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Linkage mapping, molecular cloning and functional analysis of soybean gene Fg3 encoding flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase.

Di S, Yan F, Rodas FR, Rodriguez TO, Murai Y, Iwashina T, Sugawara S, Mori T, Nakabayashi R, Yonekura-Sakakibara K, Saito K, Takahashi R - BMC Plant Biol. (2015)

Bottom Line: GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee.Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan. dishaokang@affrc.go.jp.

ABSTRACT

Background: Flavonol glycosides (FGs) are major components of soybean leaves and there are substantial differences in FG composition among genotypes. The first objective of this study was to identify genes responsible for FG biosynthesis and to locate them in the soybean genome. The second objective was to clone the candidate genes and to verify their function. Recombinant inbred lines (RILs) were developed from a cross between cultivars Nezumisaya and Harosoy.

Results: HPLC comparison with authentic samples suggested that FGs having glucose at the 2″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Nezumisaya, whereas FGs of Harosoy were devoid of 2″-glucose. Conversely, FGs having glucose at the 6″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Harosoy, whereas these FGs were absent in Nezumisaya. Genetic analysis suggested that two genes control the pattern of attachment of these sugar moieties in FGs. One of the genes may be responsible for attachment of glucose to the 2″-position, probably encoding for a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase. Nezumisaya may have a dominant whereas Harosoy may have a recessive allele of the gene. Based on SSR analysis, linkage mapping and genome database survey, we cloned a candidate gene designated as GmF3G2″Gt in the molecular linkage group C2 (chromosome 6). The open reading frame of GmF3G2″Gt is 1380 bp long encoding 459 amino acids with four amino acid substitutions among the cultivars. The GmF3G2″Gt recombinant protein converted kaempferol 3-O-glucoside to kaempferol 3-O-sophoroside. GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].

Conclusion: GmF3G2″Gt encodes a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase and corresponds to the Fg3 gene. GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee. Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

No MeSH data available.


Related in: MedlinePlus

Identification of reaction product of GmF3G2″Gt-a (cultivar Nezumisaya). (A) Elution profiles of the standards (kaempferol 3-O-glucoside and kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. (B) UV spectra of the standard (kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. Mass spectra (C) and MS/MS spectra (D) of the standard (kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. E, The MS/MS fragmentation for kaempferol 3-O-sophoroside. K3Glc, kaempferol 3-O-glucoside; K3Glc2″Glc, kaempferol 3-O-sophoroside.
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Fig6: Identification of reaction product of GmF3G2″Gt-a (cultivar Nezumisaya). (A) Elution profiles of the standards (kaempferol 3-O-glucoside and kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. (B) UV spectra of the standard (kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. Mass spectra (C) and MS/MS spectra (D) of the standard (kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. E, The MS/MS fragmentation for kaempferol 3-O-sophoroside. K3Glc, kaempferol 3-O-glucoside; K3Glc2″Glc, kaempferol 3-O-sophoroside.

Mentions: The GmF3G2″Gt recombinant protein of Nezumisaya (GmF3G2″Gt-a) and Harosoy (GmF3G2″Gt-b) were expressed in E. coli as a His/ProS2 fusion and purified. The GmF3G2″Gt proteins were used for enzymatic assays after cleavage of the His/ProS2 tag. GmF3G2″G-a and GmF3G2″G-b converted kaempferol 3-O-glucoside to kaempferol 3-O-sophoroside as confirmed by comparison of retention time, UV spectra and MS/MS ionization with the standard compound (Figure 6, Additional file 2: Figure S2). GmF3G2″Gt-a showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives (Table 2). However, GmF3G2″Gt-a did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside]. GmF3G2″Gt-a had a higher preference for UDP-glucose than UDP-galactose, with only 3% activity relative to that for UDP-galactose. No UGT activity was detected for UDP-arabinose and UDP-glucuronic acid. GmF3G2″Gt-b also showed similar substrate specificity (Additional file 3: Table S1). Accordingly, GmF3G2″Gt-a and GmF3G2″Gt-b were defined as flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferases.Figure 6


Linkage mapping, molecular cloning and functional analysis of soybean gene Fg3 encoding flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase.

