Molecular and cellular study of flavonoid metabolism and detection of relating genes in soybean

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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℃). Chilling injury was more severe in the second trifoliate leaves of To7G than in those of To7B. Lipid peroxidation was enhanced by chilling in To7G. Chilling-induced enhancement of antioxidant activity was more prominent in To7B than in To7G. High performance liquid chromatography (HPLC) 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 (RT-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.