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Phenolic biosynthesis during grain development in wheat: changes in phenylalanine Ammonia-lyase activity and soluble phenolic content
Journal oj Cereal Science
11 (1990) 35--49
Phenolic Biosynthesis during Grain Development in Wheat: Changes in Phenylalanine Ammonia-lyase Activity and Soluble Phenolic Content JOHN A.
McCALLUM* and JOHN R. L. WALKERt
University of Canterbury Department of Plant and Microbial Science, Christchurch, New Zealand Received 23 May 1989 Changes in phenylalanine ammonia lyase (PAL) activity and the total phenolic content of several wheat cultivars have been monitored during grain development. PAL activity was maximal during the early milk stage of development and then declined. The level of soluble phenolics peaked 20-24 days after the PAL peak, just prior to the appearance of mature grain colour. Red cuItivars showed higher PAL activities during early grain development and lower germination rates during maturation.
Introduction The mature wheat grain contains a complex mixture of compounds, derived biosynthetically from phenylpropanoids, which have yet to be characterised fully. It includes simple phenols such as hydroxybenzoic and hydroxycinnamic acids in a variety of soluble and bound forms 1 ,2, glucosides of methoxy-p-hydroquinone 3 , \ 5-alkylresorcinol derivatives 5 and uncharacterised aminophenolics 6 • In addition to these simple phenols, wheat grain contains a variety of flavonoids, mainly C-glycosyl-flavones derived from apigenin 7 ,8. The phenolic composition of wheat grain is qualitatively similar to that of other cereal seeds and tissues, although it has been found that wheat usually contains lower concentrations of phenolics compared to other cereal grains. The bulk of the soluble phenolics are concentrated in the germ (embryo and scutellum), with lesser amounts in the bran (testa, pericarp and aleurone) and only traces in the flour (endosperm)9. There is now considerable evidence that the phenolic content of wheat and other cereal grains may have a marked effect on the physiological, nutritional and
* Present address: c/o Department of Botany, University of British Columbia, Vancouver BC, Canada V6T 2Bl. t To whom correspondence should be addressed. Abbreviations used: ANOVA = analysis of variance; BAW = n-butanol, acetic acid, water; CIMMMYT = International Centre for Maize and Wheat Improvement, Mexico; CRD = Crop Research Division; DF = degrees of freedom; HPLC = high performance liquid chromatography; MS = mean square; PAL = phenylalanine ammonia-lyase; L-Phe = L-phenylalanine; Pr = probability; PVPP = polyvinylpolypyrrolidone; SS = sum of squares; TAL = tyrosine ammonia-lyase; TFA = trifluoroacetic acid. 0733-5210/90/010035 + 15 $03.00
© 1990 Academic Press Limited 2-2
36
J. A. McCALLUM AND 1. R. L. WALKER
technological properties of the grain and its products. Phenolic co:-npounds may contribute to dormancy, coat pigmentation and fungal resistance of the grain and to the colour, flavour and consistency of cereal products 9 • Although cereal seedlings have long been used to study the enzymology and regulation of phenolic biosynthesis, little is known of these processes in the developing grains. Conchie et al. s characterised two phenol glycosyl-transferases present in wheat germ. Changes in concentrations of flavanols during wheat grain development have been examined by Gordon 1o . The biosynthesis of these compounds has been examined in barley and sorghum grain u -14 • Phenylalanine ammonia-lyase (PAL, BC No.4. 3.1. 5) catalyses the reductive deamination of L-phenylalanine (L-Phe) to form trans-cinnamic acid, the first committed step in the biosynthesis of plant phenylpropanoid compounds including lignin, flavonoids and hydroxycinnamic acids. Activity of this enzyme is closely related to the physiological or developmental status of a plant and concomitant increases in levels of PAL and phenolic compounds have been demonstrated in many plant tissues 15 • In many cases this is also coordinated with the appearance of other enzymes associated with the pathways of phenylpropanoid and flavonoid biosynthesis. PAL from wheat seedlings has been purified and characterised by Nari et al. 16 who observed that the purified enzyme also exhibited L-tyrosine ammonium-lyase (TAL) activity. In this study, changes in PAL activity and phenolic content during wheat grain development have been investigated and compared for several red- and white-grained cultivars. Experimental
Materials L-Phenylalanine (L-Phe), trifluoroacetic acid (TFA), Amberlite XAD-2 resin, polyvinylpolypyrrolidone (PVPP), gallic acid, chlorogenic acid and cinnamic acid were obtained from Sigma Chemical Co., U.S.A. Cinnamic acid was recrystallised from aqueous ethanol before use. XAD-2 resin and PVPP were washed before use to remove UV-absorbing materiaP7. Trichloroacetic acid and FoJin-Ciocalteau reagent were obtained from BDH Ltd. All wheats were grown in irrigated plots at the Crop Research Division (CRD), DSIR, Lincoln. Fungicide application has been reported to affect PAL activity in cereal seedlings 1B ; therefore none was applied to these plants.
Plot design and sampling For a preliminary study in 1986/87 of spring bread wheats the red-grained cultivars Otane and Alcala, and the white-grained cultivars Veery and Cook, were sown in two blocks. Bulked samples were collected from these from 14 days after 50 % ear emergence (4-5 days post-anthesis) and at 5-6 day intervals until harvest ripeness. Seven spring bread wheats were examined in the subsequent 1987/88 trial: cv. Otane and the lines CRSWI and CR5.179 were red-grained while cvs Veery, Cook, Oxley and Egret were whitegrained. The red lines CRSW1 and CR5.179 were from the CRD breeding programme *. Veery is of Mexican origin from the CIMMYT (International Centre for Maize and Wheat Improvement, Mexico) programme and the other white cultivars are Australian. All cultivars were sown in single 1·5 x 3 m blocks. In order to define accurately the population age, ears were tagged on the day when they became free of their flag leaf, this corresponded to 5-6 days before anthesis for all cultivars.
* Although these numbered lines are not, strictly speaking, cultivars they will be referred to as such for the purpose of this paper.
PHENOLICS IN DEVELOPING WHEAT GRAIN
37
All sampling was carried out between 9 am and 11 am. Whole ears were frozen in liquid N 2 and stored at -20°C in double polythene bags. PAL activity was stable for at least two months under these conditions. Samples for analysis of phenolics were freeze-dried and stored at - 20°C prior to analysis. The moisture content of samples (50 seeds) was estimated by weighing before and after drying at 60 °C for 24 h. Seed germination rate was assessed by placing 20 seeds on 7 cm discs of Whatman No.1 filter paper in a Petri dish and moistened with water (2 ml). Sprouting was measured after 5 days incubation in the dark at 16°C.
