TWAMLEY - Genetics

BIOCHEMICAL ASPECTS OF THE INHERITANCE OF FLORAL ANTHOCYANINS IN DIPLOID ALFALFA

S. B. GUPTAl

Department of Genetics, University of Alberta, Edmonton, Canada

Received October 27, 1969

URPLE color in the flower petals of alfalfa (Medicago sativa L., M . falcata L. I)and their hybrids) is due to the presence of sap-soluble anthocyanins. The floral anthocyanins of this species have been studied by several workers ( LESINS 1956; BUKER and DAVIS 1961; COOPER and ELLIOTT 1964, GUPTA 1968). LESINS (1956) identified these pigments as glycosides of delphinidin, petunidin and malvidin, and this was further confirmed by COOPER and ELLIOTT (1964) who identified the three anthocyanins as 3,5-diglucosides of the above three anthocyanidins. DAVIES (1958) also found three anthocyanidins but identified as cyanidin the pigment identified as petunidin by the above workers. BUKER and DAVIS ( 1961 ) , and COOPER and ELLIOTT (1 964) found co-occurrence of the three anthocyanins in alfalfa. However, there is considerable evidence that alfalfa populations are polymorphic for presence us. absence of malvidin glycoside, since LESINS (1956) found segregation for this pigment in one of the crosses, and GUPTA (1968) found two kinds of plants, one containing and the other lacking the 3,5-diglucoside of malvidin.

TWAMLEY (1955) and LESINS (1956) have found that when certain genotypes of white-flowered alfalfa are intercrossed, purple-flowered F, hybrids are obtained, and have suggested that complementary gene action may be responsible for the biosynthesis of anthocyanins. In order to establish the total number of anthocyanins and identify their aglycones, a detailed biochemical analysis, by means of two-dimensional thin-layer chromatography (TLC) of these pigments in segregating progenies as well as in natural populations, has been undertaken in the present study, and a scheme indicating genetic control of the biosynthesis of anthocyanins in alfalfa has been proposed.

MATERIALS A N D METHODS

Plants belonging to several University of Alberta accessions of Medicago sativa L. whose origin is given in Table 1, were grown in open sunlight and studied for their anthocyanin compo- sition. Variety Revoluta of yellow-flowered M. falcata L. which contains anthocyanin at the tips of floral buds, was also examined. A detailed study was undertaken, on the three diploid purple- flowered plants of M. sativa accession No. 508, denoted as s,, s,, and s, (Table I), and on the purple-flowered F, hybrids, derived from the intercrosses of white-flowered alfalfa which were of M . sativa and M . falcata origin. Three anthocyaninless diploid plants, white-1, white-2, and creamy, were used for hybridization. Plants white-2 (254-ZOX254-8,Flp,,F,p,,), and creamy

Present address: Department of Biology. Kenyon College, Gambier, Ohio 43022 U.S.A.

Genetics 65: 867-278 June 1970

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268 S . B. GUPTA

(254-20x254-8,F1p,,F,p,,) were closely related in their origin while plant white-I (Saskatchewan white) was of an independent origin. All the F, hybrids, obtained from the crosses, white-I x white-2 and white-I x creamy, were purple-flowered, and only four plants of the former cross and one of the latter were used for the studies of the F, generation. Two white-flowered tetraploid plants, white-3 and R,,, were also crossed, and purple-flowered F, hybrids were obtained.

TABLE 1

Distribution of anthocyanidins' in flower petals of various accessions, cultivars, and hybrids of Medicago sativa L.

