Dynamics of the Tissue-Specific Metabolism of Luteolin Glucuronides
in the Mesophyll of Rye Primary Leaves (Secale cereale)
M a r g o t Schulz* a n d G o t t f r i e d W e i s s e n b ö c k
Botanisches Institut der Universität zu Köln, Gyrhofstraße 15, 5000 Köln 41,
Bundesrepublik Deutschland
Z. Naturforsch. 43c, 187-193 (1988); received November 19/December 30, 1987
Secale cereale L., UDP-glucuronate: flavone-glucuronosyltransferases,
Luteolin 7-0-Diglucuronosyl-4'-0-Glucuronide
ß-Glucuronidase,
Developing primary leaves of Secale cereale L. exhibit a dynamic metabolism of the major
flavonoid luteolin 7-0-[ß-D-glucuronosyl(l—»2)ß-D-glucuronide]-4'-0-ß-D-glucuronide (Ri). Final steps of R, biosynthesis are sequential glucuronidations of luteolin, which are catalyzed by
three specific UDP-glucuronate: flavone glucuronosyltransferases. These enzymes reach highest
activities at the fourth and fifth day of leaf development, coinciding with maximal R, accumulation. The activities decrease with advancing age of the leaves. In contrast, a R,-specific ßglucuronidase, responsible for the hydrolysis of glucuronic acid in position 4', shows increasing
activity up to the 5th or 6th day; but this activity, leading to luteolin 7-O-diglucuronide (R 2 ), is not
reduced in later developmental stages. In this phase of leaf development, the amount of R,
drastically drops, whereas R 2 accumulates only slightly. From in vitro results and from feeding
experiments using [ 14 C]cinnamic acid, a precursor of R, biosynthesis, we conclude that the
anabolic sequential glucuronidation takes place in young and expanding leaf tissue, whereas
deglucuronidation occurs in nearly mature and mature tissue. The three glucuronosyltransferases
as well as the ß-glucuronidase, and the flavonoids R) and R 2 are localized in the mesophyll.
Introduction
D u r i n g t h e last t h r e e d e c a d e s , s u b s t a n t i a l p r o g r e s s
h a s b e e n m a d e in t h e e l u c i d a t i o n of b i o c h e m i c a l a n d
p h y s i o l o g i c a l p r o c e s s e s of s e c o n d a r y p l a n t p r o d u c t s
i n c l u d i n g p h e n y l p r o p a n o i d s a n d f l a v o n o i d s [1, 2].
T h e i r b i o s y n t h e s i s , a c c u m u l a t i o n , t u r n o v e r a n d deg r a d a t i o n a r e o f t e n i n t e g r a t e d i n t o t h e o n t o g e n e s i s of
h i g h e r p l a n t s . S e c o n d a r y m e t a b o l i s m can reflect t h e
p h y s i o l o g i c a l s t a t e of a w h o l e p l a n t , its o r g a n s , certain tissues o r e v e n p a r t i c u l a r cells. In p a r t i c u l a r , t h e
r e l a t i o n b e t w e e n d i f f e r e n t i a t i o n p r o c e s s e s of seedlings a n d t h e b i o s y n t h e s i s of p h e n y l p r o p a n o i d s a n d
f l a v o n o i d s h a s b e e n s t u d i e d extensively [1—6, 11].
Abbreviations:
2-ME,
2-mercaptoethanol;
-glur,
glucuronide; LGT, UDP-glucuronate: luteolin 7-0glucuronosyltransferase; LMT, UDP-glucuronate: luteolin
7-O-glucuronide glucuronosyltransferase; LDT, UDPglucuronate:
luteolin
7-0-diglucuronide-4'-0-glucuronosyltransferase; ßGL, ß-glucuronidase; L, luteolin; N,
luteolin 7-O-glucuronide; R 2 , luteolin 7-0-[ß-D-glucuronosyl(l—»2)ß-D-glucuronide]; R,, luteolin 7-0-[ß-D-glucuronosyl(l—»2)ß-D-glucuronide]-4'-0-ß-D-glucuronide.
* Present address: Institut für Landwirtschaftliche Botanik
Universität Bonn, Meckenheimer Allee 176, D-5300
Bonn.
