Identification of a novel flavonoid glycoside sulfotransferase in Arabidopsis thaliana

Abstract

The discovery of sulfated flavonoids in plants suggests that sulfation may play a regulatory role in the physiological functions of flavonoids. Sulfation of flavonoids is mediated by cytosolic sulfotransferases (SULTs), which utilize 3 0 -phosphoadenosine 5 0 -phosphosulfate (PAPS) as the sulfate donor. A novel SULT from Arabidopsis thaliana, designated AtSULT202B7 (AGI code: At1g13420), was cloned and expressed in Escherichia coli. Using various compounds as potential substrates, we demonstrated, for the first time, that AtSULT202B7 displayed sulfating activity specific for flavonoids. Intriguingly, the recombinant enzyme preferred flavonoid glycosides (e.g. kaempferol-3-glucoside and quercetin-3-glucoside) rather than their aglycone counterparts. Among a series of hydroxyflavones tested, AtSULT202B7 showed the enzymatic activity only for 7-hydroxyflavone. pH-dependency study showed that the optimum pH was relatively low (pH 5.5) compared with those (pH 6.0 8.5) previously reported for other isoforms. Based on the comparison of high performance (pressure) liquid chromatography (HPLC) retention times between sulfated kaempferol and the deglycosylated product of sulfated kaempferol-3-glucoside, the sulfation site in sulfated kaempferol-3-glucoside appeared to be the hydroxyl group of the flavonoid skeleton. In addition, by using direct infusion mass spectrometry, it was found that the sulfated product had one sulfonate group within the molecule. These results indicated that AtSULT202B7 functions as a flavonoid glycoside 7-sulfotransferase.Keywords: Arabidopsis thaliana/AtSULT202B7/ flavonoid glycoside/sulfation/sulfotransferase.Abbreviations: HPLC, high performance (pressure) liquid chromatography; IPTG, isopropyl b-D-thiogalactopyranoside; PAP, 3 0 -phosphoadenosine 5 0 -phosphate; PAPS, 3 0 -phosphoadenosine 5 0 -phosphosulfate; PCR, polymerase chain reaction; TLC, thin-layer chromatography; SDS PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; SULT, cytosolic sulfotransferase.Flavonoids are a group of secondary metabolites distributed in a wide range of plant species. They share a common phenyl benzopyrone structure and are divided into major subclasses (e.g. flavanone, flavonol, flavone, isoflavone and anthocyanidin) based on the numbers and positions of the hydroxyl group and the C-ring structure. Flavonoids have been implicated in a variety of physiological functions, e.g. provision of colors attractive to pollinators (1); protection of the plant body from external stress such as fungal infection and UV irradiation (2); communication with the symbiont Rhizobia (3); and influence in the transport of the plant hormone, auxin (4). To date, nearly 9000 structural variants of flavonoids have been reported (5). In different plants, flavonoids occur as glycosides (e.g. glucoside, galactoside, rhamnoside and arabinoside), which, except for flavanols such as catechins and proanthocyanidins, are generated upon glycosylation by uridine-diphosphate glucose glycosyltransferases (6). Glycosylation increas...

The multi-protein family of sulfotransferases in plants: composition, occurrence, substrate specificity, and functions

“…AtSOT8 was also characterized by Hashiguchi et al (2014) . The pH optimum at 5.5 was lower than for previously characterized flavonoid SOTs.…”

“…Only 17 AtSOTs were found to be present on 24 k Affymetrix chips and for many of those the absolute signal was quite low ( Klein and Papenbrock, 2008 ). Hashiguchi et al (2013 , 2014 ) reported that microarray database research suggested that AtSOT8 is mainly expressed in roots, while At SOT13 is expressed in the early stages of the embryonic development. Interestingly, Hashiguchi et al (2013) cloned AtSOT13 from 2-week old seedlings.…”

“…Even when comparing SOTs that use the same class of substrates from different plant species, not only differences in kinetic values, but also variation of specificity toward different substrates and specific hydroxyl groups are noticed. For example, flavonol SOTs (AtSOT5, AtSOT8, AtSOT12, AtSOT13) from A. thaliana prefer kaempferol or flavonol glycosides as substrate and sulfate the hydroxyl groups at 3- and 7-position ( Hashiguchi et al, 2013 , 2014 ). But flavonol SOTs from F. chloraefolia and F. bidentis (L.) Kuntze prefer quercetin as substrates and sulfate at 3′- and 4′-position ( Varin et al, 1992 , 1997b ).…”

Secondary sulfur metabolism in cellular signalling and oxidative stress responses

“…Flavonoids have diverse roles including UV protection, growth regulation and plant-microbe interactions (Falcone Ferreyra et al, 2012). At least four SOTs are capable of sulfating a variety of flavonoids but with subtly different substrate preferences and enzymatic rates (Hashiguchi et al, 2013;Hashiguchi et al, 2014). Both SOT13 and SOT5 showed strong preference for the flavonol galangin, although the latter also has significant affinity for kaempferol.…”

Detoxification function of the Arabidopsis sulphotransferase AtSOT12 by sulphonation of xenobiotics

“…AtSOT5 and AtSOT13 were shown to sulphonate flavonol while AtSOT8 preferred flavonol glycosides as substrates and AtSOT12 favoured falvonone for sulphonation (Hashiguchi et al . , ). AtSOT16, AtSOT17 and AtSOT18 are three SOTs that sulphonate a broad range of desulphoglucosinolates in the last step of glucosinolate biosynthesis (Piotrowski et al .…”