Kyoko Toda

Flavonoid 3ʹ‐hydroxylase (F3ʹH) catalyzes hydroxylation at the 3ʹ position of the B ring of flavonoids. F3ʹH is associated with the seed coat and pubescence color, antioxidant properties, and chilling stress tolerance of soybean [Glycine max (L.) Merr.]. In this study, we found that soybean F3ʹH increases the calcium (Ca) and magnesium (Mg) contents in pods and reduces the levels of these elements in seeds. Inductively coupled plasma mass spectrometry (ICP‐MS) analysis using a near‐isogenic line (NIL) derived from Japanese cultivars ‘Natto‐shoryu’ and ‘Hyoukei‐kuro 3’ for F3ʹH (Glyma.06g202300), also known as the T gene, indicated that the contents of Ca and Mg were higher in the pods but lower in the seed coats and cotyledons from soybean with active F3ʹH than from soybean with inactive F3ʹH. No such clear tendency was observed for other inorganic ions measured in this study. These data suggested that the transport of Ca and Mg from the pod to the seed was inhibited by flavonoid produced through F3ʹH activity. The hardness of cooked beans from soybean with active F3ʹH was lower than that from soybean with inactive F3ʹH, which was likely related to the increase in heat‐labile pectin caused by fewer cross‐linked divalent ions. These results were supported by ICP‐MS analysis and the hardness of cooked beans of other NILs for the T gene. Our findings demonstrate a novel and unexpected effect of flavonoids on the processing quality of soybean.

Cooked bean hardness is an important trait for the processing of soybean products such as nimame, natto, miso, and soy sauce. Previously, we showed that cooked bean hardness is primarily affected by the pectin methylesterase gene Glyma03g03360, and that calcium content has a secondary effect on this trait. To establish a simple and timely method for the evaluation of cooked bean hardness, primers of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) were designed to detect a single-nucleotide polymorphism of Glyma03g03360 and subsequently used to evaluate three soybean progeny lines. The determined genotypes were compared to those identified using the cleaved amplified polymorphic sequence (CAPS) method. Seven out of 284 lines presented different genotypes, which were determined using the two methods: A genotypes were incorrectly assigned as heterozygous by CAPS, suggesting that ARMS-PCR is more reliable. Glyma03g03360 genotypes could be used to evaluate cooked bean hardness, except for intermediate values. Cooked bean hardness within the same genotype groups was significantly correlated with calcium contents. These findings indicate that ARMS-PCR is useful for a marker-assisted selection of soybean with soft-cooked beans and that calcium content may be used for additional selection.

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We previously isolated a soybean (Glycine max (L.) Merr.) flavonoid 3′-hydroxylase (F3′H) gene (sf3′h1) corresponding to the T locus, which controls pubescence and seed coat color, from two near-isogenic lines (NILs), To7B (TT) and To7G (tt). The T allele is also associated with chilling tolerance. Here, Western-blot analysis shows that the sf3′h1 protein was predominantly detected in the hilum and funiculus of the immature seed coat in To7B, whereas sf3′h1 was not detected in To7G. A truncated sf3′h1 protein isolated from To7G was detected only upon enrichment by immunoprecipitation. An analysis using diphenylboric acid 2-aminoethyl ester (DBPA) staining revealed that flavonoids accumulated in the hilum and the funiculus in both To7B and To7G. Further, the scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in methanol extracts from the funiculus and hilum of To7B was higher than that of To7G. Moreover, the enzymatic activity of F3′H was detected using microsomal fractions from yeast transformed with sf3′h1 from To7B, but not from To7G. These results indicate that sf3′h1 is involved in flavonoid biosynthesis in the seed coat and affects the antioxidant properties of those tissues. As shown by immunofluorescence microscopy, the sf3′h1 protein was detected primarily around the vacuole in the parenchymatic cells of the hilum in To7B. Further immunoelectron microscopy detected sf3′h1 protein on the membranous structure of the vacuole. Based on these observations, we conclude that F3′H, which is a cytochrome P450 monooxygenase and has been found to be localized to the ER in other plant systems, is localized in the tonoplast in soybean.

Chilling tolerance is an important trait of soybeans [Glycine max (L.) Merr.] produced in cool climates. We previously isolated a soybean flavonoid 3′ hydroxylase (F3′H) gene corresponding to the T locus, which controls pubescence and seed coat color. A genetic link between the T gene and chilling tolerance has been reported, although the exact underlying mechanisms remain unclear. Using the soybean near-isogenic lines (NILs) To7B (TT) and To7G (tt), we examined the relationship between chilling injury, antioxidant activity and flavonoid profiles associated with chilling treatment (15°C). Chilling injury was more severe in the second trifoliate leaves of To7G than in those of To7B. Hydrogen peroxide accumulation and lipid peroxidation were enhanced by chilling in To7G. Chilling-induced enhancement of antioxidant activity was more prominent in To7B than in To7G. High performance liquid chromatography analysis indicated that the contents of quercetin glycosides and isorhamnetin glycosides (3′,4′-dihydroxylated flavonol derivatives) increase in the second trifoliate leaves of To7B after chilling treatment, whereas the same treatment increased kaempferol glycoside (4′-monohydroxylated flavonol derivatives) content in the corresponding leaves of To7G. Histochemical staining also demonstrated chilling-induced flavonoid accumulation. Microarray analysis and real-time reverse transcription-PCR demonstrated that the transcript levels of soybean F3′H are upregulated by chilling. The differences in chilling injury, antioxidant activity and flavonoid species between the two NILs support the notion that soybean F3′H affects chilling tolerance by increasing antioxidant activity via production of 3′,4′-dihydroxylated flavonol derivatives.

ABSTRACT:  The physicochemical properties of soymilk and the texture of tofu were compared with regard to 2 kinds of soymilk, one of which was prepared by squeezing homogenates before heating and the other was prepared by squeezing after heating raw soymilk with okara, residue of soymilk production. Relative particulate protein content and viscosity were higher and pH was lower in the soymilk prepared by the latter method, in which liberated lipid bodies were decreased and more lipids were precipitated with protein after centrifugation, suggesting a change in the interaction between proteins and lipids. A difference in the distribution of proteins and lipids was also implied by analysis with a laser particle size analyzer. The breaking stress of tofu made with 0.30% glucono‐delta‐lactone increased in accordance with an increase in particulate protein. The calcium and magnesium contents increased in soymilk prepared by squeezing after heating with okara. Viscosity was slightly increased and pH decreased by adding calcium to the soymilk, but the particulate protein content and breaking stress of tofu did not increase significantly. To examine the effect of macromolecules, okara was extracted by boiling and dialyzed. Viscosity and particulate protein content in soymilk increased as the dialyzed extracts of the okara were added. The breaking stress of tofu was increased by adding the dialyzed extracts but excessive amounts of the extracts resulted in softer tofu. Spectra of Fourier‐transform infrared spectroscopy and electrophoresis‐separated patterns of proteins indicated that the dialyzed extracts contained mainly polysaccharides and the Basic 7S globulin protein.