Anthocyanidins present$

Origin of Delphinidin group Cyanidin group Accession nos., cultures

cultivars or or parentage Number of Delphinidin Petunidin Malvidin Cyanidin generation 2n= of hybrids plant+ (1) ( 2 ) (4) (9)

+ 50% 16 Iran 4 + + + 504 16 Iran 1 + + - 505 16 Turkey 2 + + + + 505 16 Turkey 3 + + - + 506 16 Persia 4 + + + + 507 16 Armenia 5 + + + + + 508 16 Iran W , ) + + + + + 508 16 Iran 1 (S,) + + 508 16 Iran 2(SJ + + 509 16 U.S.S.R. 5 + + +

1838 32 Turkey 1 + + - 1840 32 Iran 1 + + + +

Hexaploid 48 Synthetic 2 + + + + + cult. Ferox 32 Canada 1 + + + cult. Grimm 32 U S A . 1 + + +

-

- -

. . . -

- Fl 16 white-I x

Fl 16 white-I x

F, 32 white-3 x

+ white-2 4 + + creamy 1 + + - + RI 3 4 + + + +

-

* Medicago falcata var. Revoluta from U.S.S.R. contained only delphinidin in the tips of

-f (SI), (S,) and (S,) indicate the groups of three different plants discussed in the text. flower buds.

+, -and . symbolize present, absent and not analyzed, respectively. The numbers ( I ) , ( 2 ) , (4) and (9) indicate the anthocyanin number from which the respective aglycone was derived.

Dried petals (.05 g) were crushed inside a glass vial, containing 0.5 ml of ice-cold 1 % HC1 in methanol, and were stored inside a refrigerator. The anthocyanin extract was ready for a chromatographic separation after 3-4 hr. Two-dimensional TLC separation of the anthocyanins was carried out on precoated cellulose Eastman Chromagram TLC sheets in two different solvent systems, (1 ) n-butanolLconc.HC1-water (5:2:1), for one direction, and water-conc.HC1-formic acid (8:4:1), for the other, and (2) n-butanol-acetic acid-water (BAW, 4:1:5, upper phase), for the first direction, and 15% acetic acid for the second. Total anthocyanidins were obtained by hydrolyzing petals with 2~ HC1 on a water bath at 100°C for 4 0 min followed by partitioning in isoamyl alcohol. The flower petals of alfalfa also contained light yellow-colored anthoxanthins which are closely related to anthocyanins. These compounds which also include the flavonols, kaempferol and quercetin, found in alfalfa (COOPER and ELLIOTT 1964), were hydrolyzed and

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ANTHOCYANINS OF ALFALFA 269

appeared as yellow spots on the chromatogram in visible light. To obtain anthocyanidins of individual anthocyanins, the latter were chromatographed one-dimensionally on Whatman NO. 3 paper with either BAW or the n-butanol-conc.HC1-water solvent. Each spot was eluted with 2N HC1, the eluate was hydrolyzed, and the anthocyanidin (aglycone) of the pigment was extracted from the hydrolysate with isoamyl alcohol and chromatographically identified.

For the identification of the anthocyanidins, both one-dimensional and two- dimensional TLC was carried out on cellulose sheets along with standard samples of delphinidin (D), petunidin (P), malvidin (M), and cyanidin (C) . Two different solvent systems, Forestal (acetic acidxonc.HC1- water, 30:3:10) and BAW (on acid-washed sheets) were used. For two-dimensional separation, a mixture of standard anthocyanidins was spotted 4 cm away from the left margin of a 20 x 20 cm TLC sheet; the mixture of anthocyanidins, extracted from the plant under investigation, was spotted at a distance of 8 cm from the same margin, and the chromatogram was run in Forestal for the first direction and in BAW for the second. The anthocyanidins were then identified by comparing their Rf values with those of the authentic anthocyanidins, chromatographed on the same chromatogram.

TABLE 2

Rf values of the anthocyanins, from the plant S, (accession 508), using two-dimensional TLC on cellulose sheets, with two solvent systems'

~~ ~ ~~ ~~

Anthocyanin Rf values Number Solvent la Solvent 1 b Solvent ea Solvent 2b

.I6 .51

.22 .59

.28 .71

.20 .91

.I4 .26

.24 .39

.33 .54

.24 .86

* Solvents: l a = BAW or n-butanol-acetic acid-water, 4:1:5, upper phase, l b = 15% acetic acid, 2a = n-butanol-HCI-water, 5:2:1; and 2b = Water-HC1-formic acid, 8:4:1.

