Journal of Heredity Advance Access originally published online on April 9, 2007
Journal of Heredity 2007 98(3):250-257; doi:10.1093/jhered/esm012
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Analysis of Flavonoids in Flower Petals of Soybean Near-isogenic Lines for Flower and Pubescence Color Genes
From Tsukuba Botanical Garden, National Science Museum, Tsukuba, Ibaraki 305-0005, Japan (Iwashina); National Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan (Githiri, Benitez, and Takahashi); Graduate School of Agriculture, Ibaraki University, Ami, Ibaraki 300-0393, Japan (Takemura); and Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan (Kitajima). Stephen M. Githiri is now at the Department of Crop Science, University of Nairobi, PO Box 30179, Nairobi, Kenya
Address correspondence to R. Takahashi at the address above, or e-mail: masako{at}affrc.go.jp.
W1, W3, W4, and Wm genes control flower color, whereas T and Td genes control pubescence color in soybean. W1, W3, Wm, and T are presumed to encode flavonoid 3'5'-hydroxylase (EC 1.14.13.88 [EC] ), dihydroflavonol 4-reductase (EC 1.1.1.219 [EC] ), flavonol synthase (EC 1.14.11.23 [EC] ), and flavonoid 3'-hydroxylase (EC 1.14.13.21 [EC] ), respectively. The objective of this study was to determine the structure of the primary anthocyanin, flavonol, and dihydroflavonol in flower petals. Primary component of anthocyanin in purple flower cultivars Clark (W1W1 w3w3 W4W4 WmWm TT TdTd) and Harosoy (W1W1 w3w3 W4W4 WmWm tt TdTd) was malvidin 3,5-di-O-glucoside with delphinidin 3,5-di-O-glucoside as a minor compound. Primary flavonol and dihydroflavonol were kaempferol 3-O-gentiobioside and aromadendrin 3-O-glucoside, respectively. Quantitative analysis of near-isogenic lines (NILs) for flower or pubescence color genes, Clark-w1 (white flower), Clark-w4 (near-white flower), Clark-W3w4 (dilute purple flower), Clark-t (gray pubescence), Clark-td (near-gray pubescence), Harosoy-wm (magenta flower), and Harosoy-T (tawny pubescence) was carried out. No anthocyanins were detected in Clark-w1 and Clark-w4, whereas a trace amount was detected in Clark-W3w4. Amount of flavonols and dihydroflavonol in NILs with w1 or w4 were largely similar to the NILs with purple flower suggesting that W1 and W4 affect only anthocyanin biosynthesis. Amount of flavonol glycosides was substantially reduced and dihydroflavonol was increased in Harosoy-wm suggesting that Wm is responsible for the production of flavonol from dihydroflavonol. The recessive wm allele reduces flavonol amount and inhibits co-pigmentation between anthocyanins and flavonols resulting in less bluer (magenta) flower color. Pubescence color genes, T or Td, had no apparent effect on flavonoid biosynthesis in flower petals.
Color of flower petal, seed coat, hypocotyl, and pubescence is caused by the deposition of various flavonoids in the respective tissues in soybean (Glycine max (L.) Merr.). Primarily, 5 genes (T, W1, I, R, and O) control the seed coat color, 5 genes (W1, W3, W4, Wm, and Wp) control the flower color, and 2 genes (T and Td) control the pubescence color in soybean (reviewed by Palmer et al. 2004). Furthermore, 4 flavonol glycoside alleles that are irrelevant to color of tissues, Fg1 (ß(1-6)-glucoside present), Fg2 (
(1-6)-rhamnoside present), Fg3 (ß(1-2)-glucoside present), and Fg4 ((1-2)-rhamnoside present), have been identified (Buzzell and Buttery 1974). The biosynthetic pathway of flavonoids is well established, and many of the structural and some of the regulatory genes have been cloned (Holton and Cornish 1995). Schematic diagram of flavonoid biosynthesis is presented in Figure 1.
