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Comparison of saponin composition and content in wild soybean (Glycine soja Sieb. and Zucc.) before and after germination a
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b
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Panneerselvam Krishnamurthy , Chigen Tsukamoto , Yuya Takahashi , Yuji Hongo , Ram c
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J. Singh , Jeong Dong Lee & Gyuhwa Chung
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Department of Biotechnology, Chonnam National University, Yeosu, Korea
b
Department of Applied Biological Chemistry, Iwate University, Morioka, Japan
c
USDA/ARS, National Soybean Research Laboratory, University of Illinois, Urbana, IL, USA d
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School of Applied Biosciences, Kyungpook National University, Daegu, Korea Published online: 15 Aug 2014.
To cite this article: Panneerselvam Krishnamurthy, Chigen Tsukamoto, Yuya Takahashi, Yuji Hongo, Ram J. Singh, Jeong Dong Lee & Gyuhwa Chung (2014) Comparison of saponin composition and content in wild soybean (Glycine soja Sieb. and Zucc.) before and after germination, Bioscience, Biotechnology, and Biochemistry, 78:12, 1988-1996, DOI: 10.1080/09168451.2014.946389 To link to this article: http://dx.doi.org/10.1080/09168451.2014.946389
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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 12, 1988–1996
Comparison of saponin composition and content in wild soybean (Glycine soja Sieb. and Zucc.) before and after germination Panneerselvam Krishnamurthy1, Chigen Tsukamoto2,*, Yuya Takahashi2, Yuji Hongo2, Ram J. Singh3, Jeong Dong Lee4 and Gyuhwa Chung1,* 1
Department of Biotechnology, Chonnam National University, Yeosu, Korea; 2Department of Applied Biological Chemistry, Iwate University, Morioka, Japan; 3USDA/ARS, National Soybean Research Laboratory, University of Illinois, Urbana, IL, USA; 4School of Applied Biosciences, Kyungpook National University, Daegu, Korea
Received February 12, 2014; accepted July 9, 2014
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http://dx.doi.org/10.1080/09168451.2014.946389
Eight wild soybean accessions with different saponin phenotypes were used to examine saponin composition and relative saponin quantity in various tissues of mature seeds and two-week-old seedlings by LC–PDA/MS/MS. Saponin composition and content were varied according to tissues and accessions. The average total saponin concentration in 1 g mature dry seeds of wild soybean was 16.08 ± 3.13 μmol. In two-week-old seedlings, produced from 1 g mature seeds, it was 27.94 ± 6.52 μmol. Group A saponins were highly concentrated in seed hypocotyl (4.04 ± 0.71 μmol). High concentration of DDMP saponins (7.37 ± 5.22 μmol) and Sg-6 saponins (2.19 ± 0.59 μmol) was found in cotyledonary leaf. In seedlings, the amounts of group A and Sg-6 saponins reduced 2.3- and 1.3-folds, respectively, while DDMP + B + E saponins increased 2.5-fold than those of mature seeds. Our findings show that the group A and Sg-6 saponins in mature seeds were degraded and/or translocated by germination whereas DDMP saponins were newly synthesized. Key words:
wild soybean; soyasaponins; germination; triterpene glycosides; Glycine soja
Wild soybean (Glycine soja Sieb. and Zucc.) is native to East Asian countries including China, the Korean Peninsula, Japan, Russia Far East, and Taiwan.1) Soybean [Glycine max (L.) Merr.], one of the most important legumes in this world, often relies on the genetical resources of wild soybean to improve its quality.1) Wild soybean, the undomesticated wild relative of soybean, has high genetic diversity and serves as a promising gene resource to produce new soybean cultivars with better quality through natural selection and conventional breeding.1) Both soybean and wild soybean seeds contain significant amount of saponins
(soyasaponins – bioactive secondary metabolites).2) Soyasaponins are triterpene glycosides consisting of a triterpene aglycone (C30) attached with one or two sugar chains. Several structurally diverse groups of soyasaponins have been identified from the seeds of the subgenus Soja which includes the cultivated and wild soybean.3–12) Based on the aglycone structure, soyasaponins have been classified into group A, DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one), group H, group I, and group J saponins (Fig. 1). The latter three groups (H, I, and J) are collectively designated as Sg-6 saponins.6) Group A saponins are bisdesmosides containing two sugar chains at the C-3 and C-22 hydroxyl positions of soyasapogenol A (SS-A).9) Based on the terminal sugar of the C-22 sugar chain of SS-A, group A saponins are further subdivided into Aa-, Ab-, and A0-series saponins.13) The A0-series saponins have no sugar at the terminal position of the C-22 sugar chain of SS-A, whereas the Aa- and Ab-series saponins have acetylxylose and acetyl-glucose, respectively, at the same position. DDMP saponins are monodesmosides with one sugar chain and one DDMP moiety at the C-3 and C-22 hydroxyl positions of soyasapogenol B (SS-B), respectively. DDMP saponins are degraded during most extraction procedures to form group B saponins having SS-B and group E saponins having soyasapogenol E (SS-E).7) Sg-6 saponins are monodesmosides with one sugar chain at the C-3 hydroxyl position of their respective soyasapogenols (SS-H, SS-I, and SS-J).6) The nomenclature of all soyasaponins was derived from these six aglycones and their sugar moiety composition (Fig. 1). In the subgenus Soja, seed hypocotyl contains all of the three major saponin groups (group A, DDMP, and Sg-6) which produce eight common distinguishable saponin phenotypes in thin layer chromatography (TLC) namely Aa, Ab, AaBc, AbBc, Aa + α, Ab + α,
*Corresponding authors. Email: [email protected] (C. Tsukamoto); [email protected] (G. Chung) Abbreviations: ATSC, average total saponin composition; DDMP, 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one; LC–PDA/MS/MS, liquid chromatography–photodiode array detector/mass spectrometry/mass spectrometry; TLC, thin layer chromatography; SS-A, soyasapogenol A; SS-B, soyasapogenol B, SS-E, soyasapogenol E; SS-H, soyasapogenol H; SS-I, soyasapogenol I; SS-J, soyasapogenol J. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Saponins variation in G. soja before and after germination
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Fig. 1. Chemical structure and nomenclature of soyasaponins.4–6) R1 at the C-3 position of soyasapogenols A, B, E, H, I, and J, and R2 at the C22 position of soyasapogenol A are shown at the bottom of Fig. 1.