Extraction and assay of PAL activity Grains were dissected from the outer florets of spikelets in the central part of individual spikes. For each extract 25 seeds were ground, first in a mortar and pestle with liquid N 2 , and then homogenised for 30 s in chilled 0·05 MK phosphate buffer (10 ml; pH 6,6) using an Ultra-Turrax homogeniser (Janke & Kunkel, West Germany). The suspension was filtered through two layers of Miracloth and centrifuged at 4°C for IS min at 13000 g. In order to remove extraneous phenolics, the supernatant was decanted and passed through a column (10 x 30 mm) of XAD2/PVPP (l : 1) previously equilibrated with the extraction buffer. The column was washed with a further 4 ml of buffer and the eluates stored on ice and used as a source of crude enzyme. PAL activity in the crude enzyme extracts was assayed by an adaptation of the methods of Zucker lO • The assay mixture consisted of 0·06 MNa borate buffer, pH 8·8 (3'5 ml) plus crude enzyme (l ml) and the reaction was initiated by the addition of a solution (l ml) of L-phenylalanine (10 mg/ml; final concentration 11 mM). Tubes were incubated at 37°C for I h. The reaction was stopped by the addition of 35 % w/v trichloracetic acid (TFA) (0'5 ml) and tubes were centrifuged for 5 min at 5000 g. The yield of cinnamic acid was estimated by measuring A200 of the supernatant in 10 mm path length acrylic cuvettes. Triplicate assays were performed for each extract, with and without substrate, in order to compensate for increases in absorbance even in the absence of added L-Phe. Protein concentrations were determined by the dye-binding method of Bradford 2o •
Analysis of PAL data Rates were calculated for each extract from the difference of the mean absorbances with and without added L-Phe. The rates were subjected to repeated-measures model analysis of variance (ANOVA) using the SAS routine Proc GLM21. Seedcoat colour and sample age/time were designated as fixed effects factors with cultivar and extract replicates as random effects.
Tissue distribution of enzyme activities The tissue distribution of PAL activity was examined initially in a sample of cv. Otane collected 25 days after ear emergence. Grain was dissected into four fractions: outer pericarp; inner pericarp plus seedcoat (' green layer'); endosperm plus aleurone; embryo. Extractions and assays of PAL activity were performed as described previously except that tissue fractions were ground with an equal weight of PVPP and the XAD-2/PVPP column step was omitted. Embryos were extracted in smaller volumes by crushing with a glass rod and then further dispersing in the extraction buffer using a 50 W ultrasonic microprobe for two 30 s periods, with cooling in an ice/salt bath. PVPP was then added to the homogenate and after 10 min standing on ice it was centrifuged for 15 min at 20000 g at 4 0c. The supernatant was then assayed by the usual methods but in a final volume of 6001-11. For examination of changes in PAL activity in tissues against time, embryos were dissected out (after soaking if necessary) and extracted as described above. The deembryonated seeds were extracted and assayed according to the usual methods.
38
J. A. McCALLUM AND J. R. L. WALKER
PAL and a-amylase activity in germinating grain Samples (25 seeds) of cv. Gtane (mature grain obtained from this trial) were placed on two 7 cm disks of Whatman No. 1 filter paper moistened with distilled water (2 ml) in Petri dishes and incubated in darkness at 25°C. Duplicate dishes were removed at 4, 21,28 and 45 h afterimbibition commenced. After removal the seeds were frozen in liquid N 2 and stored at - 20°C until analysis. Crude enzyme extracts were prepared and assayed for PAL activity as described above and for cramylase activity by the dye-labelled substrate procedure of Barnes and Blakeney 22.
Analysis of soluble phenolics Freeze-dried grains (40-60, depending on dry weight) were powdered in a mortar and pestle. The meal was then homogenised for I min in a mixture (20 ml) of acetone/water (75 % vIv) using an Ultra-Turrax homogenizer. The extractant was filtered off using gentle suction through Ford B3 filter paper and the meal was extracted a further three times. Acetone was removed from the combined filtrates at 30°C in vacuo and the volume adjusted to 20 ml with distilled water. Aliquots of this extract were assayed for soluble phenolics using Folin-Ciocalteau phenol reagent according to the methods of Swain and Hillis 23 with gallic acid as a standard. Extracts for qualititative analysis by two-dimensional paper chromatography (2D-PC) were prepared from the grain of cultivars Otane and Veery collected in the 1986/87 trial at late milk stage (12/1/87) and at maturity (2312/87). Extracts for quantitative analysis were prepared similarly and the aqueous extract obtained adjusted to pH 2'5 with 2 MTFA. This was then applied to a column (10 x IS mm) C-18 silica previously equilibrated with 5 mM TFA. The column was washed with further 5 mM TFA and phenolics were eluted with 80 % (v Iv) methanol/water. The eluate was reduced to dryness at 40°C in vacuo and redissolved in a small volume of80 % methanol for chromatography. Descending paper chromatography (PC) was performed on large (46 x 57 cm) sheets of Whatman 3MM paper. A quantity of extract corresponding to approximately 40-50 grains (or authentic phenolic standards) were applied to each sheet and these were developed successively with BAW (nbutanol: acetic acid: water; 40: 10: 22 v/v Iv) followed by 15 % (v Iv) acetic acid. Dried chromatograms were examined under UV light (360 nm) before and after fuming with ammonia vapour.
Results Significant levels of PAL activity were found in extracts of green, developing seeds during the 'milk' stage of grain development but activity could not be detected conclusively in mature seeds. An examination of the pH-dependance of PAL activity over the range 8·4-9·2 suggested that it had a broad optimum around pH 8,8-9,0. Validation by reversed-phase high performance liquid chromatography (HPLC) confirmed that net increases in absorbance at 290 nm could be accounted for by corresponding increases in the concentration of cinnamic acid in the reaction mixture. An examination of the distribution of activity by differential centrifugation revealed that over 95 % of the activity remained in the supernatant after centrifugation at 13 OOOg. A preliminary study of the 1986/87 bulked samples of four cultivars indicated that PAL activity per seed reached a maximum between 20 and 30 days after ear emergence (15-25 days post-anthesis) during the milk stage of grain development and that all four cultivars exhibited similar activity profiles when sampled in this manner (Fig. 1). ANOVA of these results (for n = 4 sampling dates, n = 3 extracts) yielded the following estimates of major contributions to the total variance: seedcoat colour 12 % ; time 54 % ; cultivar 10 %; cultivar-time interaction 12 %: blocks 3 %. High correlations were
PHENOLICS IN DEVELOPING WHEAT GRAIN
39
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J. A. McCALLUM AND J. R. L. WALKER
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FIGURE 2. Changes in PAL activity and soluble phenolics during development of cv. Otane, 1987/88 trial. (Points represent the mean ± S.E.M. of n = 2 extracts, triplicate assays).
observed between PAL specific activity, PAL activity expressed on a dry weight basis and grain % moisture content (r > 0'75, n = 16, P > 0'001 for all correlations). Analysis of soluble phenolics in cv. Otane and cv. Veery suggested that the maximum content of soluble phenolics per seed occurred 35-40 days after ear emergence and that their concentration in the immature grain was higher in cv. Otane (P < 0·001 by ANOVA). These observations were used to optimise sampling in the subsequent trial in order to facilitate comparison between cultivars and confirm that PAL activities were higher in red-grained cultivars. The profile of PAL activity and content of soluble phenolics observed in cv. Otane in 1987/88 is shown in Fig. 2. Although mean kernel weight at maturity was similar, significantly higher maximum levels of PAL activity and phenolic content per seed were observed than in the previous year, which may be due to the differences in sampling procedure and/or environmental effects.