TABLE 3

Rf values of the anthocyanidins, from the plant S , (accession 508), using two-dimensional T U : on cellulose sheet, with Forestal and BAW solvents'

Rf values Fmestal BAW

Authentic From Authentic From Anthocyanidins samples M . satiua samples M . satiua

Delphinidin .30 .33 .25 .28 Petunidin .44- .47 .36 .35 Malvidin .64 .64 .43 .43 Cyanidin .47 .47 .48 .47

' Forestal = Acetic acid-HC1-water (30:3:10), BAW = n-butanol-acetic acid-water (4:1:5, upper phase).

RESULTS A N D DISCUSSION

Analysis of anthocyanin content of plants in natural populations and of plants of hybrid origin: In an earlier one-dimensional TLC study ( GUPTA 1968), it was observed that the plants SI and S, each contained three anthocyanins (Nos. 1, 2 and 4) and the plant S, only two (Nos. 1 and 2) . During the current investigation, two-dimensional TLC uncovered an additional anthocyanin, No. 9, not so

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2 7 0 S. B. GUFTA

Frcunrs 1 -2.-Two-dimerisional T1.C of the arithocyariins of Medicnpo snfiun plant SI (Figure 1 ) arid plnrit S, (Figure 2). using two solvents n A W (upward) arid 15% acetic acid (left to right).

FJCURFS 3-4.-Orie-dimerisinrial T1.C of the totnl arithncyariitliris of plant S i dong with the authrntic samples of tlelphiriidiri (D). peturridiri (P), mnlvitlin (M) arid cyanidin (C), in Forestal (Figure 3) aricl DAW. or1 acid-washrrl shret. (Figure 4).

F I G W I ~ I ~ ',.-Onr-dimrrisiorial TIX of the anthocyanidiris (1. 2 and 3) derived from the anthocynriiris Nos. 1 . 2 iirid 4. rrsprctivrly, nloiig with the authentic snniplrs of D. P arid M, i r r 1;orrstiil.

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ANTHOCYANINS O F ALFALFA 271

far reported in this species (Table 2). Figures 1 and 2 show that the plant S, contains anthocyanin 9 in addition to 1, 2 and 4, found in plant SI. Similarly, plant S, was also found to contain anthocyanin 9, but a plant containing only anthocyanins 1 and 2 and not 9, was observed only in accession 1838 (Table 1, accession 1838). A plant of yellow-flowered M . falcata variety Revoluta contained only anthocyanin No. 1. The anthocyanin was easily visible in the purple tips of the buds, but was barely detected in fully opened flowers. However, the pigment was extractable from the buds as well as from open flowers either with water or with 1 % HCl in methanol.

One-dimensional TLC of total anthocyanidins of plant S, in Forestal and in BAW (Figures 3 and 4) , along with the standard samples, demonstrated that it contained delphinidin, petunidin, and malvidin (Table 3). However, presence or absence of cyanidin could not be determined by one-dimensional separation. Two-dimensional chromatography of the total anthocyanidins which were obtained from one tetraploid plant and three diploid plants S,, S, and S,, is shown in Figures 8-11. S, (Figure 10, spot 2) showed the same three anthocyanidins (the third spot is faint, Rf is similar to NI) which were identified by one-dimensional TLC (see Figures 3 and 4) but the tetraploid plant from accession 1840 (Figure 8, spot 2) and S, (Figure 9, spot 2) each showed an additional anthocyanidin, cyanidin, separating from petunidin. S, (Figure 11, spot 2) showed delphinidin, petunidin and cyanidin but not malvidin. Plants of several other accessions, including diploids and tetraploids, also showed cyanidin, although in some it was present only in traces and very few completely lacked it (Table 1)- The plant belonging to M . falcata variety Revoluta contained delphinidin only.

