Journal of Heredity Advance Access originally published online on September 19, 2006
Journal of Heredity 2006 97(5):438-443; doi:10.1093/jhered/esl027
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Analysis of Flavonoids in Pubescence of Soybean Near-isogenic Lines for Pubescence Color Loci
From the Tsukuba Botanical Garden, National Science Museum, Tsukuba, Ibaraki 305-0005, Japan (Iwashina); the Utsunomiya University, Utsunomiya 321-8505, Japan (Benitez); the National Institute of Crop Science, Kannondai 2-1-18, Tsukuba, Ibaraki 305-8518, Japan (Takahashi)
Address correspondence to R. Takahashi at the address above, or e-mail: masako{at}affrc.go.jp.
T and Td loci control pubescence color of soybean with epistatic effects (TT TdTd, tawny; TT tdtd, light tawny or near-gray; tt TdTd or tt tdtd, gray). The objective of this study was to investigate the nature of flavonoids in the pubescence of near-isogenic lines (NILs) for these loci. Flavonoids were extracted with methanol from pubescence of cultivar Clark with tawny pubescence (TT TdTd) and its NILs; from Clark-t with gray pubescence (tt TdTd) and Clark-td with near-gray pubescence (TT tdtd); and from a pair of NILs, To7B with tawny (TT TdTd) and To7G with gray pubescence (tt TdTd). Primary flavonoids were flavone aglycones. Luteolin and apigenin were predominant in NILs with tawny and gray pubescence, respectively. Small amount of 7-O-glucosides of the 2 flavones were also detected. Alleles at T locus were associated with 3'-hydroxylation in the B-ring of the flavones. The primary flavonoids in Clark-td were luteolin similar to Clark, but its amount was halved. High performance liquid chromatography peaks probably corresponding to isoflavonoids were found only in Clark-td in 2003. However, the peaks were not observed in 2005. The above results suggest that Td may encode a structural or a regulatory gene controlling flavone biosynthesis. Pigments remained visible in pubescence after methanol extraction, suggesting that a major part of the pigments was polymerized. Surface rinsing experiments revealed that flavone aglycones exist outside the surface of cells.
Flavonoids are important secondary metabolites in the plant kingdom. Individual plant species synthesize a variety of flavonoid compounds that function in providing flower pigmentation to attract pollinators, in defending plants against pathogens, in acting as signal molecules in plant–microbe interactions, and in protecting plants from UV radiation (Shirley 1996, 2001). Flavonoids also have significant activities when ingested by animals. In particular, isoflavonoids abundant in soybean seeds have various health-promoting activities including anti-cancer benefits of soy-based foods (Song et al. 1999). Based on structural similarity with the principal estrogen, 17ß-estradiol, isoflavonoids such as daidzein and genistein exhibit estrogenic activity and are called phytoestrogens. Their estrogenic activity is presumed to play important roles in human health.
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 (I, T, W1, R, and O) control seed coat color, 5 genes (W1, W3, W4, Wm, and Wp) control flower color, and 2 genes (T and Td) control pubescence color in soybean (reviewed by Palmer et al. 2004). Allelic combination of TT TdTd has brown pubescence, TT tdtd has light tawny or near-gray pubescence, and tt TdTd or tt tdtd has gray pubescence (Bernard 1975). Among 16 688 of US Department of Agriculture (USDA) soybean accessions, 8442 have tawny, 707 have light tawny or near-gray, and 7539 have gray pubescence (RL Nelson, personal communication). Bernard (1975) crossed near-gray pubescence cultivars with a tawny pubescence cultivar Clark and found a wide range of pubescence color in the F2 population, with classification difficult or impossible. The development of tawniness was affected by various factors including maturity and amount of pubescence.
Major anthocyanin pigments in the seed coats of black soybeans were cyanidin 3-O-glucoside and delphinidin 3-O-glucoside (Yoshikura and Hamaguchi 1969). The hydroxylation pattern of B-ring in flavonoids plays an important role in the coloration of seed coats and pubescence of soybeans. The B-ring of flavonoids can be hydroxylated either at 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 reduced nicotinamide adenine dinucleotide phosphate as a cofactor (Forkmann 1991). Chromatographic experiments suggested that T and W1 loci 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. F3'H gene was cloned and characterized in soybean (Toda et al. 2002; Zabala and Vodkin 2003). Toda et al. (2002) compared the F3'H cDNA from a pair of near-isogenic lines (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.
