Identification and functional characterization of a flavonol synthase gene from sweet potato [Ipomoea batatas (L.) Lam.]

Abstract

Flavonol synthase (FLS) is a key enzyme of the flavonoid biosynthetic pathway, which catalyzes the conversion of dihydroflavonols into flavonols. In this study, the FLS gene IbFLS1 was cloned and characterized from sweet potato. The resulting IbFLS1 protein showed a high similarity with other plant FLSs. The conserved amino acids (HxDxnH motifs) binding ferrous iron and residues (RxS motifs) binding 2-oxoglutarate were found in IbFLS1 at conserved positions, as in other FLSs, suggesting that IbFLS1 belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. qRT-PCR analysis showed an organ-specific pattern of expression of the IbFLS1 gene, which was predominantly expressed in young leaves. The recombinant IbFLS1 protein could catalyze the conversion of dihydrokaempferol and dihydroquercetin to kaempferol and quercetin, respectively. The results of subcellular localization studies indicated that IbFLS1 was found mainly in the nucleus and cytomembrane. Furthermore, silencing the IbFLS gene in sweet potato changed the color of the leaves to purple, substantially inhibiting the expression of IbFLS1 and upregulating the expression of genes involved in the downstream pathway of anthocyanin biosynthesis (i.e., DFR, ANS, and UFGT). The total anthocyanin content in the leaves of the transgenic plants was dramatically increased, whereas the total flavonol content was significantly reduced. Thus, we conclude that IbFLS1 is involved in the flavonol biosynthetic pathway and is a potential candidate gene of color modification in sweet potato.

Why the adventitious roots of poplar are so colorful: RNAseq and metabolomic analysis reveal flavonols, flavones, and anthocyanins accumulation in canker pathogens-induced adventitious roots in poplar

“…Flavonol synthase (FLS) is the key committed enzyme in the biosynthesis of class flavonols, which convert dihydroflavonols to the form of quercetin, kaempferol, and myricetin (Kou et al 2023). Considering the high accumulation (the relative intensity, number of top accumulated metabolites, and total accumulated metabolites) of flavonols, the upregulation of gene FLS (Supplementary file 1, Supplementary file 9) suggested a redirection of metabolic flux into the flavonols branching pathway.…”

Metabolomic and Transcriptomic Analyses Reveal the Molecular Mechanism Underlying the Massive Accumulation of Secondary Metabolites in Fenugreek (Trigonella foenum-graecum L.) Seeds

“…It has an important effect on anthocyanin accumulation [ 30 ]. Many FLS genes have been cloned and identified in other plants [ 31 ]. In the present research, it was found that FLSs are involved in the synthesis of many flavonoids and were significantly upregulated according to the DEG analysis of the transcriptome.…”

Conserved amino acid residues and gene expression patterns associated with the substrate preferences of the competing enzymes FLS and DFR

“…Our analysis and enzyme assay data from previous studies suggest that FLS sequences within the first dicot subclade (Fagales I, Sapindales I, Malpighiales I, and Malvales I) display a pronounced substrate specificity for DHK. In contrast, FLS sequences within the second dicot subclade show a preference for both DHK and DHQ and may have a higher specificity for DHQ (63,65,66,103,105,114,128). ANS/LDOX, which catalyses the immediate step downstream of DFR, is closely related to FLS and shows 50-60% protein sequence similarity.…”

“…This suggests a gene duplication during the evolution of land plants probably before the split of the aforementioned orders followed by a significant divergence in FLS sequences in these orders due to functional requirements, leading to the formation of distinct FLS clades. Within the Malvales order, including cotton species, an additional duplication event was observed, originating from FLS H duplication, followed by a subsequent substitution leading to the re-emergence of FLS Y (H132Y In contrast, FLS sequences within the second dicot subclade show a preference for both DHK and DHQ and may have a higher specificity for DHQ (98,100,112,113,125,126,130).…”

“…The variable preferences of flavonol synthases for 3'-hydroxylated and 3',4'-hydroxylated substrates have been investigated. Studies highlighted that some flavonol synthases, such as Oryza sativa OsFLS (59), Zea mays ZmFLS1 (60), Arabidopsis thaliana AtFLS1 (61), Citrus unshiu CitFLS (62), and Camellia sinensis CsFLSs (63) exhibit greater efficiency in using 3'-hydroxylated dihydrokaempferol (DHK) over 3',4'-hydroxylated dihydroquercetin (DHQ), while Allium cepa AcFLS (64), Fagopyrum tataricum FtFLS1 (65), Ipomoea batatas IbFLS (66), and Nicotiana tabacum NtFLS (67) show the opposite trend. Five specific residues, namely H132, F134, K202, F293, and E295, have been reported to be involved in the binding of the DHQ substrate (68).…”