Anthocyanin 5,3'-aromatic acyltransferase from Gentiana triflora, a structural insight into biosynthesis of a blue anthocyanin.

The acylation of anthocyanins contributes to their structural diversity. Aromatic acylation is responsible for the blue color of anthocyanins and certain flowers. Aromatic acyltransferase from Gentiana triflora Pall. (Gentianaceae) (Gt5,3'AT) catalyzes the acylation of glucosyl moieties at the 5 and 3' positions of anthocyanins. Anthocyanin acyltransferase transfers an acyl group to a single position, such that Gt5,3'AT possesses a unique enzymatic activity. Structural investigation of this aromatic acyl group transfer is fundamental to understand the molecular mechanism of the acylation of double positions. In this study, structural analyses of Gt5,3'AT were conducted to identify the underlying mechanism. The crystal structure indicated that Gt5,3'AT shares structural similarities with other BAHD family enzymes, consisting of N and C terminal lobes. Structural comparison revealed that acyl group preference (aromatic or aliphatic) for the enzymes was determined by four amino acid positions, which are well conserved in aromatic and aliphatic CoA-binding acyltransferases. Although a complex structure with anthocyanins was not obtained, the binding of delphinidin 3,5,3'-triglucoside to Gt5,3'AT was investigated by evaluating the molecular dynamics. The simulation indicated that acyl transfer by Gt5,3'AT preferentially occurs at the 5-position rather than at the 3'-position, with interacting amino acids that are mainly located in the C-terminal lobe. Subsequent assays of chimeric enzymes (exchange of the N-terminal lobe and the C-terminal lobe between Gt5,3'AT and lisianthus anthocyanin 5AT) demonstrated that acyl transfer selectivity may be caused by the C-terminal lobe.

Practical optimization of liquid chromatography/mass spectrometry conditions and pretreatment methods toward the sensitive quantification of auxin in plants.

RATIONALE: The plant hormone auxin, indole-3-acetic acid, regulates many aspects of plant growth and development. Auxin quantification should offer broad insights into its mechanistic action in plants. However, limited auxin content in plant tissues hampers the establishment of quantification methods without the highest graded instruments or deeply specialized experimental techniques. METHODS: In this study, we detailed optimized conditions for high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (LC/MS). We compared LC/MS conditions, such as columns, mobile phases, parameters of acquisition methods (selective or multiple ion monitoring), dwell times (DTs), and channel numbers, using differentially mixed authentic auxin and its related compounds. We further investigated pretreatment methods through the optimization of auxin recovery and irrelative compound removal from plant tissues prior to the LC/MS analysis. RESULTS: Our LC/MS analysis demonstrated the particular importance of the column, DTs, and channel numbers on detection sensitivity. Our comparative analysis developed optimal pretreatment methods, including the pulverization of plants, concentration of extract through centrifugal evaporation, and removal of irrelative metabolites using liquid-liquid extraction and a spin filter. We injected plant samples into our LC/MS system, quantified auxin and eight related compounds in a single measurement, and determined the auxin increase in an auxin over-producing mutant. CONCLUSIONS: Our practical optimization of LC/MS conditions and pretreatment methods provides detailed experimental processes toward the sensitive quantification of auxin from 10 mg of plant tissue. These processes have not always been clearly documented; therefore, our protocol could broadly contribute to technical advances in plant growth and development research.

Imaging Mass Spectrometry Analysis of Flavonoids in Blue Viola Petals and Their Enclosure Effects on Violanin during Color Expression.

The color expression of anthocyanin pigments in blue flowers is precisely controlled by their chemical and physical properties such as pH and the presence of metal ions or colorless copigments. Despite the large number of known blue flowers, their coloration mechanisms have not been examined in sufficient detail. In this work, the blue coloration of Viola cornuta petals was expressed via the copigmentation of various flavonol 3- O-glycosides. By using a combination of imaging mass spectrometry with liquid chromatography mass spectrometry, the structures and contents of flavonols colocalized with violanin in the discrete blue-colored regions of the petal were identified. The obtained data allowed the in vitro reconstruction of the color expression that was consistent with the visible spectrum of the viola petal. The results of visible spectral analysis indicated that neither the increase in the solution pH inside the vacuole cells nor the presence of metal ions affected the color development process. Ultimately, it was experimentally confirmed that the excess amounts of flavonol 3- O-glycosides complexed with violanin, which prevented violanin molecules from forming a levorotatory helical self-assembly during the blue color expression via copigmentation.