Multi-substrate flavonol O-glucosyltransferases from strawberry (Fragaria x ananassa) achene and receptacle.
In an effort to characterize fruit ripening-related genes functionally, two glucosyltransferases, FaGT6 and FaGT7, were cloned from a strawberry (Fragaria x ananassa) cDNA library and the full-length open reading frames were amplified by rapid amplification of cDNA ends. FaGT6 and FaGT7 were expressed heterologously as fusion proteins in Escherichia coli and target protein was purified using affinity chromatography. Both recombinant enzymes exhibited a broad substrate tolerance in vitro, accepting numerous flavonoids, hydroxycoumarins, and naphthols. FaGT6 formed 3-O-glucosides and minor amounts of 7-O-, 4'-O-, and 3'-O-monoglucosides and one diglucoside from flavonols such as quercetin. FaGT7 converted quercetin to the 3-O-glucoside and 4'-O-glucoside and minor levels of the 7- and 3'-isomers but formed no diglucoside. Gene expression studies showed that both genes are strongly expressed in achenes of small-sized green fruits, while the expression levels were generally lower in the receptacle. Significant levels of quercetin 3-O-, 7-O-, and 4'-O-glucosides, kaempferol 3-O- and 7-O-glucosides, as well as isorhamnetin 7-O-glucoside, were identified in achenes and the receptacle. In the receptacle, the expression of both genes is negatively controlled by auxin which correlates with the ripening-related gene expression in this tissue. Salicylic acid, a known signal molecule in plant defence, induces the expression of both genes. Thus, it appears that FaGT6 and FaGT7 are involved in the glucosylation of flavonols and may also participate in xenobiotic metabolism. The latter function is supported by the proven ability of strawberries to glucosylate selected unnatural substrates injected in ripe fruits. This report presents the first biochemical characterization of enzymes mainly expressed in strawberry achenes and provides the foundation of flavonoid metabolism in the seeds.
Griesser
Markus
M
Biomolecular Food Technology, Technical University Munich, Lise-Meitner-Str. 34, 85354 Freising, Germany.
Vitzthum
Florian
F
Fink
Barbara
B
Bellido
Mari Luz
ML
Raasch
Constanze
C
Munoz-Blanco
Juan
J
Schwab
Wilfried
W
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005419:Flavonoids / Q000737:chemistry / Q000378:metabolism*; D031985:Fragaria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D005638:Fruit / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D018506:Gene Expression Regulation, Plant; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D010802:Phylogeny; D010937:Plant Growth Regulators / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018547:Plant Stems / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D010944:Plants / Q000145:classification / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D015262:Xenobiotics / Q000378:metabolism
D018076:DNA, Complementary; D005419:Flavonoids; D010937:Plant Growth Regulators; D010940:Plant Proteins; D011994:Recombinant Proteins; D015262:Xenobiotics; D005964:Glucosyltransferases