Developmental aspects of flavonoid patterns in <i>Brassica campestris</i> var. <i>oleifera</i>: a preliminary study

Developmental aspects of flavonoid patterns in Brassica campestris var. oleifera: a preliminary study D. CLASSEN AND C. NOZZOLILLO Department of Biology, U n i v e r s i ~of Ottawa, Ottawa, Ont., Canada KIN 6N5 Received November 12, 1980

Can. J. Bot. Downloaded from www.nrcresearchpress.com by 74.121.191.133 on 06/07/13 For personal use only.

CLASSEN, D., and C. NOZZOLILLO. 1981. Developmental aspects of flavonoid patterns in Brassica campestris var. oleijera: a preliminary study. Can. J. Bot. 59: 1382-1385. Field-grown plants of Brassica campestris L. var. oleijera (summer turnip rapeseed) were harvested at various growth stages from seedling through to maturity. Cotyledons, roots, hypocotyls, leaves, stems, and flowers were sampled as available and freeze-dried prior to extraction. Chlorophyll-free, unhydrolysed alcoholic extracts were subjected to two-dimensional chromatography on polyamide thin layers to establish flavonoid patterns. Major aglycones of compounds were tentatively identified on the basis of mobility, colour reactions with a diphenylboric acid ethanolamine spray, and co-chromatography with standards. Each organ was found to have a pattern characteristic to itself which changed little with age. The greatest variety of compounds was present in leaf tissues. Only two compounds, presumably highly glycosylated derivatives of quercetin, were common to all tissues.

Introduction Brassica campestris L. var. oleifera, commonly known as rapeseed, is the fourth most important annual crop in Canada after wheat, barley, and oats (Agriculture Canada 1971). Oil is extracted from the seed and the remaining meal is used as animal feed. Glycosides of the flavonols kaempferol, quercetin, and isorhamnetin have been reported to occur in the leaves of 21 species and cultivars of Brassica (Durkee and Harborne 1973). Quercetin, isorhamnetin, and kaempferol glycosides also occur in the closely related genus Sinapis (Paris and Charles 1962; Horhammer et al. 1966; Durkee and Harborne 1973). Hoshi (1975) reported the presence of isorhamnetin and kaempferol glycosides in outer layers of the root, petals, stems, and leaves of turnip (Brassica campestris L. ssp. rapifera Sinsk.). The food-yielding varieties of Brassica oleracea L. contain kaempferol and quercetin (Wildanger and H e m a n n 1973; Sedgley 1975), and glycosides of isorhamnetin (Horhammer et al. 1966) and kaempferol (Stengel and Geiger 1976) are reported to occur in B . napus L. Acylated flavonoids have also been reported to occur in Brassica species (Harborne 1967). The developmental studies that have been done to date have beenmainly concerned with different stages of leaf tissue. Buttery and Buzzell (1973) collected leaf samples from soybeans in vegetative, flowering, and podding stages and reported that all had the same flavonol pattern. Popovici and Weissenbock (1976) studied the flavonoid pattern in the leaves, stem, and inflorescenceof the oat plant. They reported quantitative differences in leaf flavonoids depending on the point of insertion, and qualitative and quantitative differences in the flavonoids of the leaf, stem, and inflorescence. In a