AaBc + α, and AbBc + α (Supplementary Fig. 1a). Seed cotyledon also contains all of the three major saponin groups;14) however, the concentration of group A saponins is relatively low and cannot be detected by TLC (Supplementary Fig. 1b). The polymorphism of the common saponin composition (saponin phenotype) of seed hypocotyl can be well explained by the combination of six allelic genes Sg-1a/Sg-1b, Sg-4/sg-4, and Sg6/sg-6 on three loci Sg-1, Sg-4, and Sg-6, respectively.4–6) Based on these three gene loci, the genotype of the common saponin phenotypes is designated as follows: Sg-1a/sg-4/sg-6 (phenotype – Aa); Sg-1b/sg-4/ sg-6 (Ab); Sg-1a/Sg-4/sg-6 (AaBc); Sg-1b/Sg-4/sg-6 (AbBc); Sg-1a/sg-4/Sg-6 (Aa + α); Sg-1b/sg-4/Sg-6 (Ab + α); Sg-1a/Sg-4/Sg-6 (AaBc + α); and Sg-1b/Sg-4/ Sg-6 (AbBc + α). The latter four + α types were not found in soybean, so far, while all of eight types were found in wild soybean.4–6) Despite several reports on the complexity of the chemical structure diversity of saponins and seed saponin composition, the distribution of saponin composition and content as well as the physiological function of saponins in the soybean plant is not clearly understood. Jyothi et al.15) Rupasinghe et al.16), and Shimoyamada et al.17) have reported that germination upregulates the content of group B saponins and SS-B, and down-regulates the content of group A saponins and SS-A in soybean. Jyothi et al.15) extracted saponins for 36 h using soxlet and studied the changes of saponin Bb whereas Rupasinghe et al.16) performed saponin
extraction at 50 °C for 2 h and studied the changes of SS-A and SS-B concentration before and after germination. Shimoyamada et al.17) extracted saponins for 5 h at 80 °C and studied the influence of germination and light irrigation on saponin composition and content. Except Rupasinghe et al.,16) Jyothi et al.15) and Shimoyamada et al.17) are supposed to detect DDMP saponins in their study. But, they did not detect DDMP saponins given the fact that long extraction time at high temperature can degrade all DDMP saponins into group B and/or group E saponins. In general, DDMP saponins are heat labile and become unstable at ≥ 30 °C.18,19) The removal of DDMP group from saponins in water solutions becomes more pronounced, with a 50, 75, and 95% decrease in DDMP saponin concentration after 75 min at 60, 75, and 90 °C, respectively.19) The loss of DDMP group can be completely prevented for 90 min by the presence of >30% ethanol (v/v) at 65 °C. From this, anyone can readily interpret that temperature plays a vital role in DDMP saponins degradation. Hence, the studies of Jyothi et al.15) and Shimoyamada et al.17) were biased though they provide some interesting facts. To the end, there is no information available so far about the changes of DDMP and Sg-6 saponins (which are identified recently) before and after germination. Furthermore, comprehensive profiling and identification of saponins variation are essential for the functional analysis of saponins in soybean; this is the basis for exploring the specific role of any compound in plant physiology. Since the polymorphism
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of saponin composition in wild soybean is wider than that of cultivated soybean (Supplementary Fig. 1), we selected one wild soybean accession for each common saponin phenotype in this study. Saponin profile and relative saponin quantity were examined in hypocotyl, cotyledon, germinated hypocotyl, epicotyl, apical meristem, cotyledonary leaf, unifoliolate leaf, trifoliolate leaf, primary root, and secondary root of mature seeds and two-week-old seedlings of wild soybean. We believe this is the first study conducted for comprehensive profiling of saponins from wild soybean seeds and seedlings. Our findings raise the potential knowledge of soyasaponins, which would be very helpful in designing the future research work to uncover the importance of soyasaponins from plant physiology perspective.
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Material and methods Materials and chemicals. Eight wild soybean accessions [CWS4162 (saponin phenotype: Aa), CWS0101 (Ab), CWS4151 (AaBc), CWS4345 (AbBc), CWS2976 (Aa + α), CWS4780 (Ab + α), CWS4779 (AaBc + α), and CWS4852 (AbBc + α)] were obtained from Chung’s Wild Legume Germplasm Collection at the Chonnam National University, Yeosu, Chonnam, Korea. Silica gel (SiO2)-coated TLC plates (catalog No.105626) were purchased from Merck Millipore, Germany. Saponin Bb was bought from Wako Pure Chemical Industries, Ltd., Osaka, Japan. All chemicals used in this study were of analytical grade. They were purchased from Honeywell Burdick and Jackson, Seoul, Korea, and Samchun Chemicals, Seoul, Korea.
Growing conditions. Seeds were germinated triplicate under non-controlled conditions from May 29 to June 24, 2012. Each planting was done at one-week interval. Seeds were scarified using a scalpel and sown in plastic flats containing vermiculite. The flats were simply kept on the roof of Department of Biotechnology (Chonnam National University, Yeosu campus, Korea) for germination and growth. Flats were sprinkled with tap water once a day. After two weeks, 10 seedlings from each accession were selected and dissected into eight parts (tissues): germinated hypocotyl, epicotyl, apical meristem, cotyledonary leaf, unifoliolate leaf, trifoliolate leaf, primary root, and secondary root. Sampled tissues were water drained with tissue paper, immersed in liquid nitrogen, and stored at −80 °C until freeze drying. Saponin extraction. Hypocotyl and cotyledon from five mature seeds of each accession were dissected. Saponins were extracted from intact hypocotyls, cotyledon powder, and milled seedling tissues using 10-fold volumes (v/w) of 80% (v/v) aqueous methanol. Extraction was carried out at room temperature (25 °C) for 24 h for hypocotyl and 1 h for cotyledon powder and milled seedling tissues. The resulting extracts were stored at 4 °C until TLC and LC–PDA/MS/MS.
Thin-layer chromatography analysis. TLC was performed according to Krishnamurthy et al.20) Briefly, 10 μL from hypocotyl (HY) and cotyledon (CT) extracts of each accession was directly applied on silica gel-coated TLC plates with an Eppendorf micropipette and slightly dried by using a hair drier. The plates were developed in a rectangular developing chamber which was saturated with the lower phase of chloroform:methanol:water (65:35:10, v/v/v) for 2 h. Plates were dried at 100 °C for 10 min and then developed with 10% H2SO4 for 12 min in a closed chamber. Saponins were visualized by heating the plates at 115 °C for 13 min. LC–PDA/MS/MS analysis. Hypocotyl extracts were diluted 10-fold with 80% methanol prior to use. Those from seed cotyledons and seedling tissues were used directly. Ten micro liters from each extract were analyzed in a UFLC system (Prominence UFLC system, Shimadzu, Kyoto, Japan) equipped with a photodiode array (PDA) detector and a tandem mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific, Yokohama, Kanagawa, Japan) on a C30 reverse phase column (Develosil C30-UG-3, 2.0 mm I.D. × 150 mm, Nomura Chemical, Seto, Okayama, Japan) at 40 °C. Solvent A consisted of acetonitrile with 0.1% (v/v) formic acid and solvent B consisted of water with 0.1% formic acid. A linear gradient elution of solvent A was performed at a flow rate of 0.15 mL/min: solvent A was initiated at 20% (v/v), increased to 65% (v/v) in 45 min, and then increased to 100% (v/v) for 5 min. The eluent composition was returned to the initial state of 20% (v/v) solvent A for 15 min. The eluate from the column was monitored by a PDA detector at UV 205 and 292 nm and by a tandem mass spectrometer in the positive ion mode of the electrospray ionization [ESI (+)] method. An automatic full-scan mode over a massto-charge ratio (m/z) range from 300 to 1800 and the top three ion-trap mode were used to acquire MS and MS/MS data, respectively. Saponin quantification. The recorded UV and MS spectra were analyzed with Xcalibur software version 2.1 (Thermo Fisher Scientific, Yokohama, Kanagawa, Japan). Xcalibur settings used to detect saponin peaks were as follows: peak algorithm, ICIS; baseline, 150; area noise, 5; peak noise factor, 10; and the remaining factors were kept default. Relative quantifications were performed for all the identified saponins based on external standardization by employing the calibration curve of saponin Bb at a concentration of 995 pmol/ 5 μL. Quantitative analyses were based on the peak area of UV 205 nm. Microsoft Excel 2007 was used for the statistical analysis.
Results Saponin composition in various tissues of wild soybean seeds and seedlings Based on the molecular ion [M + H]+ mass of soyasaponins, a total of 38 saponin components were detected in this study (Table 1). Of these, 14
Saponins variation in G. soja before and after germination
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Table 1.
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Saponin composition in various tissues of mature seeds and two-week-old seedlings of wild soybean. Mature seed
Two-week old seedling of wild soybean
No.
