be eluates were vacuum-dried at 40 1C, and the residues rate limiting for the production of avenanthramides redissolved in 0.5 mL methanol. The solutions were (Ishihara et al., 1999). centrifuged and the supernatants diluted 2-fold with The occurrence of avenanthramides and of HHT activity methanol. There was insufficient chlorophyll in the spikelet has only been reported in mature oat grain and in elicited extracts to interfere with the HPLC analysis, so the spikelet seedling leaves. This report examines avenanthramide extracts were analyzed directly after a 10-fold dilution with contents and HHT activity in developing spikelets of methanol. Two of the three replications of the field field-grown oats from heading to maturity and avenan- experiment and both replications of the growth chamber thramide contents in the green leaves of the plants during experiment were extracted and analysed. the same stages of maturation. In addition, the presence of avenanthramides and HHT activity in unelicited seedling leaves cultivated in a growth chamber is examined. 2.3. HPLC analysis

2. Experimental Avenanthramides from spikelet, leaf and seedling samples were analyzed with HPLC according to the 2.1. Plant culture method described by Jastrebova et al. (2006). The compounds were separated (1-mL injection) on a Eight oat cultivars and one experimental line that have a 4.6 mm 150 mm, 4 mm Genesis C18 column at 23 1C. wide variation in their resistance to crown rust infection The mobile phase was a gradient of 20–60% acetonitrile in were planted on April 18, 2001 on the West Madison 10 mM formic acid over 26 min with a ﬂow rate of Farm, Madison, WI, USA in a randomized complete block 0.35 mL min1. The detector was an Agilent 1100 single design with three replications. Plots consisted of four quadrapole mass spectrometer, equipped with an API-ES 3-meter rows, 0.3 m apart. Plants were sampled by cutting a interface. Fragmentation was at 90 V and 350 1Cinan 1 section of a row at the base at approximately 5-day 8-mL min stream of N2 gas using the positive mode. intervals from panicle emergence until leaves had lost all Quantiﬁcation was performed using synthetic 2c, 2f and 2p green color. Plants were transported to the laboratory on as external standards (Bratt et al., 2003). ARTICLE IN PRESS D.M. Peterson, L.H. Dimberg / Journal of Cereal Science 47 (2008) 101–108 103