Reprint requests to G. Weissenböck.
Verlag der Zeitschrift für Naturforschung. D-7400 Tübingen
0341 - 0382/88/0003 - 0194 $ 01.30/0
In a r e c e n t s t u d y [6], w e d e s c r i b e d t h e a c c u m u l a t i o n p a t t e r n of six d i f f e r e n t f l a v o n o i d c o m p o u n d s in
d e v e l o p i n g p r i m a r y l e a v e s of r y e . A close c o r r e l a t i o n
was found between organogenesis, and complex
p r o c e s s e s in f l a v o n o i d m e t a b o l i s m . In p a r t i c u l a r , t h e
major
flavone
luteolin
7-0-[ß-D-glucuronosyl(l—>2)ß-D-glucuronide]-4'-0-ß-D-glucuronide,
Ri [7], e x h i b i t s m a r k e d q u a n t i t a t i v e c h a n g e s d u r i n g
leaf g r o w t h . M a x i m a l a c c u m u l a t i o n of R l 5 ca.
50 n m o l e s p e r l e a f , is r e a c h e d o n t h e 5 t h d a y of s e e d ling d e v e l o p m e n t . In t h e f o l l o w i n g p h a s e , a r a p i d
d e c r e a s e o c c u r s , a n d a f t e r 10 d a y s only 2 0 % of t h e
m a x i m u m a m o u n t is l e f t . In c o n t r a s t , t h e m i n o r c o m pound
luteolin
7-0-[ß-D-glucuronosyl(l—»2)ß-Dg l u c u r o n i d e ] , R 2 , slightly i n c r e a s e s c o n t i n u o u s l y o v e r
t h e g r o w t h p e r i o d s t u d i e d . B o t h c o m p o u n d s R] a n d
R 2 a r e exclusively localized in t h e m e s o p h y l l of t h e
p r i m a r y leaf [8].
R e c e n t l y w e r e p o r t e d o n a R r s p e c i f i c ßg l u c u r o n i d a s e f r o m rye p r i m a r y l e a v e s , w h i c h is res p o n s i b l e f o r t h e h y d r o l y s i s of t h e g l u c u r o n i c acid
m o i e t y in p o s i t i o n 4 ' , with R 2 as r e a c t i o n p r o d u c t [9].
T h i s d e g l u c u r o n i d a t i o n is s u p p o s e d t o b e t h e first
s t e p of t h e t u r n o v e r o r d e g r a d a t i o n of R] in vivo. In
a d d i t i o n t o t h e ß - g l u c u r o n i d a s e , t h r e e specific U D P g l u c u r o n a t e : f l a v o n e g l u c u r o n o s y l t r a n s f e r a s e s inv o l v e d in R] b i o s y n t h e s i s w e r e c h a r a c t e r i z e d [10].
T h e y c a t a l y z e t h e s e q u e n t i a l g l u c u r o n i d a t i o n of
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M. Schulz and G. Weissenböck • Luteolin Glucuronide Metabolism in Rye Mesophyll
188
luteolin via luteolin 7-O-glucuronide (N) and luteolin
7-O-diglucuronide (R 2 ) to luteolin 7-O-diglucuronide 4-O-glucuronide (Rj) as the final product.
The metabolic sequence is summarized in Fig. 1.
The rye primary leaf is a suitable model for investigations on the relation between a distinct part of the
biosynthesis and degradation of the major flavonoid,
Ri, and organogenesis, tissue specificity and cell differentiation. In this paper we present activity profiles
of the four enzymes mentioned [9, 10] with regard to
leaf development, gradient of cell differentiation
within the leaf, and distribution in tissues. The results are compared with the accumulation patterns of
R j and R 2 .
Materials and Methods
Plant material
Caryopses of Secale cereale L. var. Kustro were
purchased from F. von Lochow-Petkus (Bergen).
Seedlings were grown in a phytotron under conditions described elsewhere [6]. Primary leaves of 3 to
10 day old seedlings were harvested at the beginning
of the light phases of the photoperiod (13 h light,
11 h darkness).