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The hydroxylation pattern of B-ring in flavonoids plays an important role in the coloration of seed coats, flower, and pubescence of soybeans. Major anthocyanin pigments in the seed coats of black soybeans are cyanidin 3-O-glucoside and delphinidin 3-O-glucoside (Yoshikura and Hamaguchi 1969). The B-ring of flavonoids can be hydroxylated at either the 3' position leading to the production of cyanidin-based pigments or at both the 3' and 5' positions to produce delphinidin-based pigments.
Two key enzymes involved in this pathway are flavonoid 3'-hydroxylase (F3'H, EC 1.14.13.21 [EC] ) and flavonoid 3'5'-hydroxylase (F3'5'H, EC 1.14.13.88 [EC] ) that are both microsomal cytochrome P450–dependent monooxygenases that require NADPH as a cofactor (Forkmann 1991). Chromatographic experiments suggested that T and W1 are responsible for the formation of flavonoids with 3', 4' and 3', 4', 5' hydroxylation patterns, respectively (Buttery and Buzzell 1973; Buzzell and Buttery 1982; Buzzell et al. 1987; Todd and Vodkin 1993). Hence, T and W1 are presumed to encode F3'H and F3'5'H, respectively.
Iwashina et al. (2006) extracted and analyzed flavonoids from pubescence of soybean near-isogenic lines (NILs) for T or Td. Primary flavonoids were flavone aglycones. Luteolin and apigenin were predominant in NILs with tawny and gray pubescence, respectively. Thus, alleles at T locus were associated with 3'-hydroxylation in the B-ring of the flavones. The primary flavonoid in Clark-td was luteolin similar to, but about 50% less than, that of Clark with tawny pubescence. It suggests that Td may encode a structural or a regulating gene controlling flavone biosynthesis.
F3'H gene was cloned and characterized in soybean (Toda et al. 2002; Zabala and Vodkin 2003). Toda et al. (2002) cloned and characterized the F3'H cDNA from a pair of NILs for the T locus, To7B (TT, tawny pubescence) and To7G (tt, gray pubescence). Sequence analysis revealed that they differed by a single-base deletion of C in the coding region of To7G. The deletion generated a truncated polypeptide lacking the GGEK consensus sequence of F3'H gene and the heme-binding domain resulting in nonfunctional protein.
The W1 gene has a pleiotropic effect on flower and hypocotyl color: soybean cultivars having purple/white flower color have purple/green hypocotyls (Takahashi and Fukuyama 1919). Nozzolillo (1973) analyzed hypocotyl pigment and found that malvidin was predominantly responsible for purple color. Peters et al. (1984) observed that malvidin was present at 40- to 60-fold higher concentration than delphinidin, whereas petunidin was present at 4-fold higher concentration than delphinidin in purple hypocotyls.
W3 and W4 alleles have epistatic effects: under W1 genotype, soybean genotype with W3W4 has dark purple, W3w4 has dilute purple or purple throat, w3W4 has purple, and w3w4 has near-white flowers (Hartwig and Hinson 1962). W3 and W4 in combination condition hypocotyl color similar to W1 (Groose and Palmer 1991). Among 16855 of United States Department of Agriculture soybean accessions, 11299 have purple, 5544 have white, 4 have near-white, and 8 have dilute purple or purple throat flowers (Nelson RL, personal communication). Additional alleles at W4 locus, w4-dp (dilute purple) and w4-p (pale), were found in descendants of w4-mutable line (Palmer and Groose 1993; Xu and Palmer 2005). Restriction fragment length polymorphism of dihydroflavonol 4-reductase (DFR, EC 1.1.1.219 [EC] ) gene cosegregated with flower color variants in an F2 population segregating for W3 locus suggesting that W3 encodes DFR (Fasoula et al. 1995). Based on the similarity between the phenotypes conditioned by w4-dp and W3 alleles, Palmer and Groose (1993) presumed that W3 and W4 might be duplicate loci. Actually, low transcript levels or abnormal transcript products of DFR2 gene were associated with less pigmentation of flower petals in w4-dp or w4-p genotypes suggesting that W4 might also encode DFR (Palmer RG, personal communication). Further, alleles at I locus were associated with chalcone synthase (EC 2.3.1.74 [EC] ) gene duplications (Todd and Vodkin 1996). cDNA microarray suggested that Wp corresponds to flavanone 3-hydroxylase (EC 1.14.11.9 [EC] ) gene (Zabala and Vodkin 2005).