PHENOLICS IN DEVELOPING WHEAT GRAIN
41
TABLE 1. Distribution of PAL activity in wheat grain (cv. Otane) 25 days after ear emergence Tissue fraction Outer pericarp Inner pericarp + testa Endosperm + aleurone Embryo + scutellum
Activity per seed" (pkatjseed) 2·70 5'19 4·00 0'51
% Total activity
Specific activity (pkat/mg protein)
21·8 41'9
23'0 21'6 9·40 28·3
32-3 4
• Values represent the mean of triplicate determinations.
Distribution of PAL activity
The distribution of PAL activity was examined initially in a sample of cv. Otane collected at 25 days after ear emergence and the results (Table I) suggested that most activity was associated with the testa and pericarp tissues although significant PAL activity was present in the aleurone plus endosperm fraction. Tissue distribution of PAL was examined further by assaying its activity in embryo + scutellum and the de-embryonated grain at several stages of development (Fig. 3). This suggested that PAL activity peaked later in the embryo plus scutellum than in the other tissues, during the late milky stage and that the shoulder observed in PAL activity in the latter part of grain development may be due in part to activity in these tissues. However it is clear that the major peak of PAL activity observed during early grain development was associated with activity in the pericarp plus testa. Comparison between cultivars of PAL activities during early grain development
Comparison of PAL activity per seed in seven cultivars from 15 to 30 days after ear emergence revealed significant differences between red- and white-grained cultivars (Table II and Fig. 4). All red cultivars exhibited a marked peak in activity at 20 days after ear emergence which was absent or smaller in the white cultivars. Integration of activity per seed between 15 and 30 days after ear emergence suggested that cultivars fell into three groups with relatively narrow ranges of activity per seed x time. There was no apparent relationship between mature grain dry weight and integrated PAL activity in the developing grain (r = 0-3, n = 7, P> 0'05). ANOVA of the results (Table III), as PAL activity per seed, revealed highly significant effects due to seedcoat colour and colour x time interaction, which confirmed that red and white cultivars differed significantly both in their overall levels of PAL activity and in the way this changed over time. Paper chromatography of phenolics in developing grain
Initially these extracts were examined by reverse-phase HPLC, but resolving and identifying the large number of UV-absorbing components rendered this technique
J. A. McCALLUM AND J. R. L. WALKER
42
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FIGURE 3. Distribution of PAL activity in the developing grain of cv. Otane. (Points represent mean ± S.E.M. of n = 2 extracts, triplicate assays), . , De-embryonated seed; 0, embryo plus scutellum. TABLE II. Integrated PAL activity 15-30 days after ear emergence in grain from 7 wheat cultivars (1987/88 trial)
Cultivar
Mature seed mean dry weight (mg)
PAL activity integral (pkat. days. seed- 1 )
Otane CRSWI CR5.179 Cook Veery Oxley Egret
47-4 42·9 39·1 42'3 47-8 36'1 39'9
295 294 276 236 203 200 194
The correlation between PAL integral and mature seed dry weight was not significant r = 0'299, P> 0·05.
impractical. Two-dimensional paper chromatography was found to provide good resolution and easier identification of phenolic compounds. Clear qualitative and quantitative differences in phenolic composition were observed between chromatograms of unhydrolysed extracts of immature and mature grain (Fig. 5, Table IV). No qualitative differences could be distinguished between cvs Otane and
PHENOLICS IN DEVELOPING WHEAT GRAIN
43
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FIGURE 4. Comparison of PAL activity in (a) red and (b) white-grained cultivars, 1987/88 trial. (Points represent the mean ±S.E.M. of n = 2 extracts, triplicate assays). (a) 0, Otane; 1::::., CRSW1; D, CR5.179. (b) 0, Veery; 1::::., Cooke; 0, Oxley; 0, Egret.
44
J. A. McCALLUM AND J. R. L. WALKER
TABLE III. Analysis of variance (ANOVA) of PAL activity per seed; 15-30 days after ear emergence in grain from seven wheat cultivars (1987/88 trial) Source Colour Cu1tivar Time Time x colour Cultivar x time
DF
SS
MS
P-value
Pr>P
1 5 3
131-42 16'76 189·39 56·63 47·26
131-42 3·35 63'13 18·88 3·15
39'19
0·0015
3
15
% Variance contribution
-,-
20·04 5·99
0·0001 0'0068
34 3 32 17 12
Variance due to differences between individual extracts of the same tissue was insignificant. DF = degrees of freedom; SS = sum of squares; MS == mean square; Pr = probability.
Veery at either stage. In general it was observed that there was a marked decline in the amounts of hydroxycinnamic acid derivatives present on maturation. By contrast there appeared to be an increase in C-glycosyl-flavones as well as qualitative changes in these compounds. Chlorogenic acid was tentatively identified in chromatograms of immature grain by its R r values and characteristic fluorescence properties under UV in the presence and abscence of NH a fumes; however it could not be detected in chromatograms of mature grain.
PAL and a-amylase activity in germinating grain In a preliminary study for this project the development of PAL and o:-amylase activity was found to correlate closely during the germination of cv. Dtane (r = 0'995, P < 0'001).
Germination rate of maturing grain The germination rate of grain was examined after the mature grain colour had developed and there was no further increase in dry weight. The white-grained cultivars exhibited high germination rates (70-95 %) at harvest ripeness whereas that of the red cultivars was markedly lower (0-12 %). Discussion
PAL This is the first time-course study of PAL in a developing cereal grain. Earlier, Neish 24 reported the presence of tyrosine ammonia-lyase (TAL) activity in barley ears at milk stage but could not detect it in wheatgerm. Our results suggest that PAL activity in immature wheat grain reaches a maximum during the early milk stage of grain development and then declines as grain moisture content declines and the grain enters a linear phase of dry weight increase. Examination of the distribution of PAL activity has suggested that initially most activity is localised
45
PHENOLICS IN DEVELOPING WHEAT GRAIN I
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FIGURE 5. Comparison of soluble phenolics in cv. Otane at milk ripe stage (A) and maturity (B). Two-dimensional paper chromatograms developed with BAW (first dimension) and 15 % (vjv) acetic acid (second dimension).