The anthocyanidins of individual anthocyanins 1, 2 and 4 of plant SI were individually chromatographed, along with standard samples, in Forestal (Figure 5) and in BAW solvents, and on the basis of Rf relationship, they were identified as delphinidin, petunidin and malvidin, respectively (Table 3). Since Sz contained one additional anthocyanin (No. 9), and the hydrolysate of its anthocyanins showed a new anthocyanidin, cyanidin, it may be inferred that the aglycone of anthocyanin 9 is cyanidin. Thus, this result confirmed the findings of COOPER and ELLIOTT (1964) that the anthocyanins, separable by one-dimensional chromatography (anthocyanins 1, 2 and 4) , were the glycosides of delphinidin, petunidin and malvidin, respectively. However, an unknown glycoside of cyanidin, anthocyanin 9, also overlaps anthocyanin 2 in one-dimensional separation by the BAW solvent.

Analysis of the progenies of crosses: Table 4 shows that, among the 180 F, progeny of five diploid F,’s, four categories of plants were found: (1 ) those with delphinidin, petunidin and cyanidin (with D, P, and C); (2) those with del-

FIGURES 6-7.-0ne-dimensional TLC of the unknown anthocyanidin, “X” derived from a single anthocyanin of an F, segregate from white-1 x white 2 cross, containing anthocyanin 9, in Forestal (Figure 6) , and BAW (Figure 7) , along with authentic P and C. In Figure 7 note that anthacyanidin “X” moves higher than petunidin in BAW and so does cyanidin. Therefore, “X’ should be cyanidin. A dark spot above “X”, which goes along with the solvent front, is due to the yellow-colored anthoxanthins present in the flowers.

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2 72 S. B. CUPTA

I:rcvnr.̂ s 8-1 1 .-Two-dimrnsiorii T1,C of t..? tok. anthncyanidiris of plants from various sourrrs of 3.1. sntira. clrvrloprd i r i I"wta1 for first clirrrtion (up\rard). and RAW for the srrontl dirrrtiori (srr arrows). I'ijiurr 8. Co-rlirorrintogrn~~hrcl iiuthrritic tlrlphiriidin (I)). prturiitliri (P), riiiilvidiri (M). arid cyariidiri (C. fairit) iit spot 1 \ t ). arid total iiiithncyaiiitliris of trtrapolicl plarit clirorriiitojii-nptic.tl at sprit 2 (2 ) . 1;igurr I). Co-rhrorriiitogr:i~~lietl iiuthrtitic I). P. M arid C tit s p t 1 ( I ). i i d total iinthncyiiriitliris of plarrt S, (-hrorniitogriipht.tl at sprit 2 ( 2 ) . b'igurr 10. Co-chroma- togriiphrrl authentic D. P. arid M iit spnt 1. arid total arittincyanitliris of pliirit S, cliromatogriiphd at spnt 2. sliowirig al)srncr of cynriidiri ( 2 ) . Figurr 11. Spnt 1 shows co-rlironiiito~raphrtl nuthrritic I). 1'. and M ( 1 ). iiiid spot 2 show thr prrsrricr of D. P arid C in plarit S:,.

I~r~vnr~s 12-1 3.-Orir-tlimrnsiorial T I X of the total anthoryariidiris of four 17, hyhrids of trtraploid wliite-flo\rrred plants (spnts 1. 2. 3 arid 4) aloiig with authrritic D. P arid M a t spot Si (I'igurr 12), arid thrw F, hyhritls of diploid white-flowcrcd alfalfa (Figure 13). Note ahsriice of malricliri ( M ) iri the diploid hyhrids. T h r chromatop;rnnis were run orie-dimensionallJ- in 1:orCs t a 1.

