Pigmentation patterns of modern rose mutants throw light on the flavonoid pathway in Rosa x hybrida

Pergamon

I

0031-9422(94)EOO81-3

Phylochrmwrg. Vol. 36. Ho 5. pp. 1189. I%. 1994 Copyright ‘c: 1994 Elwwer Sncncc Ltd Pnnlcd I” Great Bntaln All t-i@ rescrvcd 003 I 9422,94 57 a, + 0.00

PIGMENTATION PATTERNS OF MODERN ROSE MUTANTSTHROW ON THE FLAVONOID PATHWAY IN ROSAxHYBRIDA

LIGHT

JEAN-PHILIPPE BIOLLEY, MAURICE JAY and GERT FORKMANN* Laboratoire de Biologic Micromokculaire et Phytochimie. I.A.S.B.S.E., Universitt Claude Bernard-LYON I, 43. bd du 11 novembre 1918, F-69622 Villeurbanne Cedex, France; *Technische UniversitHt Miinchen, Lehrstuhl fiir Zierptlanzenbau, Weihenstephan, 8050 Freising 12, Germany (Receiaed

IN HONOUR Key Word Index-Rosa

OF PROFESSOR

22 Nooemher

RAGAI

x hyhrida; Rosaceae;

1993)

K. IBRAHIM’S

rose; mutant;

SIXTY-FIFTH

flavonoids;

BIRTHDAY

biosynthesis.

Abstract-The petal flavonoid patterns of four modern rose varieties (Rosa x hybrida) and six of their spontaneous mutants were characterized. Native glycosides of cyanidin, pelargonidin, quercetin and kaempferol were precisely quantified. Mutations could strongly enhance flavonol glycoside (quercetin or kaempferol), as well as anthocyanin

concentrations. The increase of the total flavonol concentrations, without changes in the balance between kaempferol and quercetin, also led to new distributions between the different flavonol glycosides. The opening of the pelargonidin route, the decrease of anthocyanin concentration, the modification of the division between mono- and disubstituted flavonoids also arose by mutations. All these changes are discussed with special regard to the current knowledge of flavonoid pathway established in other ornamental species.

INTRODUmION Spontaneous and radiation-induced are interesting both for breeders

colour

mutations

and scientists. For breeders, mutations are considered as an easy and cheap way of varietal creation which can lead to new cultivars without strongly affecting the agronomical and physiological features of the original cultivar. Frequently spontaneous mutations have led to numerous commercial rose mutants that show flower colours different from those of the original varieties [ 11. Gamma rays, particularly efficient in inducing (with high frequency) changes in the petal pigment content of roses [2,3], could contribute actively to the flower colour diversification of those rose cultivars with especially valuable agronomical features. For scientists, colour mutants are tools for elucidating the biosynthetic pathways of flavonoids involved in the in uioo expression of petal colour [4-61. A chemicogenetic approach using enzymology can provide evidence of correlations between single genes and particular enzymes involved in individual steps of flavonoid biosynthesis [7,8]. Most of this work was carried out on Petunia [9-l 13, Matthiola [12-153, Antirrhinum [16] and Dianthus [17-201. This biochemical knowledge can be particularly helpful on one hand to drive classical breeding methods, and on the other hand to increase colour range of a plant species by genetic engineering. Advances concerning relations between flavonoid biosynthesis and genetic engineering implied in the colour of ornamental plants were recently reviewed [6,21-231.

In spite of economical importance of roses, the pathway leading lo petal accumulation of both flavonol glycosides and anthocyanins has not yet been studied in the genus Rosa. Although it has been assumed that the flavonoid pigment content of parent varieties can be a useful basis for breeding because inheritance of each pigment seems to be mainly controlled by additive gene action [24-261, genetic interpretation of this flavonoid biosynthesis could be complicated by the tetraploid level of the modern Rosa x hybrida. As a rule, the rose flavonoid pathway is supposed to be similar to that described in other ornamental species, e.g. Petunia, Matthiola or Dianthus (Fig. 1). From these investigations, some enzymatic steps are clear. Total amounts of precursors feeding the flavonoid pathway are initially controlled by two enzymes: chalcone synthase (CHS) and chalcone isomerase (CHI); however, it is noticeable that spontaneous isomerization of chalcone to flavanone can occur (in particular biochemical surroundings) without active CHI [27]. For roses, several questions are not yet elucidated: spatial and timing expressions of genes involved in the synthesis of the different classes of flavonoids, the precise step to which flavonoid 3’-hydroxylase (F3’H) preferentially works (flavanone or dihydroflavonol), substrate specificity of some key enzymes, e.g. dihydroflavonol 4-reductase (DFR) and the functioning of a hypothetical route leading lo C-3’,4’-dihydroxylated flavonoids independently of the activity of F3’H. with direct incorporation of caffeoyl-CoA into the flavonoid skeleton. The fact that the accumulation of C-3’,4’-disubstituted

1190

J.-P.

