Delphinidin-3-rutinoside (D3R), rutin and chlorogenic acid (CGA) are the major phenolic metabolites in eggplant tissues, with CGA constituting over 90% of total phenylpropanoids, and confer high nutritional value to eggplant fruits. Biochemical analysis by LC-MS was used to characterize the distribution of phenylpropanoids in different tissues (young and mature leaf, stem, root, flower, fruit skin and flesh) of Violetta Campana eggplant variety. The anthocyanin D3R was detected only in the coloured tissues (flower and fruit peel), the flavonoid rutin in all the tissues beside fruit flesh and roots, whereas a high content of CGA was found in all the epigeous tissues. Expression of key genes PAL (phenylalanine ammonia lyase), HQT (hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase), DFR (dihydroflavonol reductase) and ANS (anthocyanidin synthase) mainly involved in the biosynthesis of these compounds was evaluated by qRT-PCR to check a possible relationship between transcript accumulation and content of analysed compounds. Consistently with the high accumulation of D3R (10 ug/mg dry weight) in fruit peel and CGA (about 40 ug/mg dry weight) in fruit flesh, a higher expression of biosynthetic genes involved in anthocyanin and CGA biosynthesis was found in these tissues. Namely a significant increase (more than 100 fold) of PAL, HQT, DFR and ANS expression was detected in fruits flesh or skin compared to the other tissues, so suggesting a high biosynthetic activity for eggplant fruits. These metabolic and molecular results prompted us to use fruit flesh tissue for the isolation of PAL and HQTcoding sequences. By RACE strategy, we isolated a complete open reading frame (ORF) of 2172 bps for PAL, which encodes 720 amino acid residues, and an ORF of 1284 bps for HQT, encoding for a 430 aa protein. To gain insight in the CGA and anthocyanins biosynthesis regulation, the promoter regions of PAL, HQT and ANS genes were isolated by Genome walking strategy and confirmed by BLAST search in the draft eggplant genome (Hirakawa H. et al., 2014, DNA Research doi:10.1093/dnares/dsu027). In silico analysis of 1.2Kb promoter regions, identified several binding motifs for Myb TFs, suggesting a major involvement of these transcription factors in the transcriptional regulation of phenylpropanoids in eggplant. Therefore, we analysed the expression of two eggplant MYB genes, proposed as anthocyanin pathway specific regulators, namely SmAN1 and SmMYB1. We found SmAN1 expression barely detectable and only in fruits, while interestingly SmMyb1transcripts were found in all the tissues. A putative role of SmMyb1 also in CGA biosynthesis regulation is being investigated by functional assays in tobacco.
Phenylpropanoid compounds in S. melongena: their biosynthesis and regulation
Docimo T;De Palma M;Tucci M
2015
Abstract
Delphinidin-3-rutinoside (D3R), rutin and chlorogenic acid (CGA) are the major phenolic metabolites in eggplant tissues, with CGA constituting over 90% of total phenylpropanoids, and confer high nutritional value to eggplant fruits. Biochemical analysis by LC-MS was used to characterize the distribution of phenylpropanoids in different tissues (young and mature leaf, stem, root, flower, fruit skin and flesh) of Violetta Campana eggplant variety. The anthocyanin D3R was detected only in the coloured tissues (flower and fruit peel), the flavonoid rutin in all the tissues beside fruit flesh and roots, whereas a high content of CGA was found in all the epigeous tissues. Expression of key genes PAL (phenylalanine ammonia lyase), HQT (hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase), DFR (dihydroflavonol reductase) and ANS (anthocyanidin synthase) mainly involved in the biosynthesis of these compounds was evaluated by qRT-PCR to check a possible relationship between transcript accumulation and content of analysed compounds. Consistently with the high accumulation of D3R (10 ug/mg dry weight) in fruit peel and CGA (about 40 ug/mg dry weight) in fruit flesh, a higher expression of biosynthetic genes involved in anthocyanin and CGA biosynthesis was found in these tissues. Namely a significant increase (more than 100 fold) of PAL, HQT, DFR and ANS expression was detected in fruits flesh or skin compared to the other tissues, so suggesting a high biosynthetic activity for eggplant fruits. These metabolic and molecular results prompted us to use fruit flesh tissue for the isolation of PAL and HQTcoding sequences. By RACE strategy, we isolated a complete open reading frame (ORF) of 2172 bps for PAL, which encodes 720 amino acid residues, and an ORF of 1284 bps for HQT, encoding for a 430 aa protein. To gain insight in the CGA and anthocyanins biosynthesis regulation, the promoter regions of PAL, HQT and ANS genes were isolated by Genome walking strategy and confirmed by BLAST search in the draft eggplant genome (Hirakawa H. et al., 2014, DNA Research doi:10.1093/dnares/dsu027). In silico analysis of 1.2Kb promoter regions, identified several binding motifs for Myb TFs, suggesting a major involvement of these transcription factors in the transcriptional regulation of phenylpropanoids in eggplant. Therefore, we analysed the expression of two eggplant MYB genes, proposed as anthocyanin pathway specific regulators, namely SmAN1 and SmMYB1. We found SmAN1 expression barely detectable and only in fruits, while interestingly SmMyb1transcripts were found in all the tissues. A putative role of SmMyb1 also in CGA biosynthesis regulation is being investigated by functional assays in tobacco.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
