Functional Analysis of a Predicted Flavonol Synthase Gene Family in Arabidopsis    

Abstract

The genome of Arabidopsis (Arabidopsis thaliana) contains five sequences with high similarity to FLAVONOL SYNTHASE1 (AtFLS1), a previously characterized flavonol synthase gene that plays a central role in flavonoid metabolism. This apparent redundancy suggests the possibility that Arabidopsis uses multiple isoforms of FLS with different substrate specificities to mediate the production of the flavonols, quercetin and kaempferol, in a tissue-specific and inducible manner. However, biochemical and genetic analysis of the six AtFLS sequences indicates that, although several of the members are expressed, only AtFLS1 encodes a catalytically competent protein. AtFLS1 also appears to be the only member of this group that influences flavonoid levels and the root gravitropic response in seedlings under nonstressed conditions. This study showed that the other expressed AtFLS sequences have tissue- and cell type-specific promoter activities that overlap with those of AtFLS1 and encode proteins that interact with other flavonoid enzymes in yeast two-hybrid assays. Thus, it is possible that these “pseudogenes” have alternative, noncatalytic functions that have not yet been uncovered.

Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase

“…During our study, Owens et al (2008a) provided evidence for the same assumption. They followed a similar approach, using T-DNA insertion lines to explore the contributions of FLS1-FLS6 genes to Xavonoid biosynthesis.…”

“…This was done under two assumptions: (1) that the FLS2-FLS6 genes do not encode functional FLS and (2) that the LDOX enzyme could have FLS activity in vivo. The Wrst assumption has been shown to be correct in this work and by the work of Owens et al (2008a). The second assumption is based mainly on the work of Turnbull et al (2003Turnbull et al ( , 2004 and Yan et al (2005).…”

“…6a) indicates that LDOX can indeed exert FLS activity in the seedling. Thus, this is the Wrst direct evidence of in planta FLS activity of LDOX as suggested recently (Lillo et al 2008;Owens et al 2008a). The origin of the remaining very low amounts of Xavonol glycosides in the ldox Xs1-2 mutant remains unclear and the physiological relevance of these traces (undetectable with HPTLC, PDA-and UV-detectors) is questionable.…”

“…These protein regions are potentially important for FLS activity, separating FLS from 2-oxoglutarate-/Fe II -dependent dioxygenases with other substrate speciWcities. The importance of the PxxxIRxxxEQP motif at the N-terminus, that is missing in the FLS2-FLS6 polypeptides, was recently identiWed and further analysed by Owens et al (2008a). In their study it was shown that a N-terminal truncated FLS1 variant, lacking the Wrst 21 N-terminal amino acids did not show in vitro FLS activity.…”

“…We further present evidence that the remaining Xavonol glycosides found in the Xs1 mutant are synthesized by the in planta FLS-like site activity of LDOX. During our study, Owens et al (2008a) provided evidence that FLS1 appears to be the only member of this group that inXuences Xavonoid levels. Their Wndings will be discussed in relation to our data.…”