46
J. A. McCALLUM AND J. R. L. WALKER
TABLE IV. Tentative identities of spots; based on Rr values and fluorescence characteristics in the presence and absence of NH a fumes
Spot no. 1
2 3-4
5
6-7 8 9-12
13 14-16
17 18
Colour (UV)
Colour (UV+NH a fumes)
Tentative identity
Purple Yellow Purple Blue Blue Purple Purple Orange Light blue Purple Blue
Green Yellow Green Green Blue Blue Green Orange Light blue Green Blue
Schaftoside + other flavones Unknown C-Glycosyl-flavone Chlorogenic acid Ferulic acid p-Coumaric acid C-Glycosyl-flavones Unknown Feruloyl esters? C-GlycosyI-flavone Hydroxycinnamate ester?
in the testa-pericarp tissues (and to a lesser extent in the endosperm plus aleurone) and that decline in the total grain activity is primarily due to the decline in activity in these tissues. Wheat germ is a rich source of soluble phenolics 4 •7 whilst aleurone and endosperm cell walls contain bound phenolic acids 8 ; therefore the presence of PAL activity in these tissues is not surprising. However, the relatively high PAL activity present in the testa-pericarp is noteworth in view of the lack of knowledge of the phenolic content of wheat bran. Although the presence in bran of alkylresorcinols 6 and bound phenolic acids 25 is well established there is still a paucity of information concerning the flavonoids, aminophenolics and other phenolic pigments that may be present in the testa-pericarp of developing and mature wheat. A variety of factors could contribute to the activity profile observed in this study. The cereal seed is a composite organ consisting of genetically and morphologically distinct tissues which undergo separate but inter-related programmes of development within the developmental program of the whole seed. Therefore phenolic biosynthesis could be expected to proceed in the different tissues at varying rates, at different times and to yield diverse products. The testa and pericarp layers undergo considerable modification during grain maturation and the decline in PAL activity may reflect the effects of crushing, desiccation and other catabolic activities in these tissues. The plateau observed in the profile during the dough stage of grain development may be due to the increase in activity in the embryo and scutellum observed during this phase. The profile and distribution of PAL activity observed in this study resembles closely those observed by other workers for
PHENOLICS IN DEVELOPING WHEAT GRAIN
47
development and germination. The patterns of PAL activity during grain development may correspond to changes in concentrations of growth regulators, such as gibberellic acid and abscisic acid, since these have been shown to influence (X-amylase activities in the developing grain and phneolic biosynthesis in vitro 15 •28 • The PAL profile also resembles closely those observed in many other plant systems, such as parsley cell suspension cultures 20, following induction of PAL synthesis. A variety of plant materials have yielded fractions that enzymically inactivate PAL and therefore it has been suggested that this common pattern of activity may arise from interaction between the competing processes of PAL synthesis and inactivation 30 • The existence of such PAL inactivating systems in developing cereal seeds seems likely since they have been reported from barley 31 and rice 32 seedlings. Studies of bean cell cuItures 33 ,34 have suggested that trans-cinnamate may playa central role in coordinating such patterns of expression by both reducing transcription of PAL subunit genes and inducing synthesis of proteinaceous PAL inactivators. Phenolics
The content of soluble phenolics was maximal just prior to the appearance of mature grain colour and when grain volume was also at a maximum; about 20-25 days after the peak in PAL activity. Such a lag period between PAL activity and phenolic content has been observed in a variety of plant systems 14 • Declines in the concentrations of flavanolso and ascorbic acid 35 during maturation have been noted during previous studies of wheat grain development. A similar pattern of changes in total phenolic content was also observed in study of developing sorghum grain 30 • Such declines in concentrations of oxidizable substances are probably a result of contact with oxidative enzymes during the breakdown in cellular structure in the seedcoat tissues that accompanies grain maturation. The fact that development of mature grain colour was associated with a marked decline in phenolics supports the idea that seedcoat pigments may be the products of their oxidation. The auxin content in developing wheat grain has been found to exhibit a similar peak just prior to termination of dry weight gain and the development of mature grain colourBo • Peroxidase activity also peaks around this time 37 and may be associated with metabolism of phenolics in the testa-pericarp. On the basis of studies on wheat coleoptiles, Machakova et al. 38 have suggested that degradation of phenolics by peroxidase in vivo may be regulated by auxin levels. Comparison of cultivars
Comparison of several cultivars during early grain development has suggested that there are marked differences in the content of PAL activity in red and white cultivars at this stage. Because most PAL activity in the red cultivars was associated with the testa and pericarp layers at this stage of development it is likely that these differences are primarily the result of differences in activity in the external tissues of the grain and that the higher activity in the red cultivars may be associated with synthesis of pigment precursors. A comparison of the germination rate of these same cultivars during grain maturation
48
J. A. McCALLUM AND J. R. L. WALKER
indicated that the red cultivars exhibited significantly lower germination rate than the white cultivars. These findings support the hypothesis, expressed in various forms by earlier workers, that phenolic compounds may contribute both to the pigmentation and germination rate of wheat grain. Higher levels of PAL activity in red-grained cultivars could influence germination rate not only by leading to higher levels of pigments and their precursors, but also giving rise to higher levels of other phenolics. The authors wish to thank the following persons and organisations for their assistance during this work: the staff of the Wheat Research Institute and the Crop Research Institute (DSIR), the University of Canterbury and the University Grants Committee for the provision of research facilities.
References 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. II. 12. 13. 14. 15. 16. 17, 18. 19. 20. 21. 22. 23. 24. 25, 26. 27. 28. 29. 30. 31. 32. 33.