similar study, Blume and McClure (1979) reported auantitative changes in flavonoids and their associated enzymes but few qualitative changes with development in the leaves of barley. Using parsley, Hahlbrock et al. (1971) also reported quantitative enzyme and flavonoid changes with time. As has been demonstrated by Egger (1969), the aglycone, as well as the number, substitution, and nature of the sugars, all play very important roles in determining chromatographic mobility-of the -flavonol glycosides. For example, if the following sugars are attached as indicated to the same aglycone, then a ~redictableseries of RcYswill occur when chromatographed on polyarnide and run in a water-alcohol solvent (from low to high Rf): 3-glucuronide, 3-glucoside, 3-diglucoside, 3,7-diglucoside (Egger 1969). Acylation introduces additional variability- in chromatographic behaviour (Ribereau-Gayon 1972). These facts were used in the preliminary study described below in which flavonols from a range of developmental stages from seedling through to mature plant, and from a variety of organs of Brassica campestris var. oleifera were examined using a two-dimensional chromatographic scan. Developmental studies alone or coupled with enzyme studies, as done by Blume and McClure (1979), may yield information regarding flavonoid biosynthesis and degradation, and organ studies may provide clues to the function of these secondary plant products. Developmental studies also provide basic information that is useful when contemplating techniques in chemotaxonomic studies, for example. If species show a varied flavonoid pattern for different stages and organs, then very careful sampling must be done in order to draw valid comparisons between taxa. u

0008-4026181/081382-04$01 .OO/O 01981 National Research Council of CanadaIConseil national de recherches du Canada

CLASSEN AND NO2

I l l 1 I l l 1 I I I I I I I I I I

Can. J. Bot. Downloaded from www.nrcresearchpress.com by 74.121.191.133 on 06/07/13 For personal use only.

Materials and methods Plant material Brassica campestris var. oleifera was grown from seed under field conditions during June-August 1978 at the Central Experimental Farm, Ottawa. The plants were harvested at specific growth stages and particular organs were taken. The stages sampled were based on the key by Harper and Berkenkamp (1975). The seedling stage (stage 1.0), the rosette stage with 4 leaves present (stage 2.4), the bud stage with the inflorescence visible at the centre of the rosette (stage 3. l ) , the bud stage with the lower buds yellowing (stage 3.3), the flower stage with many flowers opened and lower pods elongating (stage 4.2), and the flower stage with flowering complete and seeds enlarging in the lower pods (stage 4.4) were all sampled. The organs sampled were the roots and hypocotyl (R + H), cotyledons (C), vegetative leaves (VL), vegetative stems (VS), fourth leaves developmentally (4L), generative leaves (GL), generative stems (GS), and inflorescence (Inf). Vegetative leaves are simple, spatulate, and divided. They are located on or subtending the lower portions of the stem. Generative leaves are simple, lanceolate, and clasping; they are located on or subtending the flowering portions of the stem. Transitional forms of leaves and stems were not sampled. The samples were freeze-dried and ground to a powder. Preparation of extracts Repeated isopropanol extractions of 0.55 g of powdered material (1: 1 by volume) were made in a 60°C water bath until no further colour went into solution. The extracts were combined, dried in vacuo, and washed with petroleum ether a number of times to remove the chlorophyll. The powder was reextracted with 80% methanol (1: 1 by volume) using the same procedure as for the isopropanol extracts. These extracts were combined with the dried isopropanol extract and the entire solution was dried in vacuo and washed again with petroleum ether. The residue in the flask was taken up in 1 mL of acetone-water (50:50). One millilitre of final extract thus represented the flavonols present in 0.55 g dry weight of tissue. No study of anthocyanin pigmentation was made. Chromatography Polyamide microlayer (ML 1515, Mandel Scientific Co. Ltd.) was used for two-dimensional chromatography of the final extracts. A 75 X 75 mm sheet was spotted with 0.006 mL of extract (= 3.3 mg dry weight of tissue) and first run in an aqueous solvent (water-pyridine-cyclohexanone, 90:5:5), dried, and then run in the second direction in an organic solvent (n-butyl acetate - methanol - formic acid - water, 60:30:5:5). Increasing glycosylation of the flavonoids yields higher Rf's in the aqueous solvent and lower Rf's in the organic solvent. The plate was then dried, sprayed with 1% diphenylboric acid ethanolamine complex (flavone reagent) in water containing 1% triethylamine, and observed under visible and UV light.' The quercetin (Q), isorhamnetin (I), and kaempferol (K) glycosides were identified on the basis of colour, and low or high levels of glycosylation were determined by mobility. The individual glycosides were distinguished by visual comparison of chromatograms.