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ANTHOCYANINS OF ALFALFA 273

TABLE 4

Distribution of the anthocyanidins, delphinidin (D), petunidin (P) , and cyanidin (C) , in plants of the F , generation of white-I x white-2 and white-I x creamy crosses

Number of plants with Number of plants Number of plants or without D, P and C segregating for C segregating for D and P

Family Genera tion* No. D,P,C D,P C t None +D,P -D,P Prob.(9:7) +C -C Prob.(9:7)

F, (w-1 X W-2, F1pl) la 14 2 3 18 16 21 .lo-.20 17 20 .20-.30 F, (w-1 X W-2, F , P ~ ) Ib 13 2 1 9 15 10 .70-30 14 11 .90-1.0 F, (w-1 X W-2, F1p3) I C 6 6 5 4 12 9 .90-1.0 11 10 .70-.80 F, (w-1 X W-2, F,p,) Id 14 3 4 13 17 17 .30-.50 18 16 .50-.70 Total 1 47 13 13 44 60 57 .25-.30 60 57 .25-.30 F, (w-1 X Cr, FIP~) 2 30 1 5 27 31 32 .20-.30 35 28 .90-1.0

* w-I = white-I, w-2 = white-2, Cr = Creamy, p = plant. + Flowers appeared almost anthocyaninless.

phinidin and petunidin (with D, P); (3) those with cyanidin only (with C), and (4) those with no anthocyanidins (none).

The plants containing only cyanidin glycoside had a very low concentration of this pigment in the petals and could easily be misclassified as anthocyaninless. But, when the petals were left in 1 % HCl in methanol, the pigment could be extracted, concentrated and chromatographed. The hydrolysate of this anthocyanin produced cyanidin which could be distinguished from petunidin in BAW (Figures 6 and 7, see “X”; the dark spot, having higher Rf value than “X”, is due to the presence of a large quantity of yellow-colored anthoxanthin).

Since cyanidin and delphinidin (with co-occurring petunidin) show independent assortment in the F, progeny, and since the precursors required for the biosynthesis of delphinidin and cyanidin appear to be different ( HARBORNE 1967), two pairs of segregating complementary factors may be assumed to control pigmentation in the F,’s. Assuming independence of the four genes, the F, should conform to the ratio of 81:63:63:49 for 256 plants or 57:44:44:35 for 180 plants but the plants segregated for 77:14:18:71 indicating close linkage between certain genes. Therefore the F,’s were reclassified under two classes: (1 ) those having or not having delphinidin and petunidin (+ or - D, P) ; (2) those having or not having cyanidin (+ or - C). The data do not significantly diverge from a 9:7 ratio in each case indicating that two pairs of independent complementary genes control synthesis of delphinidin (assuming that the petunidin-synthesizing gene is in homozygous condition) and cyanidin, respectively.

Anthocyanin synthesis and its genetic control: Anthocyanins are glycosides of anthocyanidins which may be described as a series of compounds with a carbon skeleton consisting of two six-carbon-atom units A and B (benzene rings) con- nected by a heterocyclic (pyran) ring (see anthocyanidin nucleus in Figure 14). The A and B rings of these compounds have hydroxyl groups substituted at par- ticular positions, e.g., cyanidin and delphinidin have hydroxyl groups at positions 3’ and 4’ and at 3’, 4’ and 5’, respectively. In addition, the B ring may have methoxyl groups (-OCH,) in place of one or more hydroxyl substitutions, thus

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2 74 S. B. G U P T A

petunidin (3’0-methylated) and malvidin (3’, 5)-0-methylated) are methylated compounds of the delphinidin series.

A close examination of the distribution of anthocyanins in Medicago sativa, from widely different sources and of one variety of M . falcata, variety Revoluta (Table l ) , revealed the occurrence of five categories of plants in alfalfa; those which have (1) only delphinidin ( M . falcata variety Revoluta) , (2) delphinidin and petunidin, (3) delphinidin, petunidin and cyanidin, (4) delphinidin, petunidin and malvidin, ( 5 ) delphinidin, petunidin, malvidin, and cyanidin. Thus, the current survey disclosed segregation for the glycosides of petunidin, malvidin, and cyanidin in the anthocyanin-containing flowers of various natural populations of alfalfa and, therefore, indicated that they are not inherited as a single genetic unit.