Separation of the leaf tissues and preparation
mesophyll protoplasts
of
Leaf tissues were prepared as described previously
[11]. Mesophyll protoplasts were prepared from 40
to 60, 4 and 5 day old leaves as described in [12],
Chemicals
CATABOLISM
OH
0
Luteolin 7 - 0 - d i g l u r
UDP-glur-J
OH
Common chemicals were purchased from Merck
(Darmstadt, FRG), luteolin 7-O-glucuronide was
isolated from petals of Antirrhinum majus, R j and R 2
from rye primary leaves, and purified to 95% as determined by HPLC, according to [7]. Biochemicals
were purchased from Boehringer (Mannheim,
FRG), Serva (Heidelberg, FRG) or Sigma (München, FRG). Cellulysin (Trichoderma viride, 10,000
units x g _ 1 ), and Miracloth were obtained from
Calbiochem (La Jolla, USA). [ 14 C]cinnamic acid
( 1 . 6 - 2 . 0 GBq/mmol) was purchased from Amersham (Braunschweig, FRG). Thin layer chromatography was performed on microcrystalline cellulose
(Macherey & Nagel, Düren, FRG).
t>glu
0
Luteolin 7 - 0 - d i g l u r - 4 ' - 0 - g l u r
Fig. 1. Part of luteolin glucuronide metabolism in rye primary leaves. Sequential glucuronidation of luteolin catalyzed by three specific UDP-glucuronate: flavone glucuronosyltransferases (LGT, LMT, LDT); intermediates are
luteolin 7-O-glucuronide (N), and luteolin 7-O-diglucuronide (R ? ). The end product luteolin 7-0-diglucuronosyl-4'O-glucuronide (R,) is substrate of a specific ß-glucuronidase (ßGL), catalyzing the hydrolysis of glucuronic acid in
position 4'. The product R2 might succumb further
catabolic reactions.
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189 M. Schulz and G. Weissenböck • Luteolin Glucuronide Metabolism in Rye Mesophyll
Preparation of enzyme
extracts
Unless stated otherwise, all steps were carried out
at 4 °C. Primary leaves (3 to 10 days old) or their
1.5 cm sections were frozen in liquid nitrogen and
ground to a powder. Separated leaf tissues and
mesophyll protoplasts were homogenized with
quartz sand in 1—2 ml of extraction buffer (0.1 M KPi, pH 7.0, 1 mM 2-ME). Powders were extracted
with 10 ml buffer/g leaves. Extraction mixtures contained 15% Polyclar A T and 50% Dowex A G 1 x 2
(200-400 mesh) weight to fresh weight of plant
material, for binding phenolic constituents. After
stirring (30 min), the mixtures were squeezed
through Miracloth and centrifuged for 10 min at
40,000 x g . Protein solutions used were without
significant contaminations of phenolics.
Enzyme
assays
The activity of the ß-glucuronidase was measured
as described previously [9], but reactions were stopped within linearity after three hours. UDP-glucuronate: flavone glucuronosyltransferases were assayed
as described elsewhere [10] with the following
modifications: UDP-glucuronate: luteolin 7 - 0 glucuronosyltransferase (LGT) was incubated in
10 mM citrate buffer pH 5.5, 10 mM 2-ME, and
UDP-glucuronate: luteolin 7-O-diglucuronide 4 ' - 0 glucuronosyltransferase (LDT) in 10 mM citrate buffer pH 6.5, 10 mM 2-ME. Both activities were stopped after 30 min. UDP-glucuronate: luteolin 7-Oglucuronide glucuronosyltransferase (LMT) was
assayed with 10 mM citrate buffer p H 6.0, 10 mM
2-ME, for 15 min. Incubations were performed with
5 — 15 nl protein solutions to determine the transferase activities, and with 10—30 [xl for measurements
of ß-glucuronidase activity. Assays were analyzed
and quantified according to [9, 10] by HPLC, using
Zorbax C-8 (Dupont Instruments, Bad Nauheim,
F R G ) and Lichrosorb RP-8, standard 250-4, cat.
15540 (Merck) columns. In all cases, product formation was linear with time and protein concentration.
Protein was determined by Bradford's method
[14], using BSA as standard. Chlorophyll content of
extracts prepared from epidermal layers and from
mesophyll protoplasts were estimated as described in
[15]. Chlorophyll recovery in isolated protoplasts
was used to calculate protoplast yield.