1 l l I

1 I 1 I I I l I I I l l l I I I l l I XX XXX

I l l 1 I l l I I I I I I I I l l I 1 l I / I I 1 1 I XX I

I l l / Ill I I I I I l l

X

~

I l l 1

X

I

I

X

X

I

I I

X X X X X X X X X X

I I X X X X X X X ~ I I

I I x x x l x x x x l I J X X X X X

I /

X X X X

I I x x x I I x I

I I X X I I

I I X X X I I I I

I I ' X X X X

I

I

~

X

X X X X X

I I I X X I I I

~

I

I X X X X I I X X X X I

I

I

X

1 I X

X X X X X X X X

X

I I

I I I I I I I1 I I

I I X X X I I I I I I I l x x I I I I I I l l IxxIxI I 1 I I I I I X X X X X X X I I I I I I I l l lIxXxxxxxII I I x x I l l I I IIIXXXX I I X X I I

I I I I X X X

$

kQ c

% 2 7 ,g

2 %

+ 2 X

I 1 I I

x / l I

I x 1 1 l 1 I I

X X X X X X X

X X X X X X X X X X X X X X X

I I I I x ~ ~ x x x I l l 1 I I I X X I l l I l l 1 I I I X X I I I X X X X X I X X X x I X l l l ~ l l x l Il l l X X X X

X X X X X X X X

;

X X X X I I I X X I I I

m,

X X X X

-1

6

X X X

II I I I I l l I I I IXX

I I I 1 I I I I I I 1 I

I I

X X X X ~ I I X X

1

X X

l l x l x l x l

I 1

X

X X X X X X X X X X

I I I I I 1 I I I I I I I I I I I

1 1 1 1 1 1 1 I I I I I I l l / I I I I I I I X l x x x x l I I 1 / 1 1 I l l I I l I I x I l I I I I I I I I X I I I I I I I X I I I I I l l I I I I I I I I X I l l 1 I l l I ' X X X X I I I I

X X

I I X X X X X X

1 I l l 1 1 1 I I X X X X I I l l I I I I IIx):xx X I I I I I I I 1 1 I 1 I I I 1x1 1x1 I X I X I I I I IxIxxxxxxxxII x x x I I I I I x x I x x I X I I I

I x x I x I x x I I

I l l / I I X X I I I I l l IxxxII II I I I I l l I I I I I x I I x I 1 IIIxxxxxxxxxx I I l l I l l 1 I I I I I I X I I I

zzzz ' ~ e t h o ddeveloped by F. W. Collins, Food Research Institute, Agriculture Canada, Ottawa.

I

I I I I X X X X I I X X X X X X X X

1 1 1 1 1 1 1 1 1 I l l I I I 1 1 1 1 1 1 1 XXXXX I / I

I l l I l l I I I I I I I

X

LLZ ??? 88 uuu dddd 9;' dd .a3 ?P.?b. *'?*? * ? P ? *?** 1 N NP. ? * - N ~ P N m b m e - ~ r n~

r

n m~ b b~ b m b

~

I

I

Can. J. Bot. Downloaded from www.nrcresearchpress.com by 74.121.191.133 on 06/07/13 For personal use only.

1384

CAN. 1.

BOT. VOL.

