Constitutive expression of VviNAC17 transcription factor significantly induces the synthesis of flavonoids and other phenolics in transgenic grape berry cells

Abstract

VviNAC17 is a grapevine transcription factor activated by ABA. Because ABA has been proposed as the main signal modulating the secondary metabolism in grape berry skins, here we postulated VviNAC17 as a positive regulator of secondary metabolism in grape cells. To validate the hypothesis, VviNAC17 was constitutively and stably overexpressed in grape berry suspension-cultured cells of Gamay Fréaux cv. by Agrobacterium-mediated transformation. Targeted transcriptional analyses by qPCR showed that several genes involved the phenylpropanoid (VviPAL1), stilbenoid (VviSTS1) and flavonoid pathways (VviDFR, VviLAR1, VviANR, VviLDOX, and VviUFGT1), as well as anthocyanin vacuolar transport and accumulation (VviGST4 and VvMATE1) were significantly upregulated in VviNAC17-overexpressing transgenic cells, which translated in the stimulation of a number of enzymatic activities in those pathways. This was the case of phenylalanine ammonia lyase (PAL) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) that were about 2-fold and 3.5-fold higher in VviNAC17-overexpressing cells than in control cells. VviNAC17-overexpressing cells accumulated significantly higher amounts of anthocyanins, proanthocyanidins, total flavonoids and total phenolics. These findings confirmed that VviNAC17 is an important positive regulator of secondary metabolism in grapevine contributing to the accumulation of important berry quality-related secondary metabolites.

Metabolite and transcriptome analyses reveal the effects of salinity stress on the biosynthesis of proanthocyanidins and anthocyanins in grape suspension cells

“…The genetic evidence showed that VvMYBPA1 not only activates PA branch genes, including VvLAR1 and VvANR , but also positively regulates the expression of VvCHI and VvF3’H at the upstream of flavonoid pathway ( Terrier et al., 2009 ). Consistent with the similar expression pattern as VvMYBPA1 in response to the high salt stress, VvMYBA1 , VvMYBA2 and VvNAC17 also positively target to the general flavonoid pathway ( Badim et al., 2022 ) - this might be a strategy to compensate the absence of the functions of VvMYB5a and VvMYB5b in grape suspension cells under salinity stress. Moreover, based on homology, we identified a number of potential flavonoid transcription factors whose expressions were NaCl dose-dependent in grape suspension cells, including MYB, RAV, NAC, LBD, ERF and WRKY family proteins.…”

“…Among these three NAC transcription factors, AtNAC32 represses the stress-induced anthocyanin synthesis ( Mahmood et al., 2016 ), while PpNAC25 promotes anthocyanin accumulation ( Geng et al., 2022 ). Moreover, Badim et al. (2022) demonstrated that overexpressing VvNAC17 in grape suspension cells induces the biosynthesis of both PA and anthocyanin.…”

“…Our previous studies showed that VvMYB86 promotes PA accumulation by tuning the flux between PA and anthocyanin pathways ( Cheng et al., 2021 ), while VvbHLH93 inhibits PA production by broadly targeting flavonoid pathway genes ( Cheng et al., 2022 ). Confirmed by transgenic grape suspension cells, VvNAC17 is an activator of both PA and anthocyanin pathways ( Badim et al., 2022 ). Recently, Wei et al.…”

A simplified protocol for Agrobacterium-mediated transformation of cell suspension cultures of the model species Medicago truncatula A17

“…These procedures increase the difficulty to transfer the cells in an individualized way (e.g. Wu et al 1998 ; Moniruzzaman et al 2021 ; Badim et al 2022 ).…”

Molecular regulation of apple and grape ripening: exploring common and distinct transcriptional aspects of representative climacteric and non-climacteric fruits

“…Recently, MdNAC042 was demonstrated to positively regulate anthocyanin content through a dimerization with MdMYB10 ( Zhang et al , 2020 ). In grapevine, overexpression of VviNAC17 in cultured in vitro calli was shown to induce activation of VviMYB14/VviMYB15 and downstream stilbene-related genes, as well as VviMYBA1/VviMYBA2 and downstream anthocyanin-related genes ( Badim et al , 2022 ). VviNAC60 was recently found to directly activate expression of VviMYB14 and together with VviNAC03 significantly activated VviMYBA1 promoter, increased by the heterodimeric form of these two NACs ( D’Inca et al , 2023 ).…”