Incorporation of Exogenous L-Phenylalanine into C-Glycosylflavones in Buckwheat Cotyledons

Biochem. Physiol. Pflanzen 173,2-10 (1978)

Incorporation of Exogenous L-Phenylalanine into C-Glycosylflavones in Buckwheat Cotyledons Uno MARGNA and EVI MARGNA Institute of Experimental Biology, Harju rajoon, Harku, Estonian S.S.R., U.S.S.R. Key Term Index: C-glycosylflavone biosynthesis, substrate supply; Fagopyrum esculentum.

Summary With tracer experiments it has been demonstrated that in excised buckwheat cotyledons Lphenylalanine fed exogenously incorporated predominantly into the luteolinic C-glycosylflavones orientin and iso-orientin, but not into their simpler apigeninic analogues vitexin and isovitexin, as it could be expected theoretically. Evidenee is presented that L-phenylalanine supplied exogenously does not mix with the endogenous pool of that precursor, and that the ratios of L-phenylalanine distribution between pathways of apigeninic and luteolinic C-glycosylflavones are different depending on whether the flavonoids are synthesized from endogenous or exogenous material. This phenomenon is suggested to be caused by the differences between enzyme complexes responsible for the biosynthesis of separate C-glycosylflavones in their capability of consuming common endogenous precursors.

Introduction

WALLACE and his coworkers (WALLACE et al. 1969, WALLACE 1975) have shown that the B-ring oxidation pattern of C-glycosylflavones is determined at an early stage of their biosynthesis, probably at the flavanone level prior C-glycosylation. A later oxidative conversion of 4'-OH-derivatives into their 3', 4'-di-OH-analogues, quite possible in the case of other flavonoids (GRISEBACH 1967, HAHLBROCK and GRISEBACH 1975), is thus excluded here, resulting in that the pathways of apigeninic and luteolinic C-glycosylflavones remain separated. It provides an opportunity for a direct comparison between building rates of C-glycosylflavones showing different B-ring substitution. It must be noted that the structural peculiarities of apigeninic and luteolinic Cglycosylflavones bear a natural basis for the occurrence of rate differences in their formation. The case is that the biosynthetic pathways from the common parent precursor (L-phenylalanine) up to the final luteolinic (3', 4' -dihydroxy-C-glycosylflavone) derivatives are apparently longer and require at least one more step for their completion than the routes involved in the biosynthesis of the corresponding apigeninic (4'-monohydroxy) compounds. One may suggest therefore that apigeninic C-glycosylflavones are normally formed more easily than the related luteolinic derivatives. The experimental data available seem to support that assumption. Thus, in plants capable of synthesizing both apigeninic and luteolinic C-glycosylflavones of similar structure, the apigeninic derivatives usually exhibit higher accumulation rates than their luteolinic analogues (MCCLURE 1968, 1975, MCCLURE and WILSON 1970, HALLOP and MARGNA 1970, ABYSHEVA 1972, CARLIN and MCCLURE 1973, MARGNA et al. 1973,

Incorporation of L-Phenylaline into C-GIycosyIfIavones

Vlle)(ln

3

Orlenlln

iso-vite)(ln

Iso-orienlin

Fig. 1. C-glycosylflavones of buckwheat cotyledons (MARGNA et aI. 1967). GI - glucose moiety.

