Cinnamate metabolism in ripening fruit. Characterization of a UDP-glucose:cinnamate glucosyltransferase from strawberry

Abstract

Strawberry (Fragaria x ananassa) fruit accumulate (hydroxy)cinnamoyl glucose (Glc) esters, which may serve as the biogenetic precursors of diverse secondary metabolites, such as the flavor constituents methyl cinnamate and ethyl cinnamate. Here, we report on the isolation of a cDNA encoding a UDP-Glc:cinnamate glucosyltransferase (Fragaria x ananassa glucosyltransferase 2 [FaGT2]) from ripe strawberry cv Elsanta that catalyzes the formation of 1-O-acyl-Glc esters of cinnamic acid, benzoic acid, and their derivatives in vitro. Quantitative real-time PCR analysis indicated that FaGT2 transcripts accumulate to high levels during strawberry fruit ripening and to lower levels in flowers. The levels in fruits positively correlated with the in planta concentration of cinnamoyl, p-coumaroyl, and caffeoyl Glc. In the leaf, high amounts of Glc esters were detected, but FaGT2 mRNA was not observed. The expression of FaGT2 is negatively regulated by auxin, induced by oxidative stress, and by hydroxycinnamic acids. Although FaGT2 glucosylates a number of aromatic acids in vitro, quantitative analysis in transgenic lines containing an antisense construct of FaGT2 under the control of the constitutive 35S cauliflower mosaic virus promoter demonstrated that the enzyme is only involved in the formation of cinnamoyl Glc and p-coumaroyl Glc during ripening.

Figure 1.

Phylogenetic relationship of the four glycosyltransferases cloned from Fragaria (boxed) with 42 other sequences of glycosyltransferases, all of which have already been biochemically characterized. The dendrogram was created using the Clustal sequence alignment program of the Lasergene software package (DNASTAR). Lengths of lines indicate the relative distance between nodes. Indicated are the family and the GenBank accession number and, in the case of several Arabidopsis GTs with the same accession number, also the name of the UGT according to the superfamily nomenclature system (Mackenzie et al., 1997).

Figure 2.

Sequence alignment of FaGT2 (Fragaria_AY663785_FaGT2), Satsuma mandarin GT (Citrus_AB033758), rapeseed GT (Brassica_AF287143), and three Arabidopsis (Arabidopsis_AB019232_UGT84A2, Arabidopsis_Z97339_UGT84A1, and Arabidopsis_Z97339_UGT84A3) deduced protein sequences. The ORF of FaGT2 consists of 1,668 bp encoding for a protein with 555 amino acids. The plant secondary product glycosyltransferase box, characteristic for GTs involved in natural product conjugation, is underlined.

Figure 3.

Chemical structures of some substrates and products of FaGT2. FaGT2 converts benzoic acid, cinnamic acid, and their respective derivatives to the corresponding Glc esters, using UDP-activated Glc. A hydroxyl group at position 2 in the benzoic acids has an inhibitory effect on the activity.

Figure 4.

Developmental and spatial expression pattern of FaGT2 in different plant parts of strawberry cv Elsanta, analyzed by qRT-PCR and parallel analyses of metabolites. A, Relative expression in receptacles, vegetative tissues, and achenes. The increase in mRNA values is shown relative to the root Ct value, which is referred to as 1. Ct represents the cycle at which sample crosses threshold value. Error bars show the sd of three independent experiments. B, Quantification of free and glucosylated aromatic acids by LC-ESI-UV-MSⁿ (−, not detectable).

Figure 5.

A, Effect of removing achenes and auxin treatment of green fruit on FaGT2 gene expression analyzed by qRT-PCR. The increase in FaGT2 mRNA in de-achened fruit and de-achened fruit treated with 1-NAA is shown relative to control green receptacle fruit Ct value (sampled at day 0, achenes still attached), which is referred to as 1. Ct represents the cycle at which sample crosses threshold value. Mean values ± sd of three independent experiments. B, Effect of oxidative stress caused by menadione on FaGT2 gene expression in fruits, analyzed by qRT-PCR. Relative expression in white fruit injected with menadione (black bars) in comparison with white fruit injected with water (control; white bars) is shown. The increase in mRNA values is shown relative to control white fruit Ct value, which is referred to as 1. Mean values ± sd of three independent experiments. C, Relative expression in strawberry cell cultures treated with menadione (black bars) in comparison with untreated controls (white bars). The increase in mRNA values is shown relative to control 0-h Ct value, which is referred to as 1. Mean values ± sd of three independent experiments. D, FaGT2 gene expression in strawberry cell culture treated with free acids, analyzed by qRT-PCR. The increase in mRNA values is shown relative to control at the 4- and 8-h Ct values, which are referred to as 1. Mean values ± sd of three independent experiments.

Figure 6.

A, Relative quantification of FaGT2 transcripts in AS transgenic strawberry fruits using the comparative Ct method. Expression levels of FaGT2 mRNA in control plants are compared with those from transgenic plants (FaGT2 AS6 and 9). Ct represents the cycle at which sample crosses threshold value. The dbp mRNA was used for standardization. B, Quantitative determination of Glc esters in ripe fruit of control plants cv Calypso (white bars) and strawberry plants cv Calypso transformed with AS FaGT2 constructs (FaGT2 AS6, gray bars; FaGT2 AS9, black bars). The amount of at least three different purifications from different harvests is indicated with its sd. ANOVA performed on these results revealed a significant [F(8,54) = 5.81, P < 0.05] difference in the levels of cinnamoyl- and p-coumaroyl-d-Glc in fruits among the plants transformed with the AS construct and control plants (*). Details are described in “Materials and Methods.”