Enhancement of alpha-tomatine content in Solanum lycopersicum via metabolic engineering

Tomato (Solanum lycopersicum) plants synthesize ?-tomatine, a steroidal glycoside alkaloid, whose oligosaccharide lycotetraose consists of D-glucose 1,2,3-b linked to a D-galactose, a Dglucose, and a xylose, respectively. This compound is involved in plant defence systems against microbial pathogens and insects. Moreover, it shows interesting healthy properties including anticholesterolemic, antitumoral and anti-inflammatory activities. All organs of tomato plants contain ?-tomatine, which in immature green tomato fruit accumulates up to 500 mg/kg of fresh weight. However, ?-tomatine content in tomato fruit largely decreases during ripening as a result of enzymatic conversion. Despite the importance of the biological activities of ?-tomatine its catabolic pathway is only poorly understood. Using BLAST analysis we identified a tomato ? -D-xylosidase gene (LeXyl1) that showed homology with a tomatinase gene isolated from Septoria lycopersici. Expression pattern of LeXyl1 was achieved by Real-time PCR in mature green and red ripe fruit of S. lycopersicum var. San Marzano and of S. lycopersicum "cerasiforme" that shows high ?-tomatine content also in ripe fruit. The results seemed to confirm the role of LeXy1l in the catabolic pathway of ?-tomatine. In order to increase ?-tomatine content in S. lycopersicum, LeXyl1 was silenced by RNA interference technology (RNAi). Eleven independent transformants were generated and verified for transgene integration by PCR analysis. Four T1 lines were selected on the basis of a strongly reduced LeXyl1 expression. High-performance liquid chromatography (HPLC) showed an enhancement of ?-tomatine content in the selected T1-LeXYL1lines.