BIOLLEY er a/

icyhtion...)

Fig. 1. Flavonoid biogenesis: principal enzymic steps leadmg to flavonois and anthocyanins. Only C-4’monohydroxylated and C-3’,4’-dihydroxylated routes arc presented (accumulation of C-3’,4’.5’-trihydroxylated flavonoids was never reported in the petals of Rosa x ~y~ri~u). Enzymes involved: chalcone synthase (CHS), chalcone isomerase (CHI). flavanone 3-hydroxylase (F3H). flavonol synthase (FLS). dihydroflavonol Creductase (DFR). flavonoid 3’-hydroxylase (F3.H).

flavonoids is mainly due to a C-3’-hydroxylation process at the C-15 stage due to F3’H is generally accepted for most ornamental species [7]. Moreover, there is evidence that this enzyme can catalyse the C-3’-hydroxylation of naringenin, as well as dihydrokaempferol(283. Similarly, it appears obvious that only one flavonol synthase (FLS) is involved in the enzymic conversion of dihydroflavonois to flavonols; nevertheless, according to the plant species, efficiency of the conversion can sometimes depend on the

hydroxylation pattern of the B ring [29]. If the flavonol synthesis (from dihydroflavonoi) requires only one enzymatic step, anthocyanidin formation (from the same substrate) needs at least two enzymes. The well-known step leads to flavan-3,4-diols (leucoanthocyanidin) synthesis and is cdtafysed by DFR [19,30,31 J. It was proved that the DFR specificity for either C-4’-monohydroxylated or C-3’,4’-dihydroxylated substrates can vary according to the species [31-333. In some cases, a

Flavonoid pathway in Rosa x hybrida

1191

competition process, for the use of a flavan-3,4-diol substrate, can occur between anthocyanidin formation and proanthocyanidin synthesis. Enzyme(s) implied in conversion of the leucoanthocyanidins to anthocyanidins are not yet known. At the end of the pathway, some transferases responsible for the flavonoid structural diversification by adding sugars or acyl groups to aglycones (flavonols and anthocyanidins) have already been identified [7]. The rate of synthesis of a compound from one substrate involving only one enzymatic step depends on: presence of the gene coding for the biosynthesis of the concerned enzyme, enzyme quantity controlled by specific regulatory genes, available substrate amount, specificity of the enzyme for the substrate, possible competitions between several enzymes for the same substrate. With special regard to the actual knowledge offlavonoid pathway (Fig. I), we propose a discussion on the metabolic functioning of some extreme flavonoid types illustrated by six spontaneous colour mutants from four modern rose varieties.

RESULTS

The flavonoid diversity exhibited by a large rose mutant collection [34] can be well-illustrated by only four varieties and six of their spontaneous mutants. The four original varieties are representative of three main metabolic choices characteristic of Rosa x hybrida: the cyanidin type with cultivar no. 501, the pelargonidin-kaempferol type with no. 601 and the kaempferol type with varieties no. 403 and 411. From these original flavonoid distributions, some important metabolic changes due to mutations could be pointed out: the increase in accumulation of cyanidin; the increase of flavonol accumulation with or without changes in the equilibria between the different glycosidic forms and the modification of equilibria between the monosubstituted and the disubstituted B ring compounds. For each variety or mutant, only glycosides of kaempferol, quercetin, cyanidin and pelargonidin, previously described in rose petals [35] were detected in significant amounts and precisely quantified by means of different specific HPLC analyses (Table 1). The lines investigated contained mainly anthocyanidin 3,5-diglucosides and only traces of 3-monoglucosides. Using a photodiode array detection, it was found that none of the four intermediate compounds that are dihydrokaempferol (DHK), dihydroquercetin (DHQ), naringenin and eriodictyol significantly accumulate in the original varieties; these results agree with those reported by Jain et al. [36]. In the same way, the lack of additional amounts of anthocyanidin after a butanol-hydrogen chloride hydrolysis indicated clearly that the petals of the investigated cultivars did not accumulate significant quantities of flavan-3,4-dials or proanthocyanidins. This important result demonstrated that the formation of proanthocyanidins did not compete with the formation of anthocyanidins for the use of a leucoanthocyanidin substrate.