Di S, Yan F, Rodas FR, Rodriguez TO, Murai Y, Iwashina T, Sugawara S, Mori T, Nakabayashi R, Yonekura-Sakakibara K, Saito K, Takahashi R - BMC Plant Biol. (2015)

Identification of reaction product of GmF3G2″Gt-a (cultivar Nezumisaya). (A) Elution profiles of the standards (kaempferol 3-O-glucoside and kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. (B) UV spectra of the standard (kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. Mass spectra (C) and MS/MS spectra (D) of the standard (kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. E, The MS/MS fragmentation for kaempferol 3-O-sophoroside. K3Glc, kaempferol 3-O-glucoside; K3Glc2″Glc, kaempferol 3-O-sophoroside.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494776&req=5

Fig6: Identification of reaction product of GmF3G2″Gt-a (cultivar Nezumisaya). (A) Elution profiles of the standards (kaempferol 3-O-glucoside and kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. (B) UV spectra of the standard (kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. Mass spectra (C) and MS/MS spectra (D) of the standard (kaempferol 3-O-sophoroside) and reaction product of GmF3G2″Gt-a protein. E, The MS/MS fragmentation for kaempferol 3-O-sophoroside. K3Glc, kaempferol 3-O-glucoside; K3Glc2″Glc, kaempferol 3-O-sophoroside.
Mentions: The GmF3G2″Gt recombinant protein of Nezumisaya (GmF3G2″Gt-a) and Harosoy (GmF3G2″Gt-b) were expressed in E. coli as a His/ProS2 fusion and purified. The GmF3G2″Gt proteins were used for enzymatic assays after cleavage of the His/ProS2 tag. GmF3G2″G-a and GmF3G2″G-b converted kaempferol 3-O-glucoside to kaempferol 3-O-sophoroside as confirmed by comparison of retention time, UV spectra and MS/MS ionization with the standard compound (Figure 6, Additional file 2: Figure S2). GmF3G2″Gt-a showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives (Table 2). However, GmF3G2″Gt-a did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside]. GmF3G2″Gt-a had a higher preference for UDP-glucose than UDP-galactose, with only 3% activity relative to that for UDP-galactose. No UGT activity was detected for UDP-arabinose and UDP-glucuronic acid. GmF3G2″Gt-b also showed similar substrate specificity (Additional file 3: Table S1). Accordingly, GmF3G2″Gt-a and GmF3G2″Gt-b were defined as flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferases.Figure 6

Bottom Line: GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee.Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan. dishaokang@affrc.go.jp.

ABSTRACT

Background: Flavonol glycosides (FGs) are major components of soybean leaves and there are substantial differences in FG composition among genotypes. The first objective of this study was to identify genes responsible for FG biosynthesis and to locate them in the soybean genome. The second objective was to clone the candidate genes and to verify their function. Recombinant inbred lines (RILs) were developed from a cross between cultivars Nezumisaya and Harosoy.

Results: HPLC comparison with authentic samples suggested that FGs having glucose at the 2″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Nezumisaya, whereas FGs of Harosoy were devoid of 2″-glucose. Conversely, FGs having glucose at the 6″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Harosoy, whereas these FGs were absent in Nezumisaya. Genetic analysis suggested that two genes control the pattern of attachment of these sugar moieties in FGs. One of the genes may be responsible for attachment of glucose to the 2″-position, probably encoding for a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase. Nezumisaya may have a dominant whereas Harosoy may have a recessive allele of the gene. Based on SSR analysis, linkage mapping and genome database survey, we cloned a candidate gene designated as GmF3G2″Gt in the molecular linkage group C2 (chromosome 6). The open reading frame of GmF3G2″Gt is 1380 bp long encoding 459 amino acids with four amino acid substitutions among the cultivars. The GmF3G2″Gt recombinant protein converted kaempferol 3-O-glucoside to kaempferol 3-O-sophoroside. GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].

Conclusion: GmF3G2″Gt encodes a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase and corresponds to the Fg3 gene. GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee. Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

No MeSH data available.


Related in: MedlinePlus