EI-Basyouni and Towers, G. H. N. Can. J. Biochem. 42 (1964) 203-210. Krygier, K., Sosulski, F, and Hogge, L. J. Agric. Food Chem. 30 (1982) 330-334. Conchie, J., Moreni, A. and Cardieni, E. C. Arch. Biochem. Biophys94 (1961) 342-343. Bouvier, E. and Horvath, C. Acta Biochim. Biophys. Acad. Sci, Hung. 22 (1987) 215-228, Verdeai, K. and Lorenz, K. Cereal Chem. 54 (1977) 475-483. Fulcher, R. G., O'Brien, T. P. and Wong, S. 1. Cereal Chem. 58 (1981) 130-135. Wagner, H., Obermeier, G., Chari, V. M. and Galle, K, J, Nat. Prod. 43 (1980) 583-587. Feng, Y., McDonald, C. E. and Vick, B. A. Cereal Chem. 65 (1989) 452-456. Collins, F. W. Oat Phenolics: Structure, occurrence, and function, in 'Oats: Chemistry and Technology' (F. H. Webster, ed.) AACC. St Paul, MN (1987) pp 227-295. Gordon, 1. L. Aust. J. Agric. Res. 30 (1979) 387-402. Glennie, C. W., Kaluza, W. Z. and van Niekerk, P. J. J. Agric. Food, Chem. 29 (1981) 965-968. Glennie, C, W. J. Agric. Food. Chem. 29 (1981) 33-36. Kristiansen, K. N. Carlsberg Res. Commun. 49 (1984) 503-524. Kristiansen, K. N. Carlsberg Res. Commun. 51 (1986) 51-60. Jones, D. H. Phytochemistry 23 (1984) 1349-1359. Nari, J., Mouttet, Ch., Pinna, M. H. and Richard, J. FEBS Letters 23 (1972) 220-223. Loomis, W. D., Lile, J. D., Sandstrom, R. P. and Burbott, A. J. Phytochemistry 18 (1979) 1049-1054. Swinburne, T. R. Physiol. Plant Pathol. 5 (1975) 81-88. Zucker, M. Plant Physiol. 40 (1965) 779-784. Bradford, M. M. Anal. Biochem. 72 (1976) 248-254. 'SAS/Stat Guide For Personal Computers' [Version 6], SAS Institute Inc. Cary, NC (1987). Barnes, W. C. and Blakeney, A. B. Starke 26 (1974) 193-197. Swain, T, and Hillis, W. E. J. Sci. Food Agric. 10 (1959) 63-68. Neish, A. C. Phytochemistry 1 (1961) 1-24. Pussayanawin, V. and Wetzel, D. L. J. Chromatogr. 391 (1987) 243-255. Marchylo, B. A., Lacroix, L. J. and Kruger, 1. E. Can. J. Plant Sci, 60 (1980) 433-443. Kruger, J. E. Cereal Chem. 50 (1973) 122-131. Muthukrishnan, S., Chandra, G. R, Adv. Cereal Sci. Technol. 9 (1988) 129-160. Betz. B., Schafer, E. and Hahlbrock, K. Arch. Biochem. Biophys. 190 (1978) 126-135. Creasy, L. L. Physiol. Plant. 71 (1987) 389-392. Podstolski, A. Physiol. Plant. 58 (1983) 107-113. Ying, C. Y., Ouyang, G. C., Wo, S. G. and Zue, Y. L. Acta Phytophysiol. Sin. 13 (1987) 100-106. BolweII, G, P., Manandad, M., Millar, D. 1., Edwards, K. J., Schuch, W., Dixon, R. A. Phytochemistry 27 (1988) 2109-2117,
PHENOLICS IN DEVELOPING WHEAT GRAIN 34. 35. 36. 37. 38.
Belderok, B. Proc. Int. Seed Test. Assoc. 27 (1961) 697-760. Glennie, C. W. J. Agric. Food Chem. 29 (1981) 33-36. Wheeler, A. W. Ann. Appl. Bioi. 72 (1972) 327-334. Kruger, J. E. and LaBerge, D. E. Cereal Chern. 51 (1974) 345-354. Machackova, 1., Ganceva. K. and Zmrhal. Z. Phytochemistry 14 (1975) 1251-1254.
49
11 (1990) 35--49
Phenolic Biosynthesis during Grain Development in Wheat: Changes in Phenylalanine Ammonia-lyase Activity and Soluble Phenolic Content JOHN A.
McCALLUM* and JOHN R. L. WALKERt
University of Canterbury Department of Plant and Microbial Science, Christchurch, New Zealand Received 23 May 1989 Changes in phenylalanine ammonia lyase (PAL) activity and the total phenolic content of several wheat cultivars have been monitored during grain development. PAL activity was maximal during the early milk stage of development and then declined. The level of soluble phenolics peaked 20-24 days after the PAL peak, just prior to the appearance of mature grain colour. Red cuItivars showed higher PAL activities during early grain development and lower germination rates during maturation.
Introduction The mature wheat grain contains a complex mixture of compounds, derived biosynthetically from phenylpropanoids, which have yet to be characterised fully. It includes simple phenols such as hydroxybenzoic and hydroxycinnamic acids in a variety of soluble and bound forms 1 ,2, glucosides of methoxy-p-hydroquinone 3 , \ 5-alkylresorcinol derivatives 5 and uncharacterised aminophenolics 6 • In addition to these simple phenols, wheat grain contains a variety of flavonoids, mainly C-glycosyl-flavones derived from apigenin 7 ,8. The phenolic composition of wheat grain is qualitatively similar to that of other cereal seeds and tissues, although it has been found that wheat usually contains lower concentrations of phenolics compared to other cereal grains. The bulk of the soluble phenolics are concentrated in the germ (embryo and scutellum), with lesser amounts in the bran (testa, pericarp and aleurone) and only traces in the flour (endosperm)9. There is now considerable evidence that the phenolic content of wheat and other cereal grains may have a marked effect on the physiological, nutritional and
* Present address: c/o Department of Botany, University of British Columbia, Vancouver BC, Canada V6T 2Bl. t To whom correspondence should be addressed. Abbreviations used: ANOVA = analysis of variance; BAW = n-butanol, acetic acid, water; CIMMMYT = International Centre for Maize and Wheat Improvement, Mexico; CRD = Crop Research Division; DF = degrees of freedom; HPLC = high performance liquid chromatography; MS = mean square; PAL = phenylalanine ammonia-lyase; L-Phe = L-phenylalanine; Pr = probability; PVPP = polyvinylpolypyrrolidone; SS = sum of squares; TAL = tyrosine ammonia-lyase; TFA = trifluoroacetic acid. 0733-5210/90/010035 + 15 $03.00
© 1990 Academic Press Limited 2-2
36
J. A. McCALLUM AND 1. R. L. WALKER
technological properties of the grain and its products. Phenolic co:-npounds may contribute to dormancy, coat pigmentation and fungal resistance of the grain and to the colour, flavour and consistency of cereal products 9 • Although cereal seedlings have long been used to study the enzymology and regulation of phenolic biosynthesis, little is known of these processes in the developing grains. Conchie et al. s characterised two phenol glycosyl-transferases present in wheat germ. Changes in concentrations of flavanols during wheat grain development have been examined by Gordon 1o . The biosynthesis of these compounds has been examined in barley and sorghum grain u -14 • Phenylalanine ammonia-lyase (PAL, BC No.4. 3.1. 5) catalyses the reductive deamination of L-phenylalanine (L-Phe) to form trans-cinnamic acid, the first committed step in the biosynthesis of plant phenylpropanoid compounds including lignin, flavonoids and hydroxycinnamic acids. Activity of this enzyme is closely related to the physiological or developmental status of a plant and concomitant increases in levels of PAL and phenolic compounds have been demonstrated in many plant tissues 15 • In many cases this is also coordinated with the appearance of other enzymes associated with the pathways of phenylpropanoid and flavonoid biosynthesis. PAL from wheat seedlings has been purified and characterised by Nari et al. 16 who observed that the purified enzyme also exhibited L-tyrosine ammonium-lyase (TAL) activity. In this study, changes in PAL activity and phenolic content during wheat grain development have been investigated and compared for several red- and white-grained cultivars. Experimental
Materials L-Phenylalanine (L-Phe), trifluoroacetic acid (TFA), Amberlite XAD-2 resin, polyvinylpolypyrrolidone (PVPP), gallic acid, chlorogenic acid and cinnamic acid were obtained from Sigma Chemical Co., U.S.A. Cinnamic acid was recrystallised from aqueous ethanol before use. XAD-2 resin and PVPP were washed before use to remove UV-absorbing materiaP7. Trichloroacetic acid and FoJin-Ciocalteau reagent were obtained from BDH Ltd. All wheats were grown in irrigated plots at the Crop Research Division (CRD), DSIR, Lincoln. Fungicide application has been reported to affect PAL activity in cereal seedlings 1B ; therefore none was applied to these plants.