59, 1981

!

aqueous organic +

x

RtH

x

VS

x

Inf

I

I

1

I

I

0zquercetin @ = kaempferol ABBREVIATIONS USED: R + H, root and hypocotyl; VS, vegetative stem; GS, generative stem; Inf, inflorescence; C, cotyledon 4L, fourth leaf; VL, vegetative leaf; GL, generative leaf. FIG.1. Basic chromatographic patterns of organs consisting of quercetin (Q), isorhamnetin (I) and kaempferol (K) glycosides. Under ultraviolet light and after spraying with the flavone reagent, quercetin appears yellow-orange, isorhamnetin appears yellow-green, and kaempferol appears green. Aglycones with few sugars attached have lower Rf's in aqueous solvents and higher Rf's in organic solvents (lower numbers). Aglycones with many sugars show the opposite mobility (higher numbers). Within an aglycone series, different sugars attached at the same position will usually give a predictable series Rf values as do different substitutions of the sugars.

found to be the flavonol quercetin (Q), followed by the flavonols isorhamnetin (I) and kaempferol (K) in approximately equal amounts. A total of at least 40 Determination of aglycones Half of the acetone-water extract (= 0.275 mg dry weight different flavonol glycosides was found in the plant. Each organ was found to have a characteristic of tissue) was combined with 3 mL of 2 N HCl and 3 mL of 95% ethanol in a screw-top tube and placed in a 100°C water bath flavonol pattern (Fig. 1) which was constructed by for 4 h. The solution was allowed to cool, diluted by the examining the several chromatograms of each organ addition of 6 mL of water, and extracted with ethyl acetate. regardless of the stage of growth, and including the The ethyl acetate fraction was chromatographed on polyamide glycosides occumng in all such chromatograms to arrive rnicrolayer. The sheets were run in t-butanol - glacial acetic at a "basic" pattern for that organ. Glycosides not acid - water (3:l:l) in the first direction and in chloropresent in all stages of that organ would thus not be form-ethanol-butanone-acetylacetone (16: 105:1) in the second direction. The aglycones were identified by their represented in the basic pattern, but since few developcolour reactions with the flavone reagent, by Rf values, and by mental changes occurred within a single organ, this situation did not occur frequently. cochromatography with standards. Table 1 indicates the presence or absence of the Results various glycosides in all organs and stages studied. No The most common flavonoids in the extracts were trend in levels of glycosylation with development is The above procedures were repeated for each of the 22 samples collected.

CLASSEN AND NOZZOLILLO

Can. J. Bot. Downloaded from www.nrcresearchpress.com by 74.121.191.133 on 06/07/13 For personal use only.

strongly evident but the root-shoot axis (R + H, VS) shows a tendency towards more highly glycosylated quercetin compounds in older tissues. Two highly glycosylated quercetin compounds (Q15 and 421) are the only flavonoids common to all the samples. Quercetin glycosides are the most prominent in the pattern of the root-shoot axis. Quercetin is found in all other organs as well, but not in as high a proportion, although it is conspicuous in the inflorescence. The cotyledons show the highest proportion of isorhamnetin compounds, while kaempferol glycosides are the most prominent in the leaves. Table 1 also indicates that the root-shoot axis has the simplest pattern of flavonols. The leaves show the most diverse pattern, and the generative leaves have the greatest flavonol diversity of all organs. The simple and complex patterns of the axis and leaves, respectively, are maintained throughout development. Developmental changes may not be readily observed within an organ but they are evident when the plant is examined as a whole. The seedling stage (1.0) contains 17 flavonols, and the rosette state (2.4) shows an increase to 22 flavonols. The bud stages (3.1 and 3.3) and the flower stages (4.2 and 4.4) both show the full array of 40 glycosides.

Discussion The distribution of quercetin, isorhamnetin, and kaempferol among the organs is clearly different but changes little in an organ with age. Goodwin (1976) reports that flavonoids occur in all parts of the plant, with the bulk located in the leaves. A similar state was observed in the case of rapeseed, especially in leaves subtending flowering shoots. Popovici and Weissenbock (1976), on studying oat flavonoids, found that the primary leaf pattern is repeated in subsequent leaves. In the present study with rapeseed, the cotyledon (seedling leaf) pattern was found to be repeated, for the most part, in the leaves, although the pattern of the vegetative leaves contained a few more flavonols while that of the leaves subtending floral parts was more complex still. Developmental changes are found but the changes occur within the plant as a whole. As the plant progresses from seedling to mature plant the flavonoids diversify. In other words, as the plant becomes increasingly complex so does its overall flavonol pattern. The axis tissues (root, hypocotyl, and stem) have a relatively simple pattern which becomes most complex at the time of flowering. The leaf tissues show the most diversity in flavonol glycosides, also increasing in complexity with time until the flowering stage of the plant. Acknowledgments We express our appreciation for the continued support