Magenta flower mutant of Harosoy (Harosoy-wm, T235) was found at Urbana, IL, in 1957 (Buzzell et al. 1977). Buttery and Buzzell (1976) revealed that magenta flower allele (wm) is associated with low amounts of flavonol glycosides in leaves. Takahashi et al. (2007) cloned cDNA of flavonol synthase (EC 1.14.11.23 [EC] ) from Harosoy and Harosoy-wm. Sequence analysis revealed that they differed by a single-base deletion of G in the coding region of Harosoy-wm. The deletion generated a truncated polypeptide lacking the dioxygenase domains resulting in nonfunctional protein.
Although genetic control of flower pigmentation has been well documented, little information is available on the structure of flavonoids in flower petals of soybean probably due to the small size of flowers and apparent lack of contribution to agriculture. This study was conducted to investigate the chemical structure of flavonoids in flower petals of soybean NILs having various alleles for flower or pubescence color loci to obtain further information on the genetic control of flavonoid biosynthesis in soybean.
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Materials and Methods |
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Plant Material
Soybean cv. Clark with purple flower and tawny pubescence (W1W1 w3w3 W4W4 WmWm TT TdTd) and Harosoy with purple flower and gray pubescence (W1W1 w3w3 W4W4 WmWm tt TdTd) and their NILs, Clark-w1 with white flower (L63-2373, w1w1 w3w3 W4W4 WmWm TT TdTd), Clark-w4 with near-white flower (L68-1774, W1W1 w3w3 W4W4 WmWm TT TdTd), Clark-W3w4 with dilute purple flower (L70-4422, W1W1 W3W3 w4w4 WmWm TT TdTd), Clark-t with gray pubescence (L64-483, W1W1 w3w3 W4W4 WmWm tt TdTd), Clark-td with near-gray pubescence (L66-260, W1W1 w3w3 W4W4 WmWm TT tdtd), Harosoy-wm with magenta flower (T235, W1W1 w3w3 W4W4 wmwm tt TdTd), and Harosoy-T with tawny pubescence (L66-707, W1W1 w3w3 W4W4 WmWm TT TdTd), were used (Table 1). Seeds of the NILs were provided by the USDA Soybean Germplasm Collections. The NILs except T235 were produced by crossing Clark or Harosoy with lines having the respective alleles and backcrossing the progeny up to BC6 (Bernard et al. 1991).
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Seeds were planted on 10 June 2003 and 11 June 2004 in fields at National Institute of Crop Science, Tsukuba, Japan. N, P, and K were applied at 3.0, 4.4, and 8.3 g/m2, respectively. Banner (standard) petals were collected daily at the date of opening using forceps. For qualitative analysis, 3 g of banner petals from Harosoy and Clark were collected in 90 ml of MeOH containing 0.1% (v/v) HCl (for anthocyanin) or in 90 ml of absolute MeOH (for flavonol). For qualitative analysis of dihydroflavonol, 31 g of banner petals were collected from Harosoy-wm in 450 ml of absolute MeOH. For quantitative analysis of the anthocyanins, flavonols, and dihydroflavonols, 200 mg of banner petals were collected from the 2 cultivars and the NILs in 2 ml of the respective solvents in 3 replications.
Isolation of Anthocyanins, Flavonols, and Dihydroflavonol
After concentration, crude extracts were applied to preparative paper chromatography using the following solvent systems: BAW (n-BuOH/HOAc/H2O = 4:1:5, upper phase), 15% (v/v) HOAc, and then BEW (n-BuOH/EtOH/H2O = 4:1:2.2). Isolated compounds were purified by Sephadex LH-20 column chromatography using 70% (v/v) MeOH (flavonols and dihydroflavonol) or 70% (v/v) MeOH + 1 drop HCl (anthocyanins).
