The isolation and mapping of a novel hydroxycinnamoyltransferase in the globe artichoke chlorogenic acid pathway

<p>Abstract</p> <p>Background</p> <p>The leaves of globe artichoke and cultivated cardoon (<it>Cynara cardunculus </it>L.) have significant pharmaceutical properties, which mainly result from their high content of polyphenolic compounds such as monocaffeoylquinic and dicaffeoylquinic acid (DCQ), and a range of flavonoid compounds.</p> <p>Results</p> <p>Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase (HQT) encoding genes have been isolated from both globe artichoke and cultivated cardoon (GenBank accessions <ext-link ext-link-type="gen" ext-link-id="DQ915589">DQ915589</ext-link> and <ext-link ext-link-type="gen" ext-link-id="DQ915590">DQ915590</ext-link>, respectively) using CODEHOP and PCR-RACE. A phylogenetic analysis revealed that their sequences belong to one of the major acyltransferase groups (anthranilate N-hydroxycinnamoyl/benzoyltransferase). The heterologous expression of globe artichoke HQT in <it>E. coli </it>showed that this enzyme can catalyze the esterification of quinic acid with caffeoyl-CoA or <it>p</it>-coumaroyl-CoA to generate, respectively, chlorogenic acid (CGA) and <it>p</it>-coumaroyl quinate. Real time PCR experiments demonstrated an increase in the expression level of HQT in UV-C treated leaves, and established a correlation between the synthesis of phenolic acids and protection against damage due to abiotic stress. The HQT gene, together with a gene encoding hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT) previously isolated from globe artichoke, have been incorporated within the developing globe artichoke linkage maps.</p> <p>Conclusion</p> <p>A novel acyltransferase involved in the biosynthesis of CGA in globe artichoke has been isolated, characterized and mapped. This is a good basis for our effort to understand the genetic basis of phenylpropanoid (PP) biosynthesis in <it>C. cardunculus</it>.</p

Identification of DNA methyltransferases and demethylases in Solanum melongena L., and their transcription dynamics during fruit development and after salt and drought stresses

DNA methylation through the activity of cytosine-5-methyltransferases (C5-MTases) and DNA demethylases plays important roles in genome protection as well as in regulating gene expression during plant development and plant response to environmental stresses. In this study, we report on a genome-wide identification of six C5-MTases (SmelMET1, SmelCMT2, SmelCMT3a, SmelCMT3b, SmelDRM2, SmelDRM3) and five demethylases (SmelDemethylase_1, SmelDemethylase_2, SmelDemethylase_3, SmelDemethylase_4, SmelDemethylase_5) in eggplant. Gene structural characteristics, chromosomal localization and phylogenetic analyses are also described. The transcript profiling of both C5-MTases and demethylases was assessed at three stages of fruit development in three eggplant commercial F1 hybrids: i.e. 'Clara', 'Nite Lady' and 'Bella Roma', representative of the eggplant berry phenotypic variation. The trend of activation of C5-MTases and demethylase genes varied in function of the stage of fruit development and was genotype dependent. The transcription pattern of C5MTAses and demethylases was also assessed in leaves of the F1 hybrid 'Nite Lady' subjected to salt and drought stresses. A marked up-regulation and down-regulation of some C5-MTases and demethylases was detected, while others did not vary in their expression profile. Our results suggest a role for both C5-MTases and demethylases during fruit development, as well as in response to abiotic stresses in eggplant, and provide a starting framework for supporting future epigenetic studies in the species

Isolation and functional characterization of a cDNA coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in Cynara cardunculus L