View publication stats

1385

and encouragement by Dr. F. W. Collins while he was associated with the Biosystematics Research Institute. He not only suggested the study and developed the extraction and chromatographic techniques used, but also generously trained one of us (D.C.) in their application. AGRICULTURE CANADA. 197 1. What you should know about oil-seed crops. Can. Dep. Agric. Pub. No. 1448. BLUME, D. E., and J. W. MCCLURE. 1979. Developmental changes in flavonoids and enzyme activities of primary leaves from field grown barley. Z. Pflanzenphysiol. 95: 121-128. BUTTERY, B. R., and R. I. BUZZELL. 1973. Varietal differences in leaf flavonoids of soybean. Crop Sci. 13: 103-106. DURKEE,A. B., and J. B. HARBORNE. 1973. Flavonol glycosides in Brassica and Sinapis. Phytochemistry, 12: 1085-1089. EGGER, K. 1969. In Thin-layer chromatography-a laboratory handbook. Editedby E. Stahl. Springer-Verlag, Berlin. GOODWIN, T. W. 1976. Chemistry and biochemistry of plant pigments. Vol. 1. Academic Press, London. HAHLBROCK, K., A. SUTTER, E. WELLMAN, R. ORTMANN, and H. GRISEBACH. 1971. Relationship between organ development and activity of enzymes involved in flavone glycoside biosynthesis in young parsley plants. Phytochemistry, 10: 109-1 16. HARBORNE, J. B. 1967. Comparative biochemistry of the flavonoids. Academic Press, London and New York. HARPER, F. R., and B. BERKENKAMP. 1975. Revised growthstage key for Brassica campestris and B. napus. Can. J. Plant Sci. 55: 656-658. HORHAMMER, L., H. WAGNER, H. G. ARNDT,H. KRAEMER, and L. FARKAS. 1966. Synthesis of naturally occumng polyhydroxyflavone glycosides. Tetrahedron Lett. 6: 576571. HOSHI,T. 1975. Genetical study on the formation of anthocyanins and flavonols in turnip varieties. Genetical studies on anthocyanins in Brassicaceae. 11. Bot. Mag. (Tokyo), 88: 249-254. PARIS,R. R., and A. CHARLES. 1962. Sur le "lespecapitoside," flavonoside difficilement hydrolysable des feuilles du Lespedza capitata Michx. C. R. Hebd. Seances Acad. Sci. Ser. D, 254: 352. POPOVICI, G., and G. WEISSENBOCK. 1976. Changes in flavonoid pattern during development of the oat plant (Avena sativa). Ber. Dtsch. Bot. Ges. 89: 483-489. RIBEREAU-GAYON, P. 1972. Plant phenolics. Oliver and Boyd, Edinburgh. SEDGLEY, M. 1975. Flavonoids in pollen and stigma of Brassica oleracea and their effects on pollen germination in vitro. Ann. Bot. (London), 39(164): 1091-1095. STENGEL, B., and H. GEIGER.1976. Kaempferole-3-(0sinapoly sophorosid)-7-glucoside, a new flavonoid from Brassica napus L. (Cruciferae). Z. Naturforsch. C: Biosci. 31(9/10): 622-623. WILDANGER, W., and K. HERRMANN. 1973. Flavonols and flavones of vegetables. 1. Flavonols of Brassica varieties. Z. Lebensm. Unters. Forsch. 152(3): 134- 137.