Abstract
Flavonols are colourless secondary metabolites, primarily regarded as UV-protection pigments that are deposited in plants in their glycosylated forms. The glycosylation of flavonols is mainly catalysed by UDP-sugar-dependent glycosyltransferases (UGTs). Although the structures of flavonol glycosides accumulating in Arabidopsis thaliana are known, many genes involved in the flavonol glycosylation pathway are yet to be discovered. The flavonol glycoside profiles of seedlings from 81 naturally occurring A. thaliana accessions were screened using high performance thin layer chromatography. A qualitative variation in flavonol 3-O-gentiobioside 7-O-rhamnoside (F3GG7R) content was identified. Ler × Col-0 recombinant inbred line mapping and whole genome association mapping led to the identification of a glycoside hydrolase family 1-type gene, At1g60270/BGLU6, that encodes a homolog of acyl-glucose-dependent glucosyltransferases involved in the glycosylation of anthocyanins, possibly localized in the cytoplasm, and that is co-expressed with genes linked to phenylpropanoid biosynthesis. A causal single nucleotide polymorphism introducing a premature stop codon in non-producer accessions was found to be absent in the producers. Several other naturally occurring loss-of-function alleles were also identified. Two independent bglu6 T-DNA insertion mutants from the producer accessions showed loss of F3GG7R. Furthermore, bglu6 mutant lines complemented with the genomic Ler BGLU6 gene confirmed that BGLU6 is essential for production of F3GGR7. We have thus identified an accession-specific gene that causes a qualitative difference in flavonol glycoside accumulation in A. thaliana strains. This gene encodes a flavonol 3-O-glucoside: 6″-O-glucosyltransferase that does not belong to the large canonical family of flavonol glycosyltransferases that use UDP-conjugates as the activated sugar donor substrate.
Highlights
PCR primer sets and restriction enzymes used for genotyping of the RILs are listed
1x restriction enzyme buffer in a total volume of 10 μL
The plate was incubated at suitable temperatures
Summary
PCR primer sets and restriction enzymes used for genotyping of the RILs are listed. PCR primers were designed by either comparing Col and Ler genomic sequence using TAIR Ler BLAST search or were obtained from MASC (Schmid et al, 2003; Torjek et al, 2003). For CAPS marker analysis, 5 μL of amplicons, generated by standard PCR with the given primer pairs, were digested with 0.5 units of the appropriate restriction enzyme and 1x restriction enzyme buffer in a total volume of 10 μL in a 96-well PCR plate. The plate was incubated at suitable temperatures for at least 3 h. The lengths of fragments were checked with electrophoresis on a 1%. Amplicons were sequenced with one of the PCR primers.
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