Publication
Exploring the potential of newly-identified miRNA-encoded peptides to improve the production of bioactive secondary metabolites in grape cells
| Summary: | The global quality and characteristics of grape berries, and ultimately of wine, are influenced by their polyphenolic composition. Therefore, potential strategies to improve berry quality by targeting secondary metabolism pathways of phenolic compound synthesis are useful, particularly in an ongoing context of climate change. These pathways are modulated by several molecular mechanisms, including regulation of gene transcription by specific transcription factors and post-transcriptional regulation by microRNAs. Recently, it was discovered that the primary (non-mature) miRNAs transcripts (pri-miRNAs) could encode for small regulatory peptides (micropeptides – miPEPs). In a positive loop, these miRNA-encoded peptides enhance the transcription and accumulation of their corresponding pri-miRNAs and, consequently, of their mature miRNAs, subsequently leading to an accentuated negative regulation of miRNA-regulated target genes. The objective of this work was to explore this recent discovery and to experiment the exogenous application of a micropeptide (miPEP396a) that putatively promotes the inhibition of the transcription factor VvMYB5b, an activator of the expression of several genes involved in the flavonoid pathway. Designated in this study as miPEP-MYB5b, this micropeptide may serve as a fine-tuning tool for modulation of secondary metabolic pathways in grape berry cells and, consequently, improve their global quality-traits. MiPEP-MYB5b was identified in silico and exogenously added to a Gamay Freaux grape berry cells in two different concentrations (0.1 μM and 0.5 μM). Its effect in the concentration of secondary metabolites such as anthocyanins, total flavonoids, total phenolics and stilbenes as well as in the transcription of key genes involved in biosynthetic routes that produce secondary metabolites with bioactive properties and important for grape berry quality, particularly in flavonoid- and stilbene-synthesizing pathways was analyzed. Both concentrations of miPEP-MYB5b resulted in downregulation of key genes involved in the flavonoid pathway, such as VvLAR1, VvLAR2, and VvCHI, while 0.5 μM resulted in downregulation of flavonoid-related genes VvANR, VvFLS1, VvCHS1. A parallel stimulation of the expression of the stilbene-synthesizing gene VvSTS1 was also observed in miPEP-treated cells. This upregulation of the stilbene pathway was probably due to a miPEP-MYB5b-mediated inhibition of MYB5b and, thus, of the flavonoid pathway, that competes directly with the stilbene pathway for substrate. Concordantly with the inhibition of the flavonoid pathway and stimulation of the stilbene pathway, a higher stilbene content and lower concentration of flavonoids (including anthocyanins) were quantified in grape berry cells. Thus, miPEP-MYB5b exogenous application may be a promising strategy to modulate secondary metabolic pathways in order to produce and accumulate higher quantity of stilbenes in grape berry cells in a near future, by exploring mechanisms of microRNA-mediated gene regulation in plants. |
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| Main Authors: | Rodrigues, Jéssica Alexandra Soares |
| Subject: | Ciências Naturais::Ciências Biológicas |
| Year: | 2019 |
| Country: | Portugal |
| Document type: | master thesis |
| Access type: | open access |
| Associated institution: | Universidade do Minho |
| Language: | English |
| Origin: | RepositóriUM - Universidade do Minho |
| Summary: | The global quality and characteristics of grape berries, and ultimately of wine, are influenced by their polyphenolic composition. Therefore, potential strategies to improve berry quality by targeting secondary metabolism pathways of phenolic compound synthesis are useful, particularly in an ongoing context of climate change. These pathways are modulated by several molecular mechanisms, including regulation of gene transcription by specific transcription factors and post-transcriptional regulation by microRNAs. Recently, it was discovered that the primary (non-mature) miRNAs transcripts (pri-miRNAs) could encode for small regulatory peptides (micropeptides – miPEPs). In a positive loop, these miRNA-encoded peptides enhance the transcription and accumulation of their corresponding pri-miRNAs and, consequently, of their mature miRNAs, subsequently leading to an accentuated negative regulation of miRNA-regulated target genes. The objective of this work was to explore this recent discovery and to experiment the exogenous application of a micropeptide (miPEP396a) that putatively promotes the inhibition of the transcription factor VvMYB5b, an activator of the expression of several genes involved in the flavonoid pathway. Designated in this study as miPEP-MYB5b, this micropeptide may serve as a fine-tuning tool for modulation of secondary metabolic pathways in grape berry cells and, consequently, improve their global quality-traits. MiPEP-MYB5b was identified in silico and exogenously added to a Gamay Freaux grape berry cells in two different concentrations (0.1 μM and 0.5 μM). Its effect in the concentration of secondary metabolites such as anthocyanins, total flavonoids, total phenolics and stilbenes as well as in the transcription of key genes involved in biosynthetic routes that produce secondary metabolites with bioactive properties and important for grape berry quality, particularly in flavonoid- and stilbene-synthesizing pathways was analyzed. Both concentrations of miPEP-MYB5b resulted in downregulation of key genes involved in the flavonoid pathway, such as VvLAR1, VvLAR2, and VvCHI, while 0.5 μM resulted in downregulation of flavonoid-related genes VvANR, VvFLS1, VvCHS1. A parallel stimulation of the expression of the stilbene-synthesizing gene VvSTS1 was also observed in miPEP-treated cells. This upregulation of the stilbene pathway was probably due to a miPEP-MYB5b-mediated inhibition of MYB5b and, thus, of the flavonoid pathway, that competes directly with the stilbene pathway for substrate. Concordantly with the inhibition of the flavonoid pathway and stimulation of the stilbene pathway, a higher stilbene content and lower concentration of flavonoids (including anthocyanins) were quantified in grape berry cells. Thus, miPEP-MYB5b exogenous application may be a promising strategy to modulate secondary metabolic pathways in order to produce and accumulate higher quantity of stilbenes in grape berry cells in a near future, by exploring mechanisms of microRNA-mediated gene regulation in plants. |
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