Analysis of two single trait loci affecting flavonol glycoside accumulations in Arabidopsis thaliana natural variations

Ishihara H. Analysis of two single trait loci affecting flavonol glycoside accumulations in Arabidopsis thaliana natural variations. Bielefeld (Germany): Bielefeld University; 2007.Various plant secondary metabolites, including flavonoids, are involved in plant adaptation to different environments. The needs of sessile lifestyle of plants may have increased the variation of enzymes which are required in the modification and/or accumulation of different flavonol derivatives. The probable mechanism for generating variants of the enzymes is by mutating the corresponding genes. Therefore, Arabidopsis thaliana wildtype accessions collected from different environments and locations may have differences in the accumulation of flavonoid derivatives. Using molecular markers, these differences can be investigated and used to identify genomic variations causing observed traits. The work presented in this thesis is focused on the identification of new genes involved in accumulation of flavonoid derivatives in A. thaliana wildtype accessions using both metabolomics and genomic approaches. Three different analytical methods were employed to identify flavonoid derivatives in A. thaliana seedlings. First, high performance liquid chromatography photo diode array coupling with electro spray -ionization mass spectrometry (HPLC-PDA-ESI-MS) was employed to identify molecular mass of flavonoid aglycones and conjugate sugars. Second, high performance thin layer chromatography (HPTLC) was used to rapidly screen large amounts of plant samples. HPTLC was also employed to purify flavonoid derivatives that were then used for the identification of sugar groups by gas chromatography mass spectrometry (GC-MS). Twelve different flavonoid derivatives were identified from seedlings, all were glycosylated in mono-, di-, or tri- forms with glucose and/or rhamnose. Comparison of flavonol derivatives among twelve different A. thaliana wildtype accessions revealed quantitative differences in flavonol glycosides namely and quercetin 3-O-rhamnoside (quercitrin) and flavonol 3-O-gentiobioside 7-O-rhamnoside. Quantitative trait mapping analysis of the quercitrin trait (Qr4) located a single trait locus on chromosome 5 at 99 cM. The previously characterized TRANSPARENT TESTA 10 was identified to encode the Qr4 locus when comparing quercitrin accumulation between mutants and wildtypes (WTs) seed coats. Allelic difference in the TT10 promoter sequence in Ler was identified to cause lower promoter activity than Col. The promoter difference was proposed to involve in the quercitrin accumulation. From the metabolomic data, the candidate gene involved in qualitative accumulation difference in the flavonol 3-O-gentiobioside 7-O-rhamnoside (Km1 and Qr1) was assumed to be of the UDP glycosyltransferase family 1 protein (UGT). Linkage analysis was performed to identify the molecular markers linked to the locus. The trait was tightly linked to a molecular marker mi304 located on the chromosome 1 at 86.52 cM. The 1.8 Mb interval between flanking markers located both sides of mi304 did not contain any genes encoding UGT. Hence, the interval was narrowed down to a 90 Kb interval and sequenced. Again no UGT was found in the Ler genomic sequence. However, comparison of genomic sequences of the interval between Col and Ler showed substantial modification by large insertions and deletions. This genome region may be involved to the Km1 and Qr1 trait. Finally, based on the genome annotation, four candidate genes were proposed for further investigation