2.4. HHT assay diluted aliquot was measured at its peak (333 nm), and the concentration of the p-coumaryl CoA was adjusted to The assay for HHT activity was based on the original an assumed concentration of 2.0 mM, based on its molar 1 1 procedure of Ishihara et al. (1998). Freshly ground dried extinction coefficient e333 ¼ 21000 M cm (Obel and spikelet or leaf samples (2 g) were extracted with 8 mL Scheller, 2000). Aliquots of 260 mL were frozen in 0.1 M Bis Tris buffer, pH 7.2 containing 2 mM dithio- microfuge tubes and stored at 80 1C until needed for threitol in 25-mL polypropylene centrifuge tubes on ice the HHT assay. [Subsequently, a mass spectral analysis of using a Polytron (Brinkmann) homogenizer. The extracts another batch of p-coumaryl CoA that was prepared by the were centrifuged and the supernatants removed. The assay same method as described above indicated the presence of mixture contained 10 mL of 10 mM 5-hydroxyanthranilic three contaminants, all of which absorbed at 333 nm. Thus, acid in DMSO, 30 mL of 0.1 M Bis Tris buffer, pH 7.2, and the actual concentration of p-coumaryl CoA was less than 50 mL of 2.0 mM p-coumaryl-CoA (see below). The the 2.0 mM that was assumed. On the other hand, the HHT reaction was initiated with the addition of 10 mL extract assay had been optimized for p-coumaryl CoA concentra- and was allowed to react for 50 min at 30 1C with gentle tion, and the aliquot size chosen for the assay gave shaking. The reaction was terminated with 20 mL acetic maximal enzyme activity.] acid followed by 380 mL methanol. After centrifugation, The protein concentration in the extracts was measured aliquots (100 mL) of the supernatant were analyzed by using the BioRad protein assay procedure for microtiter HPLC. Concentration of produced 2p was used as a plates. measure of HHT activity, which was expressed as pkat/mg protein (kat ¼ mol 2p formed per second). 3. Results and discussion The p-coumaryl-CoA was prepared using ligase extracted from maize (Zea mays L.) rind using a procedure 3.1. Field experiment that was adapted from Dr. Ronald Hatfield, US Dairy Forage Research Center, Madison, WI (personal commu- This is the first report on avenanthramide concentrations nication). The rind (pith) was taken from field-grown maize in developing oat grain. The concentrations of each plants of 6–8 weeks in age, whose tassels had differentiated avenanthramide (2c, 2f and 2p) in spikelets were signifi- but were not emerged. To prepare the ligase, about 55–60 g cantly different (po0.01) among the genotypes, harvest of maize rind were pulverized in liquid N2 in a steel times, and for the genotype harvest time interaction. blender. The N2 was evaporated, and 50 g of the powdered Vista and Belle had the highest avenanthramide concentra- tissue was extracted for 2 h at room temperature with tions (averaged across harvests), whereas Dane and Jim 100 mL of 200 mM MOPS buffer, pH 7.5, containing 15% had the lowest concentrations. There was a 4-fold glycerol, 0.2 g of MgCl2, 1 mM dithiothreitol, and 800 mL difference between the concentrations in Jim and Vista at of protease inhibitor cocktail (Sigma P9599). The slurry maturity. was filtered through Miracloth and centrifuged at 25,000 g Avenanthramides were evident in the spikelets at 3–5 for 15 min at 20 1C. The supernatant was used directly for days after heading, which was defined as complete panicle CoA ester production, or stored at 80 1C until needed. To emergence (Fig. 1). In seven genotypes, the total avenan- prepare the p-coumaryl-CoA, 14 mg p-coumaric acid was thramide concentration increased throughout develop- dissolved in 5 mL 95% ethanol, and 145 mL of the MOPS/ ment, with each of the three moieties contributing to the glycerol/MgCl2 buffer was quickly added. Co-enzyme A increase (Fig. 1). Initially, 2p was the predominant form, (35 mg) and ATP (300 mg) were added and the mixture was with smaller amounts of 2f present in most genotypes. stirred until they were dissolved. Then 1 mg (7000 U) Avenanthramide 2c was not detected until 21–22 days after catalase was mixed in. The reaction was initiated with the heading, but its concentration increased with time until at addition of 50 mL maize ligase extract. The reaction the final harvest it was the major avenanthramide in all mixture was gently mixed at 30 1C for about 3 h, at which genotypes except Bay. Previously, it was shown that 2c time the reaction had reached a plateau. The CoA ester was comprised the largest fraction in mature grain of Shokan 1 separated from the reaction mixture on a 20-mL ENVI-18 (Matsukawa et al., 2000), and a similar result was reported column, that had been prepared by washing with 3 column previously for three of the genotypes used in this study, volumes of methanol followed by 3 column volumes of Dane, Gem and Belle (Emmons and Peterson, 2001). 50 mM MOPS, pH 7.5. The enzyme reaction mixture was However, higher levels of 2p than 2c have been reported in applied to the column, and the column was washed with 6 some Swedish cultivars (Bryngelsson et al., 2002). column volumes of 50 mM MOPS and 1 column volume of At heading (prior to anthesis), the spikelet consists H2O. The yellow CoA ester was then eluted with 1 column entirely of maternally derived tissues, of which the green volume of methanol. The methanol was evaporated in a modified leaves (empty glumes, lemma and palea) comprise rotary evaporator at 40 1C, leaving a small volume of water most of the mass. After anthesis, the endosperm and solution. The solution was transferred to a vial, the flask embryo grow and increase in mass until at maturity the was rinsed with pH 3.5 H2O, the pH was adjusted to 4.0, groat (caryopsis) comprises about 65–70% of the total and the volume was brought to 5 mL. The absorbance of a grain weight (Doehlert et al., 2001). Because 2c was not ARTICLE IN PRESS 104 D.M. Peterson, L.H. Dimberg / Journal of Cereal Science 47 (2008) 101–108