.c s 9

su”

.s 8 ti

s&

1192

J.-P. BIOI I r:k YI a/.

About ,foutuler Cpnidin

uuriefics

rppe. The red variety no. 501 mainly accumu-

lated cyanidin 3,5-diglucoside small

amounts

weight) among aglycone.

glycosides

which quercetin

These

tlavonoids

(4X mg g

of flavonol

’ dry weight) and (8 mgg ’ dry

is the highly dominant

specific yields of C-3’.4’-disubstituted

suggested a high activity

rate of F3’H,

gcsted a high activity

of the FLS

activities of both DFR

and F3’H.

411

The mutants

enLyme responsible for large amounts of cyanidin andior

ive varieties, i.c. based on a highly dominant However,

Diaruhus [IX].

The preferential

accumulation

supported a high efficiency of DFR Nevertheless,

indicates

kaempferol

for dihydroqucrcctin.

the significant per cent of kaempferol(21%

among tlavonols) in a cyanidin activity

of cyanidm

and dihydroqucrcetin

[20].

both were characterized

crease of kaempferol

dihydro-

are possibly used by

activity

precursors

did not appear as a good one for DFR.

chalconcs are not accumulated orange-coloured

variety no. 601 mainly produced C-4’-monohydroxylatcd tlavonoids

due to the reduced performance

synthesis of both kaempferol in equal either

quantities

and pelargonidin

was noticeable

an equilibrated

competition

glycosidcs

and might between

FLS (for the substrate dihydrokaempferol)

suggest

DFR

and

or a ditfercnce

Even if spontaneous

of flavanonc

respect to variety

duced

lower

(23 mgg-

amounts

ginal variety, twice more the flavonoid

as in carnations

small

exhibited closely related flavonoid on kaempfcrol weight),

glycosidc

exceeded 2 mgg

( > 10 mgg-

three quantified

’ dry

Havonoids

’ dry weight. This flavonoid yield sug-

Fig. 2. Mean

HPLC

profiles (UV

glycosidcs accumulated computed

patterns mainly based

production

none of the other

nos 403 and 411

detection:

no. 501. mutant of

cyanidin

distrlhution

profiles

amount

quercetin F3’H

could

of 21 chromatographic

pro-

(CHS

of kaempfcrol

(demonstrated

end-products

also bc cxplaincd

being by an

of the first two cnyymcs involved

pathway

glycosidcs)

compounds)

and CflI). glycosidcs

associated

with

by large amounts

suggested

that

in

The relatively (compared

IO

large activity

of

of disubstitutcd

In a competition

process

(possible if F3’H and FLS were activ,e at the same time). dihydrokaempfcrol

350 nm) exhibiting

Each individual

5OlM

3.5-diglucoside

was a much better substrate for F3’H

the rclativc distrlbutlons

of the petal flavonol

by three rose mutants and their respective founder. Each of these mean fingerprmts

from three individual

of

the fact that

can bc regarded as secure

the sum of quantified important

increasing activity

[XI].

[27].

’ dry weight) and higher quantities ofquercetin ’ dry weight). Compared IO the ori-

llavonol synthesis followed by a later cxpresston of DFR. type. The pink cultivars

chalcone

glycosides (8X mgg

in the timing of their expression that would allow an early

Kaetnpfirol

can occur

and

isomeri/ation

evidence of high C‘LfI activity. With

of F3’H. The

caused by an increasing

possibly

of both chalconc synthasc (CIIS)

chalconc

The

production

large amounts of flavonols rcflccted a higher availability of flavonoid

was considered as a good substrate for E‘LS, tnvcrsely it rypc.

in-

blocking of their synthesis. These

isomerasc (CHI).

kuen&ro/

kacmpferol.

hy a spectacular

a more or less drastic decrease of anthocyanin

FLS at the same rate as substrates. If dihydrokaempferol

Pelorgoniditv

and

glycosidcs that was associated with

up to a quasi-complete

type with a strong F3’H

that. as m carnations

403A

B showed tlavonotd patterns similar to their rcspect-

qucrcetin in other ornamental

species. e.g. Pefurliu [37] or

with weak

Changes due to mutations /r~cwa.sit~g ,ffu~~~o/ sphesis.