1974a, 1974b, see also POPOVICI and WEISSENBOCK 1976, 1977). Similarly, L-phenylalanine, when fed exogenously, typically shows higher incorporation rates into the apigeninic C-glycosylflavones as compared with its incorporation into the corresponding luteolinic ones of the same tissue (WALLACE and ALSTON 1966, WALLACE et al. 1969; WALLACE and GruSEBACH 1973, WALLACE 1975, see also THAKUR and IBRAHIM 1974). In the present paper we report our data on studying L-phenylalanine incorporatio~ into the C-glycosylflavones of buckwheat seedling cotyledons (Fig. 1). Surprisingly enough, in that tissue the distribution of that precursor between different C-glycosylfta.vones did not follow the pattern theoretically expected, although the general quantitative relationsip of separate derivatives well agreed with the presumed differences in the length of their biosynthetic pathways. It gave us conclusive evidence that in buckwheat seedlings conditions of forming C-glycosylflavones (if not all phenylpropanoids) were markedly different depending on whether they were synthesized from endogenous or exogenous L-phenylalanine. The results were also indicative of some intracellular differences between the pathways of apigeninic and luteolinicC-glycosylflavones in their supply with endogenous substrates. Material and Methods The experiments were carried out with isolated buckwheat cotyledons excised from 80 hold etiolated seedlings raised under laboratory conditions in distilled water. The excised material was soaked for 3-5 min in a 10-2 M solution of labelled L-phenylalanine and then incubated for 40 h under continuous illumination on filter paper moistened with the same solution (illumination from white fluorescent tubes, light intersity 29,000 erg· cm-2 • sec-I; temperature +25°C). The label was introduced into the acting solution by complementing it with a radioactive preparation of [l-14 C]-D,L-phenylalanine. An incubation unit consisted of 25 pairs of cotyledons on a glass dish supplied with 60 micromols of labeled L-phenylalanine in a 6 ml volume of the solution (total radioactivity was 30pCi). In control series the material was incubated in distilled water. Samples for the assay of C-glycosyIfIavones and radioactivity measurements were taken every 4 h during the whole 40 h incubation period. The content of C-glycosylflavones was determined 1*

4

U. }IARGNA and E. }L,"RGNA

by 11 procedure of two-dimensional paper chromatography combined with a subsequent measurement of the optical density of the eluates of flavonoid spots spectrophotometrically (l\iARGNA and MARGNA 1969). The content of separate derivatives was expressed in nmols per seedling. For radioactivity measurements the mixture of flavonoids was separated by the same chromatographic procedure. The spots of C-glycosylflavones were cut off and the amount of label of the individual compounds assayed directly on these spots with using a special device adapted for direct paper radiometry. Each spot was measured from both sides. The measurements were made with rectangular Geiger counters SBT-10 having active working area of 5 X 5 cm. The radioactivity of dried plant material was measured in a Vacutronic VA-Z-310 Geiger counter. The experiments were repeated three times with a time interval of 12-14 weeks between the separate series. The results were subjected to evaluation by the statistical techniques of Student's significance test, regression analysis, and analysis of variance (SNEDECOR 1957, BAILEY 1959).

Results

A comparison of the total radioactivity of the dried plant material, resulting from feeding labelled L-phenylalanine, with the specific activity of the nutritive solution showed that during the 40 hr experimental period about 620 nmols of L-phenylalanine entered a pair of cotyledons. About 9.3-9.5 per cent of the final radioactivity of the material was found located in the C-glycosylflavones. Since a half of the total label of the cotyledonary tissues was due to the uptake, from the acting solution, of [l-14C]D-phenylalanine (originating from the racemic preparation added for label, see above) which as an unnatural compound cannot be active precursor for flavonoid biosynthesis, the true estimate of L-phenylalanine incorporation into the C-glycosylflavones should have been twice as high as that value, i.e. of the order of 18-19 per cent of the overall uptake. In absolute terms it corresponded to about 110-120 nmols of exogenous L-phenylalaRine. The average production of the sum of C-glycosylflavones during the same period was 370 nmols per seedling. It follows therefore that about one third of the all C-glycosylflavone moieties produced under these conditions had its origin form L-phenylalanine supplied exogenously. High rate of L-phenylalanine incorporation suggested stimulation of C-glycosylflavone accumulation, yet that was not generally observed. As can be seen from Table 1, vitexin and isovitexin showed virtually no response to the feeding of L-phenylalanine, while only orientin and iso-orientin exhibited an increase in their content large enough Table 1. Effect of feeding L-phenylalanine on the accumulation of C-glycosylflavones in excised buckwheat cotyledons Flavonoid

Flavonoid content, Water control

nmol/seedlingl) Treated material

Relative effect of the treatment, %

Vitexin Isovitexin Orientin Iso-orientin

95.5 203.4 74.4 161.8

96.9 208.4 84.9 179.5

+ 1.5 + 2.5 + 14.12) + 10.9 2)

1) Assayed at the end of the feeding program; experimental conditions as described in Methods. 2) Significant effect at the level of P ~ 0.05.

