Evaluation of transgenic tomatoes overexpressing the vitamin C recycling genes

Vitamin C (ascorbate) is an important essential nutrient in the human diet. Deficiencies in ascorbate can result in scurvy and sub-optimal intake has been correlated with compromised immunity, cardiovascular and neurodegenerative diseases, as well as cancer.  Ascorbate is also the most important antioxidant in plants, protecting against reactive oxygen species (ROS) which can damage membranes, protein, and DNA within the cell.  Since it functions as a major antioxidant in both plants and animals we sought to investigate the effects of overexpressing the vitamin C recycling enzymes monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) in Micro-Tom tomato plants.  Transgenic plants were generated using constructs encoding cytosolic, cis-genic sequences for DHAR and MDHAR.  The genes were driven by the constitutive FMV34S promoter and homozygous lines were identified in the T2 generation by PCR and GUS staining.  An in vitro enzyme assay was performed to compare differences between transgenic and wildtype plants for DHAR and MDHAR activity; results suggest a significant increase in the transgenic plants. However, HPLC assays showed no significant differences in the level of ascorbate, and the oxidized form dehydroascorbate. Biotic stress response using necrotrophic plant pathogen, Botrytis cinerea, was performed to test for differences in disease incidence and severity.  Results were inconclusive due to a high degree of variability.  DHAR and MDHAR recycling lines with the highest enzyme activities were crossed to generate double transgenics to perform future tests in evaluating potential synergistic effects.