High-Performance Liquid Chromatography
Qualitative and quantitative high-performance liquid chromatography (HPLC) separation of the isolated anthocyanins, flavonols and dihydroflavonol, and crude extracts was performed with Shimadzu HPLC systems using Shim-pack CLC-ODS (internal diameter 6.0 x 150 mm [Shimadzu Co., Kyoto, Japan]) at a flow rate of 1.0 ml/min, detection wavelength was 190–700 nm, and eluent was MeCN/HOAc/H2O/H3BO3 (6:8:83:3) for anthocyanins and MeCN/H2O/H3BO3 (22:78:0.2) for flavonols and dihydroflavonols.
Qualitative Analysis
Anthocyanins, flavonols, and dihydroflavonol were identified by 1H and 13C NMR, liquid chromatography–mass spectrometry (LC–MS), UV, characterization of acid hydrolyzates (aglycones and sugars), and direct paper chromatography (Iwashina et al. 2004). Further, Samples were run separately in HPLC, and retention times and UV spectral properties were compared with authentic specimens described in Iwashina et al. (2000, 2004, 2005) and Takemura et al. (2005). PC, HPLC, UV, 1H and 13C NMR, and LC–MS and fast atom bombardment - mass spectra (FAB–MS) data of isolated anthocyanins, flavonols, and dihydroflavonol were as follows.
Malvidin 3,5-di-O-glucoside
PC: Rf 0.18 (BAW), 0.62 (15% [v/v] HOAc); visible color—red purple. HPLC: retention time (Rt) 8.50 min. UV: 
max (nm) 0.1% (v/v) MeOH-HCl 275, 536; +AlCl3 275, 536.
Delphinidin 3,5-di-O-glucoside
HPLC: Rt 6.17 min. UV: max (nm) 0.1% (v/v) MeOH-HCl 268, 538.
Kaempferol 3-O-gentiobioside
PC: Rf 0.27 (BAW), 0.50 (BEW), 0.50 (15% [v/v] HOAc), 0.38 (5% [v/v] HOAc); UV—dark purple, UV/NH3—dark greenish yellow. HPLC: Rt 5.28 min. UV: max (nm) MeOH 266, 350; +NaOMe 275, 325, 399 (inc.); +AlCl3 274, 305, 354, 396; +AlCl3/HCl 275, 303, 349, 395; +NaOAc 274, 310, 388; +NaOAc/H3BO3 267, 354. LC–MS: ES– 609 [M – H]–, calcd for C27H30O16.
Kaempferol 3-O-rutinoside
PC: Rf 0.51 (BAW), 0.66 (BEW), 0.55 (15% [v/v] HOAc), 0.38 (5% [v/v] HOAc); UV—dark purple, UV/NH3—dark greenish yellow. HPLC: Rt 6.96 min. UV: max (nm) MeOH 266, 350; +NaOMe 275, 325, 400 (inc.); +AlCl3 274, 304, 352, 396; +AlCl3/HCl 275, 303, 347, 393; +NaOAc 274, 309, 388; +NaOAc/H3BO3 266, 354.
Kaempferol 3-O-glucoside
PC: Rf 0.72 (BAW), 0.78 (BEW), 0.40 (15% [v/v] HOAc), 0.24 (5% [v/v] HOAc); UV—dark purple, UV/NH3—dark greenish yellow. HPLC: Rt 8.13 min. UV: max (nm) MeOH 266, 349; +NaOMe 275, 325, 399 (inc.); +AlCl3 274, 304, 353, 394; +AlCl3/HCl 275, 302, 347, 393; +NaOAc 274, 307, 386; +NaOAc/H3BO3 267, 353.