500 BAY OGLE DANE

400 2p 300 2f

nmol/g 2c 200

100

0 010152025305550 1015202530 0 10152025303540

500 JIM GEM VISTA

400

300 nmol/g 200

100

0 05501015202530 10 15 20 25 30 35 40 0 10 15 20 25 30 5

500 MORAINE BELLE X7571-1

400

300 nmol/g 200

100

0 05 10 15 20 25 30 35 40 0 5 10 15 20 25 30 05 1015202530 Days after heading Days after heading Days after heading

Fig. 1. Concentrations of avenanthramides 2f, 2p and 2c in spikelets of ﬁeld-grown oat cultivars from heading until maturity.

found in green leaves (except in trace amounts, see below), In Gem and Vista the avenanthramide concentration its absence in spikelet samples from early stages of declined slightly at maturity, primarily the result of a development is consistent with the hypothesis that the decrease in 2f and 2p (Fig. 1). This might indicate that 2f early detected avenanthramides (largely 2p and 2f) are and 2p were degraded as the grain matured. Recent papers located in the empty glumes, lemma and palea. The have shown that avenanthramides in oat leaves might be appearance of 2c later in development indicates that metabolized and that the rates of decrease are dependent avenanthramides are being synthesized in the groats. on the species of avenanthramides. The avenanthramide 2f Activity of the biosynthetic enzyme, HHT, has been found decreased more rapidly than 2p, and a radio-labeled form in groats (Bryngelsson et al., 2003a; Matsukawa et al., 2000 of a cyclic dehydrodimer of 2f was found incorporated into see also below). The increase in 2c as a proportion of total cell walls (Okazaki et al., 2004a, b). Avenanthramides were avenanthramides corresponds to the increase in the groat also enzymatically degraded in vitro by slurries of ground mass as a proportion of the total grain mass. This is not to oats (Bryngelsson et al., 2003b), although activity was suggest that 2f and 2p are absent from endosperm/embryo greater towards 2p and 2c than towards 2f. Another tissue, as they have been observed in mature groats by explanation might be that biosynthesis of 2f and 2p was other workers (Bratt et al., 2003; Collins, 1989; Emmons terminated earlier in these genotypes and that the and Peterson, 1999; Matsukawa et al., 2000), and as they concentrations of 2f and 2p decreased because of dilution also increased during seed development (see above). by increasing dry matter. ARTICLE IN PRESS D.M. Peterson, L.H. Dimberg / Journal of Cereal Science 47 (2008) 101–108 105

HHT activity was determined for the spikelet samples It was noted that the highest HHT activity was found in for each genotype at each harvest (Fig. 2). There was no Vista, the cultivar that attained the highest concentration detectable activity until 21–22 days after heading for Bay, of avenanthramides, and the lowest HHT activity was in Vista, Moraine and X7571-1, or until 26–29 days after Ogle, which had the lowest concentration of avenanthra- heading for Ogle, Dane, Jim, Gem, and Belle. The trend for mides at maturity. The HHT activities at maturity of the all genotypes except Ogle was an increasing activity with other cultivars did not differ much (range ¼ 3.3 to maturation, although the activity in Dane peaked at 35 5.1 pkat/mg protein), despite greater differences in their days after heading. At the final harvest, Vista had the avenanthramide concentrations. These results indicate that highest activity, followed in order by Moraine, Bay, Jim, HHT activity may regulate avenanthramide content in X7571-1, Dane, Belle, Gem, and Ogle. Activities ranged developing seeds, but that other factors, such as substrate from 0 to 8 pkat/mg protein. Previously, Matsukawa et al. availabilities (Matsukawa et al., 2000), avenanthramide (2000) reported HHT activities of 3.8 and 5.4 pkat/mg metabolism (dimerization) (Okazaki et al., 2004b), and protein in embryo and endosperm tissue, respectively, of avenanthramide degradation (Bryngelsson et al., 2003b) dry seed of cultivar Shokan 1. The activities reported here may also have significant effects. Isoforms of HHT with are in line with the previous finding. The presence of HHT differing substrate specificities may account for the activity in dry seeds may indicate that during germination, differing composition of avenanthramide moieties with avenanthramides are synthesized de novo. HHT activities development or between tissues (Ishihara et al., 1997; increased slightly in the endosperm and scutellum, and Matsukawa et al., 2000). avenanthramide content of the seed increased 2.5-fold Avenanthramides were present in green leaf samples during 10–48 h of imbibition (Bryngelsson et al., 2003a; taken about 4 days before heading (Fig. 3). Generally, their Matsukawa et al., 2000). concentrations increased until about 15 days after heading.

