Functional characterization of a stilbene synthase gene using a transient expression system in planta

Abstract

The expression and functionality of a resveratrol synthase (RS) gene from peanut (Arachis hypogaea) was studied using an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana leaves. Functional analysis of RS was demonstrated by tracking its expression during 96 h. To measure the transcripts levels of RS transgene, real-time qRT-PCR was used and revealed that the highest level of transcripts was at 48 h post-transfection. Western blot analyses showed that RS protein was accumulated to the highest levels at 72 h post-transfection. Finally, HPLC and mass spectrometry analyses revealed the production of trans-piceid (resveratrol glucoside) as the major stilbenoid compound confirming the functional activity of the RS enzyme in planta. No activity of RS transgene was detected in negative controls. This strategy showed advantages over conventional systems because it does not require establishment of cell cultures, feeding with appropriate substrates or generation of stable transgenic plants. This transient system proved to be a rapid and direct approach to perform functional analysis of stilbene synthases, such as resveratrol synthase. Furthermore, this approach can be useful to study the metabolic effects of over-expressing or silencing specific genes within a short period of time.

Structural, Functional, and Evolutionary Analysis of the Unusually Large Stilbene Synthase Gene Family in Grapevine

“…In a previous work, we have used Agrobacterium tumefaciens-mediated transient transformation of Nicotiana benthamiana to characterize the activity of a resveratrol O-methyltransferase from grapevine (Schmidlin et al, 2008); we thus chose the same approach here to investigate the activity of a selection of STS proteins in planta. A similar strategy has been used to characterize STS enzymes from peanut (Condori et al, 2009). In order to represent the diversity of the STS family, nine STS full-length genes were selected in the major clades for functional analysis, namely, VvSTS5, VvSTS10, VvSTS16, VvSTS28, VvSTS29, VvSTS36, VvSTS38, VvSTS46, and VvSTS48, based on the fact that they were representative of the diversity of the STS family (Fig.…”

Research Article Coupled transcript and metabolite identification: insights on induction and synthesis of resveratrol in peanut, wild relatives and synthetic allotetraploid.

“…Biological replicates of each genotype had similar expression patterns for RSArAs2 and RSGbAhT14, suggesting those are part of a RS gene that were induced by UV treatment and they may amplify parts of the same genes. The function of these genes was previously validated by transgenic studies with Nicotiana benthamiana (Condori et al, 2009).…”

Induced biosynthesis of resveratrol and the prenylated stilbenoids arachidin-1 and arachidin-3 in hairy root cultures of peanut: Effects of culture medium and growth stage

“…Analyses were performed by reverse phase HPLC as described previously [29]. Concentrations of trans-resveratrol, trans-arachidin-1, and trans-arachidin-3 in the root and culture media extracts were determined based on peak areas of corresponding pure standards.…”