Bloemers, Duncan and Carmo-Silva, Elizabete and Lundgren, Marjorie (2023) Exploring Genetic Diversity in Rubisco Activase. PhD thesis, Lancaster Environment Centre.
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Abstract
Via photosynthesis, plants are the most important point of entry for energy into the biosphere. The Calvin-Benson-Bassham cycle is the part of the photosynthetic process responsible for carbon assimilation, which is done by Rubisco. Rubisco activase (Rca) acts as an activator of Rubisco, removing inhibitory sugar phosphates. Rca is required for the maintenance of photosynthetic activity. Since high temperatures reduce and eventually eliminate Rca activity, this can threaten plant growth. This makes Rca an attractive target for crop improvement. Chapter 1 introduces the literature surrounding Rubisco, Rca, and its applications in crop improvement. It introduces the crop species cowpea (Vigna unguiculata (L.) Walp) and how Rca may be targeted for safeguarding its production under changing climates. Chapter 2 explores the genetic diversity of Rca, and how the natural diversity of Rca may be exploited for crop improvements - as more thermostable Rca may help to protect crop growth from the effects of climate change. First, the high-level diversity of Rca was investigated; tran- scriptomic data for Rca was collected for all species available in the One Thousand Plant Tran- scriptomes Project database. Then, the scope was narrowed to focus on Fabaceae, the family to which cowpea belongs, and identifying candidate species that may have Rca mutations relevant to thermotolerance. Chapter 3 focuses on the expression and purification of recombinant, polyhistidine-tagged Rca. Some isoforms of Rca are challenging to purify and can have solubility issues during or after purification. Factors that can be changed to optimise purity, solubility, and expression levels were investigated. This work paves the way for later work that requires a considerable amount of pure Rca for characterisation of temperature and concentration response. In Chapter 4, the temperature response of cowpea Rca isoforms was evaluated. There are four isoforms of Rca in cowpea, which differed in their temperature response despite limited differences in their protein sequence. Finally, for Chapter 5, bespoke recombinant antibodies were produced targeting individual cowpea Rca isoforms. The antibodies were validated for their avidity and specificity for their target isoforms. The primary application of these antibodies is for western blotting, but potential further applications and research questions are discussed. Furthermore, the advantages and disadvan- tages of recombinant antibody technology compared to animal-derived antibodies are discussed. Chapter 6 evaluates the results shown within, and assesses the possibility of crop improve- ment by targeting Rca. Additional research suggestions are posed to further this work, and sug- gestions for how to improve experimental methods are made. In summary, this study provides a thorough examination of the thermal properties of cowpea Rca. It also improves capacity for future research into this area by optimising recombinant protein expression and purification protocols, and new tools in the form of isoform-specific antibodies.