Plot design and sampling For a preliminary study in 1986/87 of spring bread wheats the red-grained cultivars Otane and Alcala, and the white-grained cultivars Veery and Cook, were sown in two blocks. Bulked samples were collected from these from 14 days after 50 % ear emergence (4-5 days post-anthesis) and at 5-6 day intervals until harvest ripeness. Seven spring bread wheats were examined in the subsequent 1987/88 trial: cv. Otane and the lines CRSWI and CR5.179 were red-grained while cvs Veery, Cook, Oxley and Egret were whitegrained. The red lines CRSW1 and CR5.179 were from the CRD breeding programme *. Veery is of Mexican origin from the CIMMYT (International Centre for Maize and Wheat Improvement, Mexico) programme and the other white cultivars are Australian. All cultivars were sown in single 1·5 x 3 m blocks. In order to define accurately the population age, ears were tagged on the day when they became free of their flag leaf, this corresponded to 5-6 days before anthesis for all cultivars.
* Although these numbered lines are not, strictly speaking, cultivars they will be referred to as such for the purpose of this paper.
PHENOLICS IN DEVELOPING WHEAT GRAIN
37
All sampling was carried out between 9 am and 11 am. Whole ears were frozen in liquid N 2 and stored at -20°C in double polythene bags. PAL activity was stable for at least two months under these conditions. Samples for analysis of phenolics were freeze-dried and stored at - 20°C prior to analysis. The moisture content of samples (50 seeds) was estimated by weighing before and after drying at 60 °C for 24 h. Seed germination rate was assessed by placing 20 seeds on 7 cm discs of Whatman No.1 filter paper in a Petri dish and moistened with water (2 ml). Sprouting was measured after 5 days incubation in the dark at 16°C.
Extraction and assay of PAL activity Grains were dissected from the outer florets of spikelets in the central part of individual spikes. For each extract 25 seeds were ground, first in a mortar and pestle with liquid N 2 , and then homogenised for 30 s in chilled 0·05 MK phosphate buffer (10 ml; pH 6,6) using an Ultra-Turrax homogeniser (Janke & Kunkel, West Germany). The suspension was filtered through two layers of Miracloth and centrifuged at 4°C for IS min at 13000 g. In order to remove extraneous phenolics, the supernatant was decanted and passed through a column (10 x 30 mm) of XAD2/PVPP (l : 1) previously equilibrated with the extraction buffer. The column was washed with a further 4 ml of buffer and the eluates stored on ice and used as a source of crude enzyme. PAL activity in the crude enzyme extracts was assayed by an adaptation of the methods of Zucker lO • The assay mixture consisted of 0·06 MNa borate buffer, pH 8·8 (3'5 ml) plus crude enzyme (l ml) and the reaction was initiated by the addition of a solution (l ml) of L-phenylalanine (10 mg/ml; final concentration 11 mM). Tubes were incubated at 37°C for I h. The reaction was stopped by the addition of 35 % w/v trichloracetic acid (TFA) (0'5 ml) and tubes were centrifuged for 5 min at 5000 g. The yield of cinnamic acid was estimated by measuring A200 of the supernatant in 10 mm path length acrylic cuvettes. Triplicate assays were performed for each extract, with and without substrate, in order to compensate for increases in absorbance even in the absence of added L-Phe. Protein concentrations were determined by the dye-binding method of Bradford 2o •
Analysis of PAL data Rates were calculated for each extract from the difference of the mean absorbances with and without added L-Phe. The rates were subjected to repeated-measures model analysis of variance (ANOVA) using the SAS routine Proc GLM21. Seedcoat colour and sample age/time were designated as fixed effects factors with cultivar and extract replicates as random effects.
Tissue distribution of enzyme activities The tissue distribution of PAL activity was examined initially in a sample of cv. Otane collected 25 days after ear emergence. Grain was dissected into four fractions: outer pericarp; inner pericarp plus seedcoat (' green layer'); endosperm plus aleurone; embryo. Extractions and assays of PAL activity were performed as described previously except that tissue fractions were ground with an equal weight of PVPP and the XAD-2/PVPP column step was omitted. Embryos were extracted in smaller volumes by crushing with a glass rod and then further dispersing in the extraction buffer using a 50 W ultrasonic microprobe for two 30 s periods, with cooling in an ice/salt bath. PVPP was then added to the homogenate and after 10 min standing on ice it was centrifuged for 15 min at 20000 g at 4 0c. The supernatant was then assayed by the usual methods but in a final volume of 6001-11. For examination of changes in PAL activity in tissues against time, embryos were dissected out (after soaking if necessary) and extracted as described above. The deembryonated seeds were extracted and assayed according to the usual methods.
38
J. A. McCALLUM AND J. R. L. WALKER
PAL and a-amylase activity in germinating grain Samples (25 seeds) of cv. Gtane (mature grain obtained from this trial) were placed on two 7 cm disks of Whatman No. 1 filter paper moistened with distilled water (2 ml) in Petri dishes and incubated in darkness at 25°C. Duplicate dishes were removed at 4, 21,28 and 45 h afterimbibition commenced. After removal the seeds were frozen in liquid N 2 and stored at - 20°C until analysis. Crude enzyme extracts were prepared and assayed for PAL activity as described above and for cramylase activity by the dye-labelled substrate procedure of Barnes and Blakeney 22.
Analysis of soluble phenolics Freeze-dried grains (40-60, depending on dry weight) were powdered in a mortar and pestle. The meal was then homogenised for I min in a mixture (20 ml) of acetone/water (75 % vIv) using an Ultra-Turrax homogenizer. The extractant was filtered off using gentle suction through Ford B3 filter paper and the meal was extracted a further three times. Acetone was removed from the combined filtrates at 30°C in vacuo and the volume adjusted to 20 ml with distilled water. Aliquots of this extract were assayed for soluble phenolics using Folin-Ciocalteau phenol reagent according to the methods of Swain and Hillis 23 with gallic acid as a standard. Extracts for qualititative analysis by two-dimensional paper chromatography (2D-PC) were prepared from the grain of cultivars Otane and Veery collected in the 1986/87 trial at late milk stage (12/1/87) and at maturity (2312/87). Extracts for quantitative analysis were prepared similarly and the aqueous extract obtained adjusted to pH 2'5 with 2 MTFA. This was then applied to a column (10 x IS mm) C-18 silica previously equilibrated with 5 mM TFA. The column was washed with further 5 mM TFA and phenolics were eluted with 80 % (v Iv) methanol/water. The eluate was reduced to dryness at 40°C in vacuo and redissolved in a small volume of80 % methanol for chromatography. Descending paper chromatography (PC) was performed on large (46 x 57 cm) sheets of Whatman 3MM paper. A quantity of extract corresponding to approximately 40-50 grains (or authentic phenolic standards) were applied to each sheet and these were developed successively with BAW (nbutanol: acetic acid: water; 40: 10: 22 v/v Iv) followed by 15 % (v Iv) acetic acid. Dried chromatograms were examined under UV light (360 nm) before and after fuming with ammonia vapour.