Chromatographic analysis of the three diploid anthocyaninless plants. white-1, white-2 and creamy used for hybridization. revealed that they had relatively high concentrations of anthoxanthins which share the biosynthetic pathway with the anthocyanins. When white-1 was crossed with either a white-2 or a creamy plant, all the F, plants had light purple flowers, indicating that white-1 carried genetic factors for purple flowers complementary to those carried by creamy and white-2. Similarly white-flowered tetraploids, white-3 and R13, produced all purple- flowered F,’s, out of which only four plants were examined. In Figure 12, one- dimensional separation of a mixture of three standard anthocyanidins (spot s,) and the total anthocyanidins of four tetraploid F, hybrids (spots 1, 2, 3 and 4) , is shown. Delphinidin, petunidin and malvidin appear in all of the four hybrids. Cyanidin was detected in the same plants by means of two-dimensional chromatography (Table 1). These plants contained four anthocyanins, identical to those of plant S,. Four F,’s, from white-1 x white-2, and one from white-1 x creamy crosses, contained delphinidin, petunidin, cyanidin, afid lacked malvidin (Table 1, Figure 13). In these plants, anthocyanins 1 , 2 and 9 were present and 4 (3,5- diglucoside of malvidin) was missing.

BARNES and CLEVELAND (1964) studied floral bud color inheritance in diploid alfalfa and reported that two complementary genes control the synthesis of anthocyanin which accumulates at the tips of the buds of yellow-flowered alfalfa. Similar floral buds of the yellow-flowered M . falcata variety Revoluta were found to contain only delphinidin. Therefore, it is possible that synthesis of delphinidin is controlled by two complementary genes. Complementary gene action has been demonstrated in the F, hybrids of diploid and tetraploid white-flowered alfalfa in this investigation. However, the F, hybrids, of diploid white-1 x white-2 and white-1 x creamy, produced delphinidin. petunidin and cyanidin while those of tetraploid, white-3 x R13 (white) , on the other hand, produced malvidin in addition to the above three anthocyanidins (Table 1 ) . These observations, thus, suggest that two additional genes control the synthesis of petunidin and malvidin which are methylated derivatives of delphinidin besides the two complementary genes synthesizing delphinidin. Similarly, a pair of complementary genes may control the independent synthesis of cyanidin.

Therefore, the four segregating genes uncovered by the above genetic analysis

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should be (1) a gene, C,, responsible for synthesis of the trihydroxylated precursor of delphinidin (precursor-2), (2) a gene p,, controlling conversion of precursor-2 into delphinidin, ( 3 ) a gene C,, controlling production of the dihydroxylated precursor of cyanidin (precursor-3) and (4) a gene P4, responsible for the synthesis of cyanidin from precursor3 (Figure 14). Furthermore, a gene P,, responsible for petunidin, appears to be in the homozygous condition in the F, hybrids because petunidin does not segregate in the F,. Existence of a separate gene controlling petunidin synthesis is indicated by the occurrence of only delphinidin in a plant of M . falcata variety Revoluta. Segregation for an additional gene controlling synthesis of malvidin has been observed in natural populations, as well as in controlled crosses of alfalfa. The current investigation, therefore, indicates existence of six different genes which control synthesis of four anthocyanins in diploid alfalfa, in contrast to only two genes, C and P, suggested by several authors (BARNES 1966), and three genes, A, B and My proposed by LESINS (1956).