5

Incorporation of L-Phenylaline into C-Glycosylflavones

%

A

B

150

100

50

Fig. 2. Relative content of apigeninic C-glycosylflavones in excised buckwheat cotyledons compared with the content of the corresponding luteolinic derivatives. Values on the basis of absolute data of Table 2. A, 80 h old etiolated cotyledons before feeding L-phenylalanine; B, after 40 h incubation of cotyledons in a 10-2 M solution of L-phenylalanine in continuous light. 1 - orientin, 2 - vitexin, 3 - iso-orientin, 4 - isovitexin.

to be considered statistically significant. As conditioned by that differential stimulation, the great quantitative prevalence of apigeninic C-glycosylflavones over their luteolinic analogues, initially characteristic of the matherial, was considerably reduced by the end of the treatment (Fig. 2). Table 2 shows that the total absolute production of orientin and iso-orientin during the 40 h experimental period was actually equal to or even somewhat greater than the production of vitexin and isovitexin, respectively, during the same period. This was confirmed also by the close numerical values of the slopes characteristic of the fitted regression lines of vitexinjorientin or isovitexinj iso-orientin accumulation within the period of L-phenylalanine feeding (Table 2). Table 2. Time course of C-glycosylflavone accumulation in excised buckwheat cotyledons fed with L-phenylalanine in continuous light Flavonoid

Vitexin Orientin Isovitexin Iso-orientin

Initial amount in 80 hold etiolated cotyledons before feeding L-phenylalanine, nmoljseedling

Absolute increase during Final amount the feeding program af- at the end of ter different time the feeding intervals, program, nmoljseedling nmoljseedling 12 h

24 h

40 h

41.9 25.8 83.1 49.4

24.8 13.8 50.4 31.1

33.7 38.1 75.3 78.9

55.0 59.1 125.3 130.1

96.9 84.9 208.4 179.5

Accumulation rate!), nmoljseedling per hour

1.29 1.32 2.93 3.15

1) Calculated by the techniques of regression analysis and expressed as the slopes (b) of the fitted regression lines.

U.

6

MARGNA

and E.

MARGNA

B

A 1000 0'1 !:

......

'n

~ c:

~ a.. E .-_ 500 ::n

~

>

~ a o -0 a

cr

o

.lJ1 ~

I

8 12 16 20 24 28 32

40

ill lj

II

8 12 16 20 24 28 32.

~O

llme,

h Fig. 3. Time course of exogenous L-phenylalanine incorporation into buckwheat C-glycosylflavones as measured by the increase in the total radioactivity of separate derivatives. A, vitexin and orientin; B, isovitexin and iso-orientin. Black bars - apigeninic derivatives, white bars - their luteolinic analogues. Slopes of the fitted regression lines for vitexin (10.08 imp/min/seedling per h) and isovitexin (17.39) were significantly different from the corresponding slopes for orientin (14.53) and iso-orientin (25.02), respectively.

Notwithstanding the practically equal absolute increase in the content of the related apigeninic and luteolinic C-glycosylflavones, striking and rather unexpected differences in the incorporation of L-phenylalanine into the same derivatives were observed. Total radioactivities of orientin and iso-orientin were in all stages markedly (ca. 1.5 times) higher than was the amount of label found in their apigeninic analogues (Fig. 3). Since the original quantitative relationship of C-glycosylflavones can be considered as a conditional standard indicative of relative incorporation rates of endogenous L-phenylalanine (Fig. 2, A), we encountered an obvious fact that under exogenous supply of that precursor its distribution between separate C-glycosylflavones followed a pattern entirely opposite to that normally characteristic of buckwheat C-glycosylflavones formation. A comparison of specific activities of C-glycosylflavones produced and L-phenylalanine fed revealed (Table 3) that 65.3 per cent of orientin and 36.2 per cent of iso-orientin formed during the period of incubation was synthesized from the exogenous L-phenylalanine, whereas in the case of vitexin and isovitexin the corresponding incorporation characteristics were only 35.6 and 21.5 per cent, respectively. If to calculate, from these percentages, the portions of C-glycosylflavones formed either from exogenous or endogenous material, the inverse ratios of building apigeninic and luteolinic derivatives from the two sources become especially clear-cut. While the luteolinic C-glycosylflavones, as previously, remained markedly inferior to the related apigeninic ones regarding their building from endogenous precursors, their production from exogenous L-phenylalanine was about twice as high as that of the simpler apigeninic C-gIycosylflavones vitexin and isovitexin (Fig. 4).