Results Significant levels of PAL activity were found in extracts of green, developing seeds during the 'milk' stage of grain development but activity could not be detected conclusively in mature seeds. An examination of the pH-dependance of PAL activity over the range 8·4-9·2 suggested that it had a broad optimum around pH 8,8-9,0. Validation by reversed-phase high performance liquid chromatography (HPLC) confirmed that net increases in absorbance at 290 nm could be accounted for by corresponding increases in the concentration of cinnamic acid in the reaction mixture. An examination of the distribution of activity by differential centrifugation revealed that over 95 % of the activity remained in the supernatant after centrifugation at 13 OOOg. A preliminary study of the 1986/87 bulked samples of four cultivars indicated that PAL activity per seed reached a maximum between 20 and 30 days after ear emergence (15-25 days post-anthesis) during the milk stage of grain development and that all four cultivars exhibited similar activity profiles when sampled in this manner (Fig. 1). ANOVA of these results (for n = 4 sampling dates, n = 3 extracts) yielded the following estimates of major contributions to the total variance: seedcoat colour 12 % ; time 54 % ; cultivar 10 %; cultivar-time interaction 12 %: blocks 3 %. High correlations were
PHENOLICS IN DEVELOPING WHEAT GRAIN
39
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J. A. McCALLUM AND J. R. L. WALKER
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FIGURE 2. Changes in PAL activity and soluble phenolics during development of cv. Otane, 1987/88 trial. (Points represent the mean ± S.E.M. of n = 2 extracts, triplicate assays).
observed between PAL specific activity, PAL activity expressed on a dry weight basis and grain % moisture content (r > 0'75, n = 16, P > 0'001 for all correlations). Analysis of soluble phenolics in cv. Otane and cv. Veery suggested that the maximum content of soluble phenolics per seed occurred 35-40 days after ear emergence and that their concentration in the immature grain was higher in cv. Otane (P < 0·001 by ANOVA). These observations were used to optimise sampling in the subsequent trial in order to facilitate comparison between cultivars and confirm that PAL activities were higher in red-grained cultivars. The profile of PAL activity and content of soluble phenolics observed in cv. Otane in 1987/88 is shown in Fig. 2. Although mean kernel weight at maturity was similar, significantly higher maximum levels of PAL activity and phenolic content per seed were observed than in the previous year, which may be due to the differences in sampling procedure and/or environmental effects.
PHENOLICS IN DEVELOPING WHEAT GRAIN
41
TABLE 1. Distribution of PAL activity in wheat grain (cv. Otane) 25 days after ear emergence Tissue fraction Outer pericarp Inner pericarp + testa Endosperm + aleurone Embryo + scutellum
Activity per seed" (pkatjseed) 2·70 5'19 4·00 0'51
% Total activity
Specific activity (pkat/mg protein)
21·8 41'9
23'0 21'6 9·40 28·3
32-3 4
• Values represent the mean of triplicate determinations.
Distribution of PAL activity
The distribution of PAL activity was examined initially in a sample of cv. Otane collected at 25 days after ear emergence and the results (Table I) suggested that most activity was associated with the testa and pericarp tissues although significant PAL activity was present in the aleurone plus endosperm fraction. Tissue distribution of PAL was examined further by assaying its activity in embryo + scutellum and the de-embryonated grain at several stages of development (Fig. 3). This suggested that PAL activity peaked later in the embryo plus scutellum than in the other tissues, during the late milky stage and that the shoulder observed in PAL activity in the latter part of grain development may be due in part to activity in these tissues. However it is clear that the major peak of PAL activity observed during early grain development was associated with activity in the pericarp plus testa. Comparison between cultivars of PAL activities during early grain development
Comparison of PAL activity per seed in seven cultivars from 15 to 30 days after ear emergence revealed significant differences between red- and white-grained cultivars (Table II and Fig. 4). All red cultivars exhibited a marked peak in activity at 20 days after ear emergence which was absent or smaller in the white cultivars. Integration of activity per seed between 15 and 30 days after ear emergence suggested that cultivars fell into three groups with relatively narrow ranges of activity per seed x time. There was no apparent relationship between mature grain dry weight and integrated PAL activity in the developing grain (r = 0-3, n = 7, P> 0'05). ANOVA of the results (Table III), as PAL activity per seed, revealed highly significant effects due to seedcoat colour and colour x time interaction, which confirmed that red and white cultivars differed significantly both in their overall levels of PAL activity and in the way this changed over time. Paper chromatography of phenolics in developing grain
Initially these extracts were examined by reverse-phase HPLC, but resolving and identifying the large number of UV-absorbing components rendered this technique
J. A. McCALLUM AND J. R. L. WALKER
42
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FIGURE 3. Distribution of PAL activity in the developing grain of cv. Otane. (Points represent mean ± S.E.M. of n = 2 extracts, triplicate assays), . , De-embryonated seed; 0, embryo plus scutellum. TABLE II. Integrated PAL activity 15-30 days after ear emergence in grain from 7 wheat cultivars (1987/88 trial)
Cultivar
Mature seed mean dry weight (mg)
PAL activity integral (pkat. days. seed- 1 )
Otane CRSWI CR5.179 Cook Veery Oxley Egret
47-4 42·9 39·1 42'3 47-8 36'1 39'9
295 294 276 236 203 200 194
The correlation between PAL integral and mature seed dry weight was not significant r = 0'299, P> 0·05.
impractical. Two-dimensional paper chromatography was found to provide good resolution and easier identification of phenolic compounds. Clear qualitative and quantitative differences in phenolic composition were observed between chromatograms of unhydrolysed extracts of immature and mature grain (Fig. 5, Table IV). No qualitative differences could be distinguished between cvs Otane and
PHENOLICS IN DEVELOPING WHEAT GRAIN
43
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44
J. A. McCALLUM AND J. R. L. WALKER
TABLE III. Analysis of variance (ANOVA) of PAL activity per seed; 15-30 days after ear emergence in grain from seven wheat cultivars (1987/88 trial) Source Colour Cu1tivar Time Time x colour Cultivar x time
DF
SS
MS
P-value
Pr>P
1 5 3
131-42 16'76 189·39 56·63 47·26
131-42 3·35 63'13 18·88 3·15
39'19
0·0015
3
15
% Variance contribution
-,-
20·04 5·99
0·0001 0'0068
34 3 32 17 12
Variance due to differences between individual extracts of the same tissue was insignificant. DF = degrees of freedom; SS = sum of squares; MS == mean square; Pr = probability.