In past studies, when F, hybrids of purple-flowered M . sativa and yellow- flowered M . falcata were selfed or testcrossed, a 3: 1 or 1 : 1 ratio, for purple us. nonpurple flowers, was observed by several authors (reviewed by BARNES 1966). These results could, however, be explained on the basis of the assumption that the F, hybrids would have been heterozygous either for the gene C, or P, of the present study. The possibility that the F, hybrids were heterozygous for both genes P , and P,, which might yield a I2 purple (9 with D, P, M and 3 with D, P) : 4 nonpurple, cannot be ruled out.

The general mode of flavonoid (anthoxanthins and anthocyanins) biosynthesis has been determined by NEISH (1964), GRISEBACH ( 1965) and GRISEBACH et al. (1966). The B ring, with a three-carbon-atom chain attached, is contributed by a C,-C, precursor (which may be a cinnamic acid). The A ring is formed by the condensation of three acetate units. It has been demonstrated that chalcones (anthoxanthins) serve as central intermediates in flavonoid metabolism (GRISE- BACH et al. 1966). Several flavonoids are derived from the common C,, intermediate (chalcone) which produces dihydroflavonols (dihydroxylated dihydroquercetin and trihydroxylated dihydromyricetin) , flavonols, anthocyanidins and leucoanthocyanins.

It is not entirely clear whether the hydroxyl and methoxyl substitutions on the B ring occur before or after condensation with the A ring. Genetic evidence from Phaseolus uulgaris (FEENSTRA 1960) suggests that the B ring hydroxylation occurs before the specific class of flavonoids is established. This indicates that cyanidin and delphinidin may have separate dihydroflavonols as precursors (dihydroquercetin and dihydromyricetin, see WONG 1968) whose synthesis from the chalcone is controlled by separate genes. WONG (1968) has suggested that dihydroquercetin serves as the precursor of cyanidin in the Geraldton variety of subterranean clover. It may be possible that, in Medicago, dihydroquercetin and dihydromyricetin may serve as precursors for the synthesis of cyanidin and delphinidin, respectively. It is not clearly established at what stage methylation takes place in the biosynthesis of flavonoids although there are indications that

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276 S. B. GUPTA

hydroxylation precedes methylation. HESS (1965), who worked with the floral anthocyanins of Petunia hybrida, has suggested that the genes controlling methylation regulate the methylation of cinnamic acids (C,-C, precursors). However, this does not agree with the evidence that methylation occurs toward the end of flavonoid synthesis (leucoanthocyanins and some flavonols which are not methylated also co-occur with methylated anthocyanins, ALSTON 1964; HAR- BORNE 1967).

Recent studies on the genetic control of methylation of anthocyanidins in the flowers of garden plants have revealed that a sequential stepwise process is involved during biosynthesis of methylated derivatives. In petunia, the pleio- tropic gene F controls petunidin synthesis from delphinidin (3'0-methylation) and gene K the further methylation of petunidin to malvidin (5'0-methylatio11, HESS 1964, cited by HARBORNE 1967). In the potato, gene AC controls 3'0- methylation and another gene AC', 5'0-methylation (HARBORNE 1967). SEYFFERT (1959) also found a separate gene controlling methylation of petunidin to malvidin in Primula melacoides. The present observation suggests that a similar stepwise mechanism of methylation exists in alfalfa flowers.

2' 3'

5 4 ANTHOCYANIDIN NUCLEUS

PRECURSOR- 1 CYANIDIN MALVlDlN

PRECURSOR - 2 DELPHlNlDlN PETUNlDlN

FIGURE 14.-Probable biosynthetic scheme for anthocyanidins in diploid alfalfa. The genes, C,, P,, P,, P , and C, and P,, control specific biosynthetic reactions. The arrows, with broken lines, indicate more favored probable mechanism for methylation of the hydroxylated anthocyanidins.