Kaempferol 3-O-rhamnosylgentiobioside
PC: Rf 0.14 (BAW), 0.40 (BEW), 0.80 (15% [v/v] HOAc), 0.74 (5% [v/v] HOAc); UV—dark purple, UV/NH3—dark greenish yellow. HPLC: Rt 3.77 min. UV: max (nm) MeOH 267, 346; +NaOMe 275, 325, 394 (inc.); +AlCl3 275, 306, 355, 396sh; +AlCl3/HCl 275, 303sh, 350, 395sh; +NaOAc 274, 309, 380; +NaOAc/H3BO3 267, 348. LC–MS: ES– 755 [M – H]–, calcd for C33H40O20.
Kaempferol 7-O-glucoside
PC: Rf 0.46 (BAW), 0.55 (BEW), 0.11 (15% [v/v] HOAc), 0.04 (5% [v/v] HOAc); UV—yellow, UV/NH3—greenish yellow. HPLC: Rt 8.82 min. UV: max (nm) MeOH 266, 367; +NaOMe decomp.; +AlCl3 266, 303sh, 358, 425; +AlCl3/HCl 265, 303sh, 354, 422; +NaOAc 259, 400; +NaOAc/H3BO3 266, 369. LC–MS: ES–447 [M – H]–, calcd for C21H20O11.
Kaempferol 7-O-diglucoside
PC: Rf 0.20 (BAW), 0.33 (BEW), 0.27 (15% [v/v] HOAc), 0.07 (5% [v/v] HOAc); UV—yellow, UV/NH3—greenish yellow. HPLC: Rt 5.94 min. UV: max (nm) MeOH 267, 367; +NaOMe decomp.; +AlCl3 265, 301sh, 358, 424; +AlCl3/HCl 266, 300sh, 356, 421; +NaOAc 260, 404; +NaOAc/H3BO3 265, 370. LC–MS: ES– 609 [M – H]–, calcd for C27H30O16.
Quercetin 3-O-gentiobioside
PC: Rf 0.24 (BAW), 0.39 (BEW), 0.43 (15% [v/v] HOAc), 0.27 (5% [v/v] HOAc); UV—dark purple, UV/NH3—dark greenish yellow. HPLC: Rt 5.97 min. UV: max (nm) MeOH 257, 264sh, 359; +NaOMe 275, 329, 414 (inc.); +AlCl3 274, 429; +AlCl3/HCl 266, 301, 363, 397sh; +NaOAc 273, 325, 401; +NaOAc/H3BO3 263, 378.
Aromadendrin 3-O-ß-D-glucopyranoside
PC: Rf 0.60 (BAW), 0.68 (BEW), 0.74 (15% [v/v] HOAc); UV—dark blue, UV/NH3—light blue. HPLC: Rt 4.69 min. UV: max (nm) MeOH 290, 330sh; +NaOMe 242, 322 (inc.); +AlCl3 291, 365sh; +AlCl3/HCl 291, 365sh; +NaOAc 247sh, 286sh, 330; +NaOAc/H3BO3 292, 324sh. FAB–MS: m/z 451.2 [M + H]+ (calcd. for C21H22O11), 289.2 [M – glucosyl + H]+ (aromadendrin). 1H NMR (500 MHz, pyridine-d5): 
7.55 (2H, d, J = 8.2 Hz, H-2',6'), 7.01 (2H, d, J = 8.2 Hz, H-3',5'), 6.58 (1H, d, J = 1.5 Hz, H-8), 6.40 (1H, d, J = 1.5 Hz, H-6), 5.51 (1H, d, J = 7.6 Hz, glucosyl H-1), 4.2–4.4 (glucosyl protons), 3.89 (1H, d, J = 13.7 Hz, H-2), 3.52 (1H, d, J = 14.0 Hz, H-3ax). 13C NMR (125 MHz, pyridine-d5): (aromadendrin) 79.1 (C-2), 70.9 (C-3), 195.2 (C-4), 158.4 (C-5), 97.0 (C-6), 173.6 (C-7), 93.2 (C-8), 170.6 (C-9), 102.0 (C-10), 125.6 (C-1'), 132.6 (C-2',6'), 157.8 (C-4'), 115.9 (C-3',5'); (glucose) 107.4 (C-1), 74.2 (C-2), 79.0 (C-3), 70.8 (C-4), 78.4 (C-5), 62.2 (C-6).