BACKGROUND: Cynara cardunculus L. is an edible plant of pharmaceutical interest, in particular with respect to the polyphenolic content of its leaves. It includes three taxa: globe artichoke, cultivated cardoon, and wild cardoon. The dominating phenolics are the di-caffeoylquinic acids (such as cynarin), which are largely restricted to Cynara species, along with their precursor, chlorogenic acid (CGA). The scope of this study is to better understand CGA synthesis in this plant. RESULTS: A gene sequence encoding a hydroxycinnamoyltransferase (HCT) involved in the synthesis of CGA, was identified. Isolation of the gene sequence was achieved by using a PCR strategy with degenerated primers targeted to conserved regions of orthologous HCT sequences available. We have isolated a 717 bp cDNA which shares 84% aminoacid identity and 92% similarity with a tobacco gene responsible for the biosynthesis of CGA from p-coumaroyl-CoA and quinic acid. In silico studies revealed the globe artichoke HCT sequence clustering with one of the main acyltransferase groups (i.e. anthranilate N-hydroxycinnamoyl/benzoyltransferase). Heterologous expression of the full length HCT (GenBank accession DQ104740) cDNA in E. coli demonstrated that the recombinant enzyme efficiently synthesizes both chlorogenic acid and p-coumaroyl quinate from quinic acid and caffeoyl-CoA or p-coumaroyl-CoA, respectively, confirming its identity as a hydroxycinnamoyl-CoA: quinate HCT. Variable levels of HCT expression were shown among wild and cultivated forms of C. cardunculus subspecies. The level of expression was correlated with CGA content. CONCLUSION: The data support the predicted involvement of the Cynara cardunculus HCT in the biosynthesis of CGA before and/or after the hydroxylation step of hydroxycinnamoyl esters

INVESTIGATING THE ROLE OF AN ERF TRANSCRIPTION FACTOR IN MEDIATING STRESS RESPONSE AND TOMATO FRUIT RIPENING AND QUALITY

Tomato (Solanum lycopersicum L.) is one of the world's most consumed vegetables and its consumption has been associated with decreased risk of chronic degenerative diseases. Tomato fruit is an important source of antioxidant compounds such as carotenoids, particularly lycopene, ascorbic acid, vitamin E and phenolic compounds. Fruit ripening is regulated by ethylene. Ethylene biosynthesis and signaling are modulated during fruit development and ripening and are involved in several processes such as antioxidant accumulation and softening that affect fruit quality and shelf-life. To date, several strategies have been implemented in tomato to modulate ripening and enhance tomato fruit quality and shelf-life by regulating the expression of genes involved in ethylene biosynthesis, perception or signaling. Among others, AP2/ERF genes are transcription factors which play key roles in several processes, such as plant development, ethylene response, and pathogen resistance. In tomato fruit, they can act as positive or negative regulator of ripening and of ethylene production. Our goal is to elucidate the functional role of ERF F4 gene (Solyc07g053740) in mediating modulation of the tomato fruit ripening during the plant response to abiotic stress and investigatiing its impact on fruit redox balance and antioxidant accumulation. In particular, the ERF F4 locus was targeted in Microtom by CRISPR/Cas 9 technology to generate knockout tomato plants. One month-old T3 offspring mutant and wild-type plants underwent three level of irrigation, consisting in the complete restitution of water (FWR), restitution of 50% of lost water (HWR) and 30% of water restitution (TWR), respectively. Beside no variation was observed in the lycopene and total carotenoid levels in fruit at the red-ripe stage, edited plants showed increased levels of soluble solid content than wild type plants and responded to drought treatment with a higher increase in their antioxidant capacity. These results suggested the involvement of ERF F4 in modulating ripening associated metabolic processes and fruit redox balance in response to abiotic stresses. Ongoing experiments will further investigate the role of the tomato ERF F4 in regulating the expression of genes involved in controlling the metabolism of antioxidants, particularly glutathione and ascorbate pool

Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.

[EN] Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T-1 and T-2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.Research was financially supported by the project CRISPR/Cas9-mediated gene knock-out in eggplant financed by Compagnia San Paolo.Maioli, A.; Gianoglio, S.; Moglia, A.; Acquadro, A.; Valentino, D.; Milani, AM.; Prohens Tomás, J.... (2020). Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L. 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