Virus-induced gene silencing in detached tomatoes and biochemical effects of phytoene desaturase gene silencing

Virus-induced gene silencing (VIGS) is a technology that has rapidly emerged for gene function studies in plants. Many advances have been made in applying this technique in an increasing number of crops. Recently, VIGS has been successfully used to silence genes in tomato fruit through agroinfiltration of fruit attached to the plant. The phytoene desaturase (Pds) gene has been widely used as a reporter gene in VIGS experiments, although little is known about the changes that occur due to its silencing in plants. In this paper, we describe the efficient silencing of the Pds gene through the VIGS approach in detached tomato fruits, which makes the VIGS procedure even more versatile and applicable. After 16 days of agroinfiltration, approximately 75% of the tomatoes showed Pds silencing symptoms, although the distribution of silenced areas was variable among fruits. To study the potential effects caused by Pds silencing in detached tomatoes, carotenoids and other semi-polar secondary metabolites were analyzed using Liquid Chromatography-Mass Spectrometry. In addition, potential differences in primary metabolites were analyzed using Gas Chromatography-Mass Spectrometry. The results indicated that the yellow phenotype observed in Pds-silenced fruit was mainly due to the lack of the red-colored lycopene and therefore to a more pronounced contribution of the yellow-orange carotenoids (lutein, violaxanthin, and zeaxanthin) to the final color of the fruits. Furthermore, the biochemical changes observed in Pds-silenced detached tomatoes suggested that carotenoid and other pathways, e.g. leading to alkaloids and flavonoids, might be affected by the silencing of this reporter gene, and this should be taken into consideration for future experimental designs.