In addition to pubescence and seed coat color, alleles at T locus are associated with chilling tolerance (Takahashi and Asanuma 1996; Takahashi 1997; Takahashi et al. 2005) and structural integrity of seed coats (Nicholas et al. 1993). The T locus and F3'H gene were located at the same position in the molecular linkage group C2 (Toda et al. 2002). The mechanism for the relationship between pubescence color (alleles at T locus) and chilling tolerance is controversial, and it is uncertain whether pubescence color directly affects chilling tolerance (Toda et al. 2005).
In contrast to the T locus, information on Td locus is limited; its genomic location or encoding protein has not been determined. Although pubescence color of soybean has long been noticed and utilized as a marker to ascertain hybridity in crossing, responsible pigments have not been determined probably due to the small size of pubescence. We investigated the nature of flavonoids in the pubescence of soybean NILs having different alleles at 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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 Top Materials and Methods Results and DiscussionReferences |
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Plant Material
Soybean cultivar Clark with tawny pubescence (TT TdTd) and its NILs, Clark-t with gray pubescence (L64-483, tt TdTd), Clark-td with near-gray pubescence (L66-260, TT tdtd), and a pair of NILs, To7B with tawny pubescence (TT TdTd) and To7G with gray pubescence (tt TdTd), were used (Table 1). Seeds of the Clark NILs were provided by the USDA Soybean Germplasm Collections. The NILs were produced by crossing Clark with lines having the respective alleles and backcrossing the progeny up to BC6 (Bernard et al. 1991). To7B and To7G were developed at the Tokachi Agricultural Experimental Station. They were derived from a cross between T207 (tt TdTd) and Toshidai-7910 (TT TdTd) held at the Tokachi Agricultural Experimental Station in 1977. Plants heterozygous for T/t were selected in 6 succeeding generations and finally fixed at the T locus.
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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 m–2, respectively. Pubescence was collected from pods and main stems at R7 stage (Fehr et al. 1971) using razor blades. For qualitative analysis,
20 g of pubescence was collected in 90 mL of MeOH in 2004. For quantitative analysis, 50 mg of pubescence was collected from the NILs in 2 mL of MeOH in 3 replications in 2003.
Extraction and Isolation of Flavonoids
Fresh pubescence of To7B and Clark (a total of 24.21 g) and To7G and Clark-t (a total of 20.05 g) was extracted with MeOH. After concentration, the extracts were applied to preparative paper chromatography using BAW (n-BuOH/HOAc/H2O = 4:1:5, upper phase), 15% (v/v) HOAc (glycosides) or 50% (v/v) HOAc (aglycones), and then BEW (n-BuOH/EtOH/H2O = 4:1:2.2). Isolated flavonoids were purified by Sephadex LH-20 column chromatography using 70% (v/v) MeOH. To investigate the localization of flavonoids, pods were collected from mature plants of cultivars with tawny pubescence (Hyokei Kuro-3) and gray pubescence (Tachinagaha). The pods were rinsed with acetone. The acetone extracts were evaporated to dryness, dissolved with MeOH, and subjected to the following high performance liquid chromatography (HPLC) analysis.
High Performance Liquid Chromatography
Qualitative and quantitative HPLC separation of the isolated flavonoids and crude extracts was performed with Shimadzu HPLC systems (Shimadzu Co., Kyoto, Japan) using Pegasil ODS (internal diameter = 6.0 x 150 mm [Senshu Scientific. Co., Tokyo, Japan]) at a flow rate of 1.0 mL min–1, detection wavelength of 190–700 nm, and eluents of MeCN/H2O/H3PO4 (35:65:0.2) (Solvent I) for crude extracts and aglycones and MeCN/H2O/H3PO4 (22:78:0.2) (Solvent II) for glycosides. The average amount of flavonoids was calculated from the peak areas of HPLC chromatograms from the 3 replicate samples.
Identification of Flavonoids
Flavonoids were identified by UV spectra according to Mabry et al. (1970) and characterization of acid hydrolysates (aglycones and sugars). Further, samples were run separately in HPLC, and retention times and UV spectral properties were compared with the following authentic specimens: apigenin from Extrasynthese (Genay, France), luteolin from leaves of Schumalhausenia nidulans (Iwashina and Kadota 1999), and apigenin 7-O-glucoside from leaves of Helwingia japonica (Iwashina et al. 1997). HPLC and UV spectral data were as follows:
- Apigenin. HPLC retention time (Rt.) (min): 6.95 (Solvent I). UV
max (nm): MeOH 268, 334; +NaOMe 275, 325, 391 (inc.); +AlCl3 275, 302, 348, 379; +AlCl3/HCl 277, 300, 341, 375; +NaOAc 275, 307, 384; +NaOAc/H3BO3 269, 343.