the

together

profile was characterized

accordmg

signals (the sum of which was equal to 100).

was

to the relative

Flavonoid pathway than for FLS. It is also noticeable that DFR activity has been halved. With respect to the glycosylation process, the complete flavonoid pattern (from all analysed varieties), revealed by HPLC, consisted of 21 stable and easily recognizable peaks or peak clusters; such a diversity among the native flavonoids resulted from various glycosylation and acylation patterns on either kaempferol or quercetin [38]. Important modifications in the glycosidic flavonol distributions, could be observed in a mutant increasing kaempferol synthesis, as well as in a mutant increasing quercetin synthesis. Therefore, compared to their respective varieties, mutants 41 IB and 501M exhibited novel fingerprints which were not due to modification of the balance between kaempferol and quercetin (Fig. 2). These new metabolic balances could be explained by moditications in the action of some transferases (enzymes involved in conjugation processes, e.g. glycosylation, acylation, etc) that would appear as limiting factors unable to transform the substrate at the rate required for keeping stable the original phenolic equilibrium. Conversely, mean HPLC profile of mutant 403A was close to that of the original (Fig. 2). In this case, the flavonol fingerprint, warranting the link between the mutant and its founder, would become useful in the genetic pool recognition. This demonstrated, for this mutant, an unlimiting transferase activity able to manage a significant increase of precursors (substrates). Increasing cyanidin biosynthesis. This metabolic choice was illustrated by the mutant 501D that accumulated large amounts of cyanidin 3,5-diglucoside (70 mgg- ’ dry weight) without increasing quercetin glycoside synthesis: thus, the additional amount of precursors seemed to be managed by a strong activity of the concurrent DFR. This enzyme catalysing dihydroflavonol reduction is known to present distinct substrate specificity according to the considered plant species [8]. Assuming that the flavonoid skeleton resulted essentially from condensation between malonyl-CoA and coumaroyl-CoA units, it appeared obvious that DFR exhibited a greater affinity for dihydroquercetin than for dihydrokaempferol as substrate; once more, with respect to a possible competition process, dihydrokaempferol would be a better substrate for F3’H than for DFR; the great efficiency of DFR sustained by a higher cyanidin concentration, clearly confirmed this conclusion. Mutant A of cultivar 601 produced both cyanidin and kaempferol in almost equal amounts. Among the di substituted compounds cyanidin was preferentially and highly accumulated; this corroborated the supposed high specificity of DFR for dihydroquercetin. Compared to the original cultivar, it was noticeable that the quantified flavonoid pool of the mutant was larger and that the pelargonidin content was not seriously affected by the opening route of 3’,4’-disubstitution. Modification

01 the balance

between mono- and disub-

Compared with other mutants or varieties, the mutant 501 F accumulated in signilicant and almost equal quantities both C-4’-monohydroxylated and C-3’,4’-dihydroxylated flavonoids. The significant yields of

stituted

B ringflaconoids.

PHY 36:5-H

in Rosa x hybrida

1193

both pelargonidin and kaempferol indicated a lesser F3’H activity. Moreover, this mutant is particularly important for synthesizing almost equal amounts of both flavonol heterosides and anthocyanins: the main anthocyanidin is cyanidin, while the main ilavonol is kaempferol; these indicate ellicient activities of both DFR and FLS, and once more the weak affinity of DFR for dihydrokaempferol. DISCU!NON