Quantitative analysis
Samples were run in HPLC, and average amount of flavonoids was calculated from the peak areas of the chromatograms (detection wavelength of anthocyanins = 530 nm, flavonols = 351 nm, dihydroflavonols = 290 nm) from the 3 replicate samples. The peak area was subjected to 2-way analysis of variance to evaluate effect of year and NILs using the Statistica software (StatSoft, Inc., Tulsa, OK).
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Results |
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Qualitative Analysis
Two anthocyanins, malvidin 3,5-di-O-glucoside and delphinidin 3,5-di-O-glucoside, and 7 flavonol glycosides, kaempferol 3-O-gentiobioside, kaempferol 3-O-rutinoside, kaempferol 3-O-glucoside, kaempferol 3-O-rhamnosylgentiobioside, kaempferol 7-O-glucoside, kaempferol 7-O-diglucoside, and quercetin 3-O-gentiobioside, were identified by PC and UV spectral properties, characterization of aglycones and glycosidic sugars, LC–MS data, and finally direct PC and HPLC comparisons with authentic specimens (see Materials and Methods). A dihydroflavonol, aromadendrin 3-O-glucoside, was identified by PC, UV spectral properties, FAB–MS, and 1H and 13C NMR data.
Quantitative Analysis
HPLC patterns of anthocyanins, flavonols, and dihydroflavonols are presented in Figure 2. Four peaks corresponding to anthocyanins, A1–A4, were detected in NILs having purple or magenta flower. Based on comparison of retention times with authentic samples, peaks A1 and A4 were identified to be malvidin 3,5-O-glucoside and delphinidin 3,5-di-O-glucoside, respectively. Identity of anthocyanins corresponding to peaks A2 and A3 could not be determined. Amount of anthocyanins estimated by peak areas in HPLC analysis are presented in Table 2. Anthocyanin content was not substantially different among NILs with purple or magenta flower color. In contrast, no peaks corresponding to anthocyanins were detected in banner petals of Clark-w1 and Clark-w4. Trace amount of malvidin 3,5-O-glucoside was detected in Clark-W3w4 in both years. In addition, trace amounts of anthocyanins corresponding to peaks A2 and A3 were also detected in 2004.
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Eight peaks, F1–F8, corresponding to flavonols were detected in HPLC analysis (Figure 2). Based on the retention time, the flavonols comprising the peaks were determined as follows: F1 (kaempferol 3-O-gentiobioside), F2 (kaempferol 3-O-rutinoside), F3 (kaempferol 3-O-glucoside), F4 (kaempferol 3-O-glycoside), F5 (kaempferol 3-O-rhamnosylgentiobioside), F6 (quercetin 3-O-gentiobioside), F7 (kaempferol 7-O-glucoside), and F8 (kaempferol 7-O-diglucoside). Amounts of flavonols estimated by peak areas in HPLC analysis are presented in Table 3. Kaempferol 3-O-gentiobioside was predominant and comprised 64% to 82% of the total flavonols except for Harosoy-wm. Amount of flavonols was substantially reduced in Harosoy-wm (17.1% in 2003 and 9.0% in 2004 compared with Harosoy) in agreement with the leaf flavonol content reported by Buttery and Buzzell (1976). Further, F5 is missing in both years, F3 is missing in 2004, and only trace amount of F6 was found in 2003 in Harosoy-wm. Flavonol contents were largely similar among the other NILs including NILs with white and near-white flowers (Table 3).