- Luteolin. HPLC Rt. (min): 5.17 (Solvent I). UV
max (nm): MeOH 254, 266, 348; +NaOMe 269, 328, 405 (inc.); +AlCl3 272, 422; +AlCl3/HCl 262, 274, 295, 357, 382sh; +NaOAc 268, 394; +NaOAc/H3BO3 261, 372.
- Apigenin 7-O-glucoside. HPLC Rt. (min): 11.89 (Solvent II). UV
max (nm): MeOH 268, 332; +NaOMe 274, 378 (inc.); +AlCl3 275, 300, 349, 378; +AlCl3/HCl 276, 298, 341, 375; +NaOAc 267, 388; +NaOAc/H3BO3 267, 340.
- Luteolin 7-O-glucoside. HPLC Rt. (min): 7.47 (Solvent II). UV
max (nm): MeOH 256, 264sh, 351; +NaOMe 266, 392 (inc.); +AlCl3 273, 426; +AlCl3/HCl 265, 273sh, 296, 359, 386sh; +NaOAc 263, 401; +NaOAc/H3BO3 259, 374.
- Kaempferol 3-O-rutinoside. HPLC Rt. (min): 8.38 (Solvent II). UV
max (nm): MeOH 265, 348; +NaOMe 274, 322, 398 (inc.); +AlCl3 273, 305, 358, 387sh; +AlCl3/HCl 273, 303, 355, 387sh; +NaOAc 273, 305, 392; +NaOAc/H3BO3 265, 351.
- Quercetin 3-O-rutinoside. HPLC Rt. (min): 5.95 (Solvent II). UV
max (nm): MeOH 257, 263sh, 358; +NaOMe 274, 327, 412 (inc.); +AlCl3 274, 429; +AlCl3/HCl 267, 302, 362, 395sh; +NaOAc 273, 326, 403; +NaOAc/H3BO3 263, 378.
- Luteolin. HPLC Rt. (min): 5.17 (Solvent I). UV
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Results and Discussion |
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TopMaterials and MethodsResults and DiscussionReferences |
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Qualitative Analysis
Six kinds of flavonoids were isolated from soybean pubescence. Four of them were identified as apigenin, luteolin, apigenin 7-O-glucoside, and luteolin 7-O-glucoside. Two minor flavonoids were characterized as kaempferol 3-O-rutinoside and quercetin 3-O-rutinoside by UV spectral data and direct HPLC comparisons with authentic specimens. However, they did not appear on HPLC chromatograms. Luteolin was obtained as yellow powder (
50 mg). Apigenin and apigenin 7-O-glucoside were obtained as pale yellow powders (30 and 20 mg). The biosynthetic pathway for flavones and isoflavonoids is summarized in Figure 1.
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Quantitative Analysis
HPLC chromatograms of flavonoids in soybean pubescence are presented in Figure 2. A total of 5 peaks, D1–D5, were detected at UV 349 nm. They were identified as follows: D1 = apigenin, D2 = luteolin, D3 = luteolin derivative, D4 = apigenin derivative, and D5 = apigenin 7-O-glucoside. Additional 2 peaks, D6 and D7, were detected at UV of 261 nm only in Clark-td. Based on the UV spectral properties, they were presumed as isoflavonoids. However, the 2 peaks were not observed in 2005, although the other peaks were similarly reproduced (data not shown).
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Three peaks, D1, D2, and D3, were found in To7B and Clark. Based on the peak areas, D2 comprises 95–96% of the total flavonoids (Table 2). D1 and D3 comprised small portions (D1: less than 1%, D3: 3–4% of the total flavonoids). Luteolin and its derivatives accounted for most of the flavonoids in NILs with tawny pubescence. Four peaks, D1, D2, D4, and D5, were found in To7G and Clark-t. D1 comprised
90% of the total flavonoids. The other flavonoids, D2 (1%), D4 (2–3%), and D5 (6%), comprised minor portions. Thus, apigenin and its derivatives accounted for most of the flavonoids in NILs with gray pubescence. Alleles at the T locus were associated with the hydroxylation pattern of the 3'-position in the B-ring of flavones in accordance with the previous report that T gene encodes F3'H (Buttery and Buzzell 1973; Buzzell et al. 1987; Todd and Vodkin 1993; Toda et al. 2002; Zabala and Vodkin 2003).