Incorporation of L-Phenylaline into C-Glycosylflavones

7

Table 3. Specific activities of buckwheat C-glycosylflavones as resulting from the incorporation of labelled L-phenylalanine 1 ) Flavonoid

In respect to the total amount of separate derivatives at the end of the 40 h feeding program, imp/min/nmol

In that portion of the compounds produced during the feeding program, imp/min/nmoI2)

Vitexin Orientin Isovitexin Iso-orientin

4.41 8.41 3.27 5.82

7.81 14.32 4~71

7.94

1) Specific activity - 21.94 imp/min/nmol. 2) Average data over the whole period of feeding L-phenylalanine; the range of [1_14C]-L-phenylalanine incorporation into separate derivatives remained practically unchanged during the incubation period so that specific activities calculated at different stages of the feeding program showed only random variation without any definite tendency to be increased or decreased.

Discussion

Reliable interpretation of the data described here is possible only provided that no interfering processes such as interconversions, rapid turnover etc. are involved in buckwheat C-glycosylflavone metabolism. Biosynthetic studies on flavones in Lemnaceae have demonstrated that 4'-hydroxylated C-glycosylflavones cannot be converted into their 3', 4'-substituted analogues, reflecting complete independence of pathways of these derivatives (WALLACE et al. 1969, WALLACE 1975). As there is no reason to suspect biosynthetic reactions of a different kind in buckwheat cotyledons, an interfering influence at the level of such interconversions does not appear to be very likely. Flavonoids are now generally considered metabolically active compounds nmoV

seedling

A

B

iDO 80 60 1.0

Fig. 4. Absolute amounts of buckwheat C-glycosylflavones formed from either endogenous (A) or exogenous (B) L-phenylalanine during the 40 h period of feeding L-phenylalanine. 1 - orientin, 2 - vitexin, 3 - iso-orientin, 4 - isovitexin.

8

U.

MARGNA

and E.

MARGNA

(BARZ and HOSEL 1975), yet only limited indirect data are available which indicate that catabolic degradation of C-glycosylflavones may occur in plant tissues (MCCLURE and WILSON 1970, WALLACE 1975, POPOVICI and WEISSENBi:icK 1977). Recent tracer studies of our laboratory showed that buckwheat C-glycosylflavones did not undergo any measurable turnover during at least 96 h (MARGNA and VAINJARV 1976; and unpublished results of their extended study on this subject). There are all grounds to believe therefore that the C-glycosylflavone changes documented in the present report can be solely accounted for the changes in their building. Hence, the most intriguing aspect is that in buckwheat L-phenylalanine fed exogenously incorporated predominantly into the luteolinic C-glycosylflavones but not into the simpler apigeninic ones of similar structure, as it could be expected theoretically. This was in contrast with the characteristics of L-phenylalanine incorporation observed in other C-glycosylflavone producing plants (WALLACE and ALSTON 1966, WALLACE and GRISEBACH 1973, WALLACE 1975), and with the natural quantitative relationship of apigeninic and luteolinic C-glycosylflavones in buckwheat itself, as well. Since natural levels of accumulation, in that case, obviously reflect distribution ratio of endogenous precursors, a conclusion must be drawn that in buckwheat cotyledons ratios of forming 4'-OH- and 3',4'-di-OH-C-glycosylflavones substantially differ from each other depending on whether the flavonoids are built up from L-phenylalanine supplied exogenously or the biosynthesis from endogenous material is involved. This fact seems clearly to indicate that: i) L-phenylalanine, when supplied exogenously, does not mix freely with the endogenous pool of that precursor; and ii) exogenous and endogenous L-phenylalanine have different affinity to the enzymic systems responsible for the biosynthesis of C-glycosylflavones. It looks as if exogenous and endogenous L-phenylalanine are transferred to the sites of C-glycosylflavone biosynthesis via different intracellular channels or are transported there on some kind of rather different carriers, which make the conditions of their capture, by the enzymic systems functioning at these sites, markedly different. At the same time it is clear that exogenous L-phenylalanine without any difficulties reaches biosynthetic centres of C-glycosylflavone building and effectively competes there with the endogenous substrate. It is not easy to reconcile these results yet the simplest explanation seems to be that enzyme complexes responsible for the biosynthesis of separate C-glycosylflavones are not equal in their capability of consuming common endogenous precursors (see also MARGNA 1977b). First of all it must be emphasized that enzyme complexes for building 4'-OH-derivatives are obviously simpler (consist of fewer links) than those for synthesizing 3',4'-diOH-C-glycosylflavones, and should be able therefore to transform, during a time unit, much more molecules of their common substrate. Further it seems that the former are in some way (may be due to some specific differences in their spatial orientation) closer, than the latter, to the intracellular channels supplying endogenous precursor, so that substrate molecules are preferentially captured by those of the complexes which lead to the formation of simpler compounds, 4'-OH-C-glycosylflavones. Since there are all probabilities that the amounts of L-phenylalanine, which endogenously become accessible for polyphenol synthesis, never reach the level of the total

