Enhanced accumulation of atropine in Atropa belladonna transformed by Rac GTPase gene isolated from Scoparia dulcis

Abstract

Leaf tissues of Atropa belladonna were transformed by Sdrac2, a Rac GTPase gene, that is isolated from Scoparia dulcis, and the change in atropine concentration of the transformants was examined. Re-differentiated A. belladonna overexpressing Sdrac2 accumulated considerable concentration of atropine in the leaf tissues, whereas the leaves of plants transformed by an empty vector accumulated only a very low concentration of the compound. A. belladonna transformed by CASdrac2, a modified Sdrac2 of which translate was expected to bind guanosine triphosphate (GTP) permanently, accumulated very high concentrations of atropine (approximately 2.4-fold excess to those found in the wild-type plant in its natural habitat). In sharp contrast, the atropine concentration in transformed A. belladonna prepared with negatively modified Sdrac2, DNSdrac2, expected to bind guanosine diphosphate instead of GTP, was very low. These results suggested that Rac GTPases play an important role in the regulation of secondary metabolism in plant cells and that overexpression of the gene(s) may be capable of enhancing the production of natural products accumulated in higher plant cells.

Rho of plant GTPases with geranylgeranylation motif modulate monoterpene indole alkaloid biosynthesis inCatharanthus roseus

“…In fact, the effect of CrROP3 and CrROP5 overexpression was similar to the effect observed in silencing and overexpression of CrGGPPS2 , which provides GGPP precursor for CrPGGT-I (Kumar et al, 2020). In agreement with our results, overexpression of a type-I ROP from S. dulcis Sdrac2 enhanced the biosynthesis of tropane alkaloid atropine in A. bellodona (Asano et al, 2013), indicating a possibility of a similar scenario in other alkaloid producing plants. Although the transcript level of CrROP2 was significantly enhanced in both transient and stable overexpression backgrounds, it had no effect on either the transcript levels of TFs and genes related to MIA biosynthesis or the levels of secologanin and MIA (Figure 7).…”

“…These studies highlight the significance of geranylgeranylation and indicate the involvement of geranylgeranylated proteins in the regulation of specialized metabolism in plants. Given this information, and considering that ROP proteins are known to undergo prenylation (Sorek et al, 2010; Sorek et al, 2011), and that overexpression of a ROP from S. dulcis ( Sdrac2 ) enhanced atropine content in A. belladonna (Asano et al, 2013), we hypothesized specific geranylgeranylated ROPs may play a role in the regulation of MIA biosynthesis in C. roseus . Hence, the current study aimed to conduct a detailed investigation on the role of ROPs in C. roseus MIA biosynthesis.…”

“…So far, there has been only a couple of reports that suggest the participation of ROPs in the modulation of plant specialized metabolism. It was shown that overexpression of Scoparia dulcis ROP gene ( Sdrac2) in Atropa belladonna , a plant from the Solanaceae family, increased the accumulation of the specialized metabolite atropine, a tropane alkaloid (Asano et al, 2013). Similarly, overexpression of rac2 encoding a Rac GTPase in cell cultures of Aquilaria microcarpa activated the transcription of a sesquiterpene synthase gene encoding δ-guaiene synthase (Kurosaki & Taura, 2015).…”

Press for MIA production! The role of the Rho of plant GTPases in plant-specialized metabolism

“…1A ). Interestingly, CrROP3 and CrROP5 (group 1) clustered together with SdRac2 and AmRac2, GTPases that positively regulated the production of specific alkaloids and sesquiterpenes, respectively ( Asano et al 2013 ; Kurosaki and Taura 2015 ). Additionally, the sequence alignment showed that all CrROPs possess characteristic G-box motifs and a hyper-variable C-terminal region.…”