None of the studied mutants showed a novel colour unknown in the current wide collection of Rosa x hybrida varieties. The blueing of the red-magenta colour of mutant 501 M is probably due to a strong co-pigmentation process between cyanidin 3,5-diglucoside and the large amounts of quercetin glycosides. Nevertheless, the lack of a true colour novelty reflected the absence of new llavonoid mixtures in the petals. Mutations made some flavonoid leading routes increase or decrease up to a complete blocking; however, the metabolic boundaries previously evidenced in Rosa x hybrida [38] could not be broken down by the mutations. It is evident that any mutant can yield, in significant and almost equal quantities, the four quantified flavonoids. In the same way, the co-occurrences of either kaempferol-quercetin-pelargonidin, or quercetin-pelargonidin-cyanidin appears to be impossible. Moreover, it seemed obvious that a dominant pelargonidin (among anthocyanins) is never associated with a dominant quercetin (among flavonol glycosides) [Fig. 33. This result could be expected since these distributions could not be achieved by the numerous classical breeding assays during the last century. Thus, the current existence of these forbidden flavonoid arrangements must be regarded as the result of a very complex and rigid genetic control which cannot be disturbed by a few genetic modifications inlluencing activity of some enzymes of the flavonoid pathway. Spatial and temporal expression of enzymes involved in llavonol and anthocyanidin synthesis could be responsible for the observed metabolic schemes; but there is also good evidence that the flavonoid pattern is directed by a distinct substrate specilicity of DFR which seems to prefer dihydroquercetin (rather than dihydrokaempferol) as a substrate. Moreover, from an evolutionary point of view, the weak afftnity of the rose DFR for dihydrokaempferol as substrate is in agreement with the recent origin of pelargonidin in modern roses; in fact, it is assumed that this pigment arose recently by mutation in polyantha rose varieties [39]. Most of these mutants should be regarded as useful to test some hypothesis about the rose flavonoid pathway. Thus, mutants, e.g. 501 M and 501D, notable for accumulating large amounts of C3’J’disubstituted compounds could be used to verify the hypothesis of possible incorporation of caffeoyl-CoA into the flavonoid skeletons. These mutants, e.g. mutants 403A and 41 IB which synthesize large quantities of kaempferol, are also interesting for measuring the activity rate of CHI to determine if spontaneous isomerization (of chalcone to flavanone) is weak enough to allow chalcone accumulation due to the blocked activity of CHI. It would also be particularly interesting to

J.-P. BIOLLEY er a/.

1194

OBSERVED FLAVONOID

El

DlSTRlBUTlONS

FLAVONOID DlSlRlWllONS

NEVER OBSERVED

Fig. 3. Flavonoid balances in the petals of Rosa x hphridu. Some varieties or mutants (no. 501, 501F. 601. 601A) presenting extreme metabolic eqmlibria are pointed out. This scheme was validated by a survey of more than 100 rose cultivars containing anthocyanins [38]. Cy, Pg, Qu. Km. DHQ and DHK correspond to cyanidin, pelargonidin, qucrcetin, kaempfcrol, dihydroquercctin and dihydrokaempferol, respectively.

compare the rate of F3’H activity in mutants 501M and 403A which correspond to two distinct metabolic choices according to the B ring hydroxylation pattern of their respective llavonoids. Moreover, these mutants could also be used to study the spatial expression of the flavonol pathway to know whether most flavonol glycosides mainly originate from the coloured epidermal cells (in the vacuoles of which they are supposed to act as co-pigments) or belong to the colourless mesophyll (sub-epidermic cellular layer) always devoid of anthocyanins. Mutant 501F that exhibited a dominant kaempferol together with a dominant cyanidin would be a good model to study the expression timing of some key enzymes. e.g. FLS, DFR and F3’H.

EXPERIMENTAL

material.

The analysis was carried out on 4 cvs of and 6 of their spontaneous mutants belonging to the germplasm of ‘PCpinieres et Roseraies Georges Delbard’ (F-03600, Malicorne. France). Among them, 2 varieties are commercialized: Eterna 3:. delic (no. 403) and Madame G. Dclbard $ deladel (no. 501). The other ones are genotypes of the breeder’s private collcction. Rose flowers were picked in the experimental greenhouses of ‘Pkpiniires et Roseraies Georges Delbard’ (F83400 Hyt?res), at an early stage of the anthesis: bud with the first 2 sticking out petals. Each variety or mutant was Phnt

Rosaxhybrida

sampled by 3 individual flowers which were immediately frozen and kept, until flavonoid analysis, at - 18’. Phenolic

analysis.

Extraction

of the polyphenolic

pd.

For each flower, all petals were ground in a mortar with liquid N,. The polyphenolic pool was twice extracted. under reflux. with a boiling neutral alcoholic mixt. (MeOH--EtOH; I : 1).This extract was used for the analysis described hereafter. The plant residue was weighed after drying at 80 Almond glycosides. Using HPLC, the flavonol glycoside analysis was performed on a reverse phase column (Nucleosil C18,5 p). The chromatograms were developed by a gradient of acetonitrile in H,O with 4% HOAc. UV detection was carried out at 350 nm. Twenty-one peaks (or peak clusters). representative of cu 30 different llavonol glycosides (some of them being acylated), were detected. The area of each peak was computed and expressed as a percentofthesumofthearcasofthe21 pcaksrecordedin the same individual prolile. According to this sum of 21 areas, llavonol glycosides of each individual flower extract were quantified and expressed as mg of rutin (quercetin 3rhamnosylglucoside) g ’ of petal dry wt. The agreement between the peak area and the mg of quercetin 3-rhamnosylglucoside was achieved by analysing a standardized soln of this llavonoid. Flmonol aglycones. An aliquot of each phenolic extract was submitted to hot 2 N HCI treatment in a boiling H,Obath. Then, the aglycones were extracted with Et,0 and taken up in a small vol. of MeOH. Chromatographic analysis was achieved by HPLC (C,, ~1Bondapak column;

