Root-specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum

Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2 gene (TB-mas2). This gene is similar to the Agrobacterium rhizogenes A4-mas2 gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2 is expressed at very low levels in N.tomentosiformis and in most N.tabacum cultivars; however, some cultivars show high TB-mas2 expression levels. The TB-mas2 promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2-GUS and pA4-mas2-GUS reporter genes were stably introduced in N.benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2 expression contain detectable levels of DFG. Significance Statement Several naturally occurring GMO plants have been described, belonging to the genera Nicotiana, Linaria and Ipomoea, several carry opine synthesis genes. Sofar, however, detectable levels of opines have not been demonstrated in such natural GMOs. We find that the cT-DNA TB-mas2' gene is expressed at very low levels in most Nicotiana tabacum cultivars. However, in some cultivars TB-mas2' is highly expressed in root tips giving detectable levels of the Amadori opine deoxyfructosyl-glutamine.