Saponins
m/z used for quantification [M+H]+
HY
CT
GH
EC
AM
MR
LR
CL
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Group A Aa Au Ae Ax Ag Ab Ac Af Ad Az Ah A0-αg A0-βg A0-αa
1365.63 1349.64 1203.58 1335.62 1173.57 1437.65 1421.66 1275.60 1407.64 1391.65 1245.59 1107.56 1091.56 1077.55
+++ + ++ ++
++
+++
++
++
++
++
+++ + ++
++ ++ ++
++ + +
++
++
+++
+++
++
15 16 17 18 19
DDMP αg βg γg βa γa
1085.55 1069.56 923.50 1039.55 893.49
+++ +++ +++ ++ +
20 21 22 23 24 25
Group B Ba Bb Bb’ Bx Bc Bc’
959.52 943.53 797.47 929.51 913.52 767.46
+++ +++
26 27
Group E Bd Be
957.51 941.51
+++ +
28 29 30 31 32 33 34 35 36 37 38
Sg-6 H-αg I-αg I-βg I-γg I-αa I-βa I-γa J-αg J-γg J-αa J-γa
973.50 987.48 971.48 825.43 957.47 941.47 795.42 1059.50 897.45 1029.49 867.44
+++ +++
+++ ++ +++ ++ +
+ +++ + +++ +
+++
UL
TL + +
++ +
+ +++
++ ++
+++
+ ++
+++ +++ +++ +++ ++
+++ +++ ++ ++ +
+++ +++ ++ ++
+++ +
+++ +++ +
+ +++ +
+++ +
++
+
+
++ +
+++ + +++ ++
+ +++
++ +++ +++ ++ +
+++
+++
++
+
+ +++ +++ +++
+++ +++ +++
+++ +++ +++ +++
+++ + ++ +
+++ ++ ++ +
+++ +++
+++ +++
+ +++ +
+++ +
+++ +
+++
+++
++ +
++
++
+++
+++
++ ++
+++ +++
+++
+
+++ + +++ ++ + ++
+
+
+ ++
+ +
+
Notes: HY, hypocotyl; CT, cotyledon; GH, germinated hypocotyl; EC, epicotyl; AM, apical meristem; MR, main root; LR, lateral root; CL, cotyledonary leaf; UL, unifoliolate leaf; TL, trifoliolate leaf; +++: mostly detected (75 - 100%); ++: moderately detected (25 - 74%); +: rarely detected (40 °C) and heat treatment, our primary concern was to reduce the DDMP saponins degradation. It has been reported that extraction times longer than 1 h did not affect the amount of saponins extraction and the ratio of DDMP and group B saponins remained constant in 24 h extracted samples at < 30 °C.19) From our preliminary experiments using 10-fold volumes of 80% aqueous methanol at room temperature (25 °C), we found that 12–24 h for intact hypocotyls and 30–60 min for milled cotyledons and seedling tissues gives maximum extraction of group A and DDMP saponins without degradation (data not shown). In addition, we found that (i) saponins were not extracted well from intact cotyledons or sliced cotyledon (1 mm thick) in the period of
12–24 h and (ii) a higher extraction time (>30 h) would cause saponins degradation. Hence, with regard to extract maximum saponins amount with low degradation ratio, we extracted saponins from intact hypocotyl for 24 h and from milled tissues for 1 h. Epistatic activity of soyasaponin genes is specific to accession and tissue Although several genes are epistatically participating in soyasaponins biosynthesis, only three gene loci (Sg1, Sg-4, and Sg-6) are polymorphic in nature and contribute in saponin phenotype polymorphism.4) The Sg-1 locus adds xylose and glucose at the terminal position of the C-22 sugar chain of SS-A by co-dominant alleles Sg-1a and Sg-1b, respectively.21) The Sg-4 locus attaches the second sugar (arabinose) of the C-3 sugar chain of all soyasapogenols.4–6) The Sg-6 locus is assumed to directly and/or indirectly control the presence of SS-H, SS-I, and SS-J.6) We attempt to describe the soyasaponin genes activity in qualitative manner on the basis of the presence of diverse saponin components. Based on the three polymorphic gene loci, we divided the eight analyzed accessions into four groups: (i) Sg-1/sg-4/sg-6 (saponin phenotype: Aa, Ab), (ii) Sg-1/Sg-4/sg-6 (AaBc, AbBc), (iii) Sg-1/sg-4/Sg-6 (Aa + α, Ab + α), and (iv) Sg-1/Sg-4/Sg-6 (AaBc + α, AbBc + α). The Sg-1 gene activity (evaluated by saponins having xylose and/or glucose at the terminal position of the C-22 sugar chain of SS-A) was detected in all of the four groups according to the presence of co-dominant alleles Sg-1a and Sg-1b. The Sg-4 gene activity (evaluated by saponins having arabinose at the second position of C-3 sugar chain of the soyasapogenols) was not detected in the hypocotyl of group (i) and (iii), but was detected in the cotyledon, primary root, secondary root, and cotyledonary leaf of those groups (Fig. 2, 3). The Sg-4 gene activity was detected in all tissues of group (ii) and (iv). Unlike Sg-4, Sg-6 gene activity (evaluated by saponins having either SS-H, SS-I or SS-J) was only found in groups (iii) and (iv) (Fig. 2, 3). These findings showed that the activity
Saponins variation in G. soja before and after germination a
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of Sg-1 , Sg-1 , and Sg-6 genes is accession specific followed by tissue specific while the activity of Sg-4 gene is merely specific to tissue. Regulation of soyasaponin composition and content is specific to accession and tissue Thus far, 54 saponin components are reported in soybean and wild soybean (Fig. 1). Of these, we detected 38 components in this study (Table 1). It implies that certain saponins which are not detected in given tissue/accession are absent and/or low concentrated in that given tissue/accession. In some cases, different saponins have same retention time (for example: saponin DDMP-αa has the almost same retention time to DDMP-βg). Furthermore, the contents of certain saponins in Fig. 1 are very low; we can detect them by only LC-MS analysis not by UV chromatogram. Noteworthy, none of soybean and wild soybean contains all of 54 saponin components. Presence or absence of saponin components varies based on the soybean and wild soybean variety. Significant qualitative and quantitative differences in saponin profiles were observed among different tissues of wild soybean (Table 1, 2). It has been reported that group A saponins exist only in seed hypocotyl.22) Perhaps, researchers have detected group A saponins in the roots of eight-day-old soybean seedlings17) and cotyledons of mature seeds.14) Rupasinghe et al.16) detected considerable amounts of SS-A in plumule and radical of five-day-old soybean seedlings. Similar to previous results, we detected group A saponins in many tissues other than seed hypocotyl (Table 1, 2; Fig. 2, 3). Based on our results, we concluded that group A saponins can be detectable in any tissues other than seed hypocotyl, but their concentration is significant only in seed hypocotyl. The hypocotyl of wild soybean, which represent only 15% of the seed weight, contained > 74% of the ATSC and > 93% of the total group A saponins (Table 2). This agrees the results of Tsukamoto et al.2) Hypocotyl had the highest ATSC measured at 16.08 ± 3.13 μmol of 1 g mature dry seeds (Table 2). This amount is approximately 1.6 times that in cotyledonary leaf, 3 times that in cotyledon, 4 times that in trifoliolate leaf, 6 times that in primary root and germinated hypocotyl, 8 times that in unifoliolate leaf, and 12 times that in epicotyl and apical meristem. When we compare the ATSC of hypocotyl and cotyledon (Table 2), before germination (HY and CT) and after germination (GH and CL), the results suggest that germination induces saponin biosynthesis in cotyledon while, concurrently, it enhances degradation and/or translocation of stored saponins in hypocotyl. During germination, the content of group A and Sg-6 saponins was markedly reduced (Table 2). Similar to our finding, Rupasinghe et al.16) found that SS-A content was significantly reduced by germination. Contradict to our results, Shimoyamada et al.17) reported that germination does not cause any changes to the content of group A saponins. Takada et al.13) reported that group A saponins deficient accession (B01082 – Japanese wild soybean mutant) had grown well and produced fertile seeds under normal growth condition. The
