Difference in chilling-induced flavonoid profiles, antioxidant activity and chilling tolerance between soybean near-isogenic lines for the pubescence color gene

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Abstract
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.