RESUMO
Background: Carotenoids, which are secondary metabolites derived from isoprenoids, play a crucial role in photo-protection and photosynthesis, and act as precursors for abscisic acid, a hormone that plays a significant role in plant abiotic stress responses. The biosynthesis of carotenoids in higher plants initiates with the production of phytoene from two geranylgeranyl pyrophosphate molecules. Phytoene synthase (PSY), an essential catalytic enzyme in the process, regulates this crucial step in the pathway. In Daucus carota L. (carrot), two PSY genes (DcPSY1 and DcPSY2) have been identified but only DcPSY2 expression is induced by ABA. Here we show that the ectopic expression of DcPSY2 in Nicotiana tabacum L. (tobacco) produces in L3 and L6 a significant increase in total carotenoids and chlorophyll a, and a significant increment in phytoene in the T1L6 line. Tobacco transgenic T1L3 and T1L6 lines subjected to chronic NaCl stress showed an increase of between 2 and 3- and 6-fold in survival rate relative to control lines, which correlates directly with an increase in the expression of endogenous carotenogenic and abiotic-related genes, and with ABA levels. Conclusions: These results provide evidence of the functionality of DcPSY2 in conferring salt stress tolerance in transgenic tobacco T1L3 and T1L6 lines.
RESUMO
Carotenoids are pigments with important nutritional value in the human diet. As antioxidant molecules, they act as scavengers of free radicals enhancing immunity and preventing cancer and cardiovascular diseases. Moreover, α-carotene and ß-carotene, the main carotenoids of carrots (Daucus carota) are precursors of vitamin A, whose deficiency in the diet can trigger night blindness and macular degeneration. With the aim of increasing the carotenoid content in fruit flesh, three key genes of the carotenoid pathway, phytoene synthase (DcPSY2) and lycopene cyclase (DcLCYB1) from carrots, and carotene desaturase (XdCrtI) from the yeast Xanthophyllomyces dendrorhous, were optimized for expression in apple and cloned under the Solanum chilense (tomatillo) polygalacturonase (PG) fruit specific promoter. A biotechnological platform was generated and functionally tested by subcellular localization, and single, double and triple combinations were both stably transformed in tomatoes (Solanum lycopersicum var. Microtom) and transiently transformed in Fuji apple fruit flesh (Malus domestica). We demonstrated the functionality of the S. chilense PG promoter by directing the expression of the transgenes specifically to fruits. Transgenic tomato fruits expressing DcPSY2, DcLCYB1, and DcPSY2-XdCRTI, produced 1.34, 2.0, and 1.99-fold more total carotenoids than wild-type fruits, respectively. Furthermore, transgenic tomatoes expressing DcLCYB1, DcPSY2-XdCRTI, and DcPSY2-XdCRTI-DcLCYB1 exhibited an increment in ß-carotene levels of 2.5, 3.0, and 2.57-fold in comparison with wild-type fruits, respectively. Additionally, Fuji apple flesh agroinfiltrated with DcPSY2 and DcLCYB1 constructs showed a significant increase of 2.75 and 3.11-fold in total carotenoids and 5.11 and 5.84-fold in ß-carotene, respectively whereas the expression of DcPSY2-XdCRTI and DcPSY2-XdCRTI-DcLCYB1 generated lower, but significant changes in the carotenoid profile of infiltrated apple flesh. The results in apple demonstrate that DcPSY2 and DcLCYB1 are suitable biotechnological genes to increase the carotenoid content in fruits of species with reduced amounts of these pigments.