Abstract
Temperature sensitive mutants have been widely used to study structure, biogenesis and function of a large variety of essential proteins. However, this method has not yet been exploited for the study of photosynthesis. We used negative selection to isolate temperature-sensitive-photoautotrophic (TSP) mutants in Chlamydomonas reinhardtii. From a population of randomly mutagenized cells (n=12,000), a significant number of TSP mutants (n=157) were isolated. They were able to grow photoautotrophically at 25°C, but lacked this ability at 37°C. Further phenotypic characterization of these mutants enabled the identification of three unique and highly interesting mutant strains. Following, the selected strains were genetically characterized by extensive crossing and whole genome sequencing. Correspondingly, the single amino acid changes P628F in the Chloroplast-Elongation-Factor-G (CEF-G), P129L in Phosphoribulokinase (PRK), and P101H in an essential subunit of Photosystem II (PsbO) were identified. These key changes alter the proteins in such way that they were functional at the permissive temperature, however, defective at the restrictive temperature. These mutants are presented here as superb and novel tools for the study of a wide range of aspects relevant to photosynthesis research, tackling three distinct and crucial photosynthetic processes: Chloroplast translation, PET-chain, and CBB-cycle.
Highlights
Photosynthesis is one of the most important reactions on planet earth
We identified single amino acid changes in the chloroplast elongation factor G (CEF-G), phosphoribulokinase (PRK) and an essential subunit of photosystem II (PSII) (PsbO) in TSP1, TSP2 and TSP4 respectively
PSII mediated oxygen production in TSP1, TSP2, and TSP4 was only 30, 30, and 0% compared to the wild type (Figure 1A)
Summary
Photosynthesis is one of the most important reactions on planet earth. It provides the necessary energy and organic building blocks for almost all living beings and it may be critical for the development of sustainable fuel production. It is a very complex process which requires the tight coordination. Temperature-Sensitive CEF-G, PRK, and PsbO Mutants of a large variety of active components. It can be divided in two major steps: the light reactions on the thylakoid membranes and the carbon fixation reactions in the stroma (Taiz and Zeiger, 2002)
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