Feeding experiments with [I4CJcinnamic
determination of radioactivity
acid
and
3, 4 and 5 day old primary leaves (10 of each age)
were prepared and floated on a 50 [xm [ 14 C]cinnamic
acid solution for three hours (13). After the treatment, the leaves were washed three times with water
and cut into sections (see legend of Fig. 4). The leaf
sections were extracted for flavonoids [7, 13]. Separations of the different phenolic compounds were
performed by chromatography on SC 6 polyamide
columns using sequentially H 2 0 , C H 3 O H , and
CH3OH with 0.01% NH4OH as eluents. C H 3 O H /
NH4OH fractions, containing R] and R 2 , were
evaporated and dissolved in 0.4 ml H 2 0 . Aliquots
(100 ^1) were separated by TLC, using two solvent
systems: 1. C H C 1 3 / H O A C ( 3 : 2 , H 2 O S A T . ) and 2. 15%
H O A c . R] and R 2 were identified by cochromatography with authentic compounds. Radioactively labelled compounds on TLC plates were located using a
T L C scanner with a TLC linear analyzer LB 2820/1
and a display unit model 7940 from Berthold (Wildbad, F R G ) . Radioactivity was further determined
with a Packard Instruments liquid scintillation spectrometer Tricarb model 3380, using Unisolve type I
cocktail from Zinsser (Frankfurt, F R G ) .
Result and Discussion
1. Profiles of enzyme
activities
Previous work demonstrated that each U D P glucuronate: flavone glucuronosyltransferase has a
high affinity for its natural substrate luteolin, luteolin
7-O-glucuronide (N), and luteolin-7-O-diglucuronide (R 2 ), respectively [10]. No other transferase
capable of catalyzing one of these reactions was detected. High affinity and specifity to the natural substrate R j was also found for the ß-glucuronidase, that
produces R 2 [9]. The latter compound is an intermediate of the anabolic as well as of the catabolic
sequence ( c f . Fig. 1). Using the standard assays,
there was no evidence of reverse reactions, catalyzed
by the corresponding enzymes.
These results were prerequisits to the study of
enzyme activities in developing primary leaves and
their specific distribution in the leaf. The activities of
the four enzymes were measured over a period of
7 days, starting with 3 day old seedlings. In crude
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190
M. Schulz and G. Weissenböck • Luteolin Glucuronide Metabolism in Rye Mesophyll
protein extracts of the leaves, the product of LGT,
luteolin 7-O-glucuronide (N), did not substantially
accumulate, since it was further glucuronidated by
LMT and LDT. Therefore the over all activity was
measured under optimized conditions for LGT in
crude extracts. Fig. 2a, b, c exhibits the activity profiles of the glucuronosyltransferases from the primary leaves as a function of plant age. The over all
activity (Fig. 2 a) reaches its maximum (38 pkat) on
the fourth day, declining to 45% of the maximal
amount by the tenth day. A similar profile of activity, but with a less rapid decrease was found for the
L D T (Fig. 2c), (4 d: 43 pkat, 10 d: 30%). Individual
determinations of the LMT activity with R 2 as the
major reaction product could be made using short
incubation times. The LMT enzyme exhibits highest
activity at the fifth day (Fig. 2b), (107 pkat, 10 d:
27%).
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Maximal activity of the sequential glucuronidation
is revealed in leaf stages with nearly linear R,
accumulation [6]. In older leaves, after day 5, R]
biosynthesis is considerably reduced, and the amount
of R j decreases rapidly. Nevertheless the UDPglucuronate: flavone glucuronosyltransferases are
probably not the rate limiting enzymes of R! biosynthesis. Chalcone synthase (CHS), the key enzyme of
flavonoid biosynthesis may be rate limiting as was
shown with oat primary leaves [11].
The reduction of glucuronosyltransferase activities
alone cannot explain the rapid decrease of Rj after
the 5 th day of development, but the R r reduction
would be explained by a prevailence of ß-glucuronidase activity at later stages in development. In fact,
this activity increases during early developmental
phase reaching its maximum between day 5 and 6
(Fig. 2d). No reduction was observed up to day 10.