Flavonoid pathway in Rosa x hyhrida eluent: MeOH-H,O-HOAc; UV detection: 365 nm). Using classical procedures [40] the 2 eluted compounds could be identified as quercetin and kaempferol. Area integration of their corresponding peaks allowed per cent calculation of each aglycone. Anthocyanins. Anthocyanins were resolved by HPLC on a Nucleosil Cl8-5 p column using a gradient of MeOH in H,O with 5% HCO,H. Detection: 510nm. Just before injection, phenolic extracts were diluted in MeOH-HOAc-H,O (50:8:42). The 4 pigments clearly detected were investigated by classical methods [41,42] and HPLC co-chromatography with standards. The anthocyanins were identified as (with respect to increasing R,: cyanidin 3,5-diglucoside, pelargonidin 3,5_diglucoside, cyanidin 3-monoglucoside and pelargonidin 3-monoglucoside. Each anthocyanin was quantified by the integrated area of its corresponding peak and expressed as a per cent of all the detected anthocyanins in the chromatogram. For each individual anthocyanin profile, the sum of the 4 areas (corresponding to the 4 anthocyanin peaks) was taken into account to calculate the anthocyanin amount which was expressed in mg of cyanidin 3-monoglucoside g- ’ of petal dry wt. The agreement between the peak area and the mg of cyanidin 3-monoglucoside was achieved by analysing a standardized soln of this pigment. DihydroJauonols. The R, (in the used HPLC system) and the characteristic UV spectra of both dihydrokaempferol and dihydroquercetin have been defined with standards. Then, using HPLC with a photodiode array detection it was pointed out that none of these dihydroflavonols was significantly accumulated in the rose petal extracts. Proanthocyanidins. An aliquot of each phenolic extract was submitted to hot BuOH-HCl (5% HCl in BuOH) treatment for 2 hr [43], the oxidation of colourless proanthocyanidins to coloured anthocyanidins being enhanced by addition of metallic ions [44]. Using the process detailed by Brun [45], no additional amounts of anthocyanidins were detected after oxidation.

, . ..

Acknowledgements-We are grateful to ‘Pepmteres et Roseraies Georges Delbard, S.C.A.’ (F-03 600 Malicorne, France) and especially to Guy Delbard for the kind supply of cultivated plant material. REFERENCES

1. Zykov, K. I., Rat’kin, A. V: and Klimenko, Z. K. (1991) Izvestiya Akademii Nauk SSSR 4, 524. 2. Datta, S. K. and Gupta, M. N. (1983) J. Nucl. A@. Biol. 12, 79. 3. Smilansky, Z., Umiel, N. and Zieslin, N. (1986) Environ. Exp. Botany 26, 279. 4. Kho, K. F. F. (1978) Phytochemistry 17, 245. 5. Forkmann, G. (1989) in The Genetics of Flavonoids, Proceedings of a Post Congress, Meeting of the XVI International Congress of Genetics (Styles, D. E., Gavauy, G. A. and Racchi, M. L., eds), p. 49. Edizioni Unicopoli, Milan. 6. Forkmann, G. (1991) Plant Breeding 106, 1. 7. Heller, W. and Forkmann, G. (1988) in The Flavon-

8. 9. 10.

11.

12. 13. 14. 15. 16.

17. 18. 19. 20. 21. 22. 23.

24. 25. 26. 27. 28. 29.

30. 31. 32. 33. 34.

