The regulation of ascorbate accumulation in plants

Abstract

Ascorbate (Vitamin C) is vital to human health. We are unable to synthesise ascorbate and attain the majority of ascorbate from plants. Ascorbate accumulation is a complex, regulated and environmentally responsive trait in plants with many essential roles. Ascorbate content varies over 1000-fold between different plants and little is known about the genetics behind this variation. The biosynthesis and degradation of ascorbate are controlled at the transcriptional, translational and post-translational levels of regulation. The focus of this thesis was on understanding the mechanisms regulating the different levels of ascorbate accumulation in plants and using this knowledge to generate a biofortified tomato plant with elevated levels of ascorbate. The mRNA of the rate limiting enzyme of ascorbate biosynthesis, \textit{GGP} contains an upstream open reading frame (uORF) which negatively regulates the translation of \textit{GGP} in elevated ascorbate conditions. Plants with super-high levels of ascorbate were used to test the role of the uORF translational control element in very high ascorbate accumulation. While subtle changes in the sequences of the uORF were found and could contribute to differences in ascorbate levels, I was able to show that the uORF was not found to be the main cause of high ascorbate. Instead, elevated transcription of three key genes, \textit{GMP}, \textit{GME} and \textit{GGP} was found to be important. Furthermore, transcriptional analysis of transiently elevated ascorbate showed the same three genes to respond in a feedback manner. Many other genes were also differentially expressed, including transcriptional regulators. Therefore, the work on ascorbate regulation has shown the importance of transcriptional control and feedback in modifying ascorbate levels. Finally, the regulatory information attained was used in a biofortification approach to specifically increase ascorbate in the fruit of tomato. High levels of ascorbate lead to feedback regulation and have also been shown to alter fruit development. Therefore a system was used to specifically elevate ascorbate in ripening tomato fruit. Ascorbate was elevated 4.7-fold to give maximal ascorbate of 207 mg/100 gFW, the highest recorded ascorbate content for fresh tomato fruit. This study provided insights into the regulation of the essential plant trait of ascorbate accumulation with important implications for biofortification strategies.