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-hydroxyanthra- nilic 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 minÀ1. 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 À1 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 À1 À1 procedure of Ishihara et al.