Genetic determinants of Vitamin C content in higher plants

Ascorbic acid (AsA, VitC) is the most abundant water-soluble antioxidant in plants and it plays a plethora of biological roles including resistance to abiotic stress. Hence, including VitC as a trait to improve in breeding programs is not only a way to enhance food quality but also to increase resistance to expected environmental alterations due to global change like drought, salinity or heat. Although all components of the Smirnoff-Wheeler pathway of AsA in plants are known, little information is available about how their regulation at the biochemical and cellular levels is. We have generated a number of molecular tools such as tagged constructs, stable transgenics and mutant lines with the aim of getting detailed information about how this pathway operate in plants. We will present data on protein localization, interaction among different components and their role in affecting VitC levels using a heterologous system such as Nicotiana bethamiana. This research was supported by a grant from the Spanish Ministerio de Educación, Cultura y Deporte para la formación del Profesorado Universitario (FPU014/01974), as well as by a project funded by the Spanish Ministerio de Ciencia e Innovación (BIO2014-55380R; BIO2014-56153-REDT). We also acknowledge the support by the Plan Propio from University of Malaga, Campus de Excelencia Internacional de Andalucía.This research was supported by a grant from the Spanish Ministerio de Educación, Cultura y Deporte para la formación del Profesorado Universitario (FPU014/01974), as well as by a project funded by the Spanish Ministerio de Ciencia e Innovación (BIO2014-55380R; BIO2014-56153-REDT). We also acknowledge the support by the Plan Propio from University of Malaga, Campus de Excelencia Internacional de Andalucía

Organisation and control of ascorbate biosynthesis pathway in plants

Ascorbate, also known as vitamin C, plays fundamental roles in human health. However, we are unable to synthesise ascorbate due to a series of accumulated mutations in the last enzyme of the pathway. Therefore, fruits and vegetables become the dietary source of ascorbate, hence being called a vitamin. In plants, ascorbate plays a plethora of roles provinding both biotic and abiotic stress resistance in plants. Although it is known that VTC2 (GDP-L-Galactose Phosphorylase) is the bottleneck of the pathway, little information is available on the basis of how it happens. In this thesis, I confirmed it by several two-by-two combinations as well as expressing the whole pathway. Furthermore, I have shown that this translation fusions are functional and locate in cytosol and nuclei, with the exception of GME and GLDH. Since ascorbate is essential in the response to stress in plants, and the enzymes involved in its biosynthesis locate in the same subcellular region, it was tempting to suggest that they may associate like in other pathways. Our protein-protein association assays suggest that they associate, further supported by the fact that the first and the last cytosolic enzymes immunoprecipitate together, although it deserves further investigation. Since VTC2 was found to be limiting ascorbate biosynthesis, we sought to understand the basis behind this fact. We found that the level of VTC2 expression is very low compared to the expression of the other components of the pathway. However, it was high enough to complement vtc2 phenotype therefore suggesting that keeping its protein amount little expressed in the first control point of the pathway. In addition, we observed that VTC2 is degraded rapidly just after entering the night period, which was further elucidated to be controlled by 26S proteasome

Regulación del contenido en Vitamina C en plantas superiores

Es una comunicación a un Congreso Internacional de primera línea en el campo objeto de la Tesis Doctoral de Mario FenechAscorbic Acid (AsA, vitamin C) has multiple biological roles in plants. Although several pathways for the novo synthesis of AsA have been reported, it is the L-Gal, also known as the Smirnoff-Wheeler (SW), the main pathway operating in photosynthetic tissues in plants. Although there is abundant information of how these genes are regulated at the transcriptional level, little is known about the regulation and the compartmentation of this pathway in higher plants. In order to investigate the localization of dynamics of proteins involved in AsA biosynthesis in vivo at the cellular level, we have generated GFP fusions using the genomic region of the last five genes of the pathway, expected to be localized in the cytoplasm. These constructs have been transformed in Arabidopsis thaliana (Col- 0) plants and generated stable transgenic lines. Detailed localization and dynamics of the proteins are currently being investigated. In parallel, we have analyzed whether these proteins form a complex in planta using co-immunoprecipitation assays using Nicotiana benthamiana. Our data indicate that most protein of the SW pathway show a nucleocytoplasmic localization and that they associate in vivo. Details of the results obtained will be presented.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This research was supported by a grant from the Spanish Ministerio de Educación, Cultura y Deporte para la formación del Profesorado Universitario (FPU014/01974), as well as by a project funded by the Spanish Ministerio de Ciencia e Innovación (BIO2014- 55380R)

REGULATION OF VITAMIN C BIOSYNTHESIS IN HIGHER PLANTS

Ascorbate, also known as vitamin C, plays fundamental roles in biotic and abiotic stress resistance in plants. In green tissues, ascorbate is mainly synthesized through the Smirnoff-Wheeler pathway, whose regulation is not fully understood. Thus, it is necessary a thorough understanding of the regulation of biosynthetic pathway of ascorbate. It is known that VTC2 (GDP-L-Galactose Phosphorylase) is the bottleneck of the pathway, but little information is available on the regulation of the different biosynthetic enzymes at the biochemical and cellular level. Results were presented regarding protein regulation, localization and interaction among different biosynthetic components in Arabidopsis and Nicotiana plants.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This research was supported by a grant from the Spanish Ministerio de Educación, Cultura y Deporte para la formación del Profesorado Universitario (FPU014/01974), as well as by the Ministerio de Economía, Industria y Competitividad (cofinanced by the European Regional Development Fund; grant no. BIO2016-81957-REDT and BIO2017-82609-R). We also acknowledge the support by the Plan Propio from University of Malaga, Campus de Excelencia Internacional de Andalucía