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Fig. 2. Leaf age dependent
profiles of the enzyme activities: a) LGT (over all activity), b) LMT, c) LDT, and
d) ß-GL. Values are the averages of three different series
of experiments.
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191 M. Schulz and G. Weissenböck • Luteolin Glucuronide Metabolism in Rye Mesophyll
The major part of the R 2 quantity accumulating in
the primary leaf may be due to this hydrolytic activity. Deglycosilation of flavonoid glycosides is a
known mechanism to initiate further catabolic reactions of the aglyca [16, 17]. Deglucuronidation of R,
to R2 might be the first step of a catabolic sequence.
Since R 2 remains a minor compound during all stages
of leaf development (cf. [6]), and further catabolites
of R[ or R 2 were not detected, R 2 may turnover or
degradate rapidly by still unknown reactions. From
results of in vitro measurements, polymerization of
R 2 , catalyzed by peroxidases, cannot be excluded
(data not shown).
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2. Distribution of enzyme
activities
Rye primary leaves exhibit a continuously proceeding cell differentiation and expansion from the
meristematic basal region towards the tip as a result
of basiplastic growth. The major amount of R t was
found in the middle and upper part of the leaf (4 to 6
day old), and it was concluded, that R] is synthesized
and accumulated primarily in the expansion zone
of the leaf [6], In 4 and 4.5 day old leaves, LGT
(over all activity) is present in similar amounts
throughout the leaf. At day 6, the activity is retained
in the middle to the lower leaf parts, but is reduced in
the upper ones (Fig. 3a). A comparable distribution
was found for individual measurements of LMT and
LDT activities (data not shown). The activity of ßglucuronidase increases from the base to the tip, and
it is not reduced during further development
(Fig. 3b). These in vitro data may indicate that the
hydrolysis of R] to R 2 occurs in nearly mature or
mature tissue. The results obtained from feeding
experiments with [ 14 C]cinnamic acid, a precursor of
flavonoid biosynthesis, support this interpretation.
Highest amounts of labelled R] plus R 2 were found in
the middle sections of the leaf. In the upper part, the
yield of labelled luteolin glucuronides was drastically
diminished (Fig. 4).
Luteolinglucuronides (R! and R 2 ) are only present
in the mesophyll of rye primary leaves [6, 8]. The
intermediate, N, is not detectable at all. To study the
tissue distribution of the transferases and of the ßglucuronidase, peeled lower and upper epidermal
layers and the corresponding leaf complements including mesophyll cells were measured for enzyme
activities. Fig. 5 a presents the tissue-specific distribution of LGT (over all) activity for 4 day old
3
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Fig. 3. Rye primary leaves of different ages were cut into
1.5 cm sections and extracted to determine enzyme activities. Fig. 3a represents the distribution of the relative
glucuronosyltransferase activity (LGT, over all reaction).
Comparable data were obtained for individual measurements of LMT and LDT activities (not shown). Fig. 3b
shows the distribution of relative ß-glucuronidase activity
throughout the leaf. For 100% values of the different ages,
compare profiles of activities in Fig. 2. Values represent
the means of five (3 a) and three (3 b) different series of
experiments with maximal variation of ±5%.
leaves. This result is representative also for the separately measured LMT and LDT activities as well as
for other stages of the leaf development. The enzymes showed a clear localization in the mesophyll;
extracts of epidermal layers contained only ca. 5% of
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192
M. Schulz and G. Weissenböck • Luteolin Glucuronide Metabolism in Rye Mesophyll
©
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Fig. 4. Feeding experiments with [ C]cinnamic acid using 3, 4, and 5 day old leaves. Ten treated leaves of each stage were
cut into 1 cm section and the remaining tip. Identical sections were extracted and labelled flavonoids R, and R 2 were
determined (dpm, nmol). The distribution of radioactively labelled R, plus R : is shown in 4a. b, and c; 4d exhibits the
total amount of labelled R, plus R 2 (nmol) per 10 leaves vs. leaf age.
the total activity. A similar distribution was found for
the ß-glucuronidase; about 90% of the activity was
located in the autotrophic mesophyll and ca. 10% in
each epidermal layer (Fig. 5 b). The activities present in epidermal extracts are probably due to con-
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Fig. 5. Tissue-specific distribution of a) LGT (over all activity) for 4 day old leaves and b) ß-glucuronidase for 5 day
old leaves (%). T: total leaf, LC: leaf complement (mesophyll plus upper epidermis, lower epidermis peeled off),
UC: leaf complement (mesophyll plus lower epidermis, upper epidermis peeled off), LE: lower epidermis, UE: upper
epidermis.
taminations with some remaining mesophyll, since
mesophyll cells were observed microscopically sticking to the peels. The remaining activities found in
epidermal preparations coincided with chlorophyll
contaminations.