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Three peaks, D1, D2, and D3, were found in Clark-td under UV of 349 nm. Proportion of the peaks (D1< 1%, D2
94%, D3 5%) was largely similar to Clark and To7B. However, the amount of D2 was halved compared with To7B and Clark. Two additional peaks, D6 and D7, that probably correspond to isoflavonoids were detected in Clark-td under UV of 261 nm. The most plausible hypothesis is that a gene controlling the production of flavones from flavanones is partially defective in Clark-td. The remaining flavanones were catalyzed by isoflavone synthase into isoflavonoids. Td may encode a structural or a regulatory gene responsible for flavone biosynthesis. The 2 peaks, D6 and D7, were not reproduced in 2005. Effects of environments on seed isoflavonoid contents have been studied in soybean (Kitamura et al. 1991; Tsukamoto et al. 1995; Hoeck et al. 2000; Lozovaya et al. 2005; Primomo et al. 2005). They revealed that year, location, and environments including temperatures and soil moisture at seed-filling period significantly affect seed isoflavonoid contents. Isoflavonoid biosynthesis in soybean pubescence may also be subject to environmental factors. Biosynthetic pathways of flavones differ among plant species (Akashi et al. 1998). In parsley, 2-oxoglutarate–dependent flavone synthase I (EC 1.14.11.22 [EC] ) abstracts hydrogens from C-2 and C-3 of a flavanone. In snapdragon and soybean, a cytochrome P450 monooxygenase catalyzes the formation of a 2-hydroxyflavanone from a flavanone, and the subsequent dehydration produces a flavone. This process is called flavone synthase II (FNSII). FNSII gene of soybean should be cloned to investigate the relationship between Td and FNSII.
Histological Localization
In gray pubescence cultivar Tachinagaha, the acetone extract from pod surfaces contained almost exclusively apigenin aglycone with a small amount of aromatic acids (data not shown). In tawny pubescence cultivar Hyokei Kuro-3, HPLC analysis of the acetone extract detected 3 major flavonoid peaks, one of which was identified as luteolin aglycone. Components of the other 2 peaks have not been determined. However, based on retention times, one of them probably corresponded to flavanone or dihydroflavonol and the other corresponded to flavone or flavonol (data not shown). Thus, only aglycone type of flavones was detected and flavone glucosides were not detected outside the surface of pods. The results are consistent with the previous reports that flavonoid glycosides accumulate in vacuoles, whereas aglycones, the so-called surface flavonoids, are deposited outside the surface of the cells (Wollenweber 1994).
Pigments remained visible in pubescence of all the NILs after MeOH extraction, suggesting that major part of the pigments in soybean pubescence was polymerized. The recessive td allele produced lighter pubescence color and lower amount of flavones in pubescence compared with Td allele. The recessive t allele produced gray pubescence color and flavones not hydroxylated at the 3'-position in the B-ring. These results suggest that structure and amount of flavones might be responsible for pubescence color and that flavones may be integral components of the polymers. Color of luteolin (yellow) and apigenin (pale yellow) may not be directly associated with gray and tawny pubescence color. Tawny and gray color may be derived from polymers containing luteolin and apigenin derivatives, respectively. Near-gray color is probably caused by the reduction in polymers containing luteolin derivatives. Generally, brown color is derived from polymerization of proanthocyanidins or other phenolic compounds (Tanner 2004). To our knowledge, there has been no previous report on the polymers of flavones. Components and structure of the polymers in soybean pubescence should to be determined.
C-Glycosylflavones such as vitexin were detected in soybean roots (Jay et al. 1984). However, this may be the first report of flavone aglycones and O-glycosides in soybean tissues. Flavonoids are tissue-specific in soybean, and this study describes flavonoids specific to pubescence. Further studies may be necessary to investigate the function of flavone aglycones and glycosides in soybean pubescence including the defensive roles against attacks by herbivorous insects (Simmonds 2003).
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Acknowledgments |
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We thank Dr R. L. Nelson at USDA-ARS at University of Illinois and the staff of Tokachi Agricultural Experimental Station for supplying the seeds of the NILs. We are grateful to 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 April 6, 2006
Accepted July 26, 2006
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References |
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