Phenylpropanoid compounds, being naturally occurring antioxidants, are beneficial for human health when consumed in the diet and hence play a major role in determining fruit quality. They are largely occurring in eggplant, with chlorogenic acid accounting for about 80% of total phenolics. Since little genomic information is available on phenylpropanoids biosynthesis for this species, we addressed our interest both to elucidating mechanisms underlying chlorogenic acid accumulation in the fruits and to understanding transcriptional regulation of the phenylpropanoid pathway. The biosynthetic pathway leading to the accumulation of chlorogenic acid is known in many plant species, including Solanaceae, and ortholog sequences retrieved from tobacco and tomato were used for data mining in the eggplant ESTs database. The putative genes coding for phenylalanine ammonia lyase (PAL) and hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) were isolated from eggplant fruit cDNA through a RT-PCR approach, starting from primers designed on eggplant ESTs and followed by 5'-3' RACE-PCR. The two novel eggplant PAL and HQT genes share high sequence similarity with orthologs from Solanaceae and the encoded proteins possess all the characteristic features for a proper functional activity. Sequences upstream of both PAL and HQT were isolated by a genome walking strategy and their scan through PlantCare and Place bioinformatic tools detected several recognition motifs, highlighting the presence of different MYB Transcription factors (TF) binding sites. Interestingly, the presence of cis acting elements for the StMTF1 transcription factor, known as chlorogenic acid and flavonoid regulator in Solanum tuberosum, in the PAL and HQT putative promoters suggested that an ortholog of StMTF1 might also have a role in CGA biosynthesis in eggplant. In order to isolate eggplant MYB TFs, the sequence of the S. tuberosum MTF1 was used to screen the eggplant EST database. By RACE strategy, a novel putative transcription factor gene belonging to the MYB family was isolated. ClustalW alignment and phylogenetic analysis showed that the novel MYB has high sequence homology with the potato MTF1 and other Solanaceae TFs, such as SlAN1 and StMYB113. Then, we investigated the possible relation between transcription of the eggplant MYB and biosynthetic genes and accumulation of selected phenylpropanoids through qRT-PCR and LC-MS analyses in several eggplant tissues. Expression of the eggplant MYB and of the biosynthetic genes as well as the accumulation of chlorogenic acid in different tissues suggest that the novel isolated eggplant MYB might be a good candidate for transcriptional regulation of CGA biosynthesis also in eggplant. Further studies, including gene promoters transactivation and DNA-protein interaction could be helpful to evaluate the function of this novel MYB and its interaction with phenylpropanoid biosynthetic genes. This work was partly supported by the Project GenHORT PON02_00395_3215002 of the Italian Ministry of Education, University and Scientific Research (MIUR).
A genetic insight into the chlorogenic acid biosynthetic pathway in eggplant
Docimo T;De Palma M;Tucci M
2014
Abstract
Phenylpropanoid compounds, being naturally occurring antioxidants, are beneficial for human health when consumed in the diet and hence play a major role in determining fruit quality. They are largely occurring in eggplant, with chlorogenic acid accounting for about 80% of total phenolics. Since little genomic information is available on phenylpropanoids biosynthesis for this species, we addressed our interest both to elucidating mechanisms underlying chlorogenic acid accumulation in the fruits and to understanding transcriptional regulation of the phenylpropanoid pathway. The biosynthetic pathway leading to the accumulation of chlorogenic acid is known in many plant species, including Solanaceae, and ortholog sequences retrieved from tobacco and tomato were used for data mining in the eggplant ESTs database. The putative genes coding for phenylalanine ammonia lyase (PAL) and hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) were isolated from eggplant fruit cDNA through a RT-PCR approach, starting from primers designed on eggplant ESTs and followed by 5'-3' RACE-PCR. The two novel eggplant PAL and HQT genes share high sequence similarity with orthologs from Solanaceae and the encoded proteins possess all the characteristic features for a proper functional activity. Sequences upstream of both PAL and HQT were isolated by a genome walking strategy and their scan through PlantCare and Place bioinformatic tools detected several recognition motifs, highlighting the presence of different MYB Transcription factors (TF) binding sites. Interestingly, the presence of cis acting elements for the StMTF1 transcription factor, known as chlorogenic acid and flavonoid regulator in Solanum tuberosum, in the PAL and HQT putative promoters suggested that an ortholog of StMTF1 might also have a role in CGA biosynthesis in eggplant. In order to isolate eggplant MYB TFs, the sequence of the S. tuberosum MTF1 was used to screen the eggplant EST database. By RACE strategy, a novel putative transcription factor gene belonging to the MYB family was isolated. ClustalW alignment and phylogenetic analysis showed that the novel MYB has high sequence homology with the potato MTF1 and other Solanaceae TFs, such as SlAN1 and StMYB113. Then, we investigated the possible relation between transcription of the eggplant MYB and biosynthetic genes and accumulation of selected phenylpropanoids through qRT-PCR and LC-MS analyses in several eggplant tissues. Expression of the eggplant MYB and of the biosynthetic genes as well as the accumulation of chlorogenic acid in different tissues suggest that the novel isolated eggplant MYB might be a good candidate for transcriptional regulation of CGA biosynthesis also in eggplant. Further studies, including gene promoters transactivation and DNA-protein interaction could be helpful to evaluate the function of this novel MYB and its interaction with phenylpropanoid biosynthetic genes. This work was partly supported by the Project GenHORT PON02_00395_3215002 of the Italian Ministry of Education, University and Scientific Research (MIUR).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
