Expression of the Stilbene Synthase (StSy) Gene from Grapevine in Transgenic White Poplar Results in High Accumulation of the Antioxidant Resveratrol Glucosides

Abstract

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.

Bioassay for the Response of Resveratrol Transgenic Rice Lines to Bacterial and Fungal Diseases

“…Most studies on transgenic crops overexpressing stilbene synthase reported increased resistance against bacterial and fungal diseases (Hain et al 1993;Stark-Lorenzen et al 1997;Tian et al 1998;Hipskind et al 2000;Liang et al 2000;Coutos-Thevenot et al 2001;Zhu et al 2004;Lim et al 2005;Serazetdinova et al 2005;Liu et al 2011). However, no resistance or increased susceptibility conferred by stilbene synthase gene transformation has been reported in transgenic tomato, kiwifruits, white poplar, or strawberry Kobayashi et al 2000;Giorcelli et al 2004;Hanhineva et al 2009). Here, we present the contradictory conclusions from previous studies about piceid production and concentrations, hostpathogen relationships, desirable promoters, and alternations of biosynthesis pathways.…”

“…However, some STS GM plants produced piceid or failed to produce resveratrol. For instance, the transgenic kiwifruit produced by Kobayashi et al (2000), transgenic white poplar generated by Giorcelli et al (2004) and transgenic strawberry produced by Hanhineva et al (2009) were more susceptible to fungi. Four hypotheses have been proposed to explain stilbene GM plants that lack resistance or are more susceptible to fungal pathogens.…”

“…This affects stilbene production and may interfere with secondary biosynthetic pathways (Hain et al 1990(Hain et al , 1993Thomzik et al 1997). Second, these GM plants may produce resveratrol glucoside (piceid), which has little or no antifungal activities compared with resveratrol and its dehydrodimer δ-viniferin, which are more effective against fungus (P. viticola) zoospores in grapevines (Kobayashi et al 2000;Giorcelli et al 2004;Chang et al 2011). Third, host-pathogen relationships determine the antifungal activity of foreign resveratrol production .…”

Soil persistence of DNA from transgenic poplar

“…The elite cultivar 'Villafranca' (Populus alba L.) used in the present study has become a model system in tree biotechnology, and transgenic plants have been produced using, not only conventional gene transfer techniques Giorcelli et al, 2004;Zelasco et al, 2006), but also innovative vector systems for marker-free applications (Zelasco et al, 2007).…”

“…The transgenic lines 5EAC1 and 12EAC1 (Giorcelli et al, 2004) carried a single copy of the StSy gene from Vitis vinifera (Sparvoli et al, 1994) encoding the stilbene synthase enzyme responsible for resveratrol production. Both the bar and StSy genes were placed under the control of the constitutive 35SCaMV promoter.…”