To obtain further evidence for tissue-specificity,
mesophyll protoplasts were isolated and enzyme activities were determined. Nearly 80% of the glucuronosyltransferase activities, but only 20—25% of the
ß-glucuronidase activity could be recovered, compared to whole leaves (protoplast yield was 90%).
Mixing experiments did not point to the presence of
endogenous inhibitors for either glucuronosyltransferases or ß-glucuronidase. The reason for ßglucuronidase loss is still unclear. Vascular bundles
did not contain any of the activities.
The age dependent profiles of enzyme activities
and their specific distribution reflect the dynamic accumulation pattern of the luteolin glucuronides in rye
primary leaves. A strong relationship between a major part of R) metabolism and the ontogenesis of the
leaf was demonstrated. The properties of the glucuronosyltransferases point to a channeling of the
luteolin glucuronidation [10]. Neither luteolin nor
luteolin 7-O-glucuronide (N) are detectable in the
primary leaves of any stage. An ordered interaction
between anabolic and catabolic sequences of the Ri
metabolism is only possible by a strict subcellular
separation of the different reactions. Therefore, our
future studies will concentrate on the subcellular
localization of R] metabolism in leaf mesophyll cells.
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193 M. Schulz and G. Weissenböck • Luteolin Glucuronide Metabolism in Rye Mesophyll
T h e f u r t h e r c a t a b o l i s m of R 2 will be a n o t h e r c e n t r e
of interest. F u n c t i o n s of t h e luteolin g l u c u r o n i d e s in
rye p r i m a r y leaves a r e still u n c l e a r . K n o w n f u n c t i o n s
of f l a v o n o i d s in g e n e r a l a r e f o r e x a m p l e , p r o t e c t i o n
against h a r m f u l U V r a d i a t i o n o r microbial a t t a c k
Acknowledgements
[18]. R e c e n t l y , a novel f u n c t i o n of luteolin w a s
f o u n d . This c o m p o u n d m a y serve to c o n t r o l t h e expression of Rhizobium
meliloti
nodulation genes
[19]. F u r t h e r studies of e x o g e n o u s i n f l u e n c e s o n R j
Financial s u p p o r t by t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t is gratefully a c k n o w l e d g e d . W e a r e g r a t e f u l t o P r o f . T . O s b o r n ( M a d i s o n , W I , U S A ) f o r revising t h e English version of the m a n u s c r i p t .
1] K. Hahlbrock, The Biochemistry of Plants, Vol. 7,
Chapt. 14 (P. K. Stumpf and E. E. Conn, eds.),
Academic Press, New York, London 1981.
2] R. Wiermann, The Biochemistry of Plants, Vol. 7,
Chapt. 4 (P. K. Stumpf and E. E. Conn, eds.),
Academic Press, New York, London 1981.
3] G. Weissenböck, Z. Pflanzenphysiol. 74, 298-326
(1971).
4] G. Weissenböck and H. Reznik, Z. Pflanzenphysiol.
63, 114-130 (1970).
5] N. Amrhein and M. H. Zenk, Z. Pflanzenphysiol. 63,
384-388 (1970).
6] D. Strack, B. Meurer, and G. Weissenböck, Z. Pflanzenphysiol. 108, 131-141 (1982).
7] M. Schulz, D. Strack, G. Weissenböck, K. R. Markham, G. Dellamonica, and J. Chopin, Phytochemistry
24, 343-345 (1985).
8] M. Schulz and G. Weissenböck, Z. Naturforsch. 41c,
2 2 - 2 7 (1986).
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m e t a b o l i s m will h e l p to elucidate t h e role of t h e
luteolin g l u c u r o n i d e s in rye p r i m a r y leaves.
Unauthenticated
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