1195

aids: Adoances in Research since 1980 (Harbome, J. B., ed.), p. 399. Chapman and Hall, London. Lancaster, J. E. (1992) Critical Reviews in Plant Sciences 10, 487. Wiering, H. (1974) Genen. Phaenen. 17, 117. Schram, A., Jonsson, L. and Bennink, G. (1984) in Petunia, Monographs on Theor. Appl. Genet. (Sink, K. C., ed.), Vol. 9, p. 68. Springer, Berlin. Gerats, A. G. M., Huits, H. and Beld, M. (1990) in Proceedings of the XVth International Conference of Groupe Polyphenols Vol. 15, p. 2. Strasbourg, France. Forkmann, G. (1977) Planta 137, 159. Rall, S. and Hemleben, V. (1984) Plant Mol. Biol. 3, 137. Spribille, R. and Forkmann, G. (1984) Z. Naturforsch. 39c, 714. Teusch, M., Forkmann, G. and Seyffert, W. (1987) Phytochemistry 26, 99 1. Martin, C., Carpentier, R., Coen, E. and Gerats, T. (1987) in Developmental Mutants in Higher Plants (Thomas, H. and Grierson, D., eds), p. 20. Cambridge University Press. Forkmann, G. and Dangelmayr, B. (1980) Biochem. Genet. 18, 519. Spribille, R. and Forkmann, G. (1982) Planta 155,176. Stich, K., Eidenberger, T., Wurst, F. and Forkmann, G. (1992) PIanta 187, 103. Stich, K., Eidenberger, T., Wurst, F. and Forkmann, G. (1992) Z. Naturforsch. 47c, 553. Mol, J. N. M., Stuitje, A. R. and Van der Krol, A. (1989) Plant Molec. Biol. 13, 287. Mol, J. N. M. (1991) Endeauour, New Series 15, 42. Kooter, J. M., Van Blokland, R., de Lange, P., Maike, Stam and Mol. J. N. M. (1992) in Proceedings of the XVIth International Conference of Groupe Polyphenols Vol. 16, p. 261. Lisboa, Portugal. De Vries, D. P., Van Keulen, H. A. and De Bruyn, J. W. (1974) Euphytica 23, 447. De Vries, D. P., Garretsen, F., Dubois, L. A. M. and Van KeulCn, H. A. (1980) Euphytica 29, 115. Marshall, H. H., Campbell, C. G. and Collicutt, L. M. (1983) Euphytica 32, 205. Mol, J. N. M., Robbins, M. P., Dixon, R. A. and Veltkamp, E. (1985) Phytochemistry 24, 2267. Hagmann, M., Heller, W. and Grisebach, H. (1983) Eur. J. Biochem. 134, 547. Gerats, A. G. M., de Vlaming, P., Stotc G., Wiering, H., Forkmann, G. and Schram, A. W. (1982) Planta 155, 364. Heller, W., Britsch, L., Forkmann, G. and Grisebach, H. (1985) Planta 163, 191. Heller, W., Forkmann, G., Britsch, L. and Grisebach, H. (1985) Planta 165, 284. Forkmann, G. and Ruhnau, B. (1987) Z. Naturforsch. 4tc, 1146. Meyer, P., Heidmann, I., Forkmann, G. and Saedler, H. (1987) Nature 330, 677. Biolley, J. P. and Jay, M. (1992) in Proceedings of the XVIth International Conference of Groupe Polyphenols Vol. 16, p. 51. Lisboa, Portugal.

1196

J.-P. BIOLLEY et 01

35. Yokoi, M. (1975) Technical Bulletin of Faculty o_f Horticulture, Chiba University 14, 33. 36. Jain, M. C., Seshadri, T. R. and Trikha, R. K. (1971) J. Scien. Ind. Res. 30, 77. 37. Stotz, G., de Vlaming, Y., Wiering, H., Schram, A. W. and Forkmann, G. (1985) Theor. Appl. Genet. 70, 300. 38. Biolley, J. P., Jay, M. and Viricel, M. R. (1994) Phytochemistry 35, 413. 39. Rowley, G. D. (1957) J. R. Hart. Sot. 82, 484. 40. Mabry, T. J., Markham, K. R. and Thomas, M. B. (eds) (1970) The Systematic Identification of Flaoonoids. Springer, Berlin.

41. Harborne, J. B. (1973) Phytochemical Methods. Chapman and Hall, London. 42. Markakis, P. (ed) (1982) Anthocyanins as Food Colors. Academic Press, New York. 43. Bate-Smith, E. C. (1973) Phytochemistry 12, 907. 44. Porter, L. J., Hrstich. L. N. and Chan, B. G. (1986) Phytochemisrry 25, 223. 45. Brun, N. (1991) Ph.D. Thesis, Universitk Claude Bernard, Lyon I. France.