Metabolic networking in Brunfelsia calycina petals after flower opening

Abstract

Brunfelsia calycina flowers change colour from purple to white due to anthocyanin degradation, parallel to an increase in fragrance and petal size. Here it was tested whether the production of the fragrant benzenoids is dependent on induction of the shikimate pathway, or if they are formed from the anthocyanin degradation products. An extensive characterization of the events taking place in Brunfelsia flowers is presented. Anthocyanin characterization was performed using ultraperfomance liquid chromatography-quadrupole time of flight-tandem mass specrometry (UPLC-QTOF-MS/MS). Volatiles emitted were identified by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Accumulated proteins were identified by 2D gel electrophoresis. Transcription profiles were characterized by cross-species hybridization of Brunfelsia cDNAs to potato cDNA microarrays. Identification of accumulated metabolites was performed by UPLC-QTOF-MS non-targeted metabolite analysis. The results include characterization of the nine main anthocyanins in Brunfelsia flowers. In addition, 146 up-regulated genes, 19 volatiles, seven proteins, and 17 metabolites that increased during anthocyanin degradation were identified. A multilevel analysis suggests induction of the shikimate pathway. This pathway is the most probable source of the phenolic acids, which in turn are precursors of both the benzenoid and lignin production pathways. The knowledge obtained is valuable for future studies on degradation of anthocyanins, formation of volatiles, and the network of secondary metabolism in Brunfelsia and related species.

Fig. 1.

Brunfelsia flowers during anthocyanin degradation. D0 is the day of flower opening and D1–3 are consecutive days after flower opening. Flowers were detached at D0 and grown in sucrose media as described in Materials and methods. (A) Detached flowers at D0–D3. (B) A photomicrograph of petal tissues at D0, D1, and D3 (×40 enlargement).

Fig. 2.

Characterization of the main anthocyanins in Brunfelsia flower, using UPLC-QTOF-MS/MS. (A) A UV chromatogram at 530 nm of the different anthocyanins in Brunfelsia flower petals at D0. (B) The main anthocyanins detected in Brunfelsia flowers.

Fig. 3.

Multilevel analysis reveals metabolic processes occurring in Brunfelsia flowers during the first 2 d after opening and during the degradation of anthocyanins. The diagram shows the different pathways. Genes, proteins, and metabolites that have increased significantly are boxed. The diagram summarizes the results of GC-MS, LC-MS, microarray, and 2D gel analyses. The colour of the box indicates the method by which they were identified. The dashed line box indicates a lower level of confidence of metabolite identification. Enzymes: SAMT, salicylic acid carboxyl methyltransferase; COMT, catechol O-methyl transferase; CCoA-OMT, caffeoyl-CoA O-methyl transferase; CAD, cinnamyl-alcohol dehydrogenase; DAHP synthase, 3-deoxy-D-arabino-heptulosonate 7-phosphate; PDT, prephenate dehydratase; DXPS, 1-deoxyxylulose 5-phosphate synthase.