Date of Award
9-19-2016
Document Type
Dissertation
Degree Name
Molecular Biosciences, Ph.D.
First Advisor
Argelia Lorence
Committee Members
Carole Cramer; Elizabeth Hood; Jianfeng Xu; Maureen Dolan
Call Number
LD 251 .A566d 2016 Y33
Abstract
Vitamin C (L-ascorbic acid, AsA) is the most abundant water-soluble antioxidant in plants. Ascorbate scavenges free radicals, is an enzyme cofactor, and a donor/ acceptor of electrons in the chloroplast. Ascorbate protects tissues against damage caused by reactive oxygen species (ROS) produced through normal metabolism or generated from stress. The inositol route to AsA involves four enzymes: myo-inositol oxygenase, glucuronate reductase, gluconolactonase (GNL), and L-gulono-1,4-lactone oxidase (GulLO). The third enzyme, GNL, has been characterized in rat and bacteria but not in plants. Eighteen putative GNLs were identified in Arabidopsis, one of which, AtGNL, is interesting because it possesses a chloroplastic signal peptide. Chloroplasts can accumulate up to 50 mM AsA but until now no chloroplastic AsA biosynthetic genes have been described. This study includes the characterization of the first plant GNL enzyme in vitro and in planta. Knockouts on this gene had lower foliar AsA and stunted growth compared to controls. The functional gene restored the phenotype of the knockouts, and those plants had higher AsA content, and enhanced photosynthetic capacity. These results highlight the importance of AtGNL in AsA formation and in maintaining a healthy redox balance in the leaves particularly under low light stress. AtGNL is the first AsA biosynthetic enzyme that resides in chloroplasts. The production of complex recombinant protein in plants is a rapidly expanding area of plant biotechnology. The higher metabolic flux linked with recombinant protein expression, subjects the host plant to significant stress that disrupts redox homeostasis during protein expression. Chronic alterations in the redox status of plants may be detrimental to the performance of cellular processes including protein translation, protein folding in the endoplasmic reticulum, transfer of proteins between organelles, and export of secreted proteins from cells. Our previous results showed that Arabidopsis crosses between high AsA and human interleukin 12 (hIL-12) over-expressers can accumulate 70% more hIL-12 compared to controls. This work focuses on tobacco, the preferred platform for recombinant protein expression. This study showed that crosses between GulLO and hIL-12 over-expressers led to plants with enhanced AsA, reduction of ROS, and up to 5-fold higher hIL-12 yields in multiple tobacco generations.
Rights Management
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Yactayo-Chang, Jessica Patricia, "The Role of the Chloroplastic and Endoplasmic Reticulum Ascorbate Subcellular Pools in Plant Physiology" (2016). Student Theses and Dissertations. 660.
https://arch.astate.edu/all-etd/660