Veery at either stage. In general it was observed that there was a marked decline in the amounts of hydroxycinnamic acid derivatives present on maturation. By contrast there appeared to be an increase in C-glycosyl-flavones as well as qualitative changes in these compounds. Chlorogenic acid was tentatively identified in chromatograms of immature grain by its R r values and characteristic fluorescence properties under UV in the presence and abscence of NH a fumes; however it could not be detected in chromatograms of mature grain.
PAL and a-amylase activity in germinating grain In a preliminary study for this project the development of PAL and o:-amylase activity was found to correlate closely during the germination of cv. Dtane (r = 0'995, P < 0'001).
Germination rate of maturing grain The germination rate of grain was examined after the mature grain colour had developed and there was no further increase in dry weight. The white-grained cultivars exhibited high germination rates (70-95 %) at harvest ripeness whereas that of the red cultivars was markedly lower (0-12 %). Discussion
PAL This is the first time-course study of PAL in a developing cereal grain. Earlier, Neish 24 reported the presence of tyrosine ammonia-lyase (TAL) activity in barley ears at milk stage but could not detect it in wheatgerm. Our results suggest that PAL activity in immature wheat grain reaches a maximum during the early milk stage of grain development and then declines as grain moisture content declines and the grain enters a linear phase of dry weight increase. Examination of the distribution of PAL activity has suggested that initially most activity is localised
45
PHENOLICS IN DEVELOPING WHEAT GRAIN I
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FIGURE 5. Comparison of soluble phenolics in cv. Otane at milk ripe stage (A) and maturity (B). Two-dimensional paper chromatograms developed with BAW (first dimension) and 15 % (vjv) acetic acid (second dimension).
46
J. A. McCALLUM AND J. R. L. WALKER
TABLE IV. Tentative identities of spots; based on Rr values and fluorescence characteristics in the presence and absence of NH a fumes
Spot no. 1
2 3-4
5
6-7 8 9-12
13 14-16
17 18
Colour (UV)
Colour (UV+NH a fumes)
Tentative identity
Purple Yellow Purple Blue Blue Purple Purple Orange Light blue Purple Blue
Green Yellow Green Green Blue Blue Green Orange Light blue Green Blue
Schaftoside + other flavones Unknown C-Glycosyl-flavone Chlorogenic acid Ferulic acid p-Coumaric acid C-Glycosyl-flavones Unknown Feruloyl esters? C-GlycosyI-flavone Hydroxycinnamate ester?
in the testa-pericarp tissues (and to a lesser extent in the endosperm plus aleurone) and that decline in the total grain activity is primarily due to the decline in activity in these tissues. Wheat germ is a rich source of soluble phenolics 4 •7 whilst aleurone and endosperm cell walls contain bound phenolic acids 8 ; therefore the presence of PAL activity in these tissues is not surprising. However, the relatively high PAL activity present in the testa-pericarp is noteworth in view of the lack of knowledge of the phenolic content of wheat bran. Although the presence in bran of alkylresorcinols 6 and bound phenolic acids 25 is well established there is still a paucity of information concerning the flavonoids, aminophenolics and other phenolic pigments that may be present in the testa-pericarp of developing and mature wheat. A variety of factors could contribute to the activity profile observed in this study. The cereal seed is a composite organ consisting of genetically and morphologically distinct tissues which undergo separate but inter-related programmes of development within the developmental program of the whole seed. Therefore phenolic biosynthesis could be expected to proceed in the different tissues at varying rates, at different times and to yield diverse products. The testa and pericarp layers undergo considerable modification during grain maturation and the decline in PAL activity may reflect the effects of crushing, desiccation and other catabolic activities in these tissues. The plateau observed in the profile during the dough stage of grain development may be due to the increase in activity in the embryo and scutellum observed during this phase. The profile and distribution of PAL activity observed in this study resembles closely those observed by other workers for
PHENOLICS IN DEVELOPING WHEAT GRAIN
47
development and germination. The patterns of PAL activity during grain development may correspond to changes in concentrations of growth regulators, such as gibberellic acid and abscisic acid, since these have been shown to influence (X-amylase activities in the developing grain and phneolic biosynthesis in vitro 15 •28 • The PAL profile also resembles closely those observed in many other plant systems, such as parsley cell suspension cultures 20, following induction of PAL synthesis. A variety of plant materials have yielded fractions that enzymically inactivate PAL and therefore it has been suggested that this common pattern of activity may arise from interaction between the competing processes of PAL synthesis and inactivation 30 • The existence of such PAL inactivating systems in developing cereal seeds seems likely since they have been reported from barley 31 and rice 32 seedlings. Studies of bean cell cuItures 33 ,34 have suggested that trans-cinnamate may playa central role in coordinating such patterns of expression by both reducing transcription of PAL subunit genes and inducing synthesis of proteinaceous PAL inactivators. Phenolics
The content of soluble phenolics was maximal just prior to the appearance of mature grain colour and when grain volume was also at a maximum; about 20-25 days after the peak in PAL activity. Such a lag period between PAL activity and phenolic content has been observed in a variety of plant systems 14 • Declines in the concentrations of flavanolso and ascorbic acid 35 during maturation have been noted during previous studies of wheat grain development. A similar pattern of changes in total phenolic content was also observed in study of developing sorghum grain 30 • Such declines in concentrations of oxidizable substances are probably a result of contact with oxidative enzymes during the breakdown in cellular structure in the seedcoat tissues that accompanies grain maturation. The fact that development of mature grain colour was associated with a marked decline in phenolics supports the idea that seedcoat pigments may be the products of their oxidation. The auxin content in developing wheat grain has been found to exhibit a similar peak just prior to termination of dry weight gain and the development of mature grain colourBo • Peroxidase activity also peaks around this time 37 and may be associated with metabolism of phenolics in the testa-pericarp. On the basis of studies on wheat coleoptiles, Machakova et al. 38 have suggested that degradation of phenolics by peroxidase in vivo may be regulated by auxin levels. Comparison of cultivars
Comparison of several cultivars during early grain development has suggested that there are marked differences in the content of PAL activity in red and white cultivars at this stage. Because most PAL activity in the red cultivars was associated with the testa and pericarp layers at this stage of development it is likely that these differences are primarily the result of differences in activity in the external tissues of the grain and that the higher activity in the red cultivars may be associated with synthesis of pigment precursors. A comparison of the germination rate of these same cultivars during grain maturation
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J. A. McCALLUM AND J. R. L. WALKER
indicated that the red cultivars exhibited significantly lower germination rate than the white cultivars. These findings support the hypothesis, expressed in various forms by earlier workers, that phenolic compounds may contribute both to the pigmentation and germination rate of wheat grain. Higher levels of PAL activity in red-grained cultivars could influence germination rate not only by leading to higher levels of pigments and their precursors, but also giving rise to higher levels of other phenolics. The authors wish to thank the following persons and organisations for their assistance during this work: the staff of the Wheat Research Institute and the Crop Research Institute (DSIR), the University of Canterbury and the University Grants Committee for the provision of research facilities.
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