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Only one peak (F9) corresponding to dihydroflavonol (aromadendrin 3-O-glucoside) was found by HPLC analysis (Figure 2). Amount of dihydroflavonol estimated by peak area in HPLC analysis is presented in Table 4. Dihydroflavonol in Harosoy-wm was higher than Harosoy by 3 times in 2003 and 4 times in 2004. Amount of dihydroflavonol in the other NILs was largely similar.
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Discussion |
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Primary anthocyanins in soybean flower petals were malvidin 3,5-di-O-glucoside and delphinidin 3,5-di-O-glucoside. Peters et al. (1984) analyzed pigments in soybean hypocotyls, and found that the predominant pigment was malvidin with small amounts of delphinidin and petunidin. Pleiotropic effects of flower and hypocotyl color may be caused by similar pigment composition and existence of similar genetic control of anthocyanin biosynthesis between the 2 tissues.
No peaks corresponding to anthocyanins were detected in banner petals of Clark-w4 and Clark-w1 in both years. However, small amount of anthocyanin (0.4% of malvidin 3,5-di-O-glucoside compared with Clark) was detected in Clark-w4 in the preliminary experiment in 2002, suggesting environmental effects on the synthesis of anthocyanins in soybeans with w4 allele. It is consistent with the observation of Hartwig and Hinson (1962) that tinge of pigmentation occurs in genotypes with W1w3w4 at the base of the standard petal in some environments but indistinguishable from white flowers in most environments. Considering the similar amount of flavonols and dihydroflavonols in NILs with w1 or w4 genotypes, W1 and W4 genes affect only anthocyanin biosynthesis. Although W1 is known to be responsible for the trihydroxylation of the B-ring, it is still unclear how it conditions flower color (purple or white) and the presence/absence of anthocyanins. Substrate specificity of DFR differs among plant species (Johnson et al., 2001). It is possible that soybean DFR exclusively utilize tri-hydroxylated dihydroflavonols as a substrate. Substrate specificity of soybean DFR remains to be investigated.
Anthocyanin content of Harosoy-wm was largely similar to the NILs with purple flower. On the other hand, amount of kaempferol glycosides were substantially reduced and dihydroflavonols were greatly increased in Harosoy-wm. It suggests that Wm is responsible for the production of flavonols from dihydroflavonol and it may encode flavonol synthase. Molecular cloning and characterization of the flavonol synthase cDNA confirmed the hypothesis (Takahashi et al. 2007). Co-pigmentation of anthocyanins with tannins, flavones, and flavonol glycosides can have major effects on flower color (Scott-Moncrieff 1936). When compared over a range of physiological pH values, co-pigmented anthocyanins are usually bluer than anthocyanins alone. The recessive wm allele substantially reduces flavonol glycoside content, and it inhibits copigmentation between anthocyanins and flavonol resulting in less bluer (magenta) flower color similar to the flavonol synthase mutant in petunia (Holton et al. 1993). Thus, co-pigmentation between anthocyanins and flavonol glycosides may contribute to the purple pigmentation of flower petals in soybeans having Wm allele. Recessive wm allele reduces photosynthetic rate and seed yield and causes earlier leaf senescence (Buzzell et al. 1977). Either low amounts of flavonol glycosides or high amounts of dihydroflavonol may be responsible for the deleterious effects.
Alleles at pubescence color loci, T or Td, had no obvious effects on flavonoid biosynthesis in flower petals. The 3'-position of most flavonols were not hydroxylated even in NILs with dominant T allele encoding F3'H. The mechanism is uncertain because northern analysis revealed that F3'H gene is weakly expressed in flower petals of the NIL with dominant T allele (Toda K, Takahashi R, unpublished results).
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Acknowledgments |
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We thank Dr R. L. Nelson at USDA/ARS University of Illinois for supplying the seeds of the NILs, Dr T. Aoki (Nihon University) for advice, and Dr Joseph G. Dubouzet (National Institute of Agrobiological Science) for critical reading of the manuscript.
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Footnotes |
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Corresponding Editor: Reid Palmer
Received September 5, 2006
Accepted December 14, 2006
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References |
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