Changes in Wall-Bound Phenolic Acids, Phenylalanine and Tyrosine Ammonia-Lyases, and Peroxidases in Developing Durum Wheat Grains (Triticum turgidum L. Var. Durum) Thierry Re ´gnier and Jean-Jacques Macheix* Laboratory of Biotechnology and Plant Physiology, Case 002, Montpellier II University, 34095 Montpellier Cedex 5, France Some aspects of phenolic metabolism have been followed during the development of durum wheat grain. Bound ferulic (FA) and p-coumaric (PCA) acids have been extracted after alkaline hydrolysis and quantified by high-performance liquid chromatography. They increased to reach a maximum during the hydrical step and then decreased rapidly during grain dehydratation. Changes in phenylalanine ammonia-lyase (PAL) (E.C. 4.1.1.5) and L-tyrosine ammonia-lyase (TAL) (E.C. 4.3.1.5) activities have been monitored all along the development of the grain. TAL activity was maximal 2 days before PAL, at the beginning of the hydrical step. The presence of a maximal peroxidasic activity at the end of the hydrical step should be linked to the decrease of alkaline-resistant bound forms of FA. These results may suggest a possible role of peroxidase in the progressive changes from ester-linked forms of phenolic acids to insoluble derivatives, resistant to alkaline hydrolysis. Keywords: Phenolic acids; ammonia-lyases; peroxidases; durum wheat grain; development INTRODUCTION Durum wheat (Triticum turgidum L. var. Durum) is mainly cultivated to produce semolina and pasta. The yield depends on cultivation conditions and attacks by different pathogens. Among the diseases affecting the grain itself, black point is distinguished by areas more or less black (Miller et al., 1988), which is damaging to agroalimentary use (Dexter and Matsuo, 1982). Black point may derive from fungus attacks (King et al., 1981), but climatic factors and cultivation methods strongly influence the appearance and intensity of the disease (Dexter and Matsuo, 1982). In all cases, the blackening which distinguishes the disease results in damages on a cellular scale, probably due to the oxidative degrada- tion of endogenous phenolic compounds. This is a phenomenon generally involved when browning appears in plants (Nicolas et al., 1993). Ferulic (FA) and p-coumaric (PCA) acids are the main phenolic acids present in the cell wall of monocots and especially of Gramineae (McCallum, 1989; Hartley et al., 1990; Lam et al., 1992a; Rybka et al., 1993). Ferulic acid constitutes more than 90% of the total phenolic acids in wheat flour (Sosulski et al., 1982), and its blue autofluorescence allows its localization in the aleurone cell walls of wheat kernels (Jensen et al., 1982; Pus- sayanawin et al., 1988). In the bran of soft and durum wheat (Pussayanawin and Wetzel, 1987), phenolic acids occur in bound forms as conjugates with sugars or proteins, and the presence of feruloylated arabinoxy- lans has been reported in wheat grain (McDougall, 1993). In fact, the occurrence of FA and PCA in ester linkages with arabinoxylans, pectic polysaccharides, or xyloglucans is now well established, and various specu- lative proposals for covalent cross-links between hy- droxycinnamic acids and cell wall polysaccharides have been made (Bacic et al., 1988; Iiyama et al., 1994). Although cereal seedlings have long been used to study enzymology and regulation of phenolic biosyn- thesis (Creasy, 1987), no extensive results have been obtained up to now in the developing grain. McCallum and Walker (1990) have shown that phenylalanine ammonia-lyase (PAL; E.C. 4.1.1.5) activity was maximal during the early milk stage of soft wheat grain and then declined. In many cases, this enzyme, closely related to the physiological or developmental status of the plant, is coordinated with the presence of other enzymes associated with phenolic biosynthesis (Jones, 1984). Although it has been reported that the purified PAL also exhibited L-tyrosine ammonia-lyase (TAL; E.C. 4.3.1.5) activity (Nari et al., 1972), there are indications that some tissues contained distinct forms of the enzyme with differing PAL/TAL ratios (Hanson and Havir, 1981). Up to date, no data have been reported concern- ing variations of TAL activity during the development of durum wheat grain. The general objective of our work was to determine if the appearance of black point during ontogenesis and ripening of durum wheat grain could be related to endogenous phenolic compounds. In this general frame- work, we report here the first part of a study which compares certain aspects of phenolic metabolism in two durum wheat cultivars, Arbois and Primadur, known by French breeders to be respectively susceptible and moderately resistant to black point. Changes in esteri- fied insoluble FA or PCA as well as in PAL, TAL, and peroxidase (PO) activities have been investigated during grain development and ripening. MATERIALS AND METHODS Plant Material. Two cultivars of durum wheat, Arbois and Primadur, were sowed at the end of December in the plain of Nı ˆmes (France). The sampling started as soon as the grains contained 70% water and was repeated every 2 days during the main phases of development (Figure 1), until the complete ripening of grains. Master spikes were harvested at each sampling, and only the middle one-third part of the spikes was used (Gurnade and Malet, 1981). Fifty grains were dried for 48 h in an oven at 90 °C for estimation of dry weight. Another part of the sample (300 grains/70 spikes) was kept at -30 °C and freeze-dried before being reduced into powder and used for biochemical analysis. The whole grain, including testa, * Author to whom correspondence should be ad- dressed (fax 33 67 54 48 45; e-mail macheix@ phyveg.arpb.univ-montp2.fr). 1727 J. Agric. Food Chem. 1996, 44, 1727-1730 S0021-8561(95)00607-8 CCC: $12.00 © 1996 American Chemical Society + +