1995
Korean wild soybean mutant accessions CWS15095 and CWS16027, lacking group A saponins, were also grown well and produced fertile seeds without difficulty (unpublished data). In our previous studies,4–6) we observed that 82–98% of wild soybeans did not contain Sg-6 saponins. Also, it is noteworthy to mention that Sg-6 saponins have not been found in soybean so far. Based on the availability of null accessions completely lacking group A saponins and Sg-6 saponins, and the deleterious effects on those saponin groups during germination, we presumably propose that group A and Sg-6 saponins are not essential for normal plant growth and development. Shimoyamada et al.22) have reported various parts (leaf, stem, branch, petiole, hypocotyl, cotyledon, and seed pod) of soybean plants contain group B saponins. However, we did not compare their results with ours; because they unknowingly converted all DDMP saponins into group B and/or group E saponins prior to HPLC analysis. Table 2 shows that DDMP saponins were newly synthesized by germination in wild soybean. By contrast, germination does not change DDMP saponin content in lentils (Lens culinaris) and chickpeas (Cicer aerietinum).23) This implies that DDMP saponin regulation by germination is specific to species. Although we did not estimate the absolute saponin concentration, nevertheless, we found considerable amount of saponin DDMP-βg in all the analyzed tissues (data not shown). Of the ATSC, the content of DDMP saponins represents 15–20% of the primary and lateral roots, 40–50% of the hypocotyl and germinated hypocotyl, 60–65% of the cotyledon and unifoliolate leaf, and 70– 75% of the epicotyl, apical meristem, cotyledonary leaf, and trifoliolate leaf (Table 2). In primary and lateral roots, 75–82% of DDMP saponins were in the form of group B + E saponins (Table 2; Fig. 4). These results imply that the total saponin concentration of the shoot system of wild soybean was mainly composed of DDMP saponins, while it was mainly composed of group B + E saponins in the root system. More research is required to uncover the importance of the high degradation rate of DDMP saponins in the root system.
Future perspective Since no mutants were found to lack DDMP saponins from soybean and wild soybean germplasm collections, researchers widely suspect that DDMP saponins may have some physiological functions in the plant. We believe uncovering the importance and necessities of the high degradation ratio of DDMP and group B + E saponins in the root system would give us some clues about the functionality of DDMP saponins in plant. Previously, chromosaponin I (CSI), isolated from peas (Pisum sativum) and same chemical structure as DDMP-βg, has been reported to show positive correlation with root growth,24) elongation of cortical cells,25) increase mechanical extensibility of root cell walls,26) regulation of gravitropic response of roots27), and regulation of auxin influx.28) However, these experiments were performed by soaking the plants into medium containing a DDMP saponin (CSI) during plant growth. Hence, using these methods, the physiological
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properties of DDMP saponins in situ in plants growth and development will not be conducted because DDMP saponins are very labile. The chemical structure of DDMP saponin (CSI) may be modified during absorption into plant tissue and the chemical structure of actual components affecting plant growth may be different from the original DDMP saponin. Takagi et al.29 produced transgenic soybean plants by modifying the saponin biosynthesis through RNA interference (RNAi) mediated gene silencing targeted to β-amyrin synthase using a promoter expressed only in the seeds. The seeds were obtained with almost no saponins. This may suggest that the accumulation of saponins in the seeds may not be essential, but some saponins may be produced de novo during germination. We believe that we need to consider all of the above-mentioned circumstances and many others while planning to demonstrate the physiological properties of DDMP saponins in situ in plant growth and development.
Supplemental material The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2014.946389.
Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0013600).
References [1] Chung G, Singh RJ. Broadening the genetic base of soybean: A multidisciplinary approach. Crit. Rev. Plant Sci. 2008;27:295– 341. [2] Tsukamoto C, Kikuchi A, Kudou S, Harada K, Iwasaki T, Okubo K. Genetic improvement of saponin components in soybean. In: Huang MT, Osawa T, Ho CT, Rosen RT, editors. Food Phytochemicals for Cancer Prevention I. Washington, DC: American Chemical Society; 1994. pp. 372–379. [3] Kikuchi A, Tsukamoto C, Tabuchi K, Adachi T, Okubo K. Inheritance and characterization of a null allele for group A acetyl saponins found in a mutant soybean [Glycine max (L.) Merrill]. Breed. Sci. 1999;49:167–171. [4] Krishnamurthy P, Lee CM, Tsukamoto C, Takahashi Y, Singh RJ, Lee JD, Chung G, Evaluation of genetic structure of Korean wild soybean (Glycine soja) based on saponin allele polymorphism, Genet. Resour. Crop Evol. 2014;61:1121–1130. [5] Krishnamurthy P, Tsukamoto C, Honda N, Kikuchi A, Lee JD, Yang SH, Chung G. Saponin polymorphism in the Korean wild soybean (Glycine soja Sieb. and Zucc.). Plant Breed. 2013;132:121–126. [6] Krishnamurthy P, Tsukamoto C, Singh RJ, Lee JD, Kim HS, Yang SH, Chung G, The Sg-6 saponins, new components in wild soybean (Glycine soja Sieb. and Zucc.): Polymorphism, geographical distribution and inheritance, Euphytica, 2014;198:413–424. [7] Kudou S, Tonomura M, Tsukamoto C, Uchida T, Sakabe T, Tamura N, Okubo K. Isolation and structural elucidation of DDMP-conjugated soyasaponins as genuine saponins from soybean seeds. Biosci. Biotechnol. Biochem. 1993;57:546–550. [8] Shiraiwa M, Harada K, Okubo K. Composition and structure of “group B saponin” in soybean seed. Agric. Biol. Chem. 1991;55:911–917.