10 BAY OGLE DANE 8

pkat/mg protein 2

0 20 25 30 35 40 20 25 30 35 40 20 25 30 35 40

10 JIM GEM VISTA 8

pkat/mg protein 2

0 20 25 30 35 40 20 25 30 35 40 20 25 30 35 40

10 MORAINE BELLE X-7571-1 8

pkat/mg protein 2

0 20 25 30 35 40 20 25 30 35 40 20 25 30 35 40 Days after heading Days after heading Days after heading

Fig. 2. Activity of hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) in spikelets of oat cultivars from heading until maturity. ARTICLE IN PRESS 106 D.M. Peterson, L.H. Dimberg / Journal of Cereal Science 47 (2008) 101–108

150 BAY 125 OGLE DANE

100

75 nmol/g 50

0 -505 101520-5 05 10 15 20 -505 1015202530 150

125 JIM GEM VISTA

100

75 nmol/g 50

0 -5 0555 10 15 20 25 30 -5 0 10 15 20 -5 0 10 15 20 150 BELLE X7571- 1 125 MORAINE

100 2p 2f 75 2c nmol/g 50

0 -5 05 10 15 20 25 30 -505 101520-5 05 10 15 20 Days from heading Days from heading Days from heading

Fig. 3. Concentrations of avenanthramides 2f, 2p and 2c in leaves of ﬁeld-grown oat cultivars from just prior to heading until leaves were no longer green.