Incorporation of L-Phenylaline into C-Glycosylflavones

9

biosynthetic capacity the corresponding enzymic systems in plants actually possess (MARGNA 1977 a), much lower relative saturation with endogenous substrates should be normally characteristic of the sites of forming luteolinic C-glycosylflavones, than of the sites of forming their apigeninic analogues. That seems to be sufficient to create relatively more favourable conditions for consuming exogenous L-phenylalanine specifically at the sites of building luteolinic derivatives. It should result, quite naturally, in decreased ratios of apigeninic and luteolinic C-glycosylflavone formation from exogenous L-phenylalanine, as compared with the same ratios under formation of these C-glycosylflavones from endogenous material. As we could see, in buckwheat cotyledons that ratio shift was large enough to even bring about an absolutely prevalent formation of luteolinic C-glycosylflavones, orientin and iso-orientin, over the formation of their apigeninic analogues, vitexin and isovitexin, from L-phenylalanine supplied exogenously. At present one cannot say with certainty whether buckwheat seedlings are an exceptional organism with respect to the differential biosynthetic utilization of endogenous and exogenous precursors for building C-glycosylflavones (all phenylpropanoids ?) or similar ratio differences can be observed more generally. The point is that at other initial relationships of the relevant C-glycosylflavones the phenomenon needs not necessarily manifest so clearly and the difference may simply remain undiscovered. For example, about threefold quantitative preponderance of saponarin (6-C-glucosyl7-0-glucosyl-apigenin) over its luteolinic analogue lutonarin in barley (CARLIN and MCCLURE 1973), and about ten times higher accumulation of vitexin as compared with the accumulation of orientin in Spirodela (MCCLURE 1968), make the establishment of a relatively increased incorporation of exogenous L-phenylalanine into the corresponding luteolinic C-glycosylflavones, if it occurs, rather difficult in these objects. Recent feeding experiments of our laboratory strongly suggest that at least in barley such incorporation shifts may, in fact, occur (LAANEST 1978). To elucidate that problem more detailed incorporation studies on a wider range of C-glycosylflavone producing plants should be undertaken. Acknowledgements The authors' thanks are due to U. VAHER and M. SAUL for their valuable help in performing mathematical treatment of the experimental data.

References ABYSHEVA, L. N.: Content of glycoflavons and rutin in buckwheat leaves as dependent on plant supply with boron (in Russian, Summary in English). Physiol. Biochem. Cult. Plants (Kiev) 4, 529-534 (1972). BAILEY, N. T. J.: Statistical Methods in Biology. The English Universities Press Ltd. London·1959. BARZ, W., and HOSEL, W.: Metabolism of flavonoids. In: The Flavonoids (ed. HARBORNE, J. B., MABRY, T. J., and MABRY, H.), pp. 919-969. Chapman & Hall, London 1975. CARLIN, R. M., and MCCLURE, J. W.: Action spectra for C-glycosylflavone accumulation in Hordeum vulgare plumules. Phytochemistry 12, 1009-1015 (1973). GRISEBACH, H.: Biosynthetic Patterns in Microorganisms and Higher Plants, pp. 1-31. John . Wiley and Sons, New York-London-Sydney 1967.