[9] Shiraiwa M, Kudo S, Shimoyamada M, Harada K, Okubo K. Composition and structure of “Group A saponin” in soybean seed. Agric. Biol. Chem. 1991;55:315–322. [10] Takada Y, Tayama I, Sayama T, Sasama H, Saruta M, Kikuchi A, Ishimoto M, Tsukamoto C. Genetic analysis of variations in the sugar chain composition at the C-3 position of soybean seed saponins. Breed. Sci. 2012;61:639–645. [11] Takahashi Y, Kon T, Muraoka H, Ishimoto M, Tsukamoto C. The dominant Sg-6 synthesizes saponins with a keton function at oleanane aglycone C-22 position. Kolymvari: 11th International Meeting on Biosynthesis. Function and Biotechnology of Isoprenoids in Terrestrial and Marine Organisms (TERPNET); 2013. p. 197. [12] Tsukamoto C, Kikuchi A, Harada K, Kitamura K, Okubo K. Genetic and chemical polymorphisms of saponins in soybean seed. Phytochem. 1993;34:1351–1356. [13] Takada Y, Sasama H, Sayama T, Kikuchi A, Kato S, Ishimoto M, Tsukamoto C. Genetic and chemical analysis of a key biosynthetic step for soyasapogenol A, an aglycone of group A saponins that influence soymilk flavor. Theor. Appl. Gen. 2013;126:721–731. [14] Tsukamoto C, Takada Y, Omizu Y, Kon T, Matsuhashi T, Kikuchi A, Kato S, Ishimoto M, Kitamura K. Soyasapogenol A-conjugated saponin glycosides detected in the seed cotyledon of soybeans. Bangkok, Thailand: The 12th ASEAN Food Conference, BITEC Bangna; 2011. pp. 391–394. [15] Jyothi TC, Sindhu Kanya TC, Appu Rao AG. Influence of germination on saponins in soybean and recovery of soy sapogenol I. J. Food Biochem. 2007;31:1–13. [16] Rupasinghe HPV, Jackson CJC, Poysa V, Berardo CD, Bewley JD, Jenkinson J. Soyasapogenol A and B distribution in soybean (Glycine max L. Merr.) in relation to seed physiology, genetic variability, and growing location. J. Agric. Food Chem. 2013;51:5888–5894. [17] Shimoyamada M, Okubo K. Variation in saponin contents in germinating soybean seeds and effect of light irradiation. Agric. Biol. Chem. 1991;55:577–579. [18] Lin J, Wang C. An analytical method for soy saponins by HPLC/ELSD. J. Food Sci. 2004;69:456–462. [19] Heng L, Vincken JP, Hoppe K, et al. Stability of pea DDMP saponin and the mechanism of its decomposition. Food Chem. 2006;99:326–334. [20] Krishnamurthy P, Tsukamoto C, Yang SH, Lee JD, Chung G. An improved method to resolve plant saponins and sugars by TLC. Chromatographia. 2012;75:1445–1449. [21] Sayama T, Ono E, Takagi K, Takada Y, et al. The Sg-1 glycosyltransferase locus regulates structural diversity of triterpenoid saponins of soybean. Plant Cell. 2012;24:2123–2138. [22] Shimoyamada M, Kudo S, Okubo K, Yamauchi F, Harada K. Distributions of saponin constituents in some varieties of soybean plant. Agric. Biol. Chem. 1990;54:77–81. [23] Ruiz RG, Price K, Rose M, Rhodes M, Fenwick R. A preliminary study on the effect of germination on saponin content and composition of lentils and chick peas. Z Lebensm Unters Forsch. 1996;203:366–369. [24] Tsurumi S, Tsujino Y. Chromosaponin I stimulates the growth of lettuce roots. Physiol. Plant. 1995;93:785–789. [25] Tsurumi S, Wada S. Chromosaponin I stimulates the elongation of cortical cells in lettuce cells. Plant Cell Physiol. 1995;36:925–929. [26] Tsurumi S, Watanabe R, Kamisaka S. Chromosaponin I increases mechanical extensibility of lettuce root cell walls. Physio. Plant. 1996;97:740–746. [27] Rahman A, Ahamed A, Amakawa T, Goto N, Tsurumi S. Chromosaponin I specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots. Plant Physiol. 2001;125:990–1000. [28] Rahman A, Tsurumi S. The unique auxin influx modulator chromosaponin I: A physiological overview. Plant Tissue Cult. 2002;12:181–194. [29] Takagi K, Nishizawa K, Hirose A, Kita A, Ishimoto M. Manipulation of saponin biosynthesis by RNA interference-mediated silencing of β-amyrin synthase gene expression in soybean. Plant Cell Rep. 2011;30:1835–1846.
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Comparison of saponin composition and content in wild soybean (Glycine soja Sieb. and Zucc.) before and after germination a
b
b
b
Panneerselvam Krishnamurthy , Chigen Tsukamoto , Yuya Takahashi , Yuji Hongo , Ram c
d
J. Singh , Jeong Dong Lee & Gyuhwa Chung
a
a
Department of Biotechnology, Chonnam National University, Yeosu, Korea
b
Department of Applied Biological Chemistry, Iwate University, Morioka, Japan
c
USDA/ARS, National Soybean Research Laboratory, University of Illinois, Urbana, IL, USA d
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School of Applied Biosciences, Kyungpook National University, Daegu, Korea Published online: 15 Aug 2014.
To cite this article: Panneerselvam Krishnamurthy, Chigen Tsukamoto, Yuya Takahashi, Yuji Hongo, Ram J. Singh, Jeong Dong Lee & Gyuhwa Chung (2014) Comparison of saponin composition and content in wild soybean (Glycine soja Sieb. and Zucc.) before and after germination, Bioscience, Biotechnology, and Biochemistry, 78:12, 1988-1996, DOI: 10.1080/09168451.2014.946389 To link to this article: http://dx.doi.org/10.1080/09168451.2014.946389
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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 12, 1988–1996
Comparison of saponin composition and content in wild soybean (Glycine soja Sieb. and Zucc.) before and after germination Panneerselvam Krishnamurthy1, Chigen Tsukamoto2,*, Yuya Takahashi2, Yuji Hongo2, Ram J. Singh3, Jeong Dong Lee4 and Gyuhwa Chung1,* 1
Department of Biotechnology, Chonnam National University, Yeosu, Korea; 2Department of Applied Biological Chemistry, Iwate University, Morioka, Japan; 3USDA/ARS, National Soybean Research Laboratory, University of Illinois, Urbana, IL, USA; 4School of Applied Biosciences, Kyungpook National University, Daegu, Korea
Received February 12, 2014; accepted July 9, 2014
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http://dx.doi.org/10.1080/09168451.2014.946389
Eight wild soybean accessions with different saponin phenotypes were used to examine saponin composition and relative saponin quantity in various tissues of mature seeds and two-week-old seedlings by LC–PDA/MS/MS. Saponin composition and content were varied according to tissues and accessions. The average total saponin concentration in 1 g mature dry seeds of wild soybean was 16.08 ± 3.13 μmol. In two-week-old seedlings, produced from 1 g mature seeds, it was 27.94 ± 6.52 μmol. Group A saponins were highly concentrated in seed hypocotyl (4.04 ± 0.71 μmol). High concentration of DDMP saponins (7.37 ± 5.22 μmol) and Sg-6 saponins (2.19 ± 0.59 μmol) was found in cotyledonary leaf. In seedlings, the amounts of group A and Sg-6 saponins reduced 2.3- and 1.3-folds, respectively, while DDMP + B + E saponins increased 2.5-fold than those of mature seeds. Our findings show that the group A and Sg-6 saponins in mature seeds were degraded and/or translocated by germination whereas DDMP saponins were newly synthesized. Key words:
wild soybean; soyasaponins; germination; triterpene glycosides; Glycine soja
Wild soybean (Glycine soja Sieb. and Zucc.) is native to East Asian countries including China, the Korean Peninsula, Japan, Russia Far East, and Taiwan.1) Soybean [Glycine max (L.) Merr.], one of the most important legumes in this world, often relies on the genetical resources of wild soybean to improve its quality.1) Wild soybean, the undomesticated wild relative of soybean, has high genetic diversity and serves as a promising gene resource to produce new soybean cultivars with better quality through natural selection and conventional breeding.1) Both soybean and wild soybean seeds contain significant amount of saponins
(soyasaponins – bioactive secondary metabolites).2) Soyasaponins are triterpene glycosides consisting of a triterpene aglycone (C30) attached with one or two sugar chains. Several structurally diverse groups of soyasaponins have been identified from the seeds of the subgenus Soja which includes the cultivated and wild soybean.3–12) Based on the aglycone structure, soyasaponins have been classified into group A, DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one), group H, group I, and group J saponins (Fig. 1). The latter three groups (H, I, and J) are collectively designated as Sg-6 saponins.6) Group A saponins are bisdesmosides containing two sugar chains at the C-3 and C-22 hydroxyl positions of soyasapogenol A (SS-A).9) Based on the terminal sugar of the C-22 sugar chain of SS-A, group A saponins are further subdivided into Aa-, Ab-, and A0-series saponins.13) The A0-series saponins have no sugar at the terminal position of the C-22 sugar chain of SS-A, whereas the Aa- and Ab-series saponins have acetylxylose and acetyl-glucose, respectively, at the same position. DDMP saponins are monodesmosides with one sugar chain and one DDMP moiety at the C-3 and C-22 hydroxyl positions of soyasapogenol B (SS-B), respectively. DDMP saponins are degraded during most extraction procedures to form group B saponins having SS-B and group E saponins having soyasapogenol E (SS-E).7) Sg-6 saponins are monodesmosides with one sugar chain at the C-3 hydroxyl position of their respective soyasapogenols (SS-H, SS-I, and SS-J).6) The nomenclature of all soyasaponins was derived from these six aglycones and their sugar moiety composition (Fig. 1). In the subgenus Soja, seed hypocotyl contains all of the three major saponin groups (group A, DDMP, and Sg-6) which produce eight common distinguishable saponin phenotypes in thin layer chromatography (TLC) namely Aa, Ab, AaBc, AbBc, Aa + α, Ab + α,
*Corresponding authors. Email: [email protected] (C. Tsukamoto); [email protected] (G. Chung) Abbreviations: ATSC, average total saponin composition; DDMP, 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one; LC–PDA/MS/MS, liquid chromatography–photodiode array detector/mass spectrometry/mass spectrometry; TLC, thin layer chromatography; SS-A, soyasapogenol A; SS-B, soyasapogenol B, SS-E, soyasapogenol E; SS-H, soyasapogenol H; SS-I, soyasapogenol I; SS-J, soyasapogenol J. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Saponins variation in G. soja before and after germination
1989
Fig. 1. Chemical structure and nomenclature of soyasaponins.4–6) R1 at the C-3 position of soyasapogenols A, B, E, H, I, and J, and R2 at the C22 position of soyasapogenol A are shown at the bottom of Fig. 1.