Avenanthramide 2p was the predominant avenanthramide, These field-grown plants developed in an environment with smaller amounts of 2f. Avenanthramide 2c was that contained naturally occurring spores of the crown rust present only in trace amounts at the last sampling date in fungus and other pathogens. The susceptible cultivars some genotypes. The predominance of avenanthramide 2p developed crown rust as they matured. This experiment in leaves agrees with previous reports (Ishihara et al., was unable to indicate whether the occurrence of avenan- 1998). This is the first report of avenanthramides in leaves thramides in leaf tissues resulted from elicitation, as has of oat plants growing in a natural field environment. been indicated in seedling leaves, or is constitutive. The concentrations of 2p and 2f were significantly different (po0.01) for genotypes and harvests, and the 3.2. Seedling experiments genotype harvest interaction was significant for 2f. Belle and X7571-1, a University of Wisconsin breeding selection, In a preliminary experiment, 35-day-old seedlings of the had the highest total leaf avenanthramide concentrations same nine genotypes as examined in the field experiment (averaged across harvests), and Gem and Moraine had the were grown in a controlled environment chamber and were lowest concentrations. Concentrations in leaves were lower harvested, freeze dried, and analyzed for avenanthramide than in mature grain (except X7571-1), and leaf and concentrations. Avenanthramides 2f and 2p were detected grain concentrations were not correlated (Figs. 1 and 3). in the seedling leaves of all genotypes. The average value Leaf HHT activities were not determined for the field for 2f was 26 nmol/g, with a narrow range among experiment. genotypes of 24–30 nmol/g. For avenanthramide 2p, the ARTICLE IN PRESS D.M. Peterson, L.H. Dimberg / Journal of Cereal Science 47 (2008) 101–108 107 average value was 46 nmol/g, and the range of reported on a fresh weight basis, and the present data on 22–118 nmol/g was broader. Avenanthramide 2c was not dry weight. For example, it appears from Fig. 4 of detected. These concentrations were within the ranges Mayama et al. (1981), that about 12.5 mg/g fresh wt of found for the older leaves of field-grown plants discussed ‘‘avenalumin I’’ were accumulated in the uninduced control above. These plants, grown in controlled environmental leaf. This would convert to about 400 nmol/g dry weight chambers, were not exposed to any elicitors yet substantial (assuming 90% moisture), which is substantially higher quantities of avenanthramides were detected. than the values reported here for cultivar Gem leaves. Because previous research had indicated that in 7-day- Ishihara et al. (1997) report even higher amounts of old seedling leaves, avenanthramides were only present if ‘‘avenanthramide A’’ (which is equal to 2p) in control they were induced by an elicitor (Miyagawa et al., 1995, leaves after 60 h. In other reports, it is difficult to determine 1996), another experiment was conducted to determine the concentrations of avenanthramides in control leaves whether avenanthramides were present in unelicited young because of the scale of the graphs, e.g. Fig. 5 of Yang et al. seedling leaves. In this experiment, the avenanthramides 2f, (2004) and Fig. 1 of Ishihara et al. (1999). Therefore, there 2p and 2c were all present already 7 days after planting in is no substantial conflict between the former and the both Gem and Vista. In Gem, the concentrations of 2p and present data. The presence of avenanthramides at low 2f, but not of 2c, increased to a maximum at 21 days after levels in seedling leaves in the absence of detectable HHT planting, followed by a decline (Table 1). Avenanthramide activity indicates that they may be synthesized in the roots 2p concentrations increased to much higher levels than 2f. and transported into the leaves. Experiments will be In Vista, avenanthramide concentrations did not increase conducted to determine if HHT activity can be detected further with development. HHT activity was not detected in roots. However, it should be noted that the inducible in these seedlings. avenanthramide content and HHT activity previously This finding of substantial content of avenanthramides reported by Japanese workers was in excised seedling in unelicited seedling leaves appears to contradict the leaves. literature reports indicating that no avenanthramides are present in 7-day-old leaves unless elicited by one of a group 4. Conclusions of natural or artificial elicitors (Miyagawa et al., 1995, 1996). The previous work was done primarily with the For the first time data on the accumulation of cultivar Shokan-1, whereas the present data are for avenanthramides during development of oat spikelet and cultivars Gem and Vista. Cultivar differences are sub- leaf tissues in field-grown plants are presented. The typical stantial, as evidenced by the 10-fold greater concentrations pattern among nine cultivars was an increasing accumula- of 2p that were attained in Gem, as compared to Vista. It tion of total avenanthramides in the spikelet tissues, with could be that Gem and Vista do not require elicitation to 2c becoming the predominant moiety as the grain produce avenanthramides, whereas Shokan-1 does. approached maturity. Cultivar differences were substantial. However, the most likely explanation for the differences In leaves, 2p was predominant, and only traces of 2c were may be a matter of emphasis. The Japanese group shows a measured. Concentrations of avenanthramides in leaves substantial induction of avenanthramides in the seedling and spikelets were not correlated among the cultivars. oat leaves by elicitors. However, the unelicited controls Activities of the enzyme, HHT, which catalyzes the final were not always devoid of avenanthramides. It is difficult condensation to form the avenanthramide molecule, were to make exact comparisons, as the Japanese results are detected in the later harvests of the spikelet tissues, but avenanthramides were already present before activities Table 1 could be detected. There were indications that the activity Avenanthramide concentrations of seedling leaves from plants grown in a level might influence the avenanthramide content, but these controlled environment parameters were not significantly correlated. Avenanthramides were present in seedling leaves of two Harvest age (days) Concentration (nmol/g) cultivars at ages 7–28 days, with substantial differences in 2c 2f 2p concentrations between the cultivars and age of tissues. These plants were not induced by any elicitors, indicating Gem 7 4.0270.04 3.5670.21 3.9370.08 that as in the grain, leaf avenanthramides are present 14 3.9870.07 18.1670.40 72.9977.78 constitutively. Levels in the seedling leaves were, however, 21a 6.91 12.28 74.26 substantially lower than that previously reported for the 28a 5.00 10.62 46.63 induced segments of 7-day-old seedling leaves. Vista 7 7.8071.14 5.1471.27 5.3671.31 Acknowledgments 14 4.2170.24 3.9570.56 7.9073.19 21 4.9870.45 4.3871.96 8.3973.72 The authors would like to acknowledge Lauri Herrin, 7 7 7 28 4.32 0.06 3.35 0.57 8.63 4.63 Keith Gilchrist, and Janicka Nilsson for skillful technical aOnly 1 replication. assistance, Jelena Jastrebova and Laurie Marinac for some ARTICLE IN PRESS 108 D.M. 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