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U. MARGNA and E. MARGNA, Incorporation of L-Phenylaline into C-Glycosylflavones

HAHLBROCK, K., and GRISEBACH, H.: Biosynthesis of flavonoids. In: The Flavonoids (ed. HARBORNE, . J. B., MABRY, T. J., and MARBY, H.), pp. 866-915. Chapman & Hall, London 1975. HALLOP, L., and MARGNA, U.: On the effect of light on the formation of glycoflavones in buckwheat seedlings (in Russian, Summary in English). ENSV TA Toimet., BioI. 1D, 167 -171 (1970). LAANEST, L.: Effect of exogenous feeding on C-glycosylflavone accumulation in barley seedlings. ENSV TA Toimet., BioI. 27, in Press (1978). MARGNA, U.: Control at the level of substrate supply - an alternative in the regulation of phenylpropanoid accumulation in plant cells. Phytochemistry 16, 419-426 (1977 a). Accumulation pattern of flavonoids: quantitative phenomenology and some speculations. ENS V TA Toimet., BioI. 26, 302-316 (1977b). HALLOP, L., MARGNA, E., and TOHVER, M.: Chromatographic and spectrophotometric evidence for the occurrence of luteolin and apigenin C-glycosides in the cotyledons of buckwheat seedlings. Biochim. Biophys. Acta 136, 396-399 (1967). LAANEST, L., MARGNA, E., OTTER, M., and VAINJARV, T.: The influence of temperature on the accumulation of flavonoids in buckwheat and some other plant seedlings. ENSV TA Toimet., BioI. 22, 163-175 (1973). _ _ _ - Sugar effects on the formation of buckwheat flavonoids: some newas pects and concluding remarks. ENSV TA Toimet., BioI. 23, 19-29 (1974a). _ _ _ _ Azote-induced changes in the accumulation of buckwheat seedling flavonoids. ENSV TA Toimet., BioI. 23, 298-304 (1974 b). and MARGNA, E.: A suitable chromatographic method for quantitative assay of rutin and flavone C-glycosides in buckwheat seedlings. ENSV TA Toimet., BioI. 18, 40-50 (1969). and VAINJARV, T.: On the rate of catabolic degradation of flavonoid structures in buckwheat seedlings (in Russian). In: Regulation of Plant Growth and Nutrition (ed. ROMANOVSKAYA, O. I.), pp. 125-132. Zinatne, Riga 1976. MCCLURE, J. W.: Photo control of Spirodela inter media flavonoids. Plant PhysioI. 43, 193-200 (1968). The applicability of polyphenolic data to systematic problems in the Lemnaceae. Aquatic Botany 1, 395-405 (1975). and WILSON, K. G.: Photocontrol of C-glycosylflavones in barley seedlings. Phytochemistry D, 763-773 (1970). POPOVICI, G., and WEISSENBOCK, G.: Anderungen des Flavonoidmusters wahrend der Ontogenese von Avena sativa L. Ber. Deutsch. Bot. Ges. 8D, 483-489 (1976). _ Dynamics of C-glycosyHlavones in primary leaves of Avena sativa L. grown under field conditions. Z. PflanzenphysioI. 82, 450-454(1977). SNEDECOR, G. W.: Statistical Methods Applied to Experiments in Agriculture and Biology. The Iowa State College Press. Ames, Iowa 1957. . THAKUR, M. L., and IBRAHIM, R. K.: Biogenesis of flavonoids in flax seedlings. Z. PflanzenphysioI. 71, 391-397 (1974). WALLACE, J. W.: Biosynthetic studies on flavones and C-glycosylfla vones: B-ring oxidation patterns. Phytochemistry 14, 1765-1768 (1975). and ALSTON, R. E.: C-glycosylation of flavonoids. Plant & Cell Physiol. 7, 699-700 (1966). and GRISEBACH, H.: The in vivo incorporation of a flavanone into C-glycosylflavones. Biochim. Biophys. Acta 304, 837-841 (1973). WALLACE, G. W., MABRY, T. J., and ALSTON, R. E.: On the biogenesis of flavone O-glycosides and C-glycosides in the Lemnaceae. Phytochemistry 8,93-99 (1969). Received December 17, 1977.

Authors' address: UDO MARGNA and EVI MARGNA, Institute of Experimental Biology, 203051 Harku, Harju rajoon, Estonian S.S.R., U.S.S.R.