Genetic model for the inheritance of anthocyanins in diploid alfalfa: On the basis of the evidence gathered from this investigation and previous studies on several garden plants (ALSTON 1964; SHERRAT 1958), a model, indicating the genetic control of anthocyanin synthesis in diploid alfalfa, has been illustrated in Figure 14. Precursor-1 (chalcone) is a common central intermediate involved

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in flavonoid metabolism which, in the presence of specific enzymes, controlled by basic color factors, C, and C2, undergoes hydroxylation, producing precursor-2 (dihydromyricetin) and precursor-3 (dihydroquercetin) , respectively. These two precursors, with trihydroxy1 and dihydroxyl substitutions in the B rings, are the intermediate compounds which undergo reduction in the heterocyclic rings and are subsequently converted into respective anthocyanidins. Thus comple- mentation of C , and PI and C , and P4 can only produce delphinidin and cyanidin, respectively. A separate gene P,, responsible for 3’0-methylation of delphinidin to petunidin and gene P,, controlling methylation of petunidin to malvidin, have been also suggested. Since many plants having delphinidin and petunidin did not contain even a trace of malvidin, and since other plants which contained D, P and M, had relatively higher concentration of petunidin than that of malvidin, it appears that petunidin is not produced as a result of incomplete methylation of delphinidin to malvidin, but rather that malvidin is derived from petunidin.

According to the proposed model, synthesis of petunidin and malvidin is con- ditioned by the presence of gene P I , which controls the synthesis of delphinidin. Therefore, a plant which cannot synthesize delphinidin cannot produce either petunidin or malvidin. In the above scheme, it is assumed that the anthocyanidins belonging to the delphinidin and cyanidin series are derived from a common precursor- 1 and the genes, controlling the hydroxylation patterns of precursor-2 and -3, therefore, control the hydroxylation patterns of delphinidin and cyanidin, respectively.

The author is very thankful to Dr. K. LESINS and Mr. A. ERAC for kindly providing flowers from the hybrids and F,’s of white-flowered alfalfa. Thanks are also due to Dr. G. W. R. WALKER, University of Alberta, and to my colleague, Dr. A. WOHLPART, for critical reading of the manu- script. Financial assistance of the NRC of Canada, grant A-1425 to Dr. K. LESINS, is gratefully acknowledged.

SUMMARY

Floral anthocyanins of Medicago satiua L., originating from diverse geographic regions, and of M . falcata variety Revoluta were analyzed by means of two- dimensional thin-layer chromatography and four different anthocyanins (Nos. 1, 2, 4 and 9, after GUPTA and LESINS 1969) were found. Three of them, anthocyanins 1, 2 and 4, were identified as the glycosides of delphinidin, petunidin and malvidin and the fourth (anthocyanin 9), a new pigment, was found to be a glycoside of cyanidin. In the natural populations of M . satiua, segregation for malvidin and cyanidin was observed but delphinidin and petunidin were found to co-occur. However, the yellow-flowered M . falcata variety Revoluta contained only delphinidin in the purple tips of the flower buds. The F, hybrids of the crosses between diploid white-flowered plants of different flower color genotypes (white-I x white-2; white-1 x creamy) contained glycosides of delphinidin, petunidin and cyanidin. Those of the cross tetraploid white-3 x RI3 (white) contained, in addition to these pigments, a glycoside of ma1vidin.-The F,, for four progenies of white-1 x white-2 and one of white-I x creamy (total- ing 180 plants), all showed close fit to a 9: 7 ratio for two independent segrega- tions: presence us. absence of both delphinidin and petunidin, and presence us.

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278 S. B. GUPTA

absence of cyanidin. On the basis of the present biochemical data and the informa- tion available from studies of the inheritance of flower color in alfalfa (reviewed by BARNE 1966), a revised model for the inheritance of anthocyanins in diploid alfalfa is proposed. The existence of two separate color factors ( C , and C , ) , controlling precursors of the pigments belonging to the delphinidin and cyanidin series, respectively, and four separate genes, P,, P,, P, and P,, for the production of the individual anthocyanidins is suggested.

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