AaBc + α, and AbBc + α (Supplementary Fig. 1a). Seed cotyledon also contains all of the three major saponin groups;14) however, the concentration of group A saponins is relatively low and cannot be detected by TLC (Supplementary Fig. 1b). The polymorphism of the common saponin composition (saponin phenotype) of seed hypocotyl can be well explained by the combination of six allelic genes Sg-1a/Sg-1b, Sg-4/sg-4, and Sg6/sg-6 on three loci Sg-1, Sg-4, and Sg-6, respectively.4–6) Based on these three gene loci, the genotype of the common saponin phenotypes is designated as follows: Sg-1a/sg-4/sg-6 (phenotype – Aa); Sg-1b/sg-4/ sg-6 (Ab); Sg-1a/Sg-4/sg-6 (AaBc); Sg-1b/Sg-4/sg-6 (AbBc); Sg-1a/sg-4/Sg-6 (Aa + α); Sg-1b/sg-4/Sg-6 (Ab + α); Sg-1a/Sg-4/Sg-6 (AaBc + α); and Sg-1b/Sg-4/ Sg-6 (AbBc + α). The latter four + α types were not found in soybean, so far, while all of eight types were found in wild soybean.4–6) Despite several reports on the complexity of the chemical structure diversity of saponins and seed saponin composition, the distribution of saponin composition and content as well as the physiological function of saponins in the soybean plant is not clearly understood. Jyothi et al.15) Rupasinghe et al.16), and Shimoyamada et al.17) have reported that germination upregulates the content of group B saponins and SS-B, and down-regulates the content of group A saponins and SS-A in soybean. Jyothi et al.15) extracted saponins for 36 h using soxlet and studied the changes of saponin Bb whereas Rupasinghe et al.16) performed saponin
extraction at 50 °C for 2 h and studied the changes of SS-A and SS-B concentration before and after germination. Shimoyamada et al.17) extracted saponins for 5 h at 80 °C and studied the influence of germination and light irrigation on saponin composition and content. Except Rupasinghe et al.,16) Jyothi et al.15) and Shimoyamada et al.17) are supposed to detect DDMP saponins in their study. But, they did not detect DDMP saponins given the fact that long extraction time at high temperature can degrade all DDMP saponins into group B and/or group E saponins. In general, DDMP saponins are heat labile and become unstable at ≥ 30 °C.18,19) The removal of DDMP group from saponins in water solutions becomes more pronounced, with a 50, 75, and 95% decrease in DDMP saponin concentration after 75 min at 60, 75, and 90 °C, respectively.19) The loss of DDMP group can be completely prevented for 90 min by the presence of >30% ethanol (v/v) at 65 °C. From this, anyone can readily interpret that temperature plays a vital role in DDMP saponins degradation. Hence, the studies of Jyothi et al.15) and Shimoyamada et al.17) were biased though they provide some interesting facts. To the end, there is no information available so far about the changes of DDMP and Sg-6 saponins (which are identified recently) before and after germination. Furthermore, comprehensive profiling and identification of saponins variation are essential for the functional analysis of saponins in soybean; this is the basis for exploring the specific role of any compound in plant physiology. Since the polymorphism
1990
P. Krishnamurthy et al.
of saponin composition in wild soybean is wider than that of cultivated soybean (Supplementary Fig. 1), we selected one wild soybean accession for each common saponin phenotype in this study. Saponin profile and relative saponin quantity were examined in hypocotyl, cotyledon, germinated hypocotyl, epicotyl, apical meristem, cotyledonary leaf, unifoliolate leaf, trifoliolate leaf, primary root, and secondary root of mature seeds and two-week-old seedlings of wild soybean. We believe this is the first study conducted for comprehensive profiling of saponins from wild soybean seeds and seedlings. Our findings raise the potential knowledge of soyasaponins, which would be very helpful in designing the future research work to uncover the importance of soyasaponins from plant physiology perspective.
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Material and methods Materials and chemicals. Eight wild soybean accessions [CWS4162 (saponin phenotype: Aa), CWS0101 (Ab), CWS4151 (AaBc), CWS4345 (AbBc), CWS2976 (Aa + α), CWS4780 (Ab + α), CWS4779 (AaBc + α), and CWS4852 (AbBc + α)] were obtained from Chung’s Wild Legume Germplasm Collection at the Chonnam National University, Yeosu, Chonnam, Korea. Silica gel (SiO2)-coated TLC plates (catalog No.105626) were purchased from Merck Millipore, Germany. Saponin Bb was bought from Wako Pure Chemical Industries, Ltd., Osaka, Japan. All chemicals used in this study were of analytical grade. They were purchased from Honeywell Burdick and Jackson, Seoul, Korea, and Samchun Chemicals, Seoul, Korea.
Growing conditions. Seeds were germinated triplicate under non-controlled conditions from May 29 to June 24, 2012. Each planting was done at one-week interval. Seeds were scarified using a scalpel and sown in plastic flats containing vermiculite. The flats were simply kept on the roof of Department of Biotechnology (Chonnam National University, Yeosu campus, Korea) for germination and growth. Flats were sprinkled with tap water once a day. After two weeks, 10 seedlings from each accession were selected and dissected into eight parts (tissues): germinated hypocotyl, epicotyl, apical meristem, cotyledonary leaf, unifoliolate leaf, trifoliolate leaf, primary root, and secondary root. Sampled tissues were water drained with tissue paper, immersed in liquid nitrogen, and stored at −80 °C until freeze drying. Saponin extraction. Hypocotyl and cotyledon from five mature seeds of each accession were dissected. Saponins were extracted from intact hypocotyls, cotyledon powder, and milled seedling tissues using 10-fold volumes (v/w) of 80% (v/v) aqueous methanol. Extraction was carried out at room temperature (25 °C) for 24 h for hypocotyl and 1 h for cotyledon powder and milled seedling tissues. The resulting extracts were stored at 4 °C until TLC and LC–PDA/MS/MS.
Thin-layer chromatography analysis. TLC was performed according to Krishnamurthy et al.20) Briefly, 10 μL from hypocotyl (HY) and cotyledon (CT) extracts of each accession was directly applied on silica gel-coated TLC plates with an Eppendorf micropipette and slightly dried by using a hair drier. The plates were developed in a rectangular developing chamber which was saturated with the lower phase of chloroform:methanol:water (65:35:10, v/v/v) for 2 h. Plates were dried at 100 °C for 10 min and then developed with 10% H2SO4 for 12 min in a closed chamber. Saponins were visualized by heating the plates at 115 °C for 13 min. LC–PDA/MS/MS analysis. Hypocotyl extracts were diluted 10-fold with 80% methanol prior to use. Those from seed cotyledons and seedling tissues were used directly. Ten micro liters from each extract were analyzed in a UFLC system (Prominence UFLC system, Shimadzu, Kyoto, Japan) equipped with a photodiode array (PDA) detector and a tandem mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific, Yokohama, Kanagawa, Japan) on a C30 reverse phase column (Develosil C30-UG-3, 2.0 mm I.D. × 150 mm, Nomura Chemical, Seto, Okayama, Japan) at 40 °C. Solvent A consisted of acetonitrile with 0.1% (v/v) formic acid and solvent B consisted of water with 0.1% formic acid. A linear gradient elution of solvent A was performed at a flow rate of 0.15 mL/min: solvent A was initiated at 20% (v/v), increased to 65% (v/v) in 45 min, and then increased to 100% (v/v) for 5 min. The eluent composition was returned to the initial state of 20% (v/v) solvent A for 15 min. The eluate from the column was monitored by a PDA detector at UV 205 and 292 nm and by a tandem mass spectrometer in the positive ion mode of the electrospray ionization [ESI (+)] method. An automatic full-scan mode over a massto-charge ratio (m/z) range from 300 to 1800 and the top three ion-trap mode were used to acquire MS and MS/MS data, respectively. Saponin quantification. The recorded UV and MS spectra were analyzed with Xcalibur software version 2.1 (Thermo Fisher Scientific, Yokohama, Kanagawa, Japan). Xcalibur settings used to detect saponin peaks were as follows: peak algorithm, ICIS; baseline, 150; area noise, 5; peak noise factor, 10; and the remaining factors were kept default. Relative quantifications were performed for all the identified saponins based on external standardization by employing the calibration curve of saponin Bb at a concentration of 995 pmol/ 5 μL. Quantitative analyses were based on the peak area of UV 205 nm. Microsoft Excel 2007 was used for the statistical analysis.
Results Saponin composition in various tissues of wild soybean seeds and seedlings Based on the molecular ion [M + H]+ mass of soyasaponins, a total of 38 saponin components were detected in this study (Table 1). Of these, 14
Saponins variation in G. soja before and after germination
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Table 1.
1991
Saponin composition in various tissues of mature seeds and two-week-old seedlings of wild soybean. Mature seed
Two-week old seedling of wild soybean
No.
Saponins
m/z used for quantification [M+H]+
HY
CT
GH
EC
AM
MR
LR
CL
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Group A Aa Au Ae Ax Ag Ab Ac Af Ad Az Ah A0-αg A0-βg A0-αa
1365.63 1349.64 1203.58 1335.62 1173.57 1437.65 1421.66 1275.60 1407.64 1391.65 1245.59 1107.56 1091.56 1077.55
+++ + ++ ++
++
+++
++
++
++
++
+++ + ++
++ ++ ++
++ + +
++
++
+++
+++
++
15 16 17 18 19
DDMP αg βg γg βa γa
1085.55 1069.56 923.50 1039.55 893.49
+++ +++ +++ ++ +
20 21 22 23 24 25
Group B Ba Bb Bb’ Bx Bc Bc’
959.52 943.53 797.47 929.51 913.52 767.46
+++ +++
26 27
Group E Bd Be
957.51 941.51
+++ +
28 29 30 31 32 33 34 35 36 37 38
Sg-6 H-αg I-αg I-βg I-γg I-αa I-βa I-γa J-αg J-γg J-αa J-γa
973.50 987.48 971.48 825.43 957.47 941.47 795.42 1059.50 897.45 1029.49 867.44
+++ +++
+++ ++ +++ ++ +
+ +++ + +++ +
+++
UL
TL + +
++ +
+ +++
++ ++
+++
+ ++
+++ +++ +++ +++ ++
+++ +++ ++ ++ +
+++ +++ ++ ++
+++ +
+++ +++ +
+ +++ +
+++ +
++
+
+
++ +
+++ + +++ ++
+ +++
++ +++ +++ ++ +
+++
+++
++
+
+ +++ +++ +++
+++ +++ +++
+++ +++ +++ +++
+++ + ++ +
+++ ++ ++ +
+++ +++
+++ +++
+ +++ +
+++ +
+++ +
+++
+++
++ +
++
++
+++
+++
++ ++
+++ +++
+++
+
+++ + +++ ++ + ++
+
+
+ ++
+ +
+
Notes: HY, hypocotyl; CT, cotyledon; GH, germinated hypocotyl; EC, epicotyl; AM, apical meristem; MR, main root; LR, lateral root; CL, cotyledonary leaf; UL, unifoliolate leaf; TL, trifoliolate leaf; +++: mostly detected (75 - 100%); ++: moderately detected (25 - 74%); +: rarely detected (40 °C) and heat treatment, our primary concern was to reduce the DDMP saponins degradation. It has been reported that extraction times longer than 1 h did not affect the amount of saponins extraction and the ratio of DDMP and group B saponins remained constant in 24 h extracted samples at < 30 °C.19) From our preliminary experiments using 10-fold volumes of 80% aqueous methanol at room temperature (25 °C), we found that 12–24 h for intact hypocotyls and 30–60 min for milled cotyledons and seedling tissues gives maximum extraction of group A and DDMP saponins without degradation (data not shown). In addition, we found that (i) saponins were not extracted well from intact cotyledons or sliced cotyledon (1 mm thick) in the period of
12–24 h and (ii) a higher extraction time (>30 h) would cause saponins degradation. Hence, with regard to extract maximum saponins amount with low degradation ratio, we extracted saponins from intact hypocotyl for 24 h and from milled tissues for 1 h. Epistatic activity of soyasaponin genes is specific to accession and tissue Although several genes are epistatically participating in soyasaponins biosynthesis, only three gene loci (Sg1, Sg-4, and Sg-6) are polymorphic in nature and contribute in saponin phenotype polymorphism.4) The Sg-1 locus adds xylose and glucose at the terminal position of the C-22 sugar chain of SS-A by co-dominant alleles Sg-1a and Sg-1b, respectively.21) The Sg-4 locus attaches the second sugar (arabinose) of the C-3 sugar chain of all soyasapogenols.4–6) The Sg-6 locus is assumed to directly and/or indirectly control the presence of SS-H, SS-I, and SS-J.6) We attempt to describe the soyasaponin genes activity in qualitative manner on the basis of the presence of diverse saponin components. Based on the three polymorphic gene loci, we divided the eight analyzed accessions into four groups: (i) Sg-1/sg-4/sg-6 (saponin phenotype: Aa, Ab), (ii) Sg-1/Sg-4/sg-6 (AaBc, AbBc), (iii) Sg-1/sg-4/Sg-6 (Aa + α, Ab + α), and (iv) Sg-1/Sg-4/Sg-6 (AaBc + α, AbBc + α). The Sg-1 gene activity (evaluated by saponins having xylose and/or glucose at the terminal position of the C-22 sugar chain of SS-A) was detected in all of the four groups according to the presence of co-dominant alleles Sg-1a and Sg-1b. The Sg-4 gene activity (evaluated by saponins having arabinose at the second position of C-3 sugar chain of the soyasapogenols) was not detected in the hypocotyl of group (i) and (iii), but was detected in the cotyledon, primary root, secondary root, and cotyledonary leaf of those groups (Fig. 2, 3). The Sg-4 gene activity was detected in all tissues of group (ii) and (iv). Unlike Sg-4, Sg-6 gene activity (evaluated by saponins having either SS-H, SS-I or SS-J) was only found in groups (iii) and (iv) (Fig. 2, 3). These findings showed that the activity
Saponins variation in G. soja before and after germination a
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of Sg-1 , Sg-1 , and Sg-6 genes is accession specific followed by tissue specific while the activity of Sg-4 gene is merely specific to tissue. Regulation of soyasaponin composition and content is specific to accession and tissue Thus far, 54 saponin components are reported in soybean and wild soybean (Fig. 1). Of these, we detected 38 components in this study (Table 1). It implies that certain saponins which are not detected in given tissue/accession are absent and/or low concentrated in that given tissue/accession. In some cases, different saponins have same retention time (for example: saponin DDMP-αa has the almost same retention time to DDMP-βg). Furthermore, the contents of certain saponins in Fig. 1 are very low; we can detect them by only LC-MS analysis not by UV chromatogram. Noteworthy, none of soybean and wild soybean contains all of 54 saponin components. Presence or absence of saponin components varies based on the soybean and wild soybean variety. Significant qualitative and quantitative differences in saponin profiles were observed among different tissues of wild soybean (Table 1, 2). It has been reported that group A saponins exist only in seed hypocotyl.22) Perhaps, researchers have detected group A saponins in the roots of eight-day-old soybean seedlings17) and cotyledons of mature seeds.14) Rupasinghe et al.16) detected considerable amounts of SS-A in plumule and radical of five-day-old soybean seedlings. Similar to previous results, we detected group A saponins in many tissues other than seed hypocotyl (Table 1, 2; Fig. 2, 3). Based on our results, we concluded that group A saponins can be detectable in any tissues other than seed hypocotyl, but their concentration is significant only in seed hypocotyl. The hypocotyl of wild soybean, which represent only 15% of the seed weight, contained > 74% of the ATSC and > 93% of the total group A saponins (Table 2). This agrees the results of Tsukamoto et al.2) Hypocotyl had the highest ATSC measured at 16.08 ± 3.13 μmol of 1 g mature dry seeds (Table 2). This amount is approximately 1.6 times that in cotyledonary leaf, 3 times that in cotyledon, 4 times that in trifoliolate leaf, 6 times that in primary root and germinated hypocotyl, 8 times that in unifoliolate leaf, and 12 times that in epicotyl and apical meristem. When we compare the ATSC of hypocotyl and cotyledon (Table 2), before germination (HY and CT) and after germination (GH and CL), the results suggest that germination induces saponin biosynthesis in cotyledon while, concurrently, it enhances degradation and/or translocation of stored saponins in hypocotyl. During germination, the content of group A and Sg-6 saponins was markedly reduced (Table 2). Similar to our finding, Rupasinghe et al.16) found that SS-A content was significantly reduced by germination. Contradict to our results, Shimoyamada et al.17) reported that germination does not cause any changes to the content of group A saponins. Takada et al.13) reported that group A saponins deficient accession (B01082 – Japanese wild soybean mutant) had grown well and produced fertile seeds under normal growth condition. The
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Korean wild soybean mutant accessions CWS15095 and CWS16027, lacking group A saponins, were also grown well and produced fertile seeds without difficulty (unpublished data). In our previous studies,4–6) we observed that 82–98% of wild soybeans did not contain Sg-6 saponins. Also, it is noteworthy to mention that Sg-6 saponins have not been found in soybean so far. Based on the availability of null accessions completely lacking group A saponins and Sg-6 saponins, and the deleterious effects on those saponin groups during germination, we presumably propose that group A and Sg-6 saponins are not essential for normal plant growth and development. Shimoyamada et al.22) have reported various parts (leaf, stem, branch, petiole, hypocotyl, cotyledon, and seed pod) of soybean plants contain group B saponins. However, we did not compare their results with ours; because they unknowingly converted all DDMP saponins into group B and/or group E saponins prior to HPLC analysis. Table 2 shows that DDMP saponins were newly synthesized by germination in wild soybean. By contrast, germination does not change DDMP saponin content in lentils (Lens culinaris) and chickpeas (Cicer aerietinum).23) This implies that DDMP saponin regulation by germination is specific to species. Although we did not estimate the absolute saponin concentration, nevertheless, we found considerable amount of saponin DDMP-βg in all the analyzed tissues (data not shown). Of the ATSC, the content of DDMP saponins represents 15–20% of the primary and lateral roots, 40–50% of the hypocotyl and germinated hypocotyl, 60–65% of the cotyledon and unifoliolate leaf, and 70– 75% of the epicotyl, apical meristem, cotyledonary leaf, and trifoliolate leaf (Table 2). In primary and lateral roots, 75–82% of DDMP saponins were in the form of group B + E saponins (Table 2; Fig. 4). These results imply that the total saponin concentration of the shoot system of wild soybean was mainly composed of DDMP saponins, while it was mainly composed of group B + E saponins in the root system. More research is required to uncover the importance of the high degradation rate of DDMP saponins in the root system.
Future perspective Since no mutants were found to lack DDMP saponins from soybean and wild soybean germplasm collections, researchers widely suspect that DDMP saponins may have some physiological functions in the plant. We believe uncovering the importance and necessities of the high degradation ratio of DDMP and group B + E saponins in the root system would give us some clues about the functionality of DDMP saponins in plant. Previously, chromosaponin I (CSI), isolated from peas (Pisum sativum) and same chemical structure as DDMP-βg, has been reported to show positive correlation with root growth,24) elongation of cortical cells,25) increase mechanical extensibility of root cell walls,26) regulation of gravitropic response of roots27), and regulation of auxin influx.28) However, these experiments were performed by soaking the plants into medium containing a DDMP saponin (CSI) during plant growth. Hence, using these methods, the physiological
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properties of DDMP saponins in situ in plants growth and development will not be conducted because DDMP saponins are very labile. The chemical structure of DDMP saponin (CSI) may be modified during absorption into plant tissue and the chemical structure of actual components affecting plant growth may be different from the original DDMP saponin. Takagi et al.29 produced transgenic soybean plants by modifying the saponin biosynthesis through RNA interference (RNAi) mediated gene silencing targeted to β-amyrin synthase using a promoter expressed only in the seeds. The seeds were obtained with almost no saponins. This may suggest that the accumulation of saponins in the seeds may not be essential, but some saponins may be produced de novo during germination. We believe that we need to consider all of the above-mentioned circumstances and many others while planning to demonstrate the physiological properties of DDMP saponins in situ in plant growth and development.
Supplemental material The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2014.946389.
Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0013600).
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