Acylphloroglucinol Biosynthesis in Strawberry Fruit

Abstract

Phenolics have health-promoting properties and are a major group of metabolites in fruit crops. Through reverse genetic analysis of the functions of four ripening-related genes in the octoploid strawberry (Fragaria × ananassa), we discovered four acylphloroglucinol (APG)-glucosides as native Fragaria spp. fruit metabolites whose levels were differently regulated in the transgenic fruits. The biosynthesis of the APG aglycones was investigated by examination of the enzymatic properties of three recombinant Fragaria vesca chalcone synthase (FvCHS) proteins. CHS is involved in anthocyanin biosynthesis during ripening. The F. vesca enzymes readily catalyzed the condensation of two intermediates in branched-chain amino acid metabolism, isovaleryl-Coenzyme A (CoA) and isobutyryl-CoA, with three molecules of malonyl-CoA to form phlorisovalerophenone and phlorisobutyrophenone, respectively, and formed naringenin chalcone when 4-coumaroyl-CoA was used as starter molecule. Isovaleryl-CoA was the preferred starter substrate of FvCHS2-1. Suppression of CHS activity in both transient and stable CHS-silenced fruit resulted in a substantial decrease of APG glucosides and anthocyanins and enhanced levels of volatiles derived from branched-chain amino acids. The proposed APG pathway was confirmed by feeding isotopically labeled amino acids. Thus, Fragaria spp. plants have the capacity to synthesize pharmaceutically important APGs using dual functional CHS/(phloriso)valerophenone synthases that are expressed during fruit ripening. Duplication and adaptive evolution of CHS is the most probable scenario and might be generally applicable to other plants. The results highlight that important promiscuous gene function may be missed when annotation relies solely on in silico analysis.

Figure 1.

Fruit phenotypes and relative mRNA levels of candidate genes in the transgenic fruits. Fruit phenotypes (A) and relative mRNA levels of candidate genes (B) in agroinfiltrated fruits. Infiltration with pBI-Intron alone resulted in no color change. Fruits agroinfiltrated with pBI-CHSi served as positive control. Pigmentation was decreased in fruits agroinfiltrated with gene-silencing (RNAi) and overexpression (OE) constructs of defined genes. Transcript levels decreased in gene21343- and gene33865-RNAi agroinfiltrated fruit, and transcript levels of gene00897 and gene10776 increased in fruits agroinfiltrated with overexpression constructs relative to pBI intron controls. Data represent means ± se for triplicate technical repetitions from five cDNA preparations. Asterisks indicate significant differences (*P = 0.05 and **P = 0.01).

Figure 2.

Targeted metabolite analysis and Venn diagram. Targeted metabolite analysis (A) and Venn diagram (B) showing the results of the second order analysis of untargeted LC-MS data. Metabolites were extracted from Fragaria spp. fruits in which the candidate genes were differentially expressed due to agroinfiltration of RNAi and overexpression (OE) constructs, using chalcone synthase RNAi constructs (CHSi) as positive control. Heat map also shows the range of concentration of the individual metabolites in parenthesis. Second order (meta-)analysis was performed according to Tautenhahn et al. (2011).

Figure 3.

Structures of APGs identified in strawberry fruit. APGs were identified in strawberry ‘Elsanta,’ ‘Senga Sengana,’ ‘Mara des Bois,’ and ‘Calypso’ fruit. M1, 1-[(2-Methylbutyryl)-phloroglucinyl]-2-O-β-d-glucopyranoside; M2, 1-[(2-methylpropanoyl)-phloroglucinyl]-2-O-β-d-glucopyranoside; M3, 1-[(3-methylbutyryl)-phloroglucinyl]-2-O-β-d-glucopyranoside; M4, 1-[(3-methylbutyryl)-phloroglucinyl]-2,4-di-O-β-d-glucopyranoside.

Figure 4.

Proposed formation mechanism and in vitro products. Products formed by FvCHS2-1, FvCHS2-2, and FvCHS2-3 when 4-coumaroyl-CoA, cinnamoyl-CoA, feruloyl-CoA, isobutyryl-CoA, and isovaleryl-CoA were used as starter molecules. A number sign indicates that the product was not detected by LC-MS, and an asterisk indicates that the product represents less than 10% of total products. -SCoA and CoAS- denote Coenzyme A.

Figure 5.

APGs decrease in both transient and stable CHS-silenced fruits. Fragaria spp. fruit phenotypes and CHS gene expression levels in agroinfiltrated fruits (A), and effect of CHS gene down-regulation on APGs (M1–M3) in transiently CHS-silenced fruits (B; CHSi, strawberry ‘Mara des Bois’) and a stable CHS antisense (antis.) transgenic line (C; CHS antis.; strawberry ‘Calypso’). Fourteen days after pollination, green Fragaria spp. fruit was infiltrated with Agrobacterium tumefaciens transformed with a construct encoding CHSi. Levels of metabolites were determined by LC-MS 14 d after infiltration in CHS-silenced fruits (CHSi; n = 5) and fruits infiltrated with A. tumefaciens containing the control vector (PBI, pBI-Intron; n = 5). Down-regulation of the CHS genes results in a decrease of APGs in the CHSi fruits and fruits of the stable transgenic line (CHS antis.; strawberry ‘Calypso’) compared with the control fruits (pBI-Intron and the wild type; for each, n = 5). Relative concentration (rel. conc.) is expressed in milligram equivalent internal standard g^–1. Asterisks indicate significant differences (*P = 0.01).

Figure 6.

Ester production in both transient and stable CHS-silenced fruits. Relative concentrations of esters in CHSi agroinfiltrated fruits (A; strawberry ‘Elsanta’) and a stable transgenic CHS antisense (CHS antis.) line (B; strawberry ‘Calypso’). Metabolite levels were determined by GC-MS 14 d after infiltration with A. tumefaciens transformed with a construct encoding CHS-inverted hairpin RNA or with pBI-Intron. Metabolite levels in fruits of the CHS antisense line and wild-type strawberry ‘Calypso’ fruits were determined at the mature ripening stage (n = 5–7). Identity of the compounds was confirmed by authentic references. Relative concentration (rel. conc.) is expressed in in milligram equivalent internal standard kg^–1.

Figure 7.

The proposed APG biosynthesis pathway catalyzed by CHS2-1 and CHS2-2 in Fragaria spp. fruit. GT, Glucosyl transferase; -CoAS, Coenzyme A.