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Biochemical Systematics and Ecology, Vol. 19, No. 7, pp. 577-585, 1991. 0305-1978/91 $3.00+0.00 Printed in Great Britain. © 1991 Pergamon Press plc.
Individual Variation in the Flavonoid Aglycones Excreted on the Leaves of Alnus glutinosa: Influence of Culture Conditions and Origin of Flavonoid Natural Diversity in Wild Specimens
C. DANIERE,* J. F. GONNET~r~ and A. MOIROUD* *Laboratoire d'Ecologie Microbienne et tLaboratoire de Biologie Micromol~culaire et Phytochimie, Universit~ Claude Bernard-Lyon I, 43 Bid 11 Novembre 1918, F-69622 Villeurbanne Cedex, France
Key Word Index--Alnus glutinosa; Betulaceae; flavonoid variation; Alnus-Frankia symbiosis.
Abstract--This work presents a further step in the analysis of flavonoid aglycones excreted in the leaves of Alnus glutinosa as an aid to the characterization of natural specimens of this species. The stability in the flavonoid pattern of specimens growing under different environmental and biological conditions has been examined. The expression of the flavonoid metabolism in each surveyed individual is shown to remain unchanged over the course of a growing season and over successive years. Specimens from vegetative multiplication (cuttings, grafts) show a qualitative composition of the excreted flavonoids mixture identical to that of the parent tree. Individuals of clonal origins (three grown from in vitro bud culture) display identical flavonoid patterns; however, some minor variations are recorded. The fiavonoid profiles displayed by individuals of two clonal origins, inoculated with several strains of Frankia of different efficiency in fixing nitrogen, remain unaffected, when compared with the pattern of the reference samples without nodules.
In contrast, specimens grown from seeds (collected from different trees, a single tree, or even a single catkin) consistently display important qualitative and quantitative variations in their flavonoid content. However, in some cases, relationships between profiles of a mother tree and its progeny were detectable. It appears that the individual flavonoid content in Alnus glutinosa remains unaffected by the culture conditions and by the process of vegetative multiplication. The natural diversity observed in specimens from sexual reproduction is mainly interpreted in terms of its strictly allogamous reproductive mode. The analysis of the fiavonoid material excreted on leaves appears to be a usable tool for the characterization of trees in the selection of individuals of Alnus glutinosa for experimental symbiosis with Frankia.
Introduction A/nus g/utinosa (European black alder) has been studied for its wood characteristics and beneficial effects on wet or nutrient-poor soils [1, 2]. Its rapid biomass production, as with all other A/nus species, results from a symbiotic association with the actino- mycete Frank/a in root nodules [3], the bacteria fixing substantial amounts of atmospheric nitrogen [4-6]. The symbiotic performance of the A/nus-Frankia associ- ation is strongly dependent on the capacities of each partner [7-9]. Frank/a has been extensively investigated in order to identify and characterize suitable strains [10-12]. By contrast, only a few methods of recognition have been developed for A/nus [13, 14].
In a recent paper [15] we reported a new attempt to characterize A/nus g/utinosa individuals, by means of the analysis of the flavonoid mixture excreted by their leaves. A total of 85 individuals of A/nus g/utinosa from 11 natural stands were surveyed: 20 flavonoid aglycones (mainly methylated flavones and flavonols) were detected. In that sampling, individual flavonoid patterns appeared to be extensively and continuously variable, and there was no relation of the flavonoid pattern distribution with any readily accessible parameter, especially the geographic origin of the specimens.
This unstructured natural diversity could be interpreted in terms of two factors: firstly, as a result of the influence of the parameters of natural growth. A "wild" alder represents an heterogeneous and complex system as it results from the functioning of
~:Author to whom correspondence should be addressed.
(Received 11 January 1991 )
577
578 C. DANIERE ETAL.
several partners: all the examined alders appeared simultaneously mycorrhizal and nodulated by native strains of Frankia and additionally grew on different soils; secondly, as the consequence of the allogamous reproductive mode of Alnus glutinosa [16].
Recently, some external parameters have been tested for their possible qualitative and quantitative effects on the flavonoid aglycone pattern in individual samples in order to determine whether the flavonoid analysis is stable and therefore usable in the characterization of trees. In addition, the flavonoid profiles of specimens originated from vegetative multiplication have been compared with those of the mother trees. Finally, wild specimens and their progeny from sexual reproduction (seeds) have been surveyed for their leaf flavonoid mixture.
The leaf material analysed in this study originated from several different sources: (i) six sprouts of a black alder stump growing in Marennes (France). The leaves of each sprout and of the mother tree were collected in early spring and analysed separately. (ii) Four grafts, grown outdoors and originating from a wild alder tree, termed as #G; the leaves of each graft and of their mother tree, collected in summer, were surveyed. (iii) A field-growing black alder (at Luzinay, France) identified as tree #L was examined for its flavonoid content many times during the 1986 growing period; it was reinvesti- gated later in spring 1987 and again in spring 1988. It was also used as a source of cuttings, among which seven were sampled. (iv) Plantlets of three different clonal origins #A, #B and #C, from in vitro bud culture; the leaves from the tree from which individuals of the clone A originated, were also analysed. All the plantlets of the three clones (35 for A, 58 for B and 107 for C) were examined after they had been growing for six months under controlled conditions. In addition, 20 plantlets out of 107 of clone C were analysed at 10 weeks, eight and 10 months of age. In order to test the influence of Frankia symbiosis on the flavonoid metabolism, plantlets of clones A and C were inoculated by two pure strains of the symbiotic bacteria. The strains of Frankia ULF0401601 and ULQ0102001007 (trival names AVN16a and ACN1AG, respectively) were selected on the basis of their different efficiency in fixing nitrogen [17] and their resistance to low pH value [18]: 10 plantlets of each clone were inoculated with strain AVN16a; 10 plantlets of each clone were inoculated with strain ACN1AG; 15 plantlets of each clone were not inoculated and kept as reference samples. Four months after nodulation, increase in height of plantlets was recorded and their leaves were again analysed for their flavonoid content. (v) Seedlings from seeds collected on four black alder trees growing in field (open pollinated). The flavonoid content of these mother trees has already been analysed [15]: they appeared under #8, #10, #15 and #50 in the data table published in that paper. These trees were selected on the basis of their very different flavonoid profiles. The seeds were randomly collected: 19 from tree #10, 65 from #15 and 35 from #50. The nine seedlings from tree #8, were harvested on a single female catkin. Leaves of respective mother trees were also analysed again.
All plantlets developed in culture rooms were allowed six months of growth before they were investigated for their flavonoid content. The leaf material from field mature Alnus glutinosa was harvested and analysed the same day. The flavonoid aglycone mixture exuded from the leaves was extracted and analysed following a method recently described [15].
Materials and Methods Cultivation of plants. All plants from seeds, cuttings and in vitro bud culture were grown under the same conditions. The seeds were germinated on moistened filter paper in Petri dishes. The cuttings--consisting of one internode with one leaf and axillary bud--were treated with commercial indole butyric acid (Exuberone: Rhone-Poulenc), and rooted in perlite with about 95% ambiant relative atmospheric moisture.
The seedlings, at the cotyledons stage, the rooted cuttings and the rooted plantlets from in vitro bud culture were potted into an inert substrate (expanded clay). The plants were cultivated in a culture room with a 16-h daily illumination (daylight type, supplied by fluorescent lamps, General Electric F 48 PG 17/CWX,
FLAVONOID AGLYCONES IN ALNUS GLUTINOSA 579
supplemented by a set of 400 W Phytoclaude mercury lamps). They were supplied weekly with MAIROL (GerbL~der Maier, Heidenheim) fertilizer containing mineral nitrogen. Maximum and minimum temperatures were 24°C (day) and 18°C (night).
The Frankia culture and preparation of inoculum were performed according to the method previously described [18]. Two weeks prior to inoculation, N-fertilization of the plantlets in each clone was stopped to minimize the nitrogen inhibition of Frankia infection; then a N-free mineral solution was provided weekly. Individual extraction of flavonoids and the study of the profiles were performed following a method previously described [15].
Mathematical treatment. The Discriminant Factorial Analysis (DFA) of data has been performed by "AFD" module in the STAT ITCF (V.4.0) statistical package running on an IBM PC-AT computer.
Results The previously recorded 20 flavonoid aglycones have been detected again in this new alder sampling. Of these, 13 are fully identified and partial structural information is available for the others [15]. An individual profile was established from each flavonoid extract as previously described [15]. Compounds 19 and 20 were finally not considered because they were not always readily observable (as minor chromatographic spots). Compounds 4, 10 and 12 were consistently not detected in profiles of seedlings originated from Alnus glutinosa #10, #15 and #50. Finally, the flavonoid data in this sampling was parted in two definitive tables: one, (including 15 columns) for seedlings from Alnus glutinosa #10, #15 and #50 and the other (with 18 columns) for all other specimens analysed. A descriptive multivariate method, namely Discriminant Factorial Analysis (DFA), was applied to the data in the first table.
Influence of growing season The aglycone profile of wild Alnus glutinosa #L is presented in Table 1. The leaves
examined from spring 1986 to autumn 1986 showed no difference in the composition of their flavonoids. However, at the end of summer, the extracts became "dirty" and appeared contaminated by chlorophyll pigments making the chromatograms less easily readable. The composition of the exuded flavonoid mixture continued to remain unchanged during spring 1986, 1987 and 1988.
Effects of vegetative multiplication of samples Cuttings. The flavonoid patterns of tree # L (Luzinay), and seven cuttings originated
from it, were identical (Table 1). Sprouts. The flavonid profiles of the six sprouts developed from the same old stump
(in Marennes) were identical (Table 1). Grafts. The four grafts originated from tree #G also displayed identical profiles,
which were very close to the profile of tree #G (Table 1). In vitro bud culture. The main flavonoid profile of the plantlets from the three clones
A, B and C is presented in Table 1. The qualitative and relative quantitative distributions of the flavonoid compounds are identical in the 35 plantlets from clone A. However, their profiles differ from the pattern of mother tree #A, in the quantitative accumula- tion of some molecules (compounds 2, 6, 16 and 18 especially). For the other two clones, B and C, the leaves of mother trees were not available. Fifty-eight and 107 plantlets, respectively, were surveyed. In most cases, the flavonoid patterns of the specimens of the same clonal origin were identical, but a few exceptions are noted: four plantlets out of 58 for clone B and three plantlets out of 107 for clone C displayed aglycone profiles with minor (clone B) or major (clone C) differences (Table 1). Among the different plantlets in clone B, two are identical as are the three plantlets of clone C.
Regarding the influence of growing stage, analysis of plantlets from clone C performed after 10 weeks in culture, showed only six minor spots corresponding to compounds 4, 8, 9, 11, 16 and 17. Leaves harvested in 20 plantlets of clone C at eight and 10 months of culture displayed an identical flavonoid pattern to that recorded when they were six months old.
TA
BLE
1.
DIS
TR
IBU
TIO
N (A
S P
ER
CE
NT
AG
E) O
F F
LAV
ON
OID
AG
LYC
ON
ES
IN I
ND
IVID
UA
L P
RO
FIL
ES
OF
WIL
D A
ND
CU
LTIV
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ED
SP
EC
IME
NS
OF
AL
NU
S G
LUT
INO
SA
Agl
ycon
e
Indi
vidu
als
n x
/n*
1 2
3 4
5 6
7 8
9 10
11
12
13
14
15
16
17
18
Indi
vidu
als
fro
m v
eget
ativ
e m
ultip
licat
ion
Tre
e #
L
1 1.
3 1.
3 1.
3 1.
3 5.
1 5.
1 7.
6 7.
6 5.
1 0
19
0 3.
8 3.
8 6.
3 19
12
.7
0
Cut
tings
fro
m "
# L
" 7
7/7
1.
3 1.
3 1.
3 1.
3 5.
1 5.
1 7.
6 7.
6 5.
1 0
19
0 3.
8 3.
8 6.
3 19
12
.7
0
Spr
outs
of
stem
fro
m "
Mar
enne
s"
6 6
/6
2.6
1.3
2.6
1.3
10.5
5.
3 7.
9 7.
9 3.
9 2.
6 19
.7
0 2.
6 2.
6 2.
6 15
.8
10.5
0
Tre
e #
G
1 1.
4 0.
7 1.
4 0.
7 8.
6 2.
9 11
.4
7.1
4.3
0 21
.4
0 2.
9 1.
4 4.
3 17
.1
14.3
0
Gra
fts f
rom
# G
4
4/4
2.
7 1.
4 2.
7 2.
7 8.
1 2.
7 10
.8
6.8
4.1
0 20
.3
O
2.7
1.4
4.1
16.2
13
.5
0
Tre
e #
A
1 0
6.3
12.7
3.
2 0
6.3
9.5
9.5
6.3
0 23
.8
0 0
0 0
3.2
15.9
3.
2
Clo
ne A
35
35
/35
0 2.
9 11
.8
5.9
0 11
.8
11.8
11
.8
5.9
0 17
.6
0 0
0 0
8.8
11.8
0
Clo
ne B
58
54
/58
1.7
3.4
0 0
1.7
3.4
17.2
17
.2
1.7
0 13
.8
0 6.
9 0
0 6.
9 25
.9
0
1/5
8
2.4
4.9
0 0
2.4
9.8
14.6
14
.6
2.4
0 14
.6
0 9.
8 0
0 9.
8 14
.6
0
1/5
8
1.6
3.2
3.2
O
1.6
3.2
9.7
16.1
1.
6 3.
2 19
.4
0 6.
5 0
0 6.
5 24
.2
0
2/5
8
1.5
3 0
0 6
6 14
.9
14.9
1.
5 O
14
.9
O
6 3
0 B
22
.4
0
Clo
ne C
10
7 10
4/10
7 2.
6 5.
1 5.
1 19
.2
0 5.
1 2.
6 10
.3
6.4
0 19
.2
0 0
0 0
5.1
19.2
0
3/1
07
0
3.6
0 0
0 7.
3 3.
6 14
.5
9.1
0 27
.3
0 0
0 0
7.3
27.3
0
Indi
vidu
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fro
m s
exua
l re
prod
uctio
n
Tre
e #
8
1 2.
4 2.
4 2.
4 0
4.8
6 7.
2 7.
2 1.
2 0
18.1
1.
2 7.
2 1.
2 4.
8 18
.1
14.5
1.
2
Pla
nts
fro
m
9 1
/9
0 5.
6 2.
8 0
0 5.
6 11
.3
16.9
0
0 16
.9
0 2.
8 1.
4 2.
8 28
.2
5.6
0
seed
s o
f a
1/9
0
5.7
2.9
0 0
5.7
11.4
17
.1
0 0
28.6
0
2.9
2.9
2.9
17.1
2.
9 0
sing
le c
atki
n 1
/9
0 13
.3
6.7
0 0
6.7
6.7
20
0 0
13.3
0
0 0
3,3
26.7
3.
3 0
fro
m #
8
1/9
0
13.8
6.
9 0
0 3.
4 3.
4 13
.8
3.4
0 27
.6
0 0
0 0
20.7
6.
9 0
1/9
0
10.8
5.
4 0
0 5.
4 10
.8
21.6
0
0 16
.2
0 0
2.7
5.4
21.6
0
0
1/9
0
12.9
6.
5 0
0 0
6.5
12.9
3.
2 0
25.8
0
0 3.
2 3.
2 19
.4
6.5
0
1/9
0
9.3
4.7
0 0
9.3
9.3
18.6
4.
7 0
14
0 0
0 0
27.9
2.
3 0
1/9
0
6.2
3.1
6.2
0 9.
2 6.
2 18
.5
3.1
0 18
.5
0 0
0 1.
5 24
.6
3.1
0
1/9
0
8.3
8.3
0 0
4.2
8.3
20.8
0
0 20
.8
O
0 0
O
25
4.2
0
Tre
e #
10
1
7.1
1.2
11.8
14
.1
0 2.
4 5.
9 9.
4 7.
1 0
17.6
0
1.2
1.2
0 17
.6
1.2
2.4
Tre
e #
15
1
2.2
1.1
1.1
0 8.
6 3.
2 6.
5 2.
2 6.
5 1.
1 16
.1
3.2
3.2
5.4
6.5
16.1
16
.1
1.1
p
Tre
e #
50
1
1 2.
1 O
1
15.6
5.
2 6.
3 12
.5
O
3.1
15.6
2.
1 6.
3 4.
2 3.
1 15
.6
4.2
0.7
n*
- N
umbe
r o
f su
rvey
ed i
ndiv
idua
ls;
x -
nu
mb
er
of
indi
vidu
als
pres
entin
g th
e s
ame
flavo
no
id p
atte
rn.
FLAVONOID AGLYCONES IN ALNUS GLUTINOSA 581
Influence of nodulation In nodulated individuals of clones A and C with ACN1AG Frankia strain, the symbiosis
stimulated a noticeable increase in their height (Table 2). The qualitative composition of the excreted flavonoid mixture of the nodulated plantlets of clones A and C remained identical when compared with the pattern originally displayed before inoculation and with reference samples without nodules in both clonal samplings. However, the total amount of flavonoids produced by the ACN1AG nodulated plantlets of both clones was higher (mean value: 12.67%o vs 5.45%0 dry weight, measured on fully developed subterminal leaves).
TABLE 2. HEIGHT (cm, MEAN VALUE +S.D.) OF INDIVIDUALS OF CLONES A AND C, WITH ACN1AG AND AVN16a FRANKIA STRAINS, RECORDED FOUR MONTHS AFTER THE INOCULATION OF PLANTLETS
Clone A Clone C
Before inoculation 12.8"±1.42 10.5~_+3.1 ACN1AG 42.7t+10.8 40.51"+10.5 AVN16a 19.4t + 5.3 16.41"_+4.9 Reference sample 18.2:~±1.8 16.0~_+2.7
*35 plants; t l0 plants; :~15 plants,
Flavonoid patterns in wild samples: influence of reproductive mode The flavonoid data for seedlings originated from Alnus glutinosa trees # 10, # 15 and
#50 were recorded in a data matrix consisting of 119 rows (individual plants) and 15 columns (quantitative variables: relative amount of flavonoid aglycones). The origin of specimens (qualitative variable: tree #10, #15 or #50) was included in an additional column. Because of its size, this data matrix is not supplied here.* In order to show the particular flavonoid trend(s) in each group of individuals (from each tree), the data matrix was submitted to a factorial calculation method: Discriminant Factorial Analysis (DFA). It is a multivariate method revealing the link(s) between a qualitative variable and a set of qualitative variables. In maximizing the inter-group and intra-group variances ratio, the calculation reveals the peculiar features (here, flavonoid aglycones) which are discriminating each defined group of individuals (i.e. from a given tree) over against the ensemble of all other groups.
The coefficients of correlation of flavonoid aglycones with the two factorial axes in DFA are listed in Table 3. The most discriminative patterns along axis 1 appear to be represented by an assemblage of compounds 8, 13, 18, 17 and 1 (highest in samples at the positive end of the axis) vs those including compounds 9, 16, 11 and also 15 and 7 (negative part of the factorial axis 1). On factorial axis 2, the main contribution is supported by aglycones 14 (positive end) vs 3 and 6 (negative end).
Regarding individuals, this calculation results in the graph presented in Fig. 1: individuals from t~ee #50 definitively part from individuals from trees #15 and #10, according to the following trends: (i) #50 is discriminated by patterns featuring higher levels of flavones and/or methyl-3 flavonols with a 4'-mono hydroxylated B ring, e.g. kaempferol-3-methyl ether (8) and apigenin-4'-methyl ether (13) and their 6-methoxylated derivatives (4'-methyl ether for the first, 17, 18). (ii) Specimens from trees #15 and #10 are discriminated by lower levels of the aforementioned compounds and a stronger presence of flavonoids bearing an additional substituent at C3', i.e. with a 3",4"-O-disubstituted B ring scheme: flavone 6-hydroxy-luteolin-6,4'- dimethyl ether (9), and its corresponding flavonol-3-methyl ether, quercetagetin-
*This data matrix is available upon request from the Editorial Office of Biochemical Systematics and Ecology.
582 C. DANIERE ETAL.
TABLE 3. CORRELATION OF RELATIVE AMOUNTS OF 15 AGLYCONES WITH DISCRIMINANT FACTORIAL AXES 1 AND 2 OF DFA
Aglycone
Factorial axes 1 2 3 5 6 7 8 9 11 13 14 15 16 17 18
Axis 1 0.97 0,69 --0,02 0.65 --0,25 --0.82 0,99 --0.99 --0,89 0.98 --0,06 --0.83 --0,97 0.96 0.98 Eigen value: 5.51 Inertia: 90.9%
Axis2 0.23 1 ,00 0,55 0,24 0,28 0.20 Eigen value: 0,55 Inertia: 9,1%
--0.24 --0.72 --0.99 0.76 --0.97 0.57 --0.16 0.07 --0.45
Axe 2 i •
• •
• | • 0 0
o o o l
• o e P O • elP • •
• • • •
• •
Axe1
8 o A A • •
I •
8
8
e.
0 O 0
• Q
-It
*]
FIG. 1. DFA: GRAPHICAL DISTRIBUTION OF INDIVIDUALS FROM THE SEEDS OF ALNUS GLUTINOSA #10 (,k), #15 (O) AND #50 (A) IN THE FACTORIAL ORDINATION FI×F2. (F1 is factorial axis 1; F2 is factorial axis 2).
3,6,4'-trimethyl ether (11). Unidentified aglycone 16 probably shares close structural features with these two aglycones. The main trend in profiles of samples from tree # 15 vs those from tree # 10 is their higher investment in 4',6-dimethyl ethers of 6-hydroxy- lated flavonols: quercetagetin (7) and its 7-methyl ether (14) and 6-hydroxy-kaempferol
FLAVONOID AGLYCONES IN ALNUS GLUTINOSA 583
(15). As for samples from tree #10, they are discriminated by a higher synthesis in flavone luteolin-4'-methyl ether (6) and aglycone 3; they are also completely lacking compounds 14, 15 and 5.
The above flavonoid features describe the general distinctive metabolic trends pre- vailing in the groups of individuals developed from the seeds collected on each tree. They are illustrated by the basic statistics recorded in the distribution of molecules within each group of individuals (Table 4). They correspond to preferential investment into specific steps of biosynthetic pathways: synthesis of mono- vs di-O- substituted B ring flavonoids (tree #50 vs #10, #15) and the methylation/methoxylation of flavone/ flavonol-3-methyl ethers vs flavonols (tree #15 vs #10). However, it must be strongly emphasized that diversity exists in the individual profiles recorded in each group, which is especially important in the progeny of tree #15, since some of them display profiles close to those of tree #10. This mainly results from different combinations of substitution in the O-methylation process, originating both qualitative and quantitative variations within the general flavonoid pattern described for the individuals developed from the seeds of each tree.
Finally, the individual flavonoid profiles recorded in the nine seedlings developed from seeds collected from a single catkin appear to also vary extensively (Table 1); all also appear different from the profile recorded in the mother tree #8. Some compounds (1, 5, 12, 8) present in the mother tree (as minor compounds) are not detected in the progeny. On the other hand, other compounds (2, 4, 9) not detected, or weakly represented in the profile of tree #8, are present in larger amounts. Variations in relative amounts of common molecules (7, 8 and 17 especially) are also noted.
Discussion Careful attention to the origin of variation is necessary before flavonoid patterns can be used in individual characterization within Alnus glutinosa. Under natural conditions, the flavonoid pattern of A. glutinosa #L was stable over the course of a growing season and also over a more extended line period (three consecutive springs). Measurement of flavonoid levels on young leaves at different stages of development show that most of the flavonoid content is synthesized during early growing stages.
This agrees with Wollenweber's [19] observation that most of the glandular structures are differentiated when the leaves are still unfolded and are producing most of their slimy flavonoid secretion as a continuous coating at this stage. However, as noted before, at the end of the summer the chromatograms became less clearly readable; this mainly results from the presence of some dust lime over the flavonoid mixture and, consequently, it is better to examine the flavonoid pattern on leaves collected in spring.
With the specimens from vegetative multiplication, the qualitative composition of excreted flavonoid mixture remains generally unaffected when compared with the pattern originally displayed in spring by the mother tree. However, some exceptions
TABLE 4. BASIC STATISTICS IN THE DISTRIBUTION OF AGLYCONES (EXPRESSED AS RELATIVE AMOUNTS IN PER CENT) IN INDIVIDUALS FROM THE SEEDS OF ALNUS GLUTINOSA #10, #15 AND #50
Aglycone
Group n 1 2 3 5 6 7 8 9 11 13 14 15 16 17 18
#10 19 % 1.09 5.05 3.86 0 S.D. 2.09 2.55 2.33 0
#15 65 % 0.32 4.44 1 .49 0,79 S.D. 1 .38 2 .01 2.30 1.30
#50 35 % 1.69 4.73 1 ,56 1.35 S.D. 1 .58 2 .02 2 .17 1.80
14.87 11.71 12.41 4.94 28.41 0 0 0 13.97 3.68 0 5.80 6.43 5.15 3.97 9.27 0 0 0 4.24 0.99 0 5.78 14.43 7.75 6.89 26.14 0.15 4.04 3.05 19.35 5.37 0 6.66 8.72 4.75 4.73 9.67 1.15 2.58 2.95 8.41 5.01 0 4.33 11.47 17.72 0.99 19.38 4.00 3.43 0 9.26 18.84 1.25 3.94 5.30 3.77 1.54 6.06 5.14 2.49 0 4.39 6.09 1.70
584 C. DANIERE ETAL,
have been recorded: although there were no morphological differences noted in clonal samples of origins B and C, a few individuals (especially three in clone C) displayed "original" flavonoid patterns. In addition, the profiles of individuals in clone A differ slightly from the pattern of their mother tree.
After the nodulation of the individuals from clones A and C with ACN1AG and AVN16a Frankia strains, their flavonoid content was shown to remain identical to that originally displayed before nodulation and with that of the reference individuals with- out nodules growing simultaneously under the same conditions. Thus, the Frankia strains exert no influence on the qualitative composition of flavonoid pattern of an individual.
By contrast, the quantitative production of flavonoids is strongly dependent on the symbiotic efficiency: some nodulated specimens showed a spectacular enhancement in their flavonoid accumulation which can be correlated with a better growth and is shown by the height of the plantlets. These characteristics were especially noticeable in samples nodulated with ACN1AG, a strain recorded as a far better performer than AVN16a in fixing nitrogen. The profiles displayed by very young plantlets (10 weeks old) also showed that only the major consituents (compounds 4, 8, 9, 11, 16 and 17) of the definitive pattern are present.
These results support a gradual expression of flavonoid metabolism; when its qualitative composition is completed it seems then to be stimulated by an enhanced growth resulting from suitable nitrogen nutrition of the plant.
Individual analysis of the progeny from open pollination of three different wild trees (#10, #15 and #50) shows a generalized diversity in the flavonoid profiles of seedlings in each group. However, two groups of individuals, progenies of trees #50 and #10, are characterized by specific general metabolic trends. It must also be emphasized that the flavonoid patterns recorded in most of the samples originated from seeds of trees # 10 and #50 strongly differ from those displayed by their respective mother trees; this is specially noticeable in levels of compounds 5, 8, 11 and 16 in seedlings from tree #50. For samples from seeds of tree # 15, flavonoid profiles appeared far more extensively variable and a general flavonoid feature attached to this group of trees is not readily recordable.
Finally, flavonoid analysis of young plants from seeds issued from a single female catkin (collected from tree #8) also reveals clearly different patterns.
It was previously assumed that the origin of this variation rests on the reproductive mode of Alnus glutinosa [15]: this species uses out-breeding as its unique reproductive mode [16], controlled by sporophytic incompatibility [20]. This is a well-known system to prevent autogamy and even consanguineous relationships [21, 22]. This means that each tree is pollinated by pollen originating from one or more (probably several) tree(s) growing apart. Thus, it is not surprising that most of wild specimens from sexual reproduction are heterozygotic.
As a consequence, a full recognition of genotypes of individuals of Alnus cannot be achieved by means of their flavonoid analysis. However, since the flavonoid profile in clonal individuals remains irrespective of growth conditions, this chemical fingerprint method remains suitable for the characterization of individuals. It is now applied to the recognition and selection of individuals used as host-plants in the realization of experimental Alnus-Frankia symbiosis, especially in the evaluation of the capacities of host-plants in sorting out their endophytic strain(s) among the wild natural surrounding populations of Frankia.
Acknowledgements--Daniel Prat (ENGREF, Nancy, France) for the kind supply of the plantlets from the three clones A, B and C of A/nus g/ut/nosa, and Luc Bouvarel and Eric Teissier du Cros (INRA, Ardon, France) for the kind supply of the leaves from three #G and its grafts. Thanks are also expressed to Maurice Lalonde (Univer- sity Laval, Quebec, Canada) for allowing us to use his proprietary ACN1 AG Frankia strain.
FLAVONOID AGLYCONES